Table of Contents

Acknowledgements 1

Agenda 2

Abstracts 4

Note Pages 21

Banquet / Duling Hall Information 25

UMMC Campus Map 26

Organizing Committee Meeting Secretariats

Conference Organizers

Michael Ryan, Ph.D. (Chair) Stephanie Lucas Heather Drummond, Ph.D. Courtney Graham Christine Maric, Ph.D. David Stec, Ph.D.

Abstract Software Developer Audio Visual Support Leland D. Husband G. Brent Shows

The Gulf Coast Physiology Society would like to gratefully acknowledge the generous financial support provided by our sponsors and educational grants from:

The Department of Physiology and Biophysics

University of Mississippi Medical Center

Center for Excellence in Cardiovascular Renal Research Hypertension and Cardiorenal Diseases Research Training Program

University of Mississippi Medical Center

University of Mississippi Medical Center Faculty Scholarship Exchange Award

The American Physiological Society

Data Sciences International

Harlan Laboratories, Inc.

Kent Scientific Corporation

Phoenix Technical Services, Inc.

Fisher Scientific

Special thanks to the staff of the Norman C. Nelson Student Union University of Mississippi Medical Center

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Meeting Agenda

Friday, May 20, 2011 11:00 a.m.-5:00 p.m. Registration Open 2:00 p.m. Welcome and Introduction of Keynote Speaker 2:10 p.m. Douglas C. Eaton, Ph.D. Distinguished Professor and Chair of Physiology Emory University ENaC Regulation and Dale Benos: A Man Before His Time 3:00 p.m. Break Oral Session I

- Moderator, Richard Wainford (LSUHSC-NO)

Invited Talks 3:15 p.m. Jason Gardner, LSUHSC-NO Lysyl Oxidase, Dilatation and Dysfunction in the Volume Overloaded Heart 3:30 p.m. Anna Thalacker-Mercer, UAB

A Molecular Signature of Resistance Training-Induced Human Skeletal Muscle Hypertrophy 3:45 p.m. Khalid Matrougi, Tulane

Natural Regulatory T Cells Control Coronary Arteriolar Endothelial Dysfunction in Hypertensive Mice

Selected Abstracts 4:00 p.m. Lusha Xiang , UMMC

Impaired Blood Pressure Compensation to Hemorrhage in Obese Zucker Rats with Orthopedic Trauma

4:15 p.m. Jerry Brunson, LSU-Shreveport Cytomegalovirus Promotes Microvascular Dysfunction through Alterations in the

Cyclooxygenase Pathway of Arachidonic Acid Metabolism 4:30 p.m. Flavia Souza-Smith, LSUHSC-NO Acute Alcohol Intoxication Increases the Magnitude of Phasic Ca2+ Transients in Rat

Mesenteric Collecting Lymphatics 4:45 p.m. Break 5:00 p.m. GCPS Business Meeting 6:30 p.m. Banquet

Duling Hall 622 Duling Ave Jackson, MS

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Saturday, May 21, 2011 8:00-10:00 a.m. Poster Session

Breakfast 10:00 a.m. Break Oral Session II

- Moderator, Keisa Mathis (UMMC)

Invited Talks 10:15 a.m. Karen Stokes, LSU-Shreveport Cytomegalovirus Infection: Silently Activating the Microvasculature 10:30 a.m. Xiangming Zha, USA Maturation and Trafficking of Acid-Sensing Ion Channel 1a 10:45 a.m. Jan Williams, UMMC The Progression of Diabetes-Induced Renal Injury in Dahl Salt-Sensitive Rats Selected Abstracts 11:00 a.m. Nupur Dey, USA N-terminal Lysine Residues Target Cyclic GMP Dependent Protein Kinase for Ubiquitin-

Proteasome Mediated Degradation 11:15 a.m. Torrance Green, Tulane Opposite COX-2 Inhibition Effects on Blood Pressure and Renal Hemodynamic during Early and

Late Angiotensin II-infused Hypertension 11:30 a.m. Christopher Cottingham, UAB The Antidepressant Desipramine is an Arrestin-Biased Ligand at the Alpha2A Adrenergic

Receptor Driving Receptor Internalization and Downregulation 11:45 a.m. Jessica Bradley, LSUHSC-NO

Exposure to Tobacco Smoke Accelerates Volume Overload Induced Cardiac Remodeling and Dysfunction

12:00 p.m. Announcement of Poster Awards 12:30 p.m. Meeting Adjourns

Boxed lunches provided

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Abstracts

1 Thermosensitive Synaptic Transmission In The Brainstem Anwar IJ, Derbenev AV, Neuroscience Program, Tulane University, New Orleans, Louisiana Department of Physiology, Tulane University, New Orleans, Louisiana. Temperature sensing is vital for the maintenance of thermal homeostasis. TRP receptors are present ubiquitously in the body including the central nervous system, however their functional role in the brain is unclear. We used whole-cell patch-clamp recordings from neurons of the dorsal motor nucleus of the vagus (DMV) to test our hypothesis that temperature plays an important role in the regulation of excitatory synaptic transmission. Increase of temperature from 24 °C to 38 °C increased the frequency of action potentials. This increase was followed by an inactivation at 38 °C. The action potential frequency returned to control level after decreasing the temperature back to 24 °C. In the presence of tetrodotoxin (1μM) the rise of temperature from 24 °C to 38 °C increased excitatory neurotransmission. The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) increased significantly and an inward current was observed. The temperatures below 38 °C substantially reduced mEPSC frequency, indicating that excitatory synapses are sensitive to temperature changes in the DMV. This work was supported by NIH NHLBI R21HL091293, R21HL091293-01A1S1.

2 VEGF-C-mediated Changes in the Lymphatic Endothelial Cell Proteome Marisa Belaidi, Jesse Guidry, Chau-Wen Chou, and Jerome W. Breslin Departments of Physiology and Pharmacology, Louisiana State University Health Sciences Center, New Orleans, LA 70112 Vascular endothelial growth factor-C (VEGF-C) is a pro-lymphangiogenic factor, playing an important role in the formation of new lymphatic vessels. We hypothesized that stimulation of lymphatic endothelial cells with VEGF-C will cause upregulation of proteins that are key for lymphatic endothelial cell proliferation and migration. Human dermal lymphatic endothelial cells (HMLEC-d, passage 6) were grown to confluence in growth medium and were then starved in serum-free overnight. The cells were then treated with 10 nM VEGF-C or vehicle for 4h. Cell pellets were obtained by trypsinization and centrifugation and were delivered to the LSUHSC-NO Proteomics CORE facility for 2-D gel analysis. This analysis revealed 25 spots that consistently displayed at least a 1.5-fold change (increase or decrease in expression) between the VEGF-C and vehicle groups. The spots were cut from the gel and analyzed by mass spectrometry, and the results revealed three candidate proteins altered by VEGF-C: Programmed cell death 6-interacting protein (PDC6IP; downregulated 2.3-fold), transitional endoplasmic reticulum ATPase (TERA or VCP, downregulated 2.1 fold), and Eukaryotic translation initiation factor 3 subunit 1 (EIF3I; upregulated 2.8-fold). All three proteins have known functions with protein expression at different levels and have potential roles in cell division and apoptosis. Future studies are aimed at verifying these results by Western blot and identifying the specific lymphatic endothelial cell functions impacted by these proteins. Supported by a NIH grant P20RR018766 and a grant from the American Heart Association (0835388N).

3 Exposure to Tobacco Smoke Accelerates Volume Overload Induced Cardiac Remodeling and Dysfunction J.M. Bradley and J.D. Gardner LSU Health Science Center Cigarette smoking is an independent risk factor for heart disease and is linked to sudden cardiac death and accelerated decompensation. In this study, we examine the effects of cigarette smoke inhalation on the stressed heart. Our hypothesis is that cigarette smoke (CS) exacerbates cardiac injury by accelerating ventricular remodeling and dysfunction. Volume overload (VO) stress was surgically induced in rats by an abdominal aortocaval shunt. Male rats, with and without VO, were exposed to either room air or CS (6 cig/day) for six weeks. Echocardiogram measurements taken at baseline and weekly indicated CS exposure had significantly increased LV internal diameter (10.5 ± 0.2 vs 9.6 ± 0.2 mm), decreased posterior wall thickness (0.82 ± 0.04 vs 0.98 ± 0.05 mm), and reduced ejection fraction (63 ± 2 vs 68 ± 2 %) compared to VO rats exposed to room air. The morphological analysis of ventricular collagen using Picrosirius Red staining revealed that the VO animals exposed to CS had significantly decreased interstitial collagen compared to those exposed to room air (0.92 ± 0.11 vs 1.63 ± 0.17 %total LV area). Western blot analysis of MMP-2 expression depicted no change between groups. However, CS significantly increased the expression of MMP-9 and TIMP-1 in the VO animals compared to VO exposure to room air. These animals also showed decreased levels of ET- 1 as well as reduced expression in HIF-1, VEGF, and TGF- in the heart. These data indicate that CS inhalation worsens VO-induced cardiac injury by accelerating collagen loss and impairing collagen production, thereby promoting eccentric dilation and dysfunction.

4 Cytomegalovirus Promotes Microvascular Dysfunction through Alterations in the Cyclooxygenase Pathway of Arachidonic Acid Metabolism Jerry Brunson and Karen Y. Stokes Department of Molecular & Cellular Physiology, Louisiana State University Health Science Center, Shreveport, LA 71130 Cytomegalovirus (CMV) is a b-herpes virus that has been implicated in cardiovascular disease. Like other cardiovascular risk factors, CMV infection leads to impaired arteriolar vasodilation to acetylcholine (ACh) before large vessel disease is apparent. Furthermore, this virus exacerbates hypercholesterolemia (HC)-induced arteriolar dysfunction. We previously found that indomethacin, a cyclooxygenase (COX) inhibitor, reduced vasodilation in mock-inoculated mice but not in CMV-infected mice. We hypothesized that CMV and CMV+HC elicit microvascular dysfunction by altering the balance of COX-1 and COX-2 enzymes, and therefore their metabolites. To test this, C57Bl/6J mice were infected with CMV or mock inoculum. HC diet was introduced at 5 wks post-infection (p.i.). At 11wks p.i., intravital microscopy was performed to measure arteriolar vasoreactivity to ACh in the cremaster muscle. A specific COX-2 inhibitor (NS-398) impaired vasodilation in Mock mice, but not in either CMV group. In contrast, SC-560 inhibition of COX-1 had no effect on mocks but partially restored vasodilation in CMV mice. Our findings indicate that mCMV disrupts the balance of AA metabolites produced via COX, and suggest that mCMV infection leads to enhanced generation of vasoconstrictors from COX-1 and/or reduced COX-2-derived vasodilators. Future work will identify the metabolites involved in this process. (Supported by NIH P20RR018724 and AHA 0730294N)

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5 Genetic Basis of Altered Myogenic Response and Renal Injury in FHH RatsY M. Burke, M. Pabbidi, J.M. Williams, R. Liu, Y. Ge, J. Lazar, H.J. Jacob, and R.J. Roman Dept. of Pharmacology and Physiology, UMMC, Jackson, MS 39216 and Human and Molecular Genetics Center, MCW, Milwaukee, WI 53226 This study examined the effect of substitution of a 2.6 Mb segment from Brown Norway (BN) rat Chr 1 into the Fawn Hooded Hypertensive (FHH) rat genetic background on autoregulation of renal blood flow (RBF) and development of renal injury. FHH rats exhibited poor autoregulation of RBF and GFR rose by 60% when renal perfusion pressure was increased from 100 to 150 mmHg while FHH.1BN 3.4 and 2.6 Mb congenic strains exhibited perfect autoregulation of RBF. Protein excretion rose from 51±5 to 259±41mg/day in 9 to 21 week FHH rats but only increased to 147±24 mg/day in FHH.1BN 2.6 Mb congenic strain. Diameter of isolated perfused afferent arterioles from FHH rats did not change significantly when transmural pressure was increased from 60 to 140 mmHg. In contrast, diameter of the 2.6 Mb congenic decreased 19.4% with the same pressure increase. Patch clamping experiments indicate that large conductance Calcium activated K+ channel (BK) current was 5 fold greater in vascular smooth muscle (VSM) cells isolated from renal vessels of FHH rats than the 2.6 Mb congenic strain. Pressure induced myogenic constriction of afferent arterioles is repaired in FHH arterioles incubated with Iberiotoxin, a BK channel blocker. This data indicates that the gene responsible for restoring autoregulation of RBF lies within this 2.6 Mb region containing 11 genes and transfer of this region restores the myogenic response and slows the progression of renal disease. The impaired myogenic response is likely due to hyperpolarization of VSM cells due to increased BK channel activity. NIH HL36279, DK079306, HL69321 and 1T32HL105324-01

6 Functional Comparison of Ammonium Transport by Renal Rhbg and the Erythrocyte RhAG Tolga Caner, Solange Abdulnour-Nakhoul, Karen Brown, L. Lee Hamm and Nazih L. Nakhoul Depts of Medicine and Physiology, Tulane Medical school, New Orleans, LA The erythroid Rh protein (RhAG) is one component of the “Rh complex” that is mostly known for its immunogenic characteristics. Other functions of RhAG, including a role in transport of ions have been recently proposed. Renal Rhbg is a non-erythroid Rh protein that is involved in ammonium transport. In this study, we measured ammonium transport by RhAG expressed in Xenopus oocytes and compared it to Rhbg & H2O injected oocytes. To assess transport we used ion-selective microelectrodes to measure ammonium and methyl ammonium induced pHi changes. In oocytes expressing RhAG, 5mM NH4Cl (NH3/NH4+) decreased pHi by 0.16±0.02 at a rate of -14.2±0.01 x10-4 pH/sec and depolarized the cell by 16±1.0 mV. On the other hand, 5 mM methyl ammonium hydrochloride (MA/MA+) increased pHi by 0.07±0.007 at a rate of 4.7±0.7 x10-4 pH/sec and depolarized the cell by 8.6±0.96 mV. In Rhbg oocytes, 5mM NH4Cl decreased pHi by 0.17±0.03 at a rate of -15±2.7 x10-4 pH/sec and depolarized the cell by 38±3.9 mV. Also in Rhbg oocytes, 5mM MA/MA+ increased pHi by 0.1±0.02 at a rate of 11.2±1.8 x10-4 pH/sec & depolarized the cell by 39±3.7 mV. In H2O-injected oocytes the changes were much smaller: 5 mM NH3/NH4+ decreased pHi by only 0.07±0.007 at a rate of 6.2±1.0 x10-4 pH/sec and depolarized the cell by 18±3.8 mV; 5 mM MA/MA+ did not elicit any change in pHi or Vm. These data indicate: 1) RhAG transports NH3/NH4+ in a manner that resembles Rhbg. 2) NH3/NH4+ induced pHi changes by RhAG are comparable to those by Rhbg however the voltage change is larger in Rhbg than in RhAG. 3) MA/MA+ transport by RhAG is significantly smaller than by Rhbg.

