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GRANT NO: DAJD17-90-Z-0022

TITLE: GLYCYL-L-GLUTAMINE: A DIPEPTIDE NEUROTRANSMITTERDERIVED FROM B-ENDORPHIN

PRINCIPAL INVESTIGATOR: William R. Millington, Ph.D.

CONTRACTING ORGANIZATION: University of Missouri-Kansas City2411 Holmes StreetKansas City, Missouri 64108

REPORT DATE: March 31, 1994

TYPE OF REPORT: Final Report VnC QUA~y INSpECTED 2

PREPARED FOR: U.S. Army Medical Research, Development,Acquisition and Logistics Command (Provisional),Fort Detrick, Frederick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited

The views, opinions and/or findings contained in this report arethose of the author(s) and should not be construed as an officialDepartment of the Army position, policy or decision unless sodesignated by other documentation.

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IMarch 31t_1994 Final Report (3/1/90 -3/31/94)

Glycyl-L-Glutamine: A Dipeptide Neurotransmitte:Derived from B-Endorphin Grant No.

DAMD1 7-90- Z-00226AUTHMRS)

William R. Millington, Ph.D.

I. PERFORMING ORGANIZATION NAME(S) AND ADORESS(ES) 3. PERFORMING ORGANIZTIONREPORT NUMBER

University of Missouri-Kansas City2411 Holmes StreetKansas City, Missouri 64108

1. SPONSORING/MONTORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING /MONITORINGAGENCY REPORT NUMBER

U.S. Army Medical Research, Development,Acquisition and Logistics Command (Provisional),Fort Detrick, Frederick, MD 21702-5012

11. SUPPLEMENTARY NOTES

120. DISTRIBUTION /AVAILA3ILTY STATEMENT 12b. DISTRIBUTION CODE

Approved for public release; distribution unlimjted

13. ABSTRACT (M~mum 200 wards)Glycyl-L-glutazmine (Gly-L-Gln) is a dipeptide synthesized post -trans lationally fromI-endorphin. Gly-L-Gln is a major product of £-endorphin processing in the brain-stem, pituitary and several peripheral tissues, but little is known about itsphysiological function. The long term objective of this research is to characterizeGly-L-Gln's biological activities and test the feasibility of developing pharma-cologic agents that mimic or antagonize its effects. This research has achieved fourobjectives: (1) Demonstrated that Gly-L-Gln is centrally active; it inhibits thehypotension and respiratory depression, but not the analgesia, produced by A-endor -phin. (2) Established the feasibility of developing peripherally active Gly-L-Glnanalogs; specifically, we found that cyclo-Gly-L-Gln inhibits morphine and £-endor-phin-induced cardiorespiratory depression when injected peripherally. (3) Demon-strated that Gly-L-Gln produces trophic and neuroimmune effects in peripheraltissues. (4) Developed analytical methods for measuring Gly-L-Gln in biologicaltissues. (5) Generated initial evidence that saturable Gly-L'-Gln binding sites are.present in brain. These data support the hypothesis that Gly-L-Gln functions as a;neurotransmitter in brain and a circulating hormone in the periphe.

14. SUWECT TIRMS 15L NUMBER OF PAGESNeurotransmitter; Cardiovascular Regulation; RAD IVGlycyl-L-glutauine; B3-ndorphin; Post-Translational I&. N=c coo.Processing; Proopiomelanocortin; Peptide;________

117. SEOJUTYCLASSNICATION IL. SEOJUFY 0ASSWAIMItN1. SNCURItY a..ASSW1CAIION 120. UMITATION OP ASASTRAOF REPORT OF THIS PAM OP ABSTRACTUnclassified Unclassified lUnclassified Unlimited

NSN 754041.230400 Stanard Form 296 (Rev. 2-89)P smu by AIM Sidi. MIS.l~02a

Opinions, interpretations, conclusions and recommendations arethose of the author and are not necessarily endorsed by the USArmy.

Where copyrighted material is quoted, permission has beenobtained to use such material.

____ Where material from documents designated for limitedd--tribution is quoted, permission has been obtained to use thematerial.

