Calcium antagonist verapamil prevented pulmonary arterial hypertension inbroilers with ascites by arresting pulmonary vascular remodeling
Ying Yang a, Jian Qiao b,⁎, Huiyu Wang b, Mingyu Gao b, Deyuan Ou b, Jianjun Zhang b,Maohong Sun b, Xin Yang a, Xiaobo Zhang a, Yuming Guo a,⁎
a State Key Lab of Animal Nutrition, College of Animal Science and Technology, China Agricultural University (CAU), Beijing, 100094, P. R. Chinab Department of Basic Veterinary Medicine, College of Veterinary Medicine, CAU, P. R. China
Received 19 August 2006; received in revised form 9 January 2007; accepted 11 January 2007Available online 1 February 2007
Pulmonary arterial hypertension, characterized by vascularcell proliferation and obliteration of pulmonary small arteries(Pietra et al., 1989), is a frequent and a major complication ofmany diseases affecting the pulmonary circulation leading toprimarily or secondarily and sequentially severe right ventric-ular failure. It is recognized that change in calcium signaling,explained by Ca2+ influx and cytosolic Ca2+ concentration([Ca2+]cyt), contributed to the development and perpetuation ofpulmonary arterial hypertension by destroying the balancebetween vasodilating and vasoconstrictive forces, and prolifer-ative and anti-proliferative forces. The ascites syndrome in
⁎ Corresponding authors. China Agricultural University, Beijing, 100094,P. R. China. Tel./fax: +86 10 62732712.
broiler chickens, also known as pulmonary arterial hypertensionsyndrome, was attributed to metabolic burdening, whichresulted from intensive genetic selection for rapid growth,coupled with exposure to extreme environmental conditions andnutritional levels, such as low temperature, high altitude or highenergy diets. These conditions imposed difficulties on broilersin fulfilling tissue demands for oxygen, namely relativehypoxia, resulting in reduced blood oxygen saturation andhigh hematocrit values in broilers. It is known that pulmonaryarterial hypertension is the major course in the development ofthe disease, but the crucial mechanism is still not fullyelucidated (Currie, 1999; Enkvetchakul et al., 1995; Widemanet al., 1995). The prevention or regression of pulmonary arterialhypertension is an important goal of antihypertensive therapy inbroilers afflicted with pulmonary arterial hypertension syn-drome, right ventricular failure and ascites. In a recent study, wefound that endothelin-1 played an important role in thedevelopment of pulmonary arterial hypertension and eventual
138 Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
ascites in broilers (Yang et al., 2005). Furthermore, it has beenrecognized that the intracellular mechanism induced by thecombination of endothelin-1 and endothelin ETA receptor at thebeginning of the pulmonary arterial hypertension in mammals isCa2+ influx and [Ca2+]cyt increasing, which instigates thepulmonary artery smooth muscular cell contraction (Egan andNixon, 2004; Liu and Sturek, 1996; Nakajima et al., 1996).These findings inspired us whether or not calcium antagonistscould prevent pulmonary arterial hypertension in broilers byblocking the Ca2+ influx and further [Ca2+]cyt increasing.
Calcium antagonists are chemically and pharmacodynami-cally heterogeneous substances that block the voltage-depen-dent calcium channels entrance in the external cellularmembrane, inhibiting the transmembrane influx of calciumions into vascular smooth muscle cells, cardiac muscle cells andconductive tissue (Cubeddu et al., 1986; Hernandez-Hernandezet al., 2002).
The purpose of the study is to better understand the actions ofcalcium signaling in the development of pulmonary arterialhypertension by using verapamil, one of the calcium antago-nists, and characterize the preventive efficacy of verapamil onhemodynamics and pulmonary artery function of broilers withpulmonary arterial hypertension syndrome induced by lowtemperature. It may provide a new idea for preventing thedevelopment of pulmonary arterial hypertension and rightventricular hypertrophy, which lead to ascites in broilers.