7 Overexpression of Angiopoietin-2 Promotes Myocardial Rarefaction and Fibrosis in Diabetic DB/DB Mouse Model Jian-xiong Chen University of Mississippi Medical Center Capillary rarefaction leading to microvascular insufficiency represents major causes of end-organ failure among diabetes. We investigated the potential roles of diabetic-induced excess angiopoietin-2 (Ang-2) on cardiac fibrosis and capillary rarefaction in db/db mouse hearts. The db/db mice were intra-myocardial administration of adenovirus Ang-2 or Ad-β- gal. Myocardial Ang-2, Tie-2 phosphorylation, caspase-3, expression were measured. Myocardial apoptosis, capillary density, and cardiac fibrosis were analyzed in the db/db mouse hearts. Intra-myocardial administration of Ad-Ang-2 led to an increase in Ang-2 expression and a reduction of Tie-2 phosphorylation in db/db mouse hearts compared to control Ad-β-gal treated db/db mice. Immunehistochemical staining of Ad-Ang-2 treated db/db mouse hearts showed that Ang-2 was expressed in endothelial cell and co-localized with caspase-3. Further, forced overexpression of Ang-2 led to a dramatic increase in apoptosis together with reduction of myocardial capillary density at day 14. Forced overexpression of Ang-2 also resulted in significant increases in cardiac fibrosis and cardiac hypertrophy. These findings were further confirmed by exposure of endothelial cells to Ang-2 resulted in increases in caspase-3 activity as well as endothelial apoptosis. Taken together, our data show that increased Ang-2 in diabetic hearts promotes endothelial cell apoptosis as well as cardiac fibrosis, thus lead to myocardial rarefaction. Suppression of Ang-2 may represent a potential novel therapeutic strategy for the treatment of the diabetes-associated rarefaction and cardiac dysfunction.

8 Elk-1 Sumoylation is Involved in Cyclic GMP-dependent Protein Kinase (PKG)- induced Smooth Muscle Gene Expression in Rat Aortic Smooth Muscle Cells ChungSik Choi, Hassan Sellak, Thomas M. Lincoln Dept. of Physiology, College of Medicine, University of South Alabama, Mobile, AL 36688 In vascular smooth muscle cells (VSMC), NO/cGMP/PKG is associated with many cellular events including gene expression. It has been described that the inhibition of transcription factor, Elk-1 by sumoylation induces muscle specific gene expression. In this study, we report that PKG induces the sumoylation of Elk-1, thus increasing the expression of SM specific genes in cultured VSMC. PKG overexpression in VSMC showed 2~3-fold more myocardin-induced SM22 promoter activity and increased levels of higher molecular weight species of phospho-Elk-1 (pElk-1) compared to control cells. A higher molecular weight species was identified as a sumolylated pElk-1, which was completely abolished by pElk-1 peptide (4ug/ml) or PDGF (10 ng/ml) treatment. Furthermore, siRNA-mediated suppression of PKG or transfection of dominant negative sumo-conjugating enzyme, DNUbc9, reduced the level of sumoylated pElk-1, confirming that sumoylation of Elk-1 was mediated by PKG. This repressive PKG effect on the Elk-1 was further investigated using luciferase, gel-shift, chromatin immunoprecipitation assays. Our results showed that PKG suppressed Elk-1 inhibition of SM22 reporter gene expression in COS-7 cells in addition of reducing Elk-1 binding to the CArG-box motif of SM22 and SM-MHC promoters. Taken together, these results suggest that PKG increases smooth muscle specific gene expression through inhibition of the SRF cofactor, Elk-1, thereby allowing the access of the muscle specific cofactor, myocardin to the SMC-specific gene promoters.

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9 Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase and ERK1/2 MAP-Kinase Enhances Ischemia-Induced Neovascularization in Type 2 Diabetic Mice Sookyoung Choi, Maria Galan, Megan Partyka, Khalid Matrougui Department of Physiology, Hypertension and Renal Center of Excellence, Tulane University, 1430 Tulane Ave., New Orleans, LA, 70112 Background: In this study, we determined the inhibition of EGFR and ERK1/2 could rescue the neovascularization and subsequent blood flow recovery in the ischemic hind-limb of type 2 diabetic mice. Methods and Results: Femoral artery ligation-induced ischemia was performed in all the mice for 4 weeks and then followed by the treatment with EGFR or ERK 1/2 inhibitors (AG1478 and U0126) for 3 weeks. After 4 weeks of ligation, control mice displayed 100% in blood flow recovery compared to 60 % in diabetic mice. Mice were then treated with AG1478 or U0126 for 3 weeks. Interestingly, diabetic mice treated with inhibitors displayed a significant blood flow recovery compared to non-treated diabetic mice. X-ray data revealed diabetic mice displayed lower vessels density than control mice. Importantly, AG1478 or U0126 enhanced vessel density in diabetic mice compared to nontreated diabetic mice. Fluorescent microscopic images showed a reduction in capillary density in diabetic mice compared with control, which was reversed by EGFR and ERK1/2 inhibitors. We demonstrated that cGMP was reduced in diabetic mice compared to control mice and the treatment with AG1478 or U0126 significantly enhanced cGMP level in ischemic hind-limb of diabetic mice. Western blot analysis revealed increases in EGFR, and ERK1/2 phosphorylation associated with a decrease in eNOS phosphorylation and VEGF expression in diabetic mice, which were reversed by AG1478 or U0126. Conclusion: Our data provide the evidence that the inhibition of EGFR and ERK1/2 enhances ischemia-induced neovascularization and subsequent blood flow recovery in type 2 diabetic mice.

11 The Antidepressant Desipramine is an Arrestin-biased Ligand at the Alpha2A Adrenergic Receptor Driving Receptor Internalization and Downregulation Christopher Cottingham, Yunjia Chen, and Qin Wang Department of Physiology & Biophysics, University of Alabama at Birmingham Birmingham, Alabama 35294 The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as a norepinephrine (NE) reuptake inhibitor. Evidence suggests a mechanism more complex than simple reuptake inhibition, involving regulation of noradrenergic neurotransmission. While it has been reported that DMI can bind directly to the α2A adrenergic receptor α2AAR), a receptor with altered expression and function in depression, functional consequences of this binding and implications for the antidepressant mechanism of action are not clear. In this study, we examined this direct DMI α2AAR interaction in detail. Radioligand binding analysis revealed the intrinsic affinity of the α2AAR for DMI and identified the binding site as orthosteric with classic receptor ligands. DMI was neutral with regard to α2AAR-evoked heterotrimeric G protein-mediated signaling. However, DMI was found to drive α2AAR internalization following acute stimulation, and subsequently downregulation of α2AAR expression and signaling following prolonged stimulation. The effect of DMI on receptor trafficking are critically dependent on arrestins. Given that a physiological concentration of the endogenous agonist NE did not sustain a downregulation response, we contend that direct DMI-driven α2AAR downregulation contributes to the alterations in receptor expression levels previously associated with this antidepressant but ascribed to the action of elevated NE. Our results provide novel insight into the pharmacology of this antidepressant drug.

12 Effect of Chronic Cobalt Proroporphyrin(CoPP) Treatment on Body Weight and Metabolism in Melanocortin 4-receptor Knockout Mice Eva Csongradi, Jussara M. doCarmo, John H. Dubinion, Trinity Vera, David E. Stec University of Mississippi Medical Center Heme oxygenase-1 induction (HO-1) elicits chronic weight loss in several rodent models of obesity. In this study, we investigated the mechanism of weight loss elicited by chronic HO-1 induction in a model of genetic obesity due to melanocortin-4 receptor (MC4R) deficiency. Experiments were performed on loxTB MC4R deficient mice as well as lean controls. Mice were administered cobalt protoporphyrin (CoPP, 5 mg/kg), an inducer of HO-1, once weekly from 4 to 23 weeks of age. Body weights were measured weekly and fasted blood glucose and insulin as well as food intake were determined at 18 weeks of age. O2 consumption, CO2 production, activity, and body heat production were measured at 20 weeks of age. Chronic CoPP treatment resulted in a significant decrease in body weight from 5 weeks on in loxTB mice. Chronic CoPP treatment resulted in a significant decrease in fasted blood glucose levels, plasma insulin, and a significant increase in plasma adiponectin levels in MC4R deficient mice. Chronic CoPP treatment increased O2 consumption (47 ± 4 vs. 38 ± 3 ml/kg/min, P<0.05) and CO2 production (44 ± 7 vs. 34 ± 4 ml/kg/min, P<0.05) in treated versus non-treated, MC4R deficient mice (n=4). Heat production (10%) and activity (18%) were also significantly (P<0.05) increased in CoPP treated MC4R deficient mice. Our results suggest that chronic HO-1 induction with CoPP induction elicits weight loss by increasing metabolism and activity by an MC4R independent pathway.

10 ASIC-like Currents in Freshly Isolated Cerebral Artery Smooth Muscle Cells are Inhibited by Endogenous Oxidase Activity Wen-Shuo Chung, Jerry Farley, Heather Drummond University of Mississippi Medical Center Acid Sensing Ion Channels (ASICs) are a novel class of neuronal cation channels gated by extracellular [H+]. We recently identified the presence of ASIC-like channels in cerebral vascular smooth muscle cells (cVSMCs). In most isolated cVSMCs, however, ASIC-like channels are electrically silent. Since neuronal ASICs are inhibited by oxidizing agents, we considered the hypothesis that cVSMC ASIC currents are inhibited by oxidative stress present in VSMCs. To test this hypothesis, we used whole-cell patch clamp. We first examined the effect of reducing agents on extracellular [H+] gated currents in isolated mouse cVSMCs. Pretreatment with 2 mM DTT increased the amplitude of the current evoked by pH 6.0 from 0.4±0.1 to 14.9±3.6 pA/pF, p<0.05. The potentiated currents were inhibited by the ASIC blocker amiloride (100 μM). The effect of reducing agents was reversed by the oxidizing agent DTNB. Moreover, apocynin (50 μM), an NADPH oxidase (NOX) inhibitor, increased current amplitude evoked by pH 6.0 (0.9±0.5 to 7.0±2.6 pA/pF, p<0.05). Treatment with allopurinol, a xanthine oxidase (XO) inhibitor, potentiated the ASIC-like activity similarly. These findings demonstrate that reducing agents unmask electrically silent VSMC ASIC-like channels, which may be suppressed by endogenous NOX and XO. The role of VSMC ASIC channels in oxidative stress related vascular dysfunction remains to be determined.

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Jackson Cardiovascular-Renal Meeting 2000

CENTER FOR EXCELLENCE IN CARDIOVASCULAR-RENAL RESEARCH THE UNIVERSITY OF MISSISSIPPI MEDICAL CENTER

JACKSON MISSISSIPPI

13 Postnatal Rapid Growth in Low Birth Weight Newborn is Associated with Increased Visceral Adipose Tissue in Adulthood JH Dasinger1, L Tull2, H Zhang2, L Bufkin3, J Martin3, P Rhodes1, BT Alexander2, and NB Ojeda1,2 Pediatric Department1, Physiology Department2, Maternal Fetal Medicine3 University of Mississippi Medical Center, Jackson, MS Several studies suggest that accelerated catch up growth in low birth weight babies may induce increased visceral adipose tissue (VAT) in adulthood, yet the exact mechanism is not clear. The objective of this translational study was to determine whether changes in plasma leptin, a hormone with actions on the brain, were associated with altered postnatal growth in low birth weight (LBW) offspring. The clinical protocol used a prospective observational cohort study in newborn babies; the experimental protocol used a rodent model of LBW induced by placental insufficiency. Human LBW babies exhibited an accelerated postnatal growth when compared with normal birth weight (NBW) babies within 3 weeks of postnatal life. (15±8 vs. -56±30 g; P<0.05, LBW vs. NBW; respectively). Moreover, a negative correlation for plasma leptin with post-natal weight gain was observed in LBW (Spearman r= -0.6734). Rodents LBW offspring also demonstrated accelerated postnatal growth compared to NBW offspring within 8 weeks of postnatal life (6±0.8 vs. 4±0.9 g; P<0.05, LBW vs. NBW; respectively). Furthermore, a negative correlation was observed for plasma leptin levels at birth with postnatal growth in LBW rodents (Pearson r= -0.9454). In the experimental model, LBW rodents demonstrated an increased VAT at 6 months of age relative to NBW (23±3 vs. 36±5 g/K; P<0.05, NBW vs. LBW; respectively). Thus, the parallelisms between human and animal studies suggest that plasma leptin levels at birth are associated with accelerated postnatal growth which may contribute to increased deposition of visceral adipose tissue in adult LBW offspring.

14 N-terminal Lysine Residues Target Cyclic GMP Dependent Protein Kinase for Ubiquitin-proteasome Mediated Degradation Nupur B. Dey and Thomas M. Lincoln Department of Physiology, University of South Alabama, Mobile, AL-36688 Cyclic GMP-dependent protein kinase-1 (PKG-1) mediates nitric oxide (NO) and hormone dependent smooth muscle relaxation and stimulates smooth muscle cell-specific gene expression. PKG-I expression in cultured smooth muscle cells (SMC) is inhibited by inflammatory cytokines such as interleukin-I and tumor necrosis factor-α, which are known to stimulate Type II NO synthase (iNOS) with subsequent elevation of cGMP. Chronic elevation of cGMP triggers ubiquitination and proteasomal degradation of PKG-I in SMC. We reported that the suppression of expression of the 1α but not the Iβ isoform of PKG in SMC is triggered by the ubiquitin/26S proteasome pathway. PKG catalytic activity was required for this process. Mutation of the known autophosphorylation sites of PKG-Iα to alanine uncovered a specific role for autophosphorylation of serine-64 in cGMP-dependent ubiquitination and suppression of PKG protein. To determine the involvement of specific E3 ubiquitin ligases for this process, we observed that PKG-1α forms a high MW complex with ubiquitin E3 ligases cCbl and MDM2. We extended those studies to pin point the participation of specific lysine(s) in the ubiquitination process. Replacement of several Nterminal lysine(s) with arginine by site directed mutagenesis indicated that lysine-37 and lysine-39 are the candididate residues that are ubiquitinated and that plays an important role in PKG degradation and ubiquitination. Our results suggest that ubiquitination of PKG1-α is triggered by serine-64 autophosphorylation, subsequent binding of the E3 ligase cCbl or MDM2 and the ultimate ubiquitination of lys-37 and lys-39

15 Cell-permeable (CP)-Rnd3 Protein Reduces Hemorrhagic Shock/Resuscitation (HS/R)-induced Microvascular Leakage in the Rat Mesentery Travis M. Doggett, Kristine M. Kurtz, Jesse K. Sulzer, Annie F. Whitaker, Marisa B. Belaidi, Flavia M Souza-Smith, Patricia E. Molina, and Jerome W. Breslin. Department of Physiology, LSUHSC School of Medicine, New Orleans, LA 70112 Microvascular hyperpermeability is a significant clinical problem in trauma medicine. The underlying mechanisms involve formation of actin stress fibers in endothelial cells, leading to increased stress on intercellular junctions. We tested the hypothesis that Rnd3, which inhibits actin stress fiber formation, can inhibit hemorrhagic shock/resuscitation (HS/R)- induced microvascular leakage. We used a conscious, unrestrained rat fixed-pressure HS/R model (40 mm Hg for 1 h, followed by Lactated Ringers, 40% total blood removed (TBR) i.v. bolus + 2 X TBR over 1 h) or shams, after which the rats were anesthetized for intravital microscopy of the mesenteric microcirculation. FITC-albumin (i.v.) served as a tracer of extravasation. Integrated optical intensity (IOI) of extravasated FITC-albumin was measured. The mesentery was treated with either a cell permeable (CP)-Rnd3 protein or vehicle. The results show that HS/R caused a marked increase in IOI over time (mean IOI 110 ± 34 at 60 min. vs. 20 ± 17 at BL, P<0.05) which was not observed in sham animals. CP-Rnd3 treatment attenuated the rise in IOI (mean IOI 35 ± 14 at 60 min. vs. 21 ± 9 at BL, no difference). These data suggest that Rnd3 can resolve HS/R-induced microvascular hyperpermeability in vivo. This novel strategy could potentially improve clinical management of trauma patients. Supported by NIH P20RR018766 and a grant from the American Heart Association.