Citations of commercial organizations and trade names inthis report do not constitute an official Department of Armyendorsement or approval of the products or services of theseorganizations.

In conducting research using animals, the investigator(s)ahe~red to the wGuide for the Care and Use of LaboratoryAnimals,8 prepared by the Committee on Care and Use of LaboratoryAnimals of the Institute of Laboratory Resources, NationalResearch Council (NIH Publication No. 86-23, Revised 1985).

For the protection of human subjects, the investigator(s)adhered to policies of applicable Federal Law 45 CFR 46.

In conducting research utilizing recombinant DNA technology,the investigator(s) adhered to current guidelines promulgated bythe National Institutes of Health.

In the conduct of research utilizing recombinant DNA, theinvestigator(s) adhered to the NSH Guidelines for ResearchInvolving Recombinant DNA Molecules.

In the conduct of research involving hazardous organisms,th-einvestigator(s) adhered to the CDC-NIH Guide for Biosafety inMicrobiological and Biomedical Laboratories.

Acoossion forIUTS GRA&IDTIC TAB 0 3 Bimarr Dat0Unannounced

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Justificatlon

Disfpributioo/ .Availability Codel

Vail aoo6n

Secti Page Number

Cover Page . . . . . . . . . . ... . . . . . . 1

Report DocumenaintPage . .e. . ... . .. . . .. 2

Table ofContnts. . .. .. .. .. .. .. . . .. 4

Introduction . . . . . . . . . . . 0 0 . 0 0 . 0 . . 5-6

Results and Discussionl . . . . . . . . . . . . . . . 6-18

Summary andConlusions .... .. . . . . .. ... 18-20

Publications . . . . . . . . . . . . . . . . . . . . 21-24

References . . . . . . . . . . . . . . . . . . . o . 25-28

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INTRODUCTION

Glycyl-L-glutamine is the C-terminal dipeptide fragment ofthe opioid peptide, 8-endorphin, (S-endorphin-30-31) which issynthesized when 8-endorphin undergoes post-translationalprocessing. B-Endorphin processing has been intensely studied inrecent years because it profoundly alters the peptide's analgeticactivity, transforming B-endorphin-l-31 from a highly potent opiatereceptor agonist to an antagonist, B-endorphin-1-27, and to opiateinactive forms, B-endorphin-1-26 and the a-N-acetyl forms of allthree peptides (Deakin et al., 1980; Nicolas and Li, 1985). Glycyl-L-glutamine, co-synthesized with B-endorphin-1-27 when 3-endorphin-1-31 is endoproteolytically cleaved, has not been as thoroughlyevaluated as the larger S-endorphin forms, although existingevidence indicates that it, too, may participate in synaptictransmission. The objective of our research is to establishwhether glycyl-L-glutamine acts as a neurotransmitter in brain anda circulating hormone in the periphery.

Evidence that glycyl-L-glutamine functions in synaptictransmission first arose from electrophysiologic studies by Parishet al. (1983) showing that iontophoretic glycyl-L-glutamineapplication inhibited the firing frequencies of brainstem neurons.This activity was not blocked by either naloxone or strychnineindicating that it was not mediated by either opioid or glycinereceptors, respectively. Parish et al. (1985) also purifiedglycyl-L-glutamine from sheep brainstem and demonstrated that itis present in amounts equivalent to the sum of S-endorphin-l-27 and3-endorphin-1-26, as one would predict. Immunohistochemical

studies have also demonstrated that glycyl-L-glutamine is localizedin the intermediate, but not in the anterior lobe of the pituitary(Plishka et al., 1985) where S-endorphin does not undergo C-termi-nal cleavage (Eipper and Mains, 1980; O'Donohue and Dorsa, 1982).Hence these studies indicate that glycyl-L-glutamine is present inbrain and pituitary and that it inhibits the electrical activityof brainstem neurons.