2. Materials and methods
2.1. Animal preparation and treatment
Day-old male broiler chicks (Arbor Acre) of commercialstrain were maintained in an environmental chamber withcontinuous lighting at a normal temperature of 28–30 °C withfree access to water and a commercial chick starter diet (23%crude protein, metabolizable energy=13.4 MJ/kg, from 0–15 days) and then grower diet (20% crude protein, metaboliz-able energy=13.4 MJ/kg from 16–30 days). Slow-releaseverapamil was from Knoll AG, German. At 16 days old, birdswere randomly allocated to 6 experimental groups, one controlgroup and one saline group at normal temperature (28 °C), fourgroups at low temperature (15–18 °C), including lowtemperature, low temperature+saline, low temperature+ lowdose of verapamil (5 mg/kg body weight every 12 h) and lowtemperature+high dose of verapamil (15 mg/kg body weightevery 12 h), 100 chicks in each group. Birds were orallyadministrated with 2.0 ml solution containing verapamil 5.0 mg/kg or 15.0 mg/kg body weight every 12 h from 16 to 43 days ofage, and the same volume of 0.9% saline were orally given tobroilers at the same time interval in two saline groups. Birdswere weighed every other day to determine the dosages ofverapamil to be administrated. Twenty birds for time intervalsampling were randomly selected from each group on day 29,36 and 43. All animal protocols were reviewed and approved bythe institutional animal care and use committee of the ChinaAgricultural University and conform to National Institutes ofHealth guidelines for animal use.
2.2. Measurement of pulmonary artery pressure
At 29, 36 and 43 days old, heart rate (beat/min, bpm),pulmonary arterial systolic pressure (mm Hg) and pulmonaryarterial diastolic pressure (mm Hg) of broilers were measuredusing a right cardiac catheter based on a modified method(Guthrie et al., 1987) just before sacrificing. Briefly, birds wererestrained in a dorsal position on the operating-table and locallyanesthetized with 5% procaine chloride in the middle of theright neck. A polyethylene plastic catheter was inserted into thejugular vein after the jugular vein was separated. The catheterwas pushed forward slowly to the right ventricle for heart rateand then to pulmonary artery for pulmonary arterial systolicpressure and pulmonary arterial diastolic pressure. Pressuresignals were transmitted to the host computer of RM-6000 typePolygraph (Nihon Kohden Ltd., Japan) through a sensor andpresented on the monitor. The sensor was placed at the samelevel as the bird's heart was.
2.3. Measurement of erythrocyte packed cell volume and rightventricular hypertrophy
After the in vivo measurements were completed, birds wereanesthetized by a large dose of pentobarbital sodium andsubsequently sacrificed by bleeding from carotid artery. Bloodsamples were collected into heparinized microhematocritcapillary tubes. After centrifugation, the erythrocyte packedcell volume (%) was determined. The ratio of the weight of theright ventricle to the weight of the whole ventricle wasmeasured as ascites heart index (%).
2.4. Determination of pulmonary artery remodeling
Segments with the thickness of 0.5 cm adjacent to thebronchi were removed from the lungs and fixed with 10%formaldehyde solution for more than 24 h and dehydrated in anascending gradient of ethanol. After becoming transparent indimethylbenzene, the lung tissues were embedded in paraffinand routinely processed into sections of 5 μm in depthfollowed by Weigert–van Gieson staining for elastin (Widim-sky and Herget, 1990). Small pulmonary arterioles withexternal diameters of 50 to 100 μm and 100 to 200 μm werestudied using an automatic image analyzer (Q550IW, Leica,Germany) with the advanced software (Leica Qwin Pro,Version 2.2). Ten average regions of cross section werechosen. The adventitia and the lumen diameter were measured,following which the relative median thickness (%) and relativemedian area (%) were determined and analyzed. The relativemedian thickness and the relative median area of pulmonaryarteries with different cut angles and conditions of eithercontractile or relaxation were computed from the above mea-surements according to the methods of Barth's (Barth et al.,1993) and Wang's (Wang et al., 2001). For each bird, 5–10pulmonary median muscularized arteries with external dia-meters ranging from 50 to 100 μm and from 100 to 200 μm,were measured. The analysis was performed in a blind fashionby two experienced investigators.