16 Metabolic Changes in Mice with Forebrain Stat3 Deletion Compared to Targeted Deletion of Stat3 in POMC Neurons John Dubinion, Ahmad Adi, and John E. Hall Department of Physiology & Biophysics, University of Mississippi Medical Center, Jackson, MS Signal transducer and activator of transcription 3(Stat3) is important in leptin-mediated energy homeostasis. Recent studies suggest that neurons in addition to proopiomelanocortin (POMC) neurons may play an important role in leptin-mediated energy balance. The purpose of the current study was to test whether Stat3 deletion in forebrain neurons would have a greater impact on the metabolic phenotype compared with Stat3 deletion only in POMC neurons. Stat3flox/flox mice were crossed with POMC-Cre and CamKαL7-Cre mice to generate mice with Stat3 deletion in either POMC neurons or the entire forebrain. Body weight was measured in each group of mice (Stat3flox/flox(S); Stat3flox/flox/POMC-Cre(SP); Stat3flox/flox/CamKαL7-Cre(SC)) from weaning. At 8-10 weeks of age, each mouse was placed into metabolic cages to determine VO2, VCO2, motor activity, heat production and food intake 24hrs/d for 3 days. SP and SC were heavier than S (28.6±1.4 & 31.1±1.4 vs. 23.5±0.5 g), and were hyperphagic (4.4±0.3 & 4.6±0.5 vs. 3.5±0.2 g). Respiratory quotient was different in each group with SP being the highest and SC being the lowest (0.895±0.011 vs. 0.847±0.023 vs. 0.791±0.029). Motor activity was increased in both SP and SC (0.18±0.03 & 0.15±0.02 vs. 0.09±0.01 km/day), and heat production was increased (525.1± 69.3 & 619.5±39.8 vs. 490.9±58.0 cal/hr) compared to S. SP and SC also had an attenuated compensatory increase in food intake after 24 hr fasting (6.4 & 19.7 vs. 50.2 %). These results suggest that inactivation of Stat3 in forebrain neurons induces a greater metabolic disruption than in POMC neurons alone. (NHLBI PO1 HL51971)

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17 Generation of CYP4A1 Transgenic Rats Using Sleeping Beauty Transposon System Fan Fan¹, Howard Jacob², Aron Geurtz², Sydney Murphy¹, Richard J. Roman¹. ¹Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS; ²Human and Molecular Genetics Center , Medical College of Wisconsin, Milwaukee, WI Cytochrome P450 enzymes of the 4A family that catalyze the formation of 20-HETE have been implicated in the regulation of renal function, vascular tone on the long term control of blood pressure. To better determine the role of CYP4A1 in the development of hypertension and renal and vascular dysfunction, we utilized the Sleeping Beauty (SB) transposon system to generate CYP4A1 transgenic rats. Rat full length CYP4A1 cDNA was replaced eGFP in pT2.CAG.eGFP transposon plasmid containing a hybrid chicken β- actin promoter fused with elements of the CMV enhancer (CAG). Pronuclei of the fertilized eggs collected from Dahl SS female rats are microinjected with a circular SB transposonplasmid construct along with transposase capped RNA transcripts and incubated overnight. Then 2-cell staged oocytes were transferred into the oviducts of pseudopregnant foster Dahl SS females. Transgenic founders were identified by PCR using both CYP4A1 and transgene-specific primers. The insertion sites were checked by Ligation-mediated PCR and three-primer PCR. Heterozygous founders were backcrossed to Dahl SS rats and the progeny were brother sister mated to derive two homozygous-transgenic lines. The production of 20-HETE in the renal cortex is elevated by 3 fold in the CYP4A1 transgenic line in comparison to SS rats. These animals will be in exploring the role of CYP4A1 in the pathogenesis of hypertension and renal and cardiovascular end organ damage. HL36279 and HL29587

18 Aldosterone Blunts Tubuloglomerular Feedback by Activating Mineralocorticoid Receptors on the Macula Densa Yiling Fu, John E. Hall, Lin Lin, Celso E. Gomez-Sanchez, Luis A. Juncos, Ruisheng Liu University of Mississippi Medical Center, G.V.(Sonny) Montgomery VA Medical Center We hypothesize that mineralocorticoid receptors (MR) are expressed in the macula densa (MD) cells and that activation of MR in the MD influences glomerular filtration rate (GFR) via tubuloglomerular feedback (TGF). MR expression was determined by immunohistochemistry with a specific antibody we developed. To investigate the effects of aldosterone on TGF in vitro, we microdissected rabbit juxtaglomerular apparatus and perfused the afferent arteriole (Af-Art) and distal tubule simultaneously. TGF was assessed by switching the tubular perfusion solution from 10 to 80 mM NaCl. Under control conditions, tubular perfusate with 80 mM NaCl decreased Af-Art diameter from 18.7 ± 0.3 to 16.1 ± 0.4 μm (TGF= 2.7 ± 0.2 μm). In the presence of aldosterone (10-8M), TGF was 1.41 ± 0.4 μm (p<0.05). This effect was blocked when the MR antagonist eplerenone (10-5M) was added to the tubular perfusate. To determine the role of nitric oxide (NO), we inhibited nNOS activity and measured TGF. In the presence of 7-NI (10-5M) in the tubule, TGF was 4.0 ± 0.3 μm. When we added aldosterone to tubular perfusate in the presence of 7-NI, TGF was 2.4 ± 0.4 μm. To investigate the effect of aldosterone on TGF in vivo, we applied micropuncture and measured stop flow pressure (Psf) in SD rats. TGF was determined by maximal changes of Psf (ΔPsf ) when tubular perfusion rate increased from 0 to 40 nl/min. Aldosterone (10-7M) decreased ΔPsf from 10.1 ± 1.4 to 7.7 ± 1.2 mmHg, and the effect was blocked in the presence of eplerenone in tubular perfusate. Conclusion: aldosterone-induced attenuation of TGF is mediated by MR in MD and modulated partially by nNOS.

19 Chronic ER Stress Inhibition Improves Microvascular Function in type 1 Diabetes Mellitus Mice Maria Galan; Modar Kassan; Choi Sookyoung; Partyka Megan; Khalid Matrougui Tulane University Diabetes mellitus is a major risk factor in cardiovascular diseases. Recent reports suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of diabetes and vascular complications. This study explores the role of endoplasmic reticulum (ER) stress in microvascular function in type-1-diabetes mice. EXPERIMENTAL APPROACH. Our study was performed in streptozotocin-induced Diabetes Mellitus mice untreated or treated with ER stress inhibitor (TUDCA, 150 mg/Kg) or insulin (0.1 U/day). Vascular reactivity studies were performed in mesenteric resistance arteries (MRA) using a wired myograph to study the endothelium dependent relaxation (EDR). Expression of ER stress associated proteins in MRA and heart were determined by quantitative RT-PCR and western blot analysis. Cardiac fibrosis was determined. RESULTS. The induction of ER stress in MRA and heart from diabetic mice was evidenced by an increase in P-eIF2α, CHOP, ATF4 and ATF6 expression compared to control and diabetic mice treated with TUDCA or insulin. We reported a remarkable induction in fibrosis in heart from diabetic mice associated with enhanced collagen type-I and PAI-1 expression that were reduced with TUDCA and insulin. EDR, endothelium independent relaxations and phosphorylation of eNOS and cGMP levels in MRA were decreased in diabetic mice compared to control and diabetic mice treated with TUDCA or insulin. The incubation of MRA with apocynin improved EDR only in MRA from STZ mice while L-NAME impaired it slightly. Additionally the mRNA levels of Nox2 and Nox4 in MRA from diabetic mice were reduced by TUDCA and insulin.

20 Testosterone Dilates Renal Afferent Arteriole via a Nitric Oxide-Mediated Mechanism Ying Ge, Yan Lu, Jane F. Reckelhoff, Ruisheng Liu Physiology and Biophysics and Nephrology, University of Mississippi Medical Center, Jackson The afferent arteriole (Af-Art) is the major resistance vessel in the kidney, and plays a vital role in the control of glomerular capillary pressure, autoregulation, glomerular filtration rate, salt and water balance. Males are more susceptible to renal injury than females, and androgens may contribute to the sex difference. Our hypothesis is that testosterone dilates the Af-Art via a nitric oxide (NO) pathway. Isolated Af-Art with the attached glomerulus was micro-dissected from a mouse kidney and perfused with an array of pipettes at 60 mm Hg. We preconstricted the perfused Af-Art by about 30% with norepinephrine (10-7 M), then measured the changes in diameter of the Af-Art when stimulated with testosterone (10-10 M-10-7 M). Testosterone induced a dose-responsive relaxation of Af-Art from 6.6±0.4 μm at 10-7 M to 3.5± 0.3 μm at 10-9 M. Testosterone at 10-10 M did not induce changes in diameter of the Af-Art. NO inhibitor L-arginine analog N-nitro-L-arginine methyl ester (L-NAME; 10-4 M) blocked the testosterone-induced relaxation. We conclude that testosterone dilates the Af-Art in a NO-dependent pathway. These data suggest that the presence of androgens may promote glomerular injury by causing an increase in glomerular capillary pressure.

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23 Opposite Cox-2 Inhibition Effects On Blood Pressure and Renal Hemodynamic During Early and Late Angiotensin II-infused Hypertension Torrance Green and L. Gabriel Navar Tulane University Health Sciences Center COX-2, unlike COX-1, opposes angiotensin II (AngII) actions on renal hemodynamics and blood pressure (BP). It remains unclear if an altered balanced between the COX isoforms promotes AngII-dependent hypertension. Therefore, we explored the COX-2 renoprotective and antihypertensive roles in the prehypertensive and hypertensive phases of AngIIinfusion. Clearance experiments were performed in anesthetized rats after 3-5 days (prehypertensive; n=10) or 13-14 days (hypertensive; n=8) of AngII-infusion (80 ng/kg/min). In the prehypertensive phase COX-2 inhibition with nimesulide (NMS) decreased GFR (1.08±0.09 to 0.77±0.09 ml/min/g) and RBF (6.29±0.71 to 5.37±0.49 ml/min/g) but not BP (115.0±4.2 to 110.3±3.6 mmHg). Subsequent COX-1 inhibition with SC560 decreased BP (94.4±4.4 mmHg) but not GFR or RBF. In the hypertensive phase, NMS lowered BP (140.4±7.4 to 124.9±8.6 mmHg), GFR (0.93±0.09 to 0.65±0.07 ml/min/g) and RBF (7.65±0.71 to 6.10±0.56 ml/min/g) and raised renal vascular resistance. Subsequent SC560 lowered BP (114.5±10.2 mmHg) but not GFR and RBF. SC560 decreased prehypertensive phase BP more than NMS (-14.5±2.7% vs. -2.3±0.9), but the isoforms similarly affected hypertensive phase BP. NMS lowered RBF more during the hypertensive than prehypertensive phase (-19.9±2.9% vs. -13.8±2.2%) and lowered cortical blood flow more than SC-560 (-14.1±3.0% vs. -4.1±3.8%). These data indicate COX-1 consistently elevates BP without affecting hemodynamics. In contrast AngII-dependent hypertension involves augmented COX-2 prohypertensive systemic effects despite increased renoprotective vasodilation opposing intrarenal AngII.

24 Rescue of the Cardiac Leptin Receptor in DB/DB Mice Prevents Cardiac Lipid Accumulation and Subsequent Diastolic Dysfunction M.E. Hall, M.W. Maready, G. Smith, T. Murakami, and D.E. Stec University of Mississippi Medical Center Obesity may contribute to heart disease through “lipotoxicity”, a process whereby ectopic deposition of lipids in non-adipose tissues leads to organ dysfunction. Db/db mice which have non-functional leptin receptors are obese, hyperglycemic and have elevated cardiac triglycerides. To determine if leptin can prevent cardiac lipotoxicity and preserve cardiac function, we generated a novel mouse model in which the cardiac leptin receptor was rescued in the db/db mouse (db/db-MHC-ObR). We performed serial, high resolution echocardiography in these mice and compared them with lean controls and age-matched db/db mice until 30 weeks of age. Db/db mice exhibited significantly lower mitral inflow E/A ratios (early to late diastolic filling rates) consistent with impaired relaxation, averaging 1.4 &#177 0.08 vs. 1.9 &#177 0.13 and 1.9 &#177 0.09 in lean and rescue mice, respectively. Db/db and rescue mice exhibited no significant differences in body weights, plasma glucose or plasma triglycerides. However, cardiac triglycerides were significantly lower in rescue mice compared to db/db mice and were not significantly different than in lean controls averaging 13.4 &#177 4.2 in db/db mice vs. 3.8 &#177 1.6 vs. 3.8 &#177 0.7 mg/g in lean and rescue mice, respectively. These results show that despite obesity, hyperglycemia, and hypertriglyceridemia, db/db mice with cardiac leptin receptor rescue are protected from cardiac lipid accumulation and subsequent diastolic dysfunction.

21 Heme Oxygenase-1 Induction Attenuates sFlt-1 Induced Hypertension in Pregnant Rats Eric M George, Kathy Cockrell, Marietta Arany, Eva Csongradi, David Stec, and Joey P Granger Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, MS 39216 Pregnancy induced hypertension (PIH) is one of the leading causes of fetal and maternal morbidity, affecting 5-10% of all pregnancies, with no effective treatment. The exact etiology of the disorder is unclear, but placental ischemia has been shown to be a central causative agent. In response to placental ischemia, the antiangiogenic protein sFlt-1, a VEGF antagonist, and reactive oxygen species are secreted, leading to the maternal syndrome. One promising therapeutic approach to treat PIH is through manipulation of the heme oxygenase-1 (HO-1) protein, or its two metabolites; carbon monoxide (CO), a vasodilator, and bilirubin, a powerful antioxidant. The purpose of this study was to determine whether HO-1 induction would have beneficial effects in sFlt-1 induced PIH. Pregnant rats were continuously infused with recombinant sFlt-1 from gestational days 14-19, and circulating sFlt-1 increased ~2-fold, similar to rats with experimentally induced placental ischemia. In response, mean arterial pressure (MAP) increased 17mmHg, which was completely normalized by HO-1 induction. Unbound circulating VEGF was decreased~17% in response to sFlt-1 infusion but was increased ~50% in response to HO-1 induction. In conclusion, manipulation of HO-1 presents an intriguing therapeutic approach to the treatment of PIH.

22 Responses of Renin and Prorenin Receptor–(P)RR- in the Collecting Duct of Sprague-Dawley Rats Fed a Low Salt Diet Alexis A Gonzalez, Joel P. Womack, Christina Luffman, Dale M Seth, and Minolfa C. Prieto Tulane University School of medicine, Department of Physiology and Renal Hypertension Center of Excellence; Tulane-BIRCWH Program. Renin and its receptor, the prorenin receptor ((P)RR) in the collecting duct (CD) may provide a pathway for distal nephron Ang I generation allowing further conversion to Ang II. The (P)RR by binding renin or prorenin, increases the catalytic activity of renin and fully activates prorenin. In chronic angiotensin II (Ang II)-infused hypertensive rats, the increased intrarenal de novo formation of Ang II is associated with up-regulation of CD renin. The present study tested the hypothesis that activation of the renin angiotensin system by low salt (LS) diet stimulates renin and (P)RR expression in the CD. We examined the mRNA and protein levels of renin and (P)RR primarily in the renal medulla of rats fed a LS diet (0.03% NaCl) for 7 days. Protein and mRNA levels of (P)RR were significantly higher in medullary tissues from LS-rats as compared to control animals (142 ± 9 vs. 100 ± 22 % and 140 ± 12 vs. 100 ± 9 %, respectively; P<0.05); however, no differences were observed in kidney cortex. These changes were also associated with increased medullary renin mRNA (390 ± 99 vs. 100 ± 12 %) and intrarenal Ang II levels (435 ± 57 vs. 265 ± 12 fmol/g; P<0.05) Thus, in response to a LS diet, there is augmentation of (P)RR in medullary CD cells which may augment local Ang II levels.