Several additional lines of evidence further support theconcept that glycyl-L-glutamine acts as a neurotransmitter. Be-havioral studies revealed that glycyl-L-glutamine inhibits B-endor-phin induced grooming in rats, a response thought to reflectmechanisms of attention and arousal (Hirsch and O'Donohue, 1985).Glycyl-L-glutamine is also thought to function as a trophic agentat the neuromuscular junction (Lotwick et al., 1990) and in auto-nomic ganglia (Koelle et al., 1988). In both tissues, neuronalinnervation induces synaptic acetylcholinesterase (AChE) activity;glycyl-L-glutamine produces a comparable effect, suggesting thatit may be the neurotrophic agent mediating the response. B-endorphin has the opposite effect, reducing AChE, and may beresponsible for the subsequent lowering of AChE activity observedduring synaptic reorganization (Haynes et al., 1984).

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Glycyl-L-glutamino may also play a role in the neuroendocrineregulation of the immune system. A large body of evidence supportsthe concept that the central nervous system influences immuneresponse and much of this work focuses on the role of POMC peptidesas neuroimmune mediators (Morley et al., 1987). McCain et al.showed that low concentrations of glycyl-L-glutamine enhancephytohemagglutinin (PHA) induced T-lymphocyte proliferation (McCainet al., 1987). This was a key finding because the PHA response,which mimics antigen induced lymphocyte activation, is often usedas a measure of immune competence. Once again, S-endorphin had theopposite effect, suppressing PHA-induced proliferation (McCain etal., 1987). These observations suggest that glycyl-L-glutaminerelease from the intermediate pituitary may partially counteractstress-induced suppression of immune function.

These findings support the concept that glycyl-L-glutamine,like other 8-endorphin peptides, functions both as a neurotrans-mitter in brain and as a circulating hormone in the periphery;however, the basic studies required to firmly establish such a rolefor glycyl-L-glutamine have not been performed. Several criteriamust be fulfilled. First, glycyl-L-glutamine's pharmacologicspectrum of activity must be definitively established, emphasizingits interactions with B-endorphin and opiate drugs. Second, itmust be demonstrated that glycyl-L-glutamine is present in 8-endorphin releasing neurons and endocrine cells. And third,receptors for glycyl-L-glutamine must be unequivocally identifiedand thoroughly characterized. We predict that glycyl-L-glutaminereceptors exhibit 'synaptic specificity', meaning they are foundonly in 8-endorphin neuronal synapses. The concept of synapticspecificity is important because it means that drugs targeted onglycyl-L-glutamine receptors will act only at B-endorphin neuronalsynapses, unlike all existing opiate drugs which interact withdifferent opioid receptor subtypes present in all three opioidpeptide systems. Thus, the longer term objective of this researchis to establish the data base necessary to design therapeuticagents targeted on glycyl-L-glutamine receptors to selectivelymodify the biological effects of both glycyl-L-glutamine and 3-endorphin.

RESULTS AND DISCUSSION

A. Pharmacology: CNS Effects

1. Central Cardiovascular Reaulation (Appendix I): 8-Endorphingenerates severe hypotension, bradycardia and respiratorydepression when administered intracerebroventricularly (icy) torats and other species (Petty and Sitsen 1989; Millington andHirsch, 1994). These effects are thought to be mediated, in largepart, by mu opioid receptors the nucleus of the solitary tract andadjacent brainstem nuclei. Collectively, these observationssupport the hypothesis that A-endorphin plays an important role incardiovascular homeostasis.

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This conclusion must be tempered by the consideration that B-endorphin is extensively processed in brainstem neurons; a-N-acetyl-g-endorphin-(1-27), a-N-acetyl-g-endorphin-(1-26) and B-endorphin-(l-26) are the predominant forms (Zakarian and Smyth,1982) Unlike B-endorphin-(l-31), these N- and C-terminallymodified B-endorphin peptides display little or no affinity foropioid receptors and have no effect on peripheral hemodynamics wheninjected centrally (Hirsch and Millington, 1991). Glycyl-L-glutamine is thus a major end-product of S-endorphin processing inthe brainstem because it is produced in amounts equivalent to thecombined concentrations of S-endorphin-(1-27), 8-endorphin-(1-26),a-N-acetyl-B-endorphin-(1-27) and a-N-acetyl-B-endorphin-(1-26)(Parish et al., 1982). Consequently, brainstem glycyl-L-glutamineconcentrations substantially exceed S-endorphin levels. Thisraises the possibility that glycyl-L-glutamine may be an importantproduct of B-endorphin processing, not only quantitatively, butfunctionally as well. Despite its quantitative significance,glycyl-L-glutamine's physiological functions have not beenthoroughly evaluated and its role in cardiovascular homeostasis,if any, is unknown.