Table 1Mean pulmonary artery pressure (mPAP), Heart rate (HR), erythrocyte packed cell volume (PCV) and ascites heart index (AHI) of broilers in the different groups andinvestigated intervals, respectively
Treatment Normal temperature Low temperature Low temperature
Item Age Control (n=20) Saline (n=20) Control (n=20) Saline (n=20) Verapamil (5.0 mg/kg) (n=20) Verapamil (15 mg/kg) (n=20)
aPb0.05, bPb0.01 vs. corresponding control and cPb0.05, dPb0.01 vs. corresponding saline group. Values are expressed as mean±S.D.Mean arterial pressure = Diastolic pressure+1/3 (Systolic pressure–Diastolic pressure).
139Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
2.5. Determination of the percentage of three types of smallpulmonary vessels
Observed under 40× optical microscope, the numbers ofsmall pulmonary vessels including muscularized arteries,partially muscularized arteries, and nonmuscularized vesselswith external diameter less than 200 μm were counted and thepercentage of each type of vessels was calculated. Under opticalmicroscope, the muscularized artery had continuous externaland internal elastic lamina, the partially muscularized artery hada continuous external elastic lamina and a discontinuousinternal elastic lamina, and the nonmuscularized vessel hadonly one single elastic lamina (Barth et al., 1995).
2.6. Statistical analysis
Comparisons between groups on day 29, 36 or 43 wereperformed using one-way ANOVA followed by the LSD test.Differences were considered statistically significant at the level
Table 2Relative medial thickness (RMT) and relative medial area (RMA) of broilers with pu200 μm in the different groups and investigated intervals
aPb0.05, bPb0.01 vs. corresponding control and cPb0.05, dPb0.01 vs. correspondiexpressed as mean±S.D.
of Pb0.05, and values are means±S.D. The statistical analysiswas performed with the software of SPSS 11.0 for Windows.
3. Results
3.1. Pulmonary artery pressure and heart rate
The mean pulmonary arterial pressure was significantlyincreased in non-treated birds and those treated with saline at lowtemperature on day 36 and 43 (Pallb0.05 vs control) (Table 1).However, mean pulmonary arterial pressure of birds treated withlow dose of verapamil showed no significant differencecompared with those of the corresponding control at normaltemperature at the three ages. High dose of verapamil decreasedthe mean pulmonary arterial pressure by 10.3 mmHg comparedwith the control on day 43 (Pb0.01).
Significant increases in heart rate were observed in broilerstreated with verapamil at the two doses compared with that ofcontrol on day 29 (Pb0.05) (Table 1), however the heart rates of
lmonary arteriole external diameter ranging from 50 to 100 μm and from 100 to
ng saline group. Values are mean of twenty birds with 5–10 arterioles each and
140 Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
birds in the two groups decreased to the control's level on day36 and eventually the heart rate of broilers treated with highdose verapamil was significantly lower than that in the controlon day 43 (Pb0.05). There was a significant decrease of heartrate in the untreated broilers compared to the saline treated andlow temperature controlled broilers. The heart rates were alsolower compared with that of normal temperature controlledbroilers (Pb0.05) at day 36 and 43.
3.2. Packed cell volume and ascites heart index
The packed cell volume was significantly higher in non-pharmaceutically treated broilers at low temperature compared
Fig. 1. Microphotograph of pulmonary artery structure of broilers with pulmonary a×100). (A) Section of a pulmonary arteriole of a broiler at 43 days old in normal tem28 days (at 43 days old) in normal temperature. (C) Section of a pulmonary arteriole oof a broiler treated with saline for 28 days (at 43 days old) in low temperature. (E) Sebody weight every 12 h a day) for 28 days (at 43 days old) in low temperature. (F) Secbody weight every 12 h a day) for 28 days (at 43 days old) in low temperature.
with those in normal temperature at day 29 and 36 (Table 1)(Pb0.05). At 43 days old, the packed cell volumes of untreatedbroilers and saline treated, low temperature controlled broilerswere significantly higher than those in control (Pb0.05).Nevertheless, the packed cell volume of birds treated with lowdose of verapamil showed no significant difference comparedwith those in control. Alternatively, birds treated with higherdose of verapamil had a significantly higher packed cell volumethan the normal temperature controlled broilers and the broilersthat were treated with low dose of verapamil on day 36 and 43(Pb0.05).