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25 A Translational Study using a Novel Rat Model to Identify Biomarkers of Kidney Disease and Validation in Human CKD Patients Ashlyn C. Harmon1, Albert Driesbach2, Karen Winters3, Katina Lang-Jenkins2, Stanley Smith1, Ashley Johnson1, and Michael R. Garrett1,2 1Department of Pharmacology, 2Department of Medicine, 3School of Nursing, University of Mississippi Medical Center The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension, but is also a useful model of chronic kidney disease (CKD) because it develops concomitant proteinuria, glomerulosclerosis, interstitial fibrosis, and progressive loss of renal function. We recently developed a model of accelerated CKD through genetic modification of the S rat. The new congenic model [S.SHR] demonstrates significantly more renal injury, culminating in a more rapid decline in renal function compared to the S rat. The model is currently being utilized for two-different approaches: (1) to identify novel biomarkers associated with kidney disease progression using proteomic analysis (2-D and Mass Spec) of urinary exosomes. Biomarkers identified using the rat model will be validated in a unique human CKD population from whom urine samples are presently being collected at the University of Mississippi Medical Center. We are currently fine-tuning the isolation procedure and performing validation through electron microscopy and western blot analysis; and (2) to identify the genetic variant(s) that promote increased renal injury in the model. The region containing the causative genetic variants will be refined through developing new congenic strains, microarray analysis, and sequencing. In summary, we will employ a model of accelerated of renal disease to identify novel biomarkers for progression of CKD, initiate genetic studies that could provide important insight into genes or genetic variants that cause increased renal injury, and ultimately, validate rodent findings in a unique human population.

26 Changes in Citrate Transporters in Placenta During Gestation and Ischemia. Kathleen S. Hering-Smith, Weibo Mao, Jill Verlander and L Lee Hamm. Tulane University, University of Florida College of Medicine Placenta (Pl) express a number of transporters of metabolic substrates, including NaDC3 (Na dicarboxylate transporter 3) which was initially cloned from human Pl. We have previously demonstrated a calcium (Ca) sensitive dicarboxylate transport process (citrate and succinate) in OK kidney proximal tubule cells and in the human Pl cell line JAR. Also we demonstrated that JAR contains both NaDC1 and 3, via immunobloting and staining. NaDC1 and 3 are respectively low and high affinity dicarboxylate transporters most studied on the apical and basolateral membranes of renal proximal tubule and intestinal cells. To determine the regulation of NaDC3 and NaDC1 in Pl during gestation, we compared Pl obtained from rats at gestation of 13 days (13d) and 19 days (19d) as well as from the RUPP model (ischemia). By immunohisotochemistry, both NaDC1 and NaDC3 were present in rat Pl epithelial cells at both 13d and 19d. For NaDC1, there was a clear increase in the intensity of staining at 19d. For NaDC3, changes in the pattern of staining were not evident. In contrast, by immunoblot of Pl (containing all cell types), NaDC3 decreased (by 27%, p< 0.05) at 19d; NaDC1 increased at 19d and in RUPP. In pregnancy the Pl calcifies with increasing gestational age and in pre-eclampsia. We show here that NaDC1 is upregulated towards the end of gestation and with ischemia, leaving less citrate to complex Ca2+ thus resulting in more free Ca2+ available for tissue calcification. Together these findings raise the possibility that citrate transporters in the Pl regulate its calcification during normal and ischemic pregnancies.

27 Sex-specific Mechanisms Contribute to the Pressor Response to Acute Angiotensin II in a Rat Model of Intrauterine Growth Restriction S Intapad, FL Tull, JH Dasinger, JT Black, NB Ojeda, BT Alexander. University of Mississippi Medical Center, Jackson, MS When the endogenous renin angiotensin system (RAS) is blocked by enalapril, male and female rats exhibit a pressor response to acute angiotensin II (Ang II). Endothelin contributes to the pressor response to acute AngII in male rats; yet the importance of endothelin in in female rats is unknown. Hypersensitivity to acute Ang II in male intrauterine growth restricted (IUGR) rats is abolished by endothelin type A receptor (ETAR) blockade. Thus, we tested the hypothesis that endothelin plays a key role in mediating the pressor response to acute Ang II in female rats. Mean arterial pressure (MAP) was measured in conscious, chronically instrumented female control and female IUGR rats pretreated with enalapril (250 mg/L) for 1 week. MAP was measured for 30 minutes before or after an acute infusion of Ang II (100 ng/kg/min), and plus or minus ETAR blockade (ABT627, 10 mg/kg/min). MAP was increased in response to acute Ang II in female control (140±4 mmHg; P<0.05 vs. baseline 110±3mmHg) and female IUGR rats (141±1 mmHg; P<0.05 vs. baseline 111±2 mmHg); however, ETAR blockade had no effect on blood pressure in Ang II treated female rats, control (145±5 mmHg) or IUGR (139±6 mmHg). Thus, these data indicate that utilizing a dose of an ETAR antagonist that altered MAP responses to Ang II in male rats had no effect on the pressor response to acute Ang II in female rats. Thus, sex-specific mechanisms contribute to the acute pressor response to Ang II.

28 Inhibition of Nitric Oxide Synthase Enhances Superoxide Production in Macrophage Cells in Response to Increases in Sodium Concentration in the Culture Media Mohammed T. Islam 1, Deepmala Agarwal 2, Joseph Francis 2, Dewan S.A. Majid 1 1Dept of Physiology, Hypertension & Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, LA 70112; 2Dept of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 High salt intake enhances nitric oxide (NO) production in mononuclear phagocyte system (MPS) cells. However, the regulation of O2- production in these MPS cells in high salt condition is not yet clearly understood. To examine the hypothesis that increases in extracellular Na+ conc enhances O2- production in MPS cells, cultured macrophage cells are incubated for 24 hours in control media (Na+ conc- 110 mM) and at media containing high Na+ conc (130 and 150 mM; n=6 in each group) and O2- level was measured by Lucigenin-chemiluminescence assay. The level of O2- was higher in cells at 130 & 150 mM Na+ (1029 ± 20 and 929 ± 45 RLU/ 100 μL media) compared to that at 110 mM Na+ (361 ± 15 RLU/ 100 μL media). Nitrate/nitrite (NO3/NO2) level in the media was significantly higher at 130 mM Na+ (77.6 ± 11.4 μM) but not at 150 mM Na+ (65.8 ± 3.3 μM) compared to that at 110 mM Na+ (59.6 ± 5.5 mM). There was an upregulation of iNOS mRNA in cells at higher Na+ compared to that in control media. O2- production increases many folds (1198 ± 592 to 9570 ± 1332 and 8304 ± 2515 RLU/ 100 μL media) in cells that were pre-treated with NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME; 100 μM). Co-treatment with NADPH oxidase inhibitor, apocynin (1 mM) abolishes L-NAME induced increases in O2- production in these cells. These data demonstrate that NO synthase activity exerts a tonic inhibitory effect on NADPH oxidase activity and suggest that the MPS cells, as an initial target of stress induced by high salt intake, could play a critical role in redox imbalance that leads to the development of salt sensitive hypertension.

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29 Interleukin-10 Released by CD4+CD25+ Natural Regulatory T Cells Improves Microvascular Endothelial Function through Inhibition of NADPH Oxidase Activity in Hypertensive Mice Modar Kassan, Maria Galan, Megan Partyka, Mohamed Trebak, Khalid Matrougui Department of Physiology, Hypertension and Renal Center of Excellence, Tulane University, 1430 Tulane Ave, New Orleans LA-70112; 2Center for Cardiovascular Sciences, Mail code 8, Albany Medical College, Albany 12208, New York We demonstrated that reduced CD4+CD25+-regulatory T cells (Tregs) number apoptosisdependent mechanism was responsible for microvascular dysfunction in hypertension. The underlying mechanism that dictates on vascular endothelial function by Tregs has not been explored. control and IL-10-/- ko mice were subjected to Ang II (400 ng/Kg/min) infusion for 2 weeks (HT and HT-IL-10-/-). Endothelium-dependent relaxation (EDR) in response to ACh was reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10-/- compared to control and IL-10-/- mice. Incubation of MRA from HT mice with conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, no effect was observed in MRA from control incubated with the same media. These effects were reversed when MRA were pre-incubated with IL-10 antibody or IL-10 receptor antagonist. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10-/- reduced systolic blood pressure, NADPH oxidase activity, and improved EDR in MRA compared to untreated HT-IL-10-/- mice. In vivo treatment of HT mice with IL- 10(1000 ng/mouse) reduced SBP, NADPH oxidase activity, and improved EDR in MRA compared to untreated HT mice. The transfer of cultured Tregs, isolated from IL10-/-, into HT mice did not reduce SBP, NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, while NADPH substrate attenuated EDR in MRA from control mice, which was reversed in MRA subjected to exogenous IL- 10.These data suggest that Tregs/IL-10 release could be therapeutic targets for vasculopathy in hypertension.

30 Persistent Cytomegalovirus Infection Accelerates Non-complicated Obesity-induced Venular Inflammation in a Mouse Model Mikhail V Khoretonenko, Karen Y Stokes Department of Molecular and Cellular Physiology and Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71103 Cardiovascular diseases (CVD) are the major cause of morbidity and mortality worldwide. In response to CVD risk factors, leukocytes adhere to postcapillary venules in multiple organs before clinical disease develops. Overweight affects 65% of the US population, with half of these considered obese. Obesity is often complicated by other factors, e.g. diabetes, which themselves promote microvascular inflammation, however little is known about whether non-complicated obesity alters microvascular homeostasis. Cytomegalovirus is a beta-herpesvirus that infects a majority of people, and has been implicated in CVD. We developed a model to test whether non-complicated obesity induces leukocyte recruitment and if persistent CMV infection accelerates these responses. C57Bl/6 mice received mock or CMV and at 4 wks post-infection (wpi) were kept on normal diet or placed on a no cholesterol/low glucose/high fat diet (HFD) for 4-10 wks. The obesity (Lee) index was increased by 4 wks HFD, but blood glucose levels were unchanged. While CMV alone did not alter leukocyte recruitment, leukocyte adhesion in venules was elevated at 8-10 wks HFD. CMV+HFD mice exhibited 2-3 fold increases in leukocyte adhesion and emigration at all timepoints. Our findings suggest that mild obesity induces venular inflammation, which is accelerated by persistent CMV infection before obesity-associated complications appear. (NIH P20RR018724)

31 RHO Kinase Enhances Tone in Contractile Lymphatics KM Kurtz, FM Souza-Smith and JW Breslin Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA The intrinsic pumping of lymph by lymphatics consists of phasic contractions superimposed over tone. The underlying molecular mechanisms are poorly understood. Rho kinase (ROCK) activation promotes tonic vasoconstriction. We hypothesized that ROCK also enhances tonic constriction of lymphatic smooth muscle. We inhibited ROCK activity in isolated rat mesenteric collecting lymphatics with Y-27632 (0.5-5 μM) or ROCK Inhibitor V (RIV) (0.01-10 μM). Spontaneous contractile activity was observed in a 37 ºC albumin physiological salt solution (APSS) bath, at a luminal pressure of 2 cm H2O. End diastolic diameter (EDD), end systolic diameter (ESD), and contraction frequency (CF) were recorded and phasic contraction amplitude (AMP = EDD – ESD) was calculated. Passive maximal diameter (MaxD) was also measured in Ca2+-free APSS to determine tone [(MaxD-EDD)/MaxD)*100%]. Both ROCK inhibitors increased EDD and ESD in a dose-dependent manner. These changes were associated with decreased lymphatic tone at 5 μM Y-27632 (13.5 ± 6.3 % vs. control: 22.5 ± 4.1 %, P<0.05) and both 1 μM and 10 μM RIV (1 μM: 10.4 ± 2.1 and 10 μM: 7.2 ± 2.5 vs. control: 23.1 ± 3.1, both P<0.05), but no changes in mean AMP. Y-27632 (5 μM) also decreased CF, but not RIV. Our data suggest that ROCK signaling promotes tonic but not phasic contraction of lymphatic vessels. Supported by NIH P20RR018766 and a grant from the American Heart Association.

32 AT1-mediated Angiotensinogen Augmentation and Kidney Oxidative Stress in Chronic Ang II Infused Rats Fed a High Salt Diet. Lucienne S Lara1,2, Michael McCormack1, Laura Seprum-Prieto1, Sylvia Shenouda3, 1Physiology Department, Tulane University; 2Pharmacology Department, Louisiana State University; 3Renal Hypertension Center of Excellence, New Orleans, LA; 4Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Brazil Augmentation of intrarenal angiotensinogen synthesis, secretion and excretion has been associated with the development of hypertension, renal oxidative stress and tissue injury during Ang II-dependent hypertension. High salt (HS) exacerbates hypertension and kidney injury. In this study, we determined the consequences of HS intake alone as compared to chronic Ang II infusion on the stimulation of urinary AGT and renal oxidative stress in Sprague-Dawley rats subjected to the following protocols for 14 days: 1) normal salt diet [NS, n=5]; 2) HS diet [8% NaCl, n=5]; 3) Ang II infusion in NS rats [Ang II 80 ng/min, n=5]; 4) Ang II-infusion in HS rats [Ang II+HS, n=5]; and 5) Ang II-infusion in HS rats treated with angiotensin type 1 receptor (AT1R) blocker [Ang II+HS+ARB, n=5]. Compared to Ang II rats, the combination of Ang II infusion and HS diet led to greater exacerbation in SBP, proteinuria, and uAGT associated with the greatest degree of glomerular mesangial expansion and cell proliferation. In contrast, the rats fed a HS diet alone did not show changes in SBP, proteinuria, cell proliferation, or uAGT excretion although they did exhibit mesangial expansion and renal fibrosis compared to rats with Ang II alone. In addition, they showed decreased renal levels of O2- due to the formation of OONO- which is not observed in the Ang II and Ang II+HS rats. These results indicate that HS exacerbates uAGT excretion in Ang II infused rats which maintains intratubular Ang II formation and AT1R activation contributing to increased renal oxidative stress and further renal injury.

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36 Adenovirus-mediated Coexpression of AT1a Receptors and an Intracellular Angiotensin II Fusion Protein Selectively in Proximal Tubules of the Kidney Increases Blood Pressure in AT1a Receptor-Knockout Mice Xiao C. Li, Julia L. Cook, Isabelle Rubera, and Jia L. Zhuo Department of Pharmacology and Toxicology, University of Mississippi Medical Center The present study tested the hypothesis that proximal tubule-specific expression of AT1a receptors mediates intracellular angiotensin II (Ang II)-induced blood pressure responses in AT1a -KO mice. Proximal tubule-specific coexpression of a GFP-tagged AT1a receptor, AT1aR/GFP, and a cyan fluorescent intracellular Ang II fusion protein, ECFP/AII, was induced in the cortex via intrarenal adenoviral transfer using the sodium and glucose transporter 2 (sglt2) promoter. Intrarenal transfer of AT1aR/GFP and ECFP/AII induced a time-dependent proximal tubule-specific expression of AT1aR/GFP or ECFP/AII in the cortex with a peak expression 2 weeks after the gene transfer. No significant ectopic AT1aR/GFP or ECFP/AII expression was detected in extrarenal tissues. Systolic blood pressure was increased from 86 ± 5 mmHg to 102 ± 3 mmHg by day 14 in AT1a-KO mice with proximal tubule-specific coexpression of AT1aR/GFP and ECFP/Ang II (n=9, p<0.01). UNaV was significantly decreased from 234.7 ± 4.8 μmol/24 h in control AT1a-KO mice to 159.2 ± 10.9 μmol/24 h in AT1a-KO mice with proximal tubule-specific coexpression of AT1aR/GFP and ECFP/Ang II (p<0.01). These responses were associated with marked increases in phosphorylated ERK1/2 and NHE-3 proteins in the cortex (p<0.01). By contrast, expression of GFP or a scrambled fusion protein, ECFP/AIIc, alone in proximal tubules had no effect on blood pressure, UNaV, p-ERK1/2 or p-NHE-3 proteins. These results support a physiological role for proximal tubule-specific AT1a receptors and intracellular Ang II in the regulation of blood pressure and proximal tubule transport function.