a) Glycyl-L-glutamine Inhibits B-Endorphin-Induced HyDotensionWe found that glycyl-L-glutamine inhibited 8-endorphin-inducedhypotension, but not bradycardia, when administered icy topentobarbital anesthetized rats 15 min after 8-endorphin injection.S-Endorphin (0.5 nmol) followed by saline injection produced arapid and sustained reduction in arterial blood pressure, loweringMAP by 42.3 ± 6.2 mm Hg within 60 min. Glycyl-L-glutamine (0.3,0.6, 1.0 or 10.0 nmol) dose-dependently inhibited S-endorphin-elicited hypotension; the lowest significant inhibitory dose was1.0 nmol. In contrast, glycyl-L-glutamine had no significant effecton S-endorphin-induced bradycardia. S-Endorphin-reduced heart rateby 154 ± 14 beats/min within 60 min from a baseline of 389 ± 15beats/min. Glycyl-L-glutamine partially restored heart rate tocontrol values but its effect was neither statistically significantnor dose-dependent. Glycyl-L-glutamine had no effect on MAP orheart rate when administered icy to animals that had not receivedS-endorphin at doses as high as 100 nmol. N-Acetyl-glycyl-L-glutamine and glycyl-L-glutamate also reversed B-endorphin-inducedhypotension although they were less potent than glycyl-L-glutamine.Glycyl-D-glutamine was ineffective, suggesting that the dipeptideresponse is stereoselective.

One important caveat to these experiments, is the possibilitythat the observed effects resulted from the hydrolysis of glycyl-L-glutamine to its constituent amino acids. Indeed, icy glycinelowers MAP albeit at relatively high doses (1 umol or more)(Persson, 1980). We found that co-administration of 1 nmol of eachamino acid, glycine and glutamine, had no effect whatsoever on 8-endorphin-induced hypotension. Consistent with published reports,a higher dose (1.0 gmol of each amino acid) potentiated, ratherthan inhibited, the hypotensive response to B-endorphin. Thesefindings indicate that glycyl-L-glutamine hydrolysis does notaccount for its cardiovascular effects and further suggest that

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glycyl-L-glutamine does not act through glycine receptors.

b) ResDiratory Devression: Like morphine, 8-endorphinproduces respiratory depression when injected centrally (Shook etal., 1990). To determine if glycyl-L-glutamine inhibits therespiratory depressant effect of B-endorphin, we measured bloodgases immediately before and 45 min after S-endorphin injection;as in previous experiments, glycyl-L-glutamine or saline wasadministered 15 min after B-endorphin. As expected, 8-endorphinfollowed by saline injection increased pCO2 and lowered p02 and pHalthough HCO3 " and base excess concentrations did not changesignificantly.

Glycyl-L-glutamine attenuated the hypercapnia and hypoxiainduced by 3-endorphin. Both the rise in plasma pCO and fall inpO elicited by 3-endorphin were significantly diminished bysubsequent glycyl-L-glutamine injection; indeed, pCO2 and pO werenot significantly different than baseline values followingsequential 8-endorphin and glycyl-L-glutamine administration.Glycyl-L-glutamine did not influence the reduction in plasma pHcaused by B-endorphin, however. When administered icy to rats thathad not been pretreated with S-endorphin, glycyl-L-glutamine (1.0nmol) had no effect on pCO2 ; pO or pH measured immediately beforeand 30 min post-injection. These data indicate that glycyl-L-glutamine attenuates B-endorphin-induced hypercapnia and hypoxiabut has no effect on blood gases when administered independently.

c) Glvcyl-L-glutamine Does Not Displace 3H-Naloxone Binding:The cardiorespiratory effects of 8-endorphin are thought to bemediated by mu opioid receptors (Petty and Sitsen, 1989; Millingtonand Hirsch, 1994) raising the possibility that glycyl-L-glutamineinhibits B-endorphin's effects by acting as a mu receptor antago-nist. To test this, we conducted receptor binding experimentsusing C3H]naloxone as a ligand. [ H]Naloxone binding to rat brainhomogenates was saturable with Kd and BUx values of 1.5 nM and 138fmol/mg protein, respectively, consistent with previous reports(Wood et al , 1981). Non-specific binding was less than 25% totalbinding. [h]Naloxone binding was displaced by morphine, 8-endorphin and S-endorphin-(1-27) with K. values in the nM rangecomparable to previously published data (Wood, et al., 1981).