There was no significant difference between ascites heartindices of all the groups on day 29 (Table 1). Ascites heart
rteriole external diameter ranging from 50 to 100 μm (Weigert and van Giesonperature. (B) Section of a pulmonary arteriole of a broiler treated with saline forf a broiler at 43 days old in low temperature. (D) Section of a pulmonary arteriolection of a pulmonary arteriole of a broiler orally treated with verapamil (5 mg/kgtion of a pulmonary arteriole of a broiler orally treated with verapamil (15 mg/kg
Table 3The percentage of muscularized arteries (MA), partially muscularized arteries (PMA), and nonmuscularized vessels (NMV) in broilers with pulmonary arteriolesexternal diameter less than 200 μm in the different groups and investigated intervals, respectively
Treatment Normal temperature Low temperature Low temperature
Age (day) Item Control (n=20) Saline (n=20) Control (n=20) Saline (n=20) Verapamil (5.0 mg/kg) (n=20) Verapamil (15 mg/kg) (n=20)
aPb0.05, bPb0.01 vs. corresponding control and cPb0.05, dPb0.01 vs. corresponding saline group. Values are expressed as mean±S.D.
141Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
indices of broilers, in both non-pharmaceutically treated groups,at low temperature were significantly higher than that of thecorresponding control (Pb0.01), when the treatment periodswere prolonged from 36 to 43 days old. However, no significantdifference in ascites heart index was found between the othergroups at the above said treatment periods.
3.3. Pulmonary artery remodeling
The external diameters of pulmonary arteries were similarbetween groups, either within 50≤Φ≤100 μm or within100≤Φ≤200 μm on day 29, 36 and 43. Nevertheless, the meanexternal diameter of pulmonary arteries of birds in the high doseof verapamil group was shorter than those in the other groups(data not shown). The relative median thicknesses weresignificantly greater in the two non-pharmaceutically treatedgroups at low temperature than at normal temperature and thetwo groups treated with verapamil on day 36 and 43,respectively (Pallb0.05) (Table 2, Fig. 1). Similarly, nosignificant difference was observed between relative medianareas of birds treated with low dose of verapamil and of thebirds in control group, whereas relative median area of birdstreated with low dose of verapamil was smaller than those in thetwo non-pharmaceutically treated groups at low temperature onday 36 and 43, respectively (Pallb0.05) (Fig. 1).
The percentages of the three types of small pulmonaryvessels showed no significant difference between any of the twogroups on day 29 (Table 3). At 36 and 43 days old, thepercentages of muscularized arteries and partially muscularizedarteries in small pulmonary vessels of broilers in the two non-pharmaceutically treated groups at low temperature weresignificantly higher than those of the other groups (Pallb0.05),respectively. On the contrary, the percentages of non-muscular-ized vessels in small pulmonary vessels of broilers in the twonon-pharmaceutically treated groups at low temperature weresignificantly lower than those in other groups.
4. Discussion
According to the studies regarding pulmonary arterialhypertension and ascites syndrome in broilers, mean pulmonary
arterial pressure, ascites heart index, packed cell volume andheart rate have been determined as the pathophysiologicalparameters for assessing the development of the disease inyoung broilers. It was proved that the mean pulmonary arterialpressure, ascites heart index and packed cell volume of broilersraised at low temperature were much higher than those ofbroilers raised at normal temperature, conversely, heart rate waslower than those in normal groups (Enkvetchakul et al., 1995;Wideman et al., 1995). In the present study, the significantincreases of mean pulmonary arterial pressure accompaniedwith the increases of packed cell volume and ascites heart indexin the two non-pharmaceutically treated broilers at lowertemperature compared with those at normal temperature groupsindicated that low temperature did induce the occurrence anddevelopment of pulmonary arterial hypertension in broilers.Additionally, all 3 structural characteristics of pulmonaryarterial hypertension (right ventricular hypertrophy, increasedsmooth muscle wall areas of resistance arteries, and themuscularization of normally nonmuscular arteries) wereobserved in broilers raised at low temperature, and meanpulmonary arterial pressures were elevated. As anticipated,there was no significant increase in mean pulmonary arterialpressure, packed cell volume and ascites heart index of broilerstreated with the low dose of verapamil at lower temperaturecompared with those of control at normal temperature.