35 (Pro)renin Receptor Deletion in the Subfornical Organ Attenuates Hypertension in Human Reninangiotensinogen Double Transgenic Mice. Wencheng Li1, Hua Peng1, Sarah J. McDaniels1, Curt D. Sigmund2, Yumei Feng1 1. Department of Physiology, Tulane University School of Medicine; Tulane Hypertension and Renal Center of Excellence, New Orleans, LA. 2. Department of Pharmacology, Center on Functional Genomics of Hypertension, University of Iowa, Iowa City. IA. The (pro)renin receptor (PRR) is a new member of the brain rennin angiotensin system. To investigate the role of central PRR in hypertension, adeno-associated virus (AAV2/2)-mediated PRR short hairpin RNA (AAV-PRR-shRNA) and control virus (AAV-eGFP) were intracerebroventricularly (ICV) administrated to non-transgenic (NT) and R+A+ mice. Blood pressure (BP) was recorded using radio telemetric probes in conscious animals for 2 weeks. Autonomic function and spontaneous baroreflex sensitivity (SBRS) were also assessed. PRR expression levels were assessed by real-time PCR. At baseline, the R+A+ mice exhibited increase of BP (146.9±1.4 vs. 93.6±1.2mmHg), cardiac (delta HR: -146±27 vs. -42±6 bpm) and vascular (delta BP: -75.9 ± 1.2 vs. -38.2 ± 1.4 mmHg) sympathetic tone, and decrease of SBRS (1.5±0.1 vs. 3.2±0.3 msec/mmHg) compared to NT mice. ICV delivery of AAV-PRR-shRNA significantly reduced BP (124.4±1.3mmHg), cardiac (delta HR:-91±17 bpm) and vascular (delta BP: -58.6±6.6 mmHg) sympathetic tone, and improved SBRS (2.2±0.2 msec/mmHg) in R+A+ mice. At the end of experiments, key brain nuclei from subfornical organ (SFO), paraventricular nuclei (PVN) and nucleus tractus solitarius (NTS) were micropunched for measuring PRR mRNA levels. ICV administration of AAV-PRR-shRNA significantly reduced the PRR mRNA in SFO (0.86±0.04 vs. 1.18±0.03), but not in PVN (1.10±0.02 vs. 1.27±0.06) and NTS (1.03±0.04 vs. 0.99±0.02) compared to control virus administration in R+A+ mice. These data suggested PRR may play a role in central regulation of BP and supported PRR as a potential drug target for the treatment of hypertension.

34 Conformation Specific Small Molecules Of Estrogen Signaling As Anti Cancer Drugs Joshua Leggette, Marc Koyack and Rajendram V Rajnarayanan Natural Sciences, Tougaloo College, Jackson, MS. Pharmacology and Toxicology, University at Buffalo, NY. Estrogen receptors are present in about 70% of breast cancers and play a critical role in the biology of breast carcinoma and improves prognosis. The cognate ligand, estrogen binds to the Estrogen receptor and mediates a cascade of cellular signaling events. The administration of the antiestrogen tamoxifen to patients induces substantial regression of the tumor and an increase in disease free survival. However, breast cancers that initially respond well to tamoxifen tend to develop resistance and resume growth despite the continued presence of the antagonist. In this poster we present profiles of the molecular interfaces of Estrogen receptor bound to peptides that senses a specific receptor conformation and an approach to discover new breed of small molecular therapeutics for breast cancer therapy. Protein- protein interactions mediate liganded/unliganded estrogen receptor signaling by binding to Estrogen Receptor. The interacting proteins are regionspecific and sense specific receptor conformations, which triggers the associated biological activity. The objective is to identify the Estrogen Receptor conformation sensing regions of the interacting proteins and to discover potential small molecule sensors using state of the art bioinformatics and structure based tools. Acknowledgements: We acknowledge support from FASEB-MARC Summer Travel award (JL) and NIH-RIMI grant from NICHMD.

33 Systems Biology Analysis of Gut-Specific Mechanisms Underlying Chronic Δ9-THC Modulation of Simian Immunodeficiency Virus Infection N.J. LeCapitaine, J. Zabaleta, A. Amedee, P. Zhang, P. Winsauer, P.E. Molina Louisiana State University Health Sciences Center Δ9-THC, the primary psychoactive component in marijuana, is FDA-approved to ameliorate AIDS-associated wasting. Our studies have demonstrated that chronic THC administration results in generalized attenuation of set point viral load/viral level in general of simian immunodeficiency virus (SIV) disease progression in macaques. Gut-associated lymphoid tissue is an important site for HIV replication and inflammation, and is a possible site for THC immunomodulation. We examined the impact of chronic THC administration (0.32 mg/kg i.m., 2 X daily), starting one year prior to inoculation with SIVmac251 (100 TCID50/ml, i.v.), on this immunological site. Viral load, immunological parameters, and transcriptome was determined in gut mucosa from chronic THC- (N=4) and vehicle (VEH)-treated (N=3) SIV-infected macaques at necropsy. Gut viral load was lower in THC/SIV than in VEH/SIV macaques. There were 115 genes identified to be differentially expressed in gut mucosa samples of THC/SIV when compared to VEH/SIV macaques. A total of 12 pathway maps were identified to include 3 or more differentially expressed genes. Of these, the predominant cell processes were involved in cytoskeleton remodeling, immunomodulation, cell adhesion, and cell signaling. Gut mucosa of THC/SIV macaques had significantly more central memory cells vs. the VEH/SIV animals. These findings suggest that mechanisms involved in chronic THC modulation of SIV/HIVs disease progression may result from structural and functional modification of barrier function, immune cell phenotype, and suppressed gut viral replication.

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40 An Oxidant-sensitive TRPM2 Channel Expressed in the Afferent Arteriole Regulates Ang II-induced Vessel Constriction Yan Lu, Deyin Lu, Ying Ge, Xiaolong Zhu, Luis A. Juncos, Ruisheng Liu Physiology and Biophysics and Nephrology, University of Mississippi Medical Center, Jackson TRPM2 is oxidant-activated and non selective cation channel expressed in mammalian tissues. We hypothesized that TRPM2 is expressed in renal afferent arterioles (Af-Art) and regulates AngII-induced vasoconstriction. A superficial Af-Art with its attached glomerulus was microdissected from a mouse kidney and perfused with an array of pipettes at 60 mm Hg. Ang II (10-9 M) induced constriction of the Af-Art from 11.2 ± 0.5 to 8.7 ± 0.8 μm. This constriction was blocked by treating the Af-Arts with either 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ, 10-5M) or 3-aminobenzamide (3-AB, 10-3M), non-specific inhibitors of TRPM2. We then confirmed that these perfused Af-Arts expressed TRPM2 using RT-PCR and immunoblotting. Finally, we silenced the TRPM2 in the Af-Arts by treating them with a siRNA against TRPM2 for 24 hours. Expression of TRPM2 was decreased by 75% as measured by RT-PCR and 70% as measured by immunoblotting. Silencing TRPM2 with the anti-TRPM2 siRNA also blocked AngII-induced vasoconstriction of the perfused Af-Art, whereas Af-Arts treated with scrambled siRNA showed the same responsiveness to AngII as freshly dissected and perfused Af-Arts. Thus our results suggest that TRPM2 channels in mice Af-Art are necessary for AngII-induced vasoconstriction, and thus may play an important role in the regulation of the renal microcirculation.

39 The Local and Global Effects from Exercise Training on the Vascular Responses to Muscle Contraction Silu Lu, Lusha Xiang, Mohamad E. Sebai, Elizabeth K. Jones, and Robert L. Hester. Department of Physiology, University of Mississippi Medical Center Impaired functional vasodilation is observed in obesity. We have shown that insulin resistance impairs functional vasodilation via an increased thromboxane receptor (TP)-mediated vasoconstriction in obese Zucker rats (OZ). Exercise improves functional vasodilation in the spinotrapezius muscle of the OZ but the mechanisms remain unclear. Since we’ve shown that exercise improves insulin resistance, we hypothesized that exercise improves functional vasodilation in OZ via improved insulin sensitivity and resultant decreased TP-mediated vasoconstriction. Six-week-old lean Zucker rats (LZ) and OZ were trained on a treadmill (24 meter/min, 30 min/day, 5 days/week) for 6 weeks. We performed the oral glucose tolerance test and measured functional vasodilation in both exercised (spinotrapezius) and non–exercised (cremaster) muscles. The sedentary OZ exhibited impairments in glucose tolerance and functional vasodilation in both muscles compared with sedentary LZ. The TP antagonist SQ29548 improved the vasodilator responses in the sedentary OZ with no effect in the LZ. Exercise increased the functional vasodilation in spinotrapezius muscle with no effect in the cremaster muscle in LZ. Exercise improved glucose tolerance along with increased functional vasodilation in both spinotrapezius and cremaster muscles in OZ. SQ29548 had no effect on the vasodilator responses in either type of muscles of the exercise trained OZ. These results suggest that in the OZ there is a systemic effect of exercise to improve insulin sensitivity and increase functional vasodilation via a decreased TP-mediated vasoconstriction. (Supported by NIH HL0895811)

38 Urinary Renin Excretion is Increased in Chronic Angiotensin II-Infused Hypertensive Rats Liu Liu 1, Alexis A. Gonzalez 1, Michael McCormack 1, Dale M Seth 1, Hiroyuki Kobori 1,2, L. Gabriel Navar 1,2, Minolfa C.Prieto 1,2,3 1 Department of Physiology, 2 Hypertension and Renal Center of Excellence, 3 BIRCWHProgram, Tulane University School of Medicine, New Orleans, LA. Renin expression in principal cells of the collecting ducts (CD) is upregulated during Angiotensin II (Ang II)-dependent hypertension, but it has not been established that renin is secreted into tubular fluid. In chronic Ang II-infused male Sprague-Dawley rats [80 ng/min, SC minipumps for 14 d, n=10], we measured plasma Ang II content, plasma renin activity (PRA), urinary renin (uRen) protein levels, uRen enzymatic activity, and urinary Ang II excretion rates (uAng II). Systolic blood pressures increased progressively in the Ang II rats reaching 175±10 mmHg by day 13 compared to sham rats (n=10) at 116±2 mmHg. Although PRA was markedly suppressed in the Ang II-infused rats, renin content in renal medullary tissues was augmented (12,605 ± 1,343 vs. 7,956 ± 765 ng Ang I/h/mg; p<0.05). uRen excretion rate was markedly increased in the Ang II rats (9±3 vs.1±1x10-5 Enzyme Units/day, p<0.05). These changes were in associated with augmented renin and prorenin protein bands (33 kDa and 40 kDa) examined by Western Blot in the urine samples of Ang II rats. uAngII also increased in the Ang II rats compared to sham rats (3,813±431 vs. 2,080±361 fmol/day). Thus, augmentation of urinary renin protein levels and its enzymatic activity in Ang II-infused rats indicates that CD renin is secreted into the tubular fluid, thereby contributing to increased distal nephron Ang II formation during Ang II-dependent hypertension. NIH [IdeA (P20-RR-017659-06), Tulane-BIRCWH (K12HD043451)]; and AHA (09PRE2209079 and 09BGIA2280440).

37 Role of the Sympathetic Nervous System in Mediating Hypertension in Aged Female Spontaneously Hypertensive Rats. Roberta Lima, Mouhammad F. Mathbout, Huimin Zang, Courtney M Alexander, Jane F Reckelhoff. University of Mississippi Medical Center Hypertension (HT) is one of the major risk factors for cardiovascular disease in postmenopausal women. Various humoral systems have been proposed to play a role in mediating postmenopausal hypertension. The effect of aging on activation of the sympathetic nervous system (SNS) is controversial, but may contribute to postmenopausal hypertension. This study tested the hypothesis that activation of the SNS plays a greater role in mediating the hypertension in aging female SHR than in young females. Telemetry transmitters were placed in old, (22 mos; PMR), and young females (3 months; YF) SHR (n-5/grp). After baseline BPs were measured, rats were treated with Terazosin (20mg/Kg/d) and Propranolol (20mg/kg/d) by osmotic minipumps for 7 day infusion, BP was measured during a 7 day recovery period. Baseline mean arterial pressure (MAP) was significantly higher in PMR than in YF (181±3 vs. 127±3 mmHg; p<0.001). The sympathetic blockade reduced MAP by 23% in PMR compared with 15% in YF (140±3 vs. 108±3 mmHg; p<0.001). After 7 days recovery, MAP was back to baseline in both PMR and YF, respectively (178±3 vs. 125±2 mmHg). The data suggest that sympathetic activation is important in the hypertension of both young and old SHR females, but that the SNS plays a greater role in mediating hypertension in PMR than YF. Therefore, studies will be necessary to determine if sympathetic activation may also contribute to postmenopausal hypertension in aging women. Studies were supported by NIH HL 69194, HL66072, HL51971.

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44 Salt Sensitivity of Blood Pressure in a Rat Model of Polycystic Ovary Syndrome Mohadetheh Moulana, Roberta Lima, Jane F. Reckelhoff University of Mississippi Medical Center Polycystic ovary syndrome (PCOS) is the most common hormonal disorder among women of reproductive age. Enlarged ovaries containing numerous small cysts, elevated androgen level, and insulin resistance are among the main symptoms of PCOS. We have characterized a rat model of PCOS, the hyperandrogenemic rat (HAF), that exhibits characteristics of PCOS in women. The aim of this study was to investigate whether the hypertension in HAF rats, is salt sensitive. Dihydrotestosterone (DHT) (7.5 mg/90 days or placebo) pellets were implanted subcutaneously in female SD rats (n = 5/group) at 4 weeks of age and compared to placebo. After 4 weeks of DHT treatment, radiotelemetry probes were implanted to measure mean arterial pressure (MAP). HAF rats (10 wk old, 6 wk DHT or placebo) were placed on 0.5% NaCl drinking water, 1% NaCl or 1.5% NaCl in drinking water with 1% NaCl food, each for 7 days. Urinary sodium excretion was measured on days 11, 18, and 25. Pressure-natriuresis curves were drawn using MAP’s on days 10, 17, and 24. As shown in the figure, HAF rats have a rightward parallel shift of the pressure-natriuresis relationship compared to placebo. These data show that the hypertension in HAF rats is not salt-sensitive, and suggest that women with PCOS may also not be salt sensitive. This Study was supported by NIH HL69194, HL66072, and POL HL51971.

43 Renal Nerves Contribute to Renal Injury, but not Hypertension, During Chronic Inflammatory disease Keisa W. Mathis, Marcia Venegas-Pont, William H. Ray, Chester W. Masterson, Terry Dwyer, Michael J. Ryan University of Mississippi Medical Center, Jackson, MS Inflammation contributes to the pathogenesis of hypertension and renal disease. Systemic lupus erythematosus (SLE) is an inflammatory disorder with prevalent hypertension and renal injury. Evidence suggests that patients with SLE may have enhanced sympathetic nervous system activity. Using an established hypertensive mouse model of SLE (NZBWF1) we tested whether the renal nerves promote hypertension and renal injury during SLE. Female SLE and control (NZW) mice were subjected to bilateral renal denervation (Dnx) or sham surgery at 32 weeks of age. Successful Dnx was confirmed by measuring renal catecholamines with HPLC. At 34 weeks of age, mean arterial pressure (MAP) was assessed in conscious mice using carotid artery catheters. MAP was increased in sham SLE mice compared to sham controls (135 ± 5 vs. 120 ± 1 mmHg; p<0.02). Dnx did not alter MAP in control (117 ± 3) or SLE (133 ± 4) mice. None of the SLE Dnx mice had albuminuria (≥100 mg/dL by dipstick) compared with 40% of SLE sham mice. Renal cortex MCP-1 protein expression was lower in SLE Dnx mice compared to SLE sham mice (p<0.01) and CuZn superoxide dismutase (SOD) was increased in SLE Dnx mice compared to SLE sham mice (p<0.01). These data suggest that the renal nerves promote renal injury during SLE independent of MAP, and possibly by contributing to local inflammation and oxidative stress. Supported by 10POST4350019 (KWM) and HL085907, HL092284, and HL085907S1 (MJR).