Glycyl-L-glutamine failed to displace [3H]naloxone binding atconcentrations ranging from 1 pM to 10 mM (Table 4). For example,(3H]naloxone binding in the presence of 10 mM glycyl-L-glutamine,the highest concentration tested, was essentially the same ascontrol values (102.5 + 6.7 % control). Glycyl-L-glutamine istherefore unlikely to inhibit 8-endorphin-induced hypotension byacting as an opioid receptor antagonist.

d) Cyclo-vlycyl-L-glutamine: A Peripherally Active Analog:A longer term objective of this research is to test the feasibilityof developing peripherally active glycyl-L-glutamine analogs toselectively inhibit the cardiorespiratory depression, but not theanalgesia, produced by opiate drugs. Toward this end, we tested

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whether cyclo-glycyl-L-glutamine, a cyclic analog formed bycoupling glycyl-L-glutamine's N- and C-terminals, inhibited B-endorphin-induced hypotension. Cyclo-glycyl-L-glutamine was ofinterest in light of evidence that other cyclic dipeptides aremetabolically stable, and because they are non-polar, permeate theblood-brain barrier (Hoffman et al., 1977).

We found that cyclo-glycyl-L-glutamine effectively blocks 8-endorphin-induced hypotension. Initially, we tested whether cyclo-glycyl-L-glutamine was effective when injected centrally. We foundthat, like glycyl-L-glutamine, cyclo-glycyl-L-glutamine (0.3, 0.6or 1.0 nmol) produced a dose-related inhibition of B-endorphin-induced hypotension; indeed, it was nearly as potent as the linearform. In subsequent experiments, we found that intra-arterial (ia)cyclo-glycyl-L-glutamine injection (5 mg/kg) also inhibited thehypotension induced by central B-endorphin injection. A comparableintra-arterial dose of glycyl-L-glutamine was ineffectivesuggesting that cyclo-glycyl-L-glutamine is unlikely to act atperipheral sites. Cyclo-glycyl-L-glutamine (1, 5 or 50 mg/kg; ia),injected alone, was similarly without effect on peripheralhemodynamics. These results indicate that cyclo-glycyl-L-glutamineeffectively blocks the hypotensive effects of B-endorphin wheninjected either icy or intra-arterially.

Our final objective, was to test whether glycyl-L-glutamineblocks the cardiorespiratory depression produced by morphine andother opiates. In our initial experiments, we treated pentobar-bital anesthetized rats with a relatively high morphine dose (100nmol) icv, followed 15 min thereafter, by glycyl-L-glutamine (10nmol icy). Morphine alone produced profound hypotension, loweringMAP by up to 80 mm Hg; glycyl-L-glutamine completely blocked theresponse. Cyclo-glycyl-L-glutamine (5 mg/kg, ia) also effectivelyblocked morphine (100 nmol icy) induced hypotension. Furthermore,cyclo-glycyl-L-glutamine (5 mg/kg ia) inhibited the respiratorydepression induced by central morphine (50 nmol icv) injection.Together, these data indicate that both glycyl-L-glutamine andcyclo-glycyl-L-glutamine inhibit the cardiorespiratory depressionproduced by, not only 8-endorphin, but opiate drugs as well.

The mechanism responsible for glycyl-L-glutamine's inhibitoryeffects on opioid-induced cardiorespiratory depression remains tobe determined. However, the profound hypoxia and hypercapniaproduced by both morphine and S-endorphin suggests that thehypotensive response to opioids may be secondary to respiratorydepression in anesthetized animals. In support of this conjecture,we found that morphine and B-endorphin were substantially lesspotent hypotensive agents when administered to conscious ormechanically ventilated animals. This raises the intriguingpossibility that glycyl-L-glutamine may effectively oppose opioid-induced respiratory depression, a significant side effect of opiatedrugs. The prospect that glycyl-L-glutamine may inhibit one of theside effects of opiate analgetics prompted us to examine whetherthe dipeptide also blocks the analgetic effects of opioids.