Verapamil, a phenylalkylamine derivative, L-voltage-depen-dent calcium channel antagonist, was reported to inhibit thecontraction of rat portal vein (Dacquet et al., 1987; Singh et al.,1978), depress sinoatrial node automaticity and slow atrioven-tricular node conduction (Grossman and Messerli, 1997; Pitt,1997). Pretreatment of rats with verapamil (10–100 μg/kg/min,intravenous infusion) markedly attenuated the hypertensioninduced by Phoneutria nigriventer venom (Costa et al., 1996). Itwas also reported that, verapamil (2 mg/kg, i.v.) decreased boththe arterial pressure and cardiac output (Lay et al., 2006).
Increase in [Ca2+]cyt, which is subsequent to a hypoxiainsult, has been suggested to play a vital role in excitotoxiccascades leading to cell injury (Morley et al., 1994; Siesjo,1989; Stary et al., 2003). Abnormalities in pulmonary vascularsmooth muscle are also likely to contribute to alterations invasoreactivity during chronic hypoxic pulmonary hypertension
142 Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
(Hu and Wang, 1994; Mandegar et al., 2002; Shimoda et al.,2000b; Tirosh et al., 2006). Calcium entry into the cellsactivates contractile system in the smooth muscle, whereas,depletion in the amount of intracellular calcium tends to reducesmooth muscle contraction force, which is the basic mechanismof the clinical anti-hypertension agents being used in varioushypertension (Del Valle-Rodriguez et al., 2006; Hernandez-Hernandez et al., 2002; Wamhoff et al., 2006). The suppressionof verapamil at the dose of 5 mg/kg indicated that verapamilblocked L-voltage-dependent Ca2+ channels, subsequent largeCa2+ influx, and interdicted the abnormal contractile action ofpulmonary vessels, and prevented pulmonary arterial hypertension.
Furthermore, the results also provided other rationale bene-ficial actions of verapamil in preventing pulmonary arterialhypertension syndrome. Factors, such as cold and high altitude,increased the broiler's body demand for oxygen to enhance heatproduced for maintaining normal body temperature and basalmetabolism, which lead to a relative hypoxia status, andhypoxia had been conformed as an important pathophysiologicstimulus for erythrocytes number increment accompanied bypulmonary arterial hypertension (Yang et al., 2005). A higherpacked cell volume accounting for increased erythrocyteproduction indicated higher blood viscosity (Julian, 1993).One of the important parameters determining the right hearthypertrophy is the increase in the ascites heart index (weightratio of the right ventricle to the whole ventricle) (Shlosberg,1992). The augmentation in the above parameter occurs becauseof the formation of compensatory right ventricular hypertrophy.Verapamil at the dose of 5 mg/kg prevented the increases ofpacked cell volume and ascites heart index of broilers at lowertemperature.
Additionally, the results showing that verapamil preventedthe straightness of vessel cavity caused by pulmonary arteriolemedian thickening is in consistent with verapamil's action todilate the smooth muscle vessel by directly prohibiting theinflux of Ca2+ (Brundel et al., 2004; Pereira et al., 2004). It hasalready been proved that an increase in [Ca2+]cyt of pulmonaryartery smooth muscle cells due to activation of voltage-gatedCa2+ channels is not only one of the mechanisms underlyingchronic hypoxic pulmonary arterial hypertension (Shimodaet al., 2000a), but elevated [Ca2+]cyt is involved in stimulatingcell proliferation (Mandegar et al., 2004). Similarly, based onour results, it can be deduced that the relative hypoxia caused bylower temperature, resulted in [Ca2+]cyt enhancement, andsuccessive pulmonary arterial smooth muscle cells prolifera-tion. In contrast, verapamil with long-term administration inter-rupted the influx of Ca2+, prevented successive proliferation ofCa2+-dependent pulmonary artery smooth muscle cells. Ourfindings suggested that verapamil would be beneficial not onlyin the pulmonary vasodilation but also in modulating pul-monary arteriole remodeling.