42 Treatment with C-peptide Slows the Progression of Diabetic Renal Disease in the Streptozotocin (STZ)-induced Diabetic Rat Christine Maric University of Mississippi Medical Center Diabetic renal disease includes progressive deterioration of renal function, fibrosis, and changes in the microcirculation. C-peptide is a biologically active pro-insulin product; however, its potential role in the diabetic kidney and mechanisms of its action are unknown. After 8 weeks of untreated diabetes, male STZ-induced diabetic Sprague-Dawley rats were randomly divided in untreated (D) or treatment with C-peptide (D+Cp) for additional 4 weeks. Urine albumin excretion (UAE) and HbA1C were measured at 8 weeks, while glomerulosclerosis (GSI) and glomerular filtration rate (GFR) measured by 125I-[iothalamate] clearance, were assessed only at the end of the study. Furthermore, renal microvascular (MV) density was quantified ex vivo using a 3D micro-CT reconstruction of the microcirculation. Treatment with C-peptide was associated with a 17% reduction in HbA1C as compared with the baseline. While no difference in UAE were observed in the D+Cp between baseline and time of sacrifice, UAE was reduced by 58% compared with the untreated D at the time of sacrifice. C-peptide also preserved the renal microcirculation and attenuated glomerulosclerosis, but did not affect GFR or blood pressure. The study suggests a renoprotective effect for C-peptide in slowing the progression of diabetes-associated albuminuria and renal MV disease, possibly via improved glycemic control. Supported by RO1DK075832

41 Dual Treatment with Dihydrotestosterone and an Aromatase Inhibitor Provides Renoprotection in Male Streptozotocin (STZ)-induced Diabetic Rats Michaele B. Manigrasso, R.Taylor Sawyer, Zachary M. Hutchens Jr., Elizabeth R. Flynn, Christine Maric University of Mississippi Medical Center, Jackson, MS Our previous data showed that streptozotocin (STZ)-diabetic males have increased estradiol and decreased testosterone levels that correlate with renal injury. Supplementing dihydrotestosterone (DHT) or inhibiting estradiol biosynthesis in these diabetic rats was previously shown to provide partial renoprotection. The aim of this study was to examine if a concomitant therapy of DHT supplementation and estradiol inhibition would afford full renoprotection. The study was performed in 12 week old male STZ-diabetic (D), STZdiabetic supplemented with 0.75mg/day DHT (Dt), STZ-diabetic treated daily with 0.15mg/kg/day of the aromatase inhibitor anastrozole (Da) and STZ-diabetic that had combined therapy of DHT and anastrozole (Dta) for 12 weeks. Urine albumin excretion (UAE) was determined by ELISA and calculated by measuring urine albumin concentration and daily urine output. Glomerulosclerosis (GSI) and tubulointerstitial fibrosis (TIFI) indices were graded using a semiquantitative scoring method. Markers of inflammation (IL-6 and TNFα) and fibrous matrix deposition (collagen type IV) protein expression were determined by Western blot and normalized to total β-actin protein expression. Our data suggest that combined treatment of DHT and the aromatase inhibitor, anastrozole, further reduced diabetes-associated increases in UAE, GSI, TIFI and further decreased the amount of IL-6, TNFα and collagen type IV protein expression, suggesting that supplemental DHT along with blocking aromatase activity in diabetic males provides a more superior therapy than either of these treatments alone.

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48 Effect of Muscarinic Receptor Stimulation on Electrophysiological Parameters of Esophageal Epithelium. Solange Abdulnour-Nakhoul and Nazih L. Nakhoul Southeast Louisiana Veterans Health Care Network and Tulane School of Medicine, Dept of Medicine, New Orleans LA. Pig esophageal tissue, like the human, bears submucosal glands (SMG) that secrete bicarbonate and mucus into the esophageal lumen. This secretion is stimulated by cholinergic agonist carbachol and inhibited by atropine. We have previously demonstrated the presence of muscarinic receptors M1 in the glands’ ducts. The presence of other muscarinic receptors and the effect of cholinergic stimulation on the electrophysiology of esophageal tissue have not been investigated. The glands are numerous in the cephalic part of the pig esophagus and lacking in the caudal part, which allowed us to compare the effect of cholinergic stimulation in gland-bearing and gland-devoid mucosea from same animals. Sections of esophageal tissue were mounted in Ussing chambers. Transepithelial potential difference (Vte), short circuit current (Isc) and tissue resistance® were monitored. Other sections were processed for immunohistochemical staining with antibodies against muscarinic receptors M3 and M5, α-actin and Na,K-ATPase. In gland-bearing sections, addition of carbachol to the serosal side caused a quick transient hyperpolarization of Vte, an increase in Isc and a drop in R. In gland-devoid sections, carbachol did not cause significant changes. Staining with an antibody to M3 was positive in glands ducts and acini and co-localized with Na,K-ATPase on the basolateral side of the cells. Staining with M5 co-localized with α-actin in myoepithelial cells surrounding the glands acini. Electrical effects upon stimulation by carbachol are more pronounced in gland-bearing mucosa. Support: VA Merit grant and RO1-DK-62295.

47 Trp-230 in the Hydrophobic Pore of Rhbg is Essential for Transport of NH3/NH4+ Nazih Nakhoul, Solange Abdulnour-Nakhoul, Trang Le, Edd Rabon, Rienk Doetjes, L Lee Hamm Depts. of Medicine & Physiology, Tulane Medical School, New Orleans, LA Rhbg is a non-erythroid membrane glycoprotein that belongs to the Rh antigen family. In the kidney, Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH3/NH4+ transport. Recently, a high resolution crystal structure of AmtB, a bacterial homologue of Rh, was determined. We used the structure of AmtB to align the sequence of Rhbg to AmtB & identify important sites of Rhbg that may affect function. Our model positioned Trp-230 within the hydrophobic pore where it is likely to be critical for transport. Three mutations of Trp-230 (to Ala, Leu or Phe) were generated and expressed in oocytes. To assess function, we measured NH3/NH4+ & MA/MA+ – induced currents and pHi in oocytes expressing the mutants and compared it to measurements in control oocytes or those expressing Rhbg. In all mutants, NH4+ current was much smaller than that in oocytes expressing WT Rhbg & there was no MA+-induced current. The NH3/NH4+ and MA/MA+ – induced pHi changes in the mutants were not different from those in H2Oinjected oocytes. We confirmed by immunohistochemistry that the mutated Rhbg was adequately expressed in the membrane of the oocyte. These data indicate complete loss of function upon substituting Trp-230. The loss of function was not due to impairment of trafficking or insertion in the membrane and indicates that this site is critical for transport by Rhbg. Supported by NIH-NIDDK.

46 Upregultation of the Renal Production of 20-HETE Opposes the Development of Hypertension in CYP4A1 Transgenic Dahl S Rats Sydney Murphy, Fan Fan, Howard Jacob, Aron Guertz, Rodney Baker, Richard J. Roman University of Mississippi Medical Center, Medical College of Wisconsin The present study examined whether a genetic deficiency in the renal formation of 20-HETE contributes to the development of hypertension in Dahl S rats. We created a line of transgenic Dahl S rats utilizing the Sleeping Beauty transposon system in which the expression of the rat CYP4A1 gene was driven by a chicken -actin promoter fused with elements of the CMV enhancer (CAG). We found the production of 20-HETE in the renal cortex increased 3 fold, from 17±1 versus 67±9 pmol/mg/min in the CYP4A1 transgenic line relative to levels seen in the S rat. Mean arterial pressure (MAP) rose from 116±5 to 134±5 mmHg in S rats (n=6) fed a 8.0% high salt (HS) diet for 7 days, while blood pressure remained unaltered (106±5 to 108±5 mmHg, n=10) in the CYP4A1 transgenic rats. After 21 days on HS diet, MAP was significantly lower in the CYP4A1 transgenic rats as compared to S controls (135±7mmHg vs. 162±10 mmHg, p<0.05). Protein excretion was also significantly lower in CYP4A1 transgenic rats (332±25) relative to Dahl S rats (559± 79 mg/day, p<0.05). These results indicate that upregulation of the CYP4A1 gene into the Dahl S genetic background increases the renal production of 20-HETE, improves proteinuria and opposes the development of hypertension. NIH HL29587, HL36279

45 Unfolded Protein Response in the ER and Alterations to Mitochondrial Respiration in the Progression of Obesity-induced Renal Injury Shankar Munusamy, Jussara M. do Carmo, *Jonathan P. Hosler and John E. Hall. Departments of Physiology & Biophysics and *Biochemistry, University of Mississippi Medical Center, Jackson, MS. Obesity has been implicated as an independent risk factor for end-stage renal disease. In this study, we investigated the impact of obesity in the absence of hypertension on renal function using two normotensive genetic mouse models of obesity, leptin-deficient ob/ob-/-mice (n = 7) and hyperleptinemic melanocortin 4 receptor knockout mice (LoxTB MC4R-/-, n = 7), compared to lean wild type (WT) C57BL/6J mice (n = 10). We measured urinary albumin excretion, creatinine clearance, renal triglycerides, ATP levels, state-3 mitochondrial respiration, cytochrome C oxidase content, and C/EBP homologous protein (CHOP) expression (a marker of ER unfolded protein response) in these mice. Our findings indicate that the ob/ob-/- mice and LoxTB MC4R-/- mice exhibit significant albuminuria (76.6±6.3 and 85.9±19.2 vs. 14.5±1.1 μg/24h) and increased creatinine clearance (693±61.1 and 752.3±50.6 vs. 534±31.5 μL/min) compared to WT mice. Renal triglycerides were significantly increased in ob/ob-/- mice (8.1±0.8 vs. 4.8±0.2 mg triglyceride/g tissue), whereas LoxTB MC4R-/- mice showed no significant differences in renal lipid levels compared to WT mice. Despite significant decreases in renal ATP levels in both obese models, only the LoxTB MC4R-/- mice kidneys showed an impaired state-3 mitochondrial respiration, cytochrome C oxidase content and a 3-fold induction of CHOP expression compared to WT mice. Together, our data suggest that obesity in the absence of hypertension cause only mild renal dysfunction, and unveils the potential involvement of alterations to renal mitochondria and ER assembly in obesity-induced renal injury.

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52 Mechanism of Hypoxia-induced α1H (CaV3.2) Gene Expression: Examining the Transcriptional Regulation Hassan Sellak1, Chun Zhou2, Bainan Liu2, Hairu Chen2, Thomas M. Lincoln1, Songwei Wu2 1Department of Physiology, 2Center for Lung Biology and Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688 T-type Ca2+ channels have been proposed as a major effector mediating cellular responses to hypoxia in many tissues. Among three cloned genes encoding the poreforming subunits of the T-type Ca2+ channels, α1G, α1H, and α1I, only α1H has been reported to be upregulated under hypoxia. Despite this observation, mechanism(s) underlying such hypoxia-dependent α1H-gene expression remains unknown. The present study sought to examine the involvement of hypoxia-response elements (HREs) within the promoter of the α1H-gene in the regulation of its expression under hypoxia. We confirmed that α1H mRNA was upregulated under hypoxia (3% O2, 24 – 48 hrs) compared to normoxia. Patch-clamp studies revealed the elevation of α1H-mediated T-type current under hypoxia in PC-12 cells and rat pulmonary artery smooth muscle cells (PASMCs). A 3.1-kb rat α1H 5’-flanking region was cloned in pGL3 vector. Sequence analysis of this region revealed the presence of 11 potential HRE sites. Transient transfection of PC-12 cells and PASMCs with this construct showed a significant increase in luciferase activity under hypoxia. Serial deletions analysis of the 3.1-kb fragment combined with luciferase reporter assays revealed that the potential HRE site (CACGC) at -1075 was responsible for hypoxia-induced upregulation of α1H. Electrophoretic mobility shift assay using the HRE as a probe showed an increase in its binding activity in nuclear extract harvested from PC-12 cells and PASMCs subjected to chronic hypoxia. Taken together, these data suggest that hypoxia-induced upregulation of α1H-gene expression involves an HRE site located within the promoter.

51 Interferon gamma Biphasically Regulates Angiotensinogen Expression via a JAKSTAT Pathway and SOCS1 in Renal Proximal Tubular Cells Ryousuke Satou, Kayoko Miyata, Omar W. Acres, L. Gabriel Navar, Hiroyuki Kobori Department of Physiology, and Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, LA Intrarenal angiotensin II (Ang II) contributes to the development of hypertension and renal diseases, and also increases intrarenal interferon γ (IFN-γ) levels. Therefore, this study examined the effect of IFN-γ on angiotensinogen (AGT, a precursor of Ang II) expression, and elucidated its molecular mechanisms in renal proximal tubular cells (RPTCs) which are the major source of intrarenal AGT. Rat RPTCs were treated with 0 – 20 ng/ml IFN-γ for 0 – 48 h. At 6 h, IFN-γ treatment suppressed AGT expression (0.61 ± 0.1, ratio to control) accompanied with activation of STAT1, SOCS1 (an intracellular suppressor of the JAK-STAT pathway) augmentation and suppression of STAT3 activity. Conversely, 24 h-IFN-γ treatment augmented AGT expression (1.76 ± 0.44, ratio to control) accompanied with activation of STAT3 and with no changes in STAT1 activity and SOCS1. Suppression of STAT1 by siRNA reduced SOCS1 augmentation, induced STAT3 activity and attenuated AGT suppression by 6 h-IFN-γ treatment. SOCS1 siRNA increased STAT3 activity and attenuated AGT suppression by 6 h-IFN-γ treatment. STAT3 siRNA did not affect any change in SOCS1 augmentation by 6 h-IFN-γ treatment. The STAT3 suppression decreased the basal AGT levels. These results indicate that IFN-γ biphasically regulates AGT expression in RPTC. Furthermore, STAT3 activity is responsible for the late upregulation of AGT; however, this effect is prevented earlier by the activation of the STAT1-SOCS1 axis. These findings indicate that the JAK-STAT pathway and SOCS1 contribute to intrarenal AGT regulation by IFN-γ.

50 Enhanced BK Channel Activity Contributes to Impaired Myogenic Response in the Cerebral Circulation of Fawn Hooded Hypertensive (FHH) Rats. P.M. Reddy1, Julio J1, M.F. Burke1, Jerry M. Farley1, David R. Harder2, Richard J. Roman1 1 Univ. of Mississippi Medical Center. 2 Medical College of Wisconsin. The present study examined the role of Iberiotoxin (IBTX) sensitive BK channel activity in impaired myogenic response in the cerebral circulation of FHH rats. Middle cerebral arterioles (MCA) isolated from FHH rats exhibited impaired myogenic response when transmural pressure was increased from 40 to 140 mmHg (FHH:40mmHg-142±16μm;140mmHg-157±19μm). In contrast, vascular diameter decreased by about 49% in FHH.1BN congenic strain in which a 2.6 Mb region of BN chromosome 1 was introgressed into the FHH genetic background (FHH.1BN:40mmHg-127±16μm; 140mmHg-65±13μm). Whole-cell patch-clamp of cerebral VSMC revealed increased BK channel current densities (pA/pF) in FHH rats compared to FHH.1BN and SD rats (23.2±6;6.9±1.9 and 7.1±1.3 respectively). Additionally, in excised i/o patch clamping, BK channel exhibited similar unitary conductance in both strains (FHH-235 and FHH.1BN strain-245 pS). However, open probability of BK channel were significantly higher in FHH rats compared to minimal congenic FHH.1BN strain (FHH-0.8±0.04 and FHH.1BN-0.08±0.004). Finally, MCA treated with IBTX partially restored myogenic response in FHH rats (FHH:40mmHg-119±7μm; 140mm Hg-133 ±8μm; FHH+IBTX:40mmHg- 115±5μm;140mmHg-97±4μm) but vascular diameter is not altered in FHH.1BN strain (FHH.1BN:40mmHg-110±14μm;140mmHg-83±9μm;FHH.1BN+IBTX:40mmHg-108±13μm; 140mmHg-81 ± 9μm). These results suggest that enhanced activity of the BK channel contributes to loss of myogenic tone that contributes to the impaired autoregulation of CBF in FHH rats.