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2. AntinociceDtion: To determine whether glycyl-L-glutaminemodulates the antinociceptive effects of B-endorphin we used thetail flick test which measures the response time for a rat toremove its tail from a beam of light. Initial experiments affirmedthat the technique generated consistent baseline data by testingthe antinociceptive effects of morphine (3.5 mg/kg ip; 30 or 100nmol, icy) and S-endorphin (1, 1.5 or 3 nmol; icy). Consistentwith previous reports, S-endorphin generated a prolonged increasein tail flick response latencies, which were maximal 40 min afterinjection and persisted for up to two hours.

After establishing consistent baseline data, we tested whetherglycyl-L-glutamine modulates the antinociceptive response to s-endorphin. Our initial results indicated that glycyl-L-glutamineinhibited the 8-endorphin effect when the two peptides were co-injected icy. Glycyl-L-glutamine (15 nmol) significantly reducedthe maximal response to 8-endorphin (1.5 nmol) by 67%. A higherglycyl-L-glutamine dose (50 nmol) produced a small, but non-significant inhibitory effeLt (39%) on B-endorphin-induced anti-nociception, but neither lower (5 nmol) nor higher (150 or 500nmol) glycyl-L-glutamine doses had any effect. Glycyl-L-glutamine,administered alone (5 nmol - 1.5 pmol) was ineffective. Hence, ourresults thus far are somewhat equivocal; glycyl-L-glutamine appearsto inhibit 8-endorphin-induced antinociception over a narrow doserange but produces no effect when administered alone. Futureexperiments will test whether glycyl-L-glutamine modulates theantinociceptive response to morphine. Collectively, these resultsindicate that glycyl-L-glutamine inhibits the cardiorespiratorydepression generated by B-endorphin, but has no consistent effecton S-endorphin-induced antinociception in the same dose range.

3. Thermoreaulation (Appendix II): The results of our studies oncardiovascular function suggests that glycyl-L-glutaminespecifically modulates the effects of S-endorphin, from which itis synthesized, as well as other opioids which activate the samereceptors. Alternatively, it is possible that glycyl-L-glutaminefunctions as an inhibitory modulator of central effects producedby other peptides synthesized from the B-endorphin prohormone, pro-opiomelanocortin (POMC). Fortuitously, one of our collaborators,Dr. Garth Resch, had recently demonstrated that a-MSH induceshyperthermia when injected into specific thermoregulatory sites themedial preoptic area (mPOA) of the hypothalamus (Resch and Simpson,1991). This finding provided us with an ideal opportunity to testwhether glycyl-L-glutamine modulates the central actions of asecond POMC peptide, a-MSH.

We found that glycyl-L-glutamine completely blocked the hyper-thermic response to a-MSH (Resch and Millington, 1993). a-MSH (60pmol) injection into the mPOA generated a sustained elevation incolonic temperature (Tc), reaching a maximum of 0.85 ± 0.19 *Cwithin 45 min. Co-administration of glycyl-L-glutamine (3 nmol)completely blocked the response to a-MSH, maintaining Tc atbaseline levels throughout the 45 min test period. Glycyl-L-

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glutamine, injected alone produced no significant effect on Tc,however. The inhibitory response to glycyl-L-glutamine did notresult from hydrolysis of the peptide to its constituent aminoacids because co-administration of equimolar amounts of glycineand glutamine had no effect whatsoever on a-MSH thermogenesis.Interestingly, the modulatory effect of glycyl-L-glutamine was ofunexpectedly long duration. Pretreatment with glycyl-L-glutaminesignificantly attenuated the response to a-MSH given 3 h or 24 hthereafter although it was fully restored within 48 h. Theattenuated response did not result from desensitization becausecontrol experiments showed that two a-MSH injections, given 4 h or48 h apart, produced the same rise in Tc. Thus, glycyl-L-glutamineproduces a long-lasting inhibition of the thermoregulatory responseto a-MSH without directly altering thermoregulation when givenalone. These aggregate results indicate that glycyl-L-glutaminemodulates certain of the central effects produced by both B-endorphin and a-MSH.