As to the effect of the high dose of verapamil, the lowness ofmean pulmonary arterial pressure and heart rate on day 36 and43 compared with those in corresponding control suggested thatthe excessive pulmonary vasodilation resulted from the block-age of physiological influx of Ca2+ caused by verapamil withlong-term over dosage and the packed cell volume increasing at
the end of the experiment was due to the hypoxia induced by thelong term mean pulmonary arterial pressure decreasing.
In conclusion, our results revealed that long-term oral admi-nistration and appropriate dosage of the slow-release calciumantagonist verapamil could prevent the low-temperature-inducedacute pulmonary arterial hypertension syndrome pulmonary arte-rioles remodeling and right ventricular hypertrophy in broilers,which identifies the critical importance of calcium signaling in thedevelopment of pulmonary arterial hypertension in broilers.Ongoing trials are being conducted to investigate the intracellularcascade of calcium signaling in the cardiacmuscle and pulmonaryartery smooth muscle cells of broilers with pulmonary arterialhypertension syndrome in our group. These efforts would furtherelucidate the intracellular mechanism of pulmonary arterial hy-pertension syndrome mediated by calcium signaling.
Acknowledgements
We thank Vittal V. Kurisetty for critical reading of themanuscript. This work was supported by Scientific ResearchStart-up Foundation of The China Agricultural University (NO.2006001), Ministry of Science and Technology of P.R China(NO. 2006BAD12B07).
References
Barth, P.J., Kimpel, C., Roy, S., 1993. An improved mathematical approach forthe assessment of the medial thickness of pulmonary arteries. Pathol. Res.Pract. 189, 567.
Barth, P.J., Muller, B., Wagner, U.L., Bittinger, A., 1995. Quantitative analysisof parenchymal and vascular alterations in NO2-induced lung injury in rats.Eur. Respir. J. 8, 1115–1121.
Brundel, B.J., Kampinga, H.H., Henning, R.H., 2004. Calpain inhibitionprevents pacing-induced cellular remodeling in a HL-1 myocyte model foratrial fibrillation. Cardiovasc. Res. 62, 521–528.
Costa, S.K., Moreno, H.J., Brain, S.D., De Nucci, G., Antunes, E., 1996. Theeffect of Phoneutria nigriventer (armed spider) venom on arterial bloodpressure of anaesthetised rats. Eur. J. Pharmacol. 298, 113–120.
Cubeddu, L.X., Aranda, J., Singh, B., Klein, M., Brachfeld, J., Freis, E., Roman, J.,Eades, T., 1986. Comparison of verapamil and propranolol for the initialtreatment of hypertension: racial differences in response. JAMA 256,2214–2221.
Dacquet, C., Mironneau, C., Mironneau, J., 1987. Effects of calcium entryblockers on calcium-dependent contractions of rat portal vein. Br.J. Pharmacol. 92, 203–211.
Del Valle-Rodriguez, A., Calderon, E., Ruiz, M., Ordonez, A., Lopez-Barneo, J.,Urena, J., 2006. Metabotropic Ca2+ channel-induced Ca2+ release and ATP-dependent facilitation of arterial myocyte contraction. Proc. Natl. Acad. Sci.103, 4316–4321.
Egan, C.G., Nixon, G.F., 2004. Endothelin-1 and depolarization-induceddifferential regulation of cAMP response element-binding protein inproliferating and developed vascular smooth muscle. Cell. Signal. 16,1387–1396.
Enkvetchakul, B., Beasley, J., Bottje, W., 1995. Pulmonary arteriolehypertrophy in broilers with pulmonary hypertension syndrome (ascites).Poult. Sci. 74, 1677–1682.
Grossman, E., Messerli, F.H., 1997. Effect of calcium antagonists on plasmanorepinephrine levels, heart rate, and blood pressure. Am. J. Cardiol. 80,1453–1458.
Guthrie, A.J., Cilliers, J.A., Huchzermeyer, F.W., Killeen, V.M., 1987. Broilerpulmonary hypertension syndrome. II. The direct measurement of right
143Y. Yang et al. / European Journal of Pharmacology 561 (2007) 137–143
ventricular and pulmonary artery pressures in the closed chest domesticfowl. Onderstepoort J. Vet. Res. 54, 599–602.