49 Sex Differences in Collecting Duct Renin During High Salt Intake Rands, VF 1; Seth, D 1; Prieto MC 1,2,3 1Tulane University, Physiology Department, 2 Tulane Hypertension and Renal Center of Excellence, 3 Tulane-BIRCWH Program. In contrast to the inhibitory effect of high salt (HS) on renin from the juxtaglomerular cells, renin from the collecting duct (CD renin) is not suppressed in male rats fed HS. To determine if CD renin exhibits sex disparity in response to HS, we measured the inner medullary renin gene expression, content and urinary excretion in male (M) and female (F) Sprague-Dawley rats fed normal salt (NS) and HS (8%NaCl) diet for 14 days. Blood pressures were similar between sexes and unchanged by HS. Under NS, there were higher levels of CD renin mRNA in males compared to females (1.0±0.4 vs 0.2 ±0.1, AU). However, in response to HS, females increased renin message, (M HS 1.3±0.1 vs. F HS 2.5±0.5 AU). Protein levels were similar to mRNA except in HS females where increased (pro) renin (44 kD band) but not renin (42 kDa band) levels were found. Males had significantly more medulla renin content at baseline and it was unchanged by HS (M NS 9217 ± 1393 vs. F NS 2608 ± 356 ug/hr/g Ang I formed); however, HS increased rennin content in females (F HS 3439 ± 217 ug/g/hr Ang I formed). The renin urinary excretion levels were higher in male rats (M NS 2.6±0.7 vs. F NS 0.2±0.04 X 10-5 EU excreted/day) and increased in both sexes with HS (M HS 9.5±2.0 vs. F HS 2.3±0.6 X 10-5 EU excreted/day) These data indicate that in basal conditions, CD renin synthesis and intratubular secretion are higher in males. Although CD renin expression is more responsive to HS in females, the greater urinary renin activity in males, suggest greater capability to form intratubular Ang I and ultimately Ang II, in males. Tulane-BIRCWH (K12HD043451)

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54 Acute Alcohol Intoxication Increases the Magnitude of Phasic Ca2+ Transients in Rat Mesenteric Collecting Lymphatics. Flavia M. Souza-Smith, Kristine M. Kurtz, Patricia E. Molina, Jerome W. Breslin Department of Physiology, School of Medicine, Louisiana State University Health Sciences Center, New Orleans LA Alcohol intoxication enhances the phasic contraction amplitude of mesenteric collecting lymphatics and decreases lymphatic myogenic tone, but the mechanisms are not known. We hypothesized that the effects of alcohol at on mesenteric lymphatic pumping and myogenic response is due to altered calcium signaling. Acute alcohol intoxication was produced by intragastric administration of 30% alcohol to conscious, unrestrained rats through surgically implanted catheters. Isovolumic administration of water (vehicle) served as control. Calcium ratio measurements were made in every pressure studied, which consisted in stepwise increase of intraluminal pressure (to 4,6,8,10,and12 cm H2O) from the baseline level (2 cm H2O). We loaded isolated mesenteric lymphatic vessels with fura-2 to study intrinsic pump parameters. The results show that alcohol increased the amplitude of spontaneous Ca2+ transients, compared to vehicle. Step increases in pressure caused an elevation in [Ca2+]i during lymphatic diastole, that were equivalent for all pressures tested and were not different between the alcohol and vehicle groups. These results suggest that the increase in phasic contraction amplitude caused by alcohol is due to an increase in transient mobilization of Ca2+, but the mechanisms by which alcohol inhibits myogenic constriction may be Ca2+-independent or may involve Ca2+-sensitizing pathways. Supported by NIH P20HL018766 and a grant from the American Heart Association.

53 Mechanisms Underlying Angiotensin II Enhanced Microvascular Thrombosis. Elena Y. Senchenkova, D. Neil Granger Department of Molecular & Cellular Physiology, LSU Health Science Center – Shreveport, 71130-3932 Different mediators, cell populations, and receptors have been implicated in the elevated blood pressure mediated by angiotensin II. However, the mechanisms responsible for the prothrombotic actions of AngII remain poorly understood. We focused on defining the contribution of different receptor populations, immune cells and their products of activation, and oxidative stress, in the enhanced microvascular thrombosis induced by chronic angiotensin II (1 μg/kg/min, 14 days) administration. We demonstrated, using intravital microscopy, that arterioles exhibit an accelerated rate of thrombosis following light/dye injury, as evidenced by reductions in the time of onset of the thrombus and time to complete blood flow cessation. These variables were also monitored in WT mice (with an AngII –loaded Alzet pump) treated with antagonists to receptors for AngII (AT1, AT2, AT4), bradykinin (B1 & B2) and endothelin (ET-1), and in mutant mice deficient in lymphocytes, CD4+ or CD8+ T-cells, or gp91phox. The roles of different components of the coagulation cascade were assessed using blocking antibodies (tissue factor, activated protein C), thrombin inhibitors (hirudin, antithrombin III) and transgenic mice over-expressing the endothelial protein C receptor. The findings from these experiments are summarized in the Table. Conclusion: Of the factors assessed to date, CD4+ T-lymphocytes, NADPH oxidase, receptors for endothelin-1 and bradykinin-1, and thrombin appear to play a major role in mediating the enhanced thrombus formation induced in arterioles by chronic AngII infusion. (SUPPORTED BY MALCOLM FEIST POSTDOCTORAL FELLOWSHIP)

56 Chronic Alcohol Exposure Enhances Pulmonary Neutrophil Recruitment in Response to Intratracheal Endotoxin Xu Teng, Gregory Bagby, Kyle Happel LSU Health Sciences Center, New Orleans, LA 70112 PURPOSE: Chronic alcohol abuse is a major health problem in the U.S. and a risk factor in the development of the acute respiratory distress syndrome (ARDS). Neutrophil (PMN) influx is a histologic hallmark of ARDS and is essential to ARDS development. Acute alcohol intoxication in mice impairs alveolar PMN recruitment to the lung during local airway challenge with bacterial lipopolysaccharide (LPS), but the effects of chronic ethanol administration on alveolar PMN influx in this model are not well-characterized. METHODS: Female C57BL/6 mice were fed a Lieber-DiCarli liquid ethanol diet for 8 weeks. E. coli LPS was administered intratracheally (i.t.), and animals were sacrificed for tissue harvesting. RESULTS: Alcohol feeding significantly increased the number of PMN in BAL fluid compared to control animals 24 hrs post LPS infection. This result was associated with significantly greater increases in inflammatory cytokine and chemokine production in the lung tissue of ethanol-fed mice, as well as greater total protein leak into the alveolar space compared to controls. Plasma levels of IL-6, G-CSF, and MCP-1 were higher in ethanol-fed mice, and qRT-PCR of lung tissue mRNA confirmed the exaggerated IL-6 induction in alcohol-fed animals occurs at the transcriptional level. CONCLUSIONS: Chronic alcohol feeding enhances PMN recruitment into the lung following airway LPS challenge, possibly by increasing the local expression of cytokines. These data suggest exaggerated PMN recruitment as a possible mechanism by which chronic alcohol abuse potentiates the lung’s inflammatory response to bacterial challenge.

55 Hypertonic Saline Resuscitation Enhances Blood Pressure Recovery via AVP and Improves Tissue Perfusion in Alcohol Intoxicated Rats Jesse K Sulzer, Annie M Whitaker, Patricia E Molina Dept of Physiology and Alcohol Research Center, LSUHSC, New Orleans, LA Acute alcohol intoxication (AAI) impairs the hemodynamic response to hemorrhagic shock (HS) and fluid resuscitation (FR) with lactated Ringer’s (LR) and attenuates the HS-induced rise in plasma arginine vasopressin (AVP). AAI enhances nitric oxide (NO) inhibitory tone in the paraventricular nucleus (PVN) during HS, but not during osmotic stimulation with hypertonic saline (HTS). Thus, we hypothesized FR with HTS would decrease PVN NO and enhance AVP leading to improved blood pressure (BP) recovery and organ perfusion in AAI-HS. Sprague Dawley rats received a 15h alcohol infusion (2.5g/kg + .3g/kg/h) or dextrose (DEX) prior to HS (40mmHg x 60 min) and FR with HTS (7.5%; 4ml/kg) or LR (2.4x blood volume removed). HTS decreased PVN NO and enhanced (~66%) AVP vs LR in AAI- and DEX-HS 2h post-FR. HTS improved BP recovery vs LR in AAI (109 vs 80 mmHg) and DEX (114 vs 83 mmHg). Peripheral V1a antagonism prevented the BP response to HTS without affecting LR-treated. Organ blood flow was not altered in AAI shams or DEX-HS 2h post-FR. AAI-HS significantly reduced blood flow to liver (72% vs sham), small intestine (SI; 65% vs sham), and large intestine (LI; 67% vs sham). HTS decreased LI blood flow in DEX-HS (43% vs sham) without altering liver or SI. In AAI-HS, HTS significantly increased liver (3-fold vs LR) and SI (2-fold vs LR) blood flow. These results suggest that HTS removes central inhibition of NO restoring circulating AVP and that the pressor effect of HTS is dependent on AVP release. Furthermore, the enhanced BP recovery with HTS improved organ perfusion after AAI-HS. DOD-PR-054196, NIAAA-7577, NIAAA-19587, AMA Foundation

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58 A CNS Gαi2 Protein-gated Renal Sympathoinhibitory Mechanism is Essential to Maintain Fluid and Electrolyte Homeostasis and Normotension in Response to Increased Dietary Sodium Richard D Wainford LSUHSC-NO Hypothesis: CNS Gαi2 proteins, which mediate sympathoinhibitory and natriuretic responses to i.v. isotonic saline volume expansion and acute i.v. sodium loading, control a central renal sympathoinhibitory pathway acting to prevent salt-sensitive hypertension in Sprague-Dawley (SD) rats. Methods: Intact or bilaterally denervated (RDNX) rats receiving a continuous i.c.v. infusion of a Gαi2 oligodeoxynucleotide (ODN; 25 μg/day) were fed a normal 0.4% (NS) or high 8% NaCl (HS) diet. After 21-days, MAP, 24h metabolic balance, plasma norepinephrine (NE), plasma renin activity (PRA) & CNS Gαi2 protein levels were determined (N=6/gp). Results: Endogenous PVN Gαi2 protein levels increased 8 fold (P<0.05) in response to HS intake in naïve SD rats. ODN-mediated prevention of Gαi2 up-regulation caused renal nerve-dependent fluid and electrolyte retention (24h Na+ balance [meq] naïve HS 0.3±0.1, Gαi2 ODN + NS 0.4±0.1, Gαi2 ODN + HS 2.9±0.3*, Gαi2 ODN, RDNX + HS 0.8±0.4τ), global sympathoexcitation (plasma NE [nmol/L] naïve HS 49±5, Gαi2 ODN + NS 62±5, Gαi2 ODN + HS 98±8*, Gαi2 ODN, RDNX + HS 73±5τ) and salt-sensitive hypertension (MAP [mmHg] naïve HS 128±3, Gαi2 ODN + NS 126±3, Gαi2 ODN + HS 147±3*, Gαi2 ODN, RDNX + HS 132±2τ) without altering HS evoked suppression of the reninangiotensin system. *P<0.05 vs. Gαi2 ODN + NS gp. τP <0.05 vs. Gαi2 ODN + HS gp. Conclusion: PVN Gαi2 protein up-regulation during high salt-intake is an endogenous antihypertensive renal nerve-dependent mechanism which mediates sympathoinhibition to maintain fluid and electrolyte homeostasis and normotension in salt-resistant subjects.

57 TNF-α Increases Cardiac Fibroblast Lysyl Oxidase by ALK5, Smad3, and PI3K Dependent Mechanisms T.G. Voloshenyuk T, A. Fournett, J.D. Gardner TNF-α increases cardiac fibroblast lysyl oxidase by ALK5, Smad3, and PI3K dependent mechanisms The increase of TNF-α is associated with cardiac hypertrophy, dysfunction, dilatation and heart failure in humans. In many cases the pathological increase of TNF-α levels was accompanied by increased collagen expression and deposition. Cardiac fibroblasts are the main synthetic cells of the heart and they play a significant role in myocardial remodeling. Cardiac fibroblasts produce collagens type I and III, the main fibrillar components of the extracellular matrix. Lysyl oxidase (LOX) is a key enzyme that catalyzes the final step required for covalent cross-linking of collagen molecules in the syntheses of functional extracellular matrix. There is evidence that increased expression of LOX is associated with fibrosis and cardiac dysfunction, yet little is known about the regulation of this enzyme in the heart. In this study we evaluate the influence of TNF-α on LOX expression in cardiac fibroblasts and the molecular pathways involved. TNF-α down regulated LOX transcript, protein expression and activity at 1 ng/ml. However, TNF-α at 30 ng/ml significantly enhanced LOX mRNA and protein expression, similar to the effects observed for TGF-β1 (1-10ng/ml) which were associated with an elevation of enzymatic activity in conditioned media of cardiac fibroblasts. These TNF-α effects were ALK5, Smad3 and PI3K/Akt dependent. Blockade of PI3K also prevented the upregulation of collagens I and III, BMP-1, and attenuated the activation of Smad3 and Akt. These findings indicate that cardiac LOX expression is regulated by TNF-α, and that this regulation is mediated by PI3K, ALK5 and Smad3 signaling pathways.