Hernandez-Hernandez, R., Velasco, M., Armas-Hernandez, M.J., Armas-Padilla, M.C., 2002. Use of calcium antagonists in hypertension. Int.Congr. Ser. 1237, 109–117.
Hu, Q.H., Wang, D.X., 1994. Both hypoxic endothelial cell conditioned mediumand hypoxia elevate intracellular free calcium in pulmonary artery smoothmuscle cells. J. Tongji Med. Univ. 14, 200–203.
2006. Effect of verapamil on nitric oxide synthase in a portal vein-ligated ratmodel: role of prostaglandin. World J. Gastroenterol. 12, 2351–2356.
Liu, C.Y., Sturek, M., 1996. Attenuation of endothelin-1-induced calciumresponse by tyrosine kinase inhibitors in vascular smooth muscle cells. Am.J. Physiol. 270, C1825–C1833.
Mandegar, M., Remillard, C.V., Yuan, J.X., 2002. Ion channels in pulmonaryarterial hypertension. Prog. Cardiovasc. Dis. 45, 81–114.
Mandegar, M., Fung, Y.C., Huang, W., Remillard, C.V., Rubin, L.J., Yuan, J.X.,2004. Cellular and molecular mechanisms of pulmonary vascular remodel-ing: role in the development of pulmonary hypertension. Microvasc. Res.68, 75–103.
Nakajima, T., Hazama, H., Hamada, E., Wu, S.N., Igarashi, K., Yamashita, T.,Seyama, Y., Omata, M., Kurachi, Y., 1996. Endothelin-1 and vasopressinactivate Ca2+-permeable non-selective cation channels in aortic smoothmuscle cells: mechanism of receptor-mediated Ca2+ influx. J. Mol. CellCardiol. 28, 707–722.
Pereira, L.M., Bezerra, D.G., Mandarim-de-Lacerda, C.A., 2004. Aortic wallremodeling in rats with nitric oxide deficiency treated by enalapril orverapamil. Pathol. Res. Pract. 200, 211–217.
Pietra, G.G., Edwards, W., Kay, J., Rich, S., Kernis, J., Schloo, B., Ayres, S.,Bergofsky, E., Brundage, B., Detre, K., 1989. Histopathology of primarypulmonary hypertension. A qualitative and quantitative study of pulmonaryblood vessels from 58 patients in theNational Heart, Lung, and Blood Institute,Primary Pulmonary Hypertension Registry. Circulation 80, 1198–1206.
vasoreactivity in the chronically hypoxic lung. Physiol. Res. 49, 549–560.Shimoda, L.A., Sylvester, J.T., Sham, J.S., 2000b. Mobilization of intracellular
Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells.Am. J. Physiol., Lung Cell. Mol. Physiol. 278, L157–L164.
Shlosberg, A., 1992. The effects of poor ventilation, low temperatures, type offeed and sex of bird on development of ascites in broiler. Physiopathologicalfactors. Avian Pathol. 21, 369–382.
Siesjo, B., 1989. Calcium and cell death. Magnesium 8, 223–237.Singh, B.N., Ellrodt, G., Peter, C.T., 1978. Verapamil: a review of its pharma-
cological properties and therapeutic use. Drugs 15, 169–197.Stary, C.M., Kohin, S., Samaja, M., Howlett, R.A., Hogan, M.C., 2003.
Wang, X., Ohnishi, M., Wada, A., Tsutamoto, T., Sawaki, M., Fujii, M.,Matsumoto, T., Yamamoto, T., Kurokawa, K., Yamada, H., Kinoshita, M.,2001. Endothelin-1 promotes vascular structural remodeling during theprogression of heart failure prevention of vascular remodeling using aspecific endothelin-converting enzyme inhibitor. Life Sci. 69, 2477–2488.
Wideman, R.F.J., Ismail, M., Kirby, Y.K., Bottje, W.G., Moore, R.W.,Vardeman, R.C., 1995. Furosemide reduces the incidence of pulmonaryhypertension syndrome (ascites) in broilers exposed to cool environmentaltemperatures. Poult. Sci. 74, 314–322.