60 Impaired Blood Pressure Compensation to Hemorrhage in Obese Zucker Rats with Orthopedic Trauma Lusha Xiang, Silu Lu, and Robert Hester Univeristy of Mississippi Medical Center The obese Zucker rats (OZ) following orthopedic trauma (OZT) exhibit increased PGE2 along with a loss of skeletal arteriolar tone. We hypothesize that the elevated PGE2 production and resultant loss of arteriolar tone in OZT blunts the vasoconstrictor responses and blood pressure compensation to hemorrhage. Orthopedic trauma was induced with soft tissue injury and local injection of bone components in both hindlimbs in lean (LZ) and OZ (11-13 wk). One to three days after the orthopedic trauma, arteriolar tone and vasoconstrictor responses to phenylephrine (PE) or hemorrhage in spinotrapezius muscle were determined. In another set of experiments, the blood pressure responses to hemorrhage were measured in conscious animals. OZT exhibited elevated plasma PGE2 levels, decreased arteriolar tone, and a blunted PE-induced vasoconstriction. Treatment with glibenclamide inhibited PGE2-induced vasodilation in non-trauma animals and normalized the arteriolar tone as well as the PE-induced vasoconstriction in OZT. Hemorrhage resulted in similar blood pressure and vasoconstrictor responses in control animals and trauma-treated LZ (LZT). The recovery of blood pressure and vasoconstrictor response to hemorrhage were blunted in the OZT. These results suggest that the elevated circulating PGE2 in OZT blunted the adrenergic vasoconstriction via opening KATP channels, resulting in an impaired arteriolar constriction response to hemorrhage. These findings may reveal a possible mechanism for the increased risk following a second hemorrhage in obese patient with orthopedic trauma. (AHA SE Affiliate 0765396B, NIH HL089581 and HL51971)

59 Congenital Solitary Kidney Rats: A Novel Model to Study Cardiovascular and Renal Disease Xuexiang Wang1, Ashley Johnson1, and Michael R. Garrett1,2 1Department of Pharmacology,2Department of Medicine, University of Mississippi Medical Center Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) represent the single greatest cause of childhood renal failure. Unilateral renal agenesis (URA) or developing with single kidney is part of CAKUT and occurs at a frequency of ~1:1000 births. Some studies suggest that 20-40% of patients with URA develop hypertension, proteinuria and in some cases renal failure. However, inadequate long-term follow-up data in humans and lack of a good animal model has made it difficult for definite conclusions to be reached. Recently, our lab developed a new genetic animal model [i.e., the heterogeneous stock derived model of unilateral renal agenesis, (HSRA)] that exhibits spontaneous URA ranging from 50-75%. The model is characterized by kidney hypertrophy, proteinuria, and renal injury. We are currently performing a time course evaluation of cardiovascular and renal injury between 4 groups of animals; (1) HSRA-S (spontaneous URA), (2) HSRA-N (2-kidney control), (3) HSRA-UNX3 (uninephrectomy at 3 wks of age) and (4) HSRA-UNX8 (uninephrectomy at 8 wks of age). This study will provide detailed data on onset and progression of differences in cardiovascular and renal function in animals exhibiting spontaneous URA compared to control animals undergoing uninephrectomy in early postnatal development of the kidney (week 3) and later when development of the kidney is complete (week 8). These results will directly address the question of whether prenatal development with a solitary kidney predisposes to renal injury later in life and investigate the role of altered renal hemodynamics in the pathogenesis of cardiovascular and renal disease.

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62 Mechanisms of Leukocyte and Platelet Recruitment in the Microvasculature During Chronic Angiotensin II Administration. Alper Yildirim, D. Neil Granger Molecular & Cellular Physiology, LSU Health Sciences Center, Shreveport, 1501 Kings Hwy., Shreveport, Louisiana, 71130 Angiotensin II (AngII) contributes to the pathogenesis of hypertension and other cardiovascular diseases. AngII is also known to induce a pro-oxidative, pro-inflammatory, and pro-thrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets in the microvasculature, the mechanisms that underlie these responses remain poorly defined. In this study, we addressed the contributions of Ang II type-1 receptors (AT1r), RANTES, and interleukin-1 to the recruitment of leukocytes and platelets in skeletal muscle arterioles and venules during chronic (2 wks) infusion of AngII in wild type (WT) and mutant mice. Intravital microscopy was used to quantify the adhesion of leukocytes and platelets, and the emigration of leukocytes in the cremaster muscle microvasculature. In WT mice, AngII infusion induced the adhesion of both leukocytes and platelets in venules and only leukocytes in arterioles. These adhesion responses were not observed in AT1r-/- mice, in AT1r-/- bone marrow chimeras and in ANS treated mice (neutrophils were depleted in vivo by i.p injection of anti-mouse Gr-1 antibody) Although RANTES-/- mice and APS treated mice (platelets were depleted in vivo by i.p. injection of rabbit anti-mouse trombocyte antiserum) responded similarly to WT mice, IL-1r-/- mice exhibited significant reductions in leukocyte adhesion and emigration in venules, while platelet adhesion was unaffected. These findings implicate blood cell-associated AT1r and IL-1 in the proinflammatory and prothrombogenic phenotype assumed by microvessels exposed to chronic AngII. (Supported by HL26441)

61 Angiogenesis is Not Impaired in Mesenteric Microvascular Networks from Spontaneously Hypertensive Rats Yang, Ming and Murfee, Walter Lee Tulane University Elevated blood pressure during hypertension has been associated with microvascular rarefaction defined by loss of microvessels. However, whether rarefaction is a result of impaired angiogenesis remains unclear. The objective of this study was to compare angiogenesis across the time course of mesenteric microvascular network remodeling in adult spontaneously hypertensive versus normotensive rats. Angiogenic responses in 15-16-week-old SHR and Wistar rats at 0, 3, 5, 10 or 25 days post 20 minute exteriorization of the mesentery were quantified. Consistent with the phenomenon of rarefaction, vascular area per tissue area in unstimulated SHR was decreased compared to Wistar. By 25 days, SHR vascular area per tissue area had increased to the Wistar level and vascular length density and capillary sprouting were comparable. At 3 and 5 days, SHR and Wistar tissues displayed an increase in the capillary sprouting and vascular density relative to their unstimulated groups. At 10 days, capillary sprouting in the SHR remained elevated. The percent change in vascular density was elevated in the SHR compared to the Wistar group at 3 and 5 days and by 25 days the rate of change was more negative in SHRs. Our results suggest that SHR networks show faster growth in the beginning of remodeling process post stimulation and later an increased rate of pruning which may explain rarefaction in hypertension.

64 Protein Phosphatase 2A (PP2A) Plays a Critical Role Determining the Endothelial Cell Gene Expression Profile of Leukemia Inhibitory Factor (LIF) Carlos Zgheib, Thomas Sebastian, Rony Chidiac, Mazen Kurdi, George W. Booz Department of Pharmacology and Toxicology, School of Medicine, The University of Mississippi Medical Center, Jackson, Mississippi Vascular inflammation is initiated by stimuli acting on endothelial cells (EC) to enhance leukocyte adherence/chemotaxis and leads to vessel remodeling and endothelial dysfunction. A clinical feature of vascular inflammation is increased circulating interleukin 6 type cytokines (IL6TC) such as LIF. Yet the role of IL6TC in vascular inflammation is not fully defined. IL6TC signal mainly through the transcription factor STAT3. Canonical STAT3 gene induction is due to phosphorylation of both Y705 and S727, leading to STAT3 dimerization and DNA binding and enhancing homodimerization and DNA binding, respectively. As PP2A is responsible for dephosphorylating S727, we hypothesized that PP2A inhibitor calyculin A (CA) would enhance gene expression in human microvascular EC by LIF. Cotreatment with LIF and CA increased nuclear S727 phosphorylation 3.4 ± 0.3 fold at 60 min without affecting the fold-increase in nuclear pY705 levels: 7.6 ± 1.4 LIF vs 8.2 ± 2.2 LIF + CA. Unexpectedly CA cotreatment blocked LIF-induced expression of SOCS3 and CCL2 genes, which have a STAF_HOXF_STAT framework in their promoters. Indeed CA cotreatment reduced STAT3 binding to an oligonucleotide containing STAT3 consensus binding sites and caused marked serine phosphorylation of the peptidyl/prolyl isomerase Pin1 that was found to associate with STAT3 in the nucleus. Serine phosphorylation of Pin1 is reported to reduce its enzymatic activity. In conclusion, our results reveal a critical role for PP2A-Pin1 interplay in IL6TC inflammatory gene induction. We propose that STAT3 S727 phosphorylation acts to recruit Pin1 that enhances its DNA binding.

63 Serine Protease Effects on Paracellular Permeability in Cortical Collecting Duct Cells Miao Yu, Kathleen S Hering-Smith and L Lee Hamm Tulane University, Departments of Medicine and Physiology Luminal serine proteases activate Na channels (ENaC) in the collecting duct. Also, some of these proteases (including trypsin T) have effects on transepithelial resistance® and paracellular permeability (PP) which are opposite to those expected with ENaC activation. PP is controlled by tight junctions (TJ) that form a size- and charge- selective barrier. The present studies aimed to define the molecular mechanism(s) using M-1 cells. T decreased paracellular R (increased PP) and was also found to increase the dilution potential consistent with an increase in the Cl- to Na+ PP ratio. Expression of the TJ proteins ZO-1, claudin-4, claudin-8 and occludin were not altered by T. However, threonine phosphorylation of claudin-4 (measured using immunoprecipitation) was found to be altered by T. These data indicate that trypsin and other endogenous proteases may influence ion selectivity across the paracellular pathway via claudin-4 phosphorylation.

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66 Increasing Shear Stress on the Macula Densa Blunts Tubuloglomerular Feedback by Enhancing Macula Densa nNOS Activity Mediated by Primary Cilia Xiaolong Zhu, Deyin Lu, Yiling Fu, Haifeng Liu, Yan Lu, Luis A. Juncos, Ruisheng Liu University of Mississippi Medical Center We hypothesize that increase of sheer stress blunts tubuloglomerular feedback (TGF) by enhancing nNOS activity via bending primary cilia on the macula densa (MD). To determine the effect of shear stress on TGF, we perfused rabbit afferent arteriole (Af-Art) and distal tubule simultaneously. We increased shear stress by 1 fold in 80 mM NaCl solution via increasing viscosity by adding dextran (MW 200, 000). Osmolarity was adjusted to the same level with mannitol. Diameter of Af-Art decreased from 18.4 ± 0.6 to 14.5 ± 0.6 μm when increasing tubular NaCl from 10 to 80 mM (TGF was 3.9 ± 0.4 μm). TGF was 2.44 ± 0.1 μm (p < 0.05) in the presence of dextran. In MMDD1 cells cultured in a circular flow chamber we increased shear stress from 0.5 to 5 dynes/cm2 and measured nitric oxide (NO) production, which was elevated from 174.8 ± 21.5 to 276.1 ± 24.3 units/min (p < 0.01). To investigate whether flow-induced increases in NO involve an NKCC2- or nNOS-dependent mechanism, we repeated the double-perfusion experiment after inhibiting NKCC2 with furosemide (10–4M); nNOS with 7-NI (10–5M). The increase was blocked by 7-NI, but not by furosemide. We then determined whether primary cilia mediated the NO elevation. Cilia were found on both perfused MD and cultured MMDD1 cells by fluoresence immunoblotting. We removed the primary cilia of MMDD1 cells with an siRNA against polaris, repeated the above experiments, and found that increasing shear stress had no effect on NO (48.6 ± 17.5 vs. 62.7 ± 15.2 units/min). These data indicate that shear stress suppresses TGF by increasing macula densa NO from nNOS in a primary cilia dependent manner.

65 Assessment of Changes in Glomerular Permeability to Albumin in Rat Models of Glomerular Disease Jin Zhang, Jan M. Williams, Richard J. Roman University of Mississippi Medical Center, Jackson, MS Hypertension (HTN) and diabetes mellitus (DM) are the primary causes of proteinuria and chronic kidney disease. However, it is difficult to determine the relative importance of changes in the filtration of protein versus reuptake due to the lack of an efficient method to measure changes in glomerular permeability. The present study examined the ability of a new dilutional fluorescent technique to detect changes in the glomerular permeability to albumin in models of HTN (Fawn hooded-hypertensive (FHH) and Dahl S rats) and DM (STZ and T2DN rats) induced glomerular injury. The rats were treated with a high molecular weight marker in vivo and the glomeruli were isolated. Changes of the fluorescent signal in the glomerular capillaries in response to an imposed oncotic pressure gradient was used to determine dσAlb. The results presented below indicate that dσalb is reduced in various models of renal injury and that the changes correlate with the severity of proteinuria and renal injury. NIH HL36279, 29587 and DK.

67 Transient Receptor Potential Type C Channels Play a Critical Role in Angiogenesis Fouad A. Zouein, Thomas Sebastian, Jerry M. Farley, and George W. Booz UMMC Recent genetic studies indicate that transient receptor potential type C (TRPC) channels may play a critical though undefined role in angiogenesis. Here we sought to establish their importance in new blood vessel formation using an in vitro assay and human microvascular endothelial cells (HMEC1). RNA was isolated from HMEC1 obtained from the CDC and human coronary microvascular endothelial cells (HCEC) from a deceased individual and analyzed by RT-PCR. Results indicated that HMEC1 express mRNA for TRPC1, TRPC3, TRPC4, and TRPC6. HCEC express TRPC1 and TRPC4. Western blot analysis showed that HMEC1 express TRPC1, TRPC3, and TRPC6 protein. A Matrigel assay was carried out to assess the effect of the TRPC channel inhibitor BTP2 on angiogenesis. Treatment of HMEC1 with 1μM BTP2 reduced the number of branching intersections by 68% from 31.3 ± 2.3 for vehicle control cultures to 9.8 ± 1.8 for BTP2 treated cultures (mean ± SD, n=3, P<0.001). The density of sprouts per surface area was reduced by 48% from 1.32 ± 0.10 to 0.69 ± 0.06 (P<0.001). Finally, intracellular calcium levels in HMEC1 exhibited oscillations which were reduced by BPT2. In summary, our findings establish the utility of HMEC1 and HCEC as model systems to explore the role of TRPC channels in angiogenesis at the molecular level. Strategies aimed at exploiting their activity may be of benefit in reparative angiogenesis of the heart after myocardial infarction or in heart failure.

68 TRPV1 Receptor in the Hypothalamus of Type 1 Diabetic Mice Andrea Zsombok, Hong Gao, Kayoko Miyata, K. Doug Hebert, Muthu D. Bhaskaran, Andrei V. Derbenev Departments of Physiology and Medicine, School of Medicine and Neuroscience Program, Tulane University, New Orleans, LA, 70112 Preautonomic neurons in the paraventricular nucleus (PVN) of the hypothalamus regulate sympathetic and parasympathetic output to the liver participating in the regulation of hepatic glucose production. Recently, peripheral TRPV1 receptor (transient receptor potential vanilloid type 1) involvement in the regulation of energy and fat metabolism was discovered. However, the role of central TRPV1 on glucose metabolism is unclear. We revealed TRPV1 receptor expression levels in control and type 1 diabetic (T1D) mice by using Western blot. Patch-clamp recordings from control and T1D mice were performed to characterize synaptic properties of liver-related PVN neurons identified with PRV-152. Administration of capsaicin, a TRPV1 receptor agonist increased mEPSCs frequency in liver-related PVN neurons in control but not in T1D mice. There was no difference in total TRPV1 receptor protein expression, however increased phosphorylation of TRPV1 receptor was observed in T1D mice, suggesting acute desensitization of TRPV1 receptor. Therefore, T1D appears to affect TRPV1 receptor signaling contributing to autonomic dysfunction and dysregulation of glucose metabolism. Supported by AHA 10GRNT4540000, Tulane BIRCWH 2K12HD043451, NHLBI R21HL091293, R21HL091293-01A1S1.

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Banquet / Duling Hall Information

Transportation to and from Duling Hall

The Fondren Trolley will transport guests from the Cabot Lodge beginning at 6:15p.m. The Trolley will loop between the hotel and Duling Hall every 15 minutes until 7:30 p.m. From 7:30-9:00 pm the Trolley will loop every 30 minutes. From 9:00-10:00 pm, the Trolley will again loop every 15 minutes.

For those that would prefer to drive:

Driving Directions To Duling Hall from the Cabot Lodge

Make a left out of the Cabot Lodge parking lot onto North State Street. Stay in the Right lane. After 0.4 miles, the Road will split. Stay all the way to the Right. 0.7 miles after the split, make a left onto Duling Ave. Duling Hall will be on the Right (looks like an old school building). Duling Hall 622 Duling Ave Jackson, MS

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RonaldMcDonaldHouse

StudentsApartments

UniversityRehabilitation

Center

OfficeAnnex

Alumni House

CreditUnion

StudentUnion

School ofHealth Related �Professions

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tate

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Lakeland Drive

School of�DentistryLearning

Resource�Center

GuytonBuilding

School ofNursing

The University Hospital

ParkingGarage A

ParkingGarage B

Women's andInfant's Hospital

Children's �Cancer Clinic

Children's �Hospital

Methodist �Rehabilitation �Center

AddyMcBrydeCenter forthe Blind

UniversityMedicalPavilion

Clinical�SciencesBuilding

Woodrow Wilson Avenue

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The University of MississippiMedical Center

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