These results prompted us to consider whether, in addition toa-MSH, glycyl-L-glutamine inhibits the hyperthermia induced byother hyperthermic agents. The mPOA is an important site of actionfor interleukin 16 (IL-1B) and other pyrogens which are releasedfrom circulating lymphocytes as part of the acute phase responseto infection. The pyrogenic effects of IL-1B and related pyrogensare thought to be mediated by prostaglandin E (PGE 2) (Kluger,1991). Hence, our initial studies tested whether glycyl-L-glutamine inhibited the hyperthermic response to mPOA PGE 2injection. We found that glycyl-L-glutamine (3 nmol) completelyblocked the hyperthermia induced by PGE2 (3 nmol) (unpublisheddata). In subsequent experiments, we tested whether IL-1B-inducedfever was similarly affected. IL-1B (1 I.U.) alone, generated aprolonged rise in body temperature which lasted for the entire 2h duration of the experiment. Co-injection of glycyl-L-glutamine(3 nmol) completely blocked the response. These results indicatethat the thermomodulatory effects of glycyl-L-glutamine are notlimited to POMC peptides. Moreover, they show that glycyl-L-glutamine blocks the hyperthermia induced by typical pyrogenicagents suggesting that it may serve an antipyretic thermoregulatoryrole.

B. Pharmacology: Peripheral Effects

1. Trophic Effects on Cardiac Myocytes: The first evidence thatglycyl-L-glutamine may function as a hormone and/or neuromodulatorin peripheral tissues was the finding that glycyl-L-glutamineinduces the expression of acetylcholinesterase in sympatheticganglia (Koelle et al., 1988) and skeletal muscle cells (Lotwicket al., 1990). These tissues do not normally synthesize POMCpeptides, however, suggesting that glycyl-L-glutamine acts as acirculating hormone, following its release from the pituitary.Recent reports that B-endorphin immunoreactivity is localized incardiac tissue (Forman et al., 1989) prompted us to examine whetherglycyl-L-glutamine produces similar trophic actions on AChE in

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cardiac myocytes. Hence, our working hypothesis was that glycyl-L-glutamine may be synthesized and released from cardiac myocytesto regulate cardiac function locally.

AChE molecular forms can be divided into two classes,asymmetric or globular, based on the presence or absence of acollagen-like tail. The predominant asymmetric form, A islocalized in the basal lamina and is thought to be involved in thephysiological regulation acetylcholine hydrolysis (Rieger etal., 1980). Relatively high A12 AChE activity is detectable inintact rat heart, but not in cardiac myocytes in tissue culturesuggesting that a trophic factor maintains AU AChE expression invivo. To determine whether glycyl-L-glutamine induces expressionof A1 AChE, we prepared myocyte cultures by enzymaticallydissociating ventricles from 2-4 day old rats; the myocytes wereconfluent and beating for at least 48 h prior to experimentation.AChE molecular forms were analyzed by sucrose density gradientfractionation followed by analysis of AChE activity in theresulting fractions.

Previous studies by our collaborator, Dr. Nyquist-Battie(1987), showed that A12 AChE is a relatively minor component oftotal AChE activity in adult rat heart, comprising approximately5% of ventricular AChE activity. We found, in myocyte cultures,that a comparable proportion of total AChE was attributable to theA12 AChE form in myocyte prepared from pre-natal rat ventricles (8± 0.3 %). As in the adult, globular forms predominated, includingmonomeric, G, (58 ± 1 %), and tetrameric, G (34 + 3 %) (Nyquist-Battie et al., 1993). However, in post-natal myocyte cultures, A12AChE was not detectable. Co-incubation with glycyl-L-glutamine (1AM) for 72h increased the proportional amount of A12 AChE, restoringits activity to that of pre-natal cultures (8 ± 0.8 %). Correspond-ing decreases occurred in G, (53 + 3 %) and G4 (33 ± 3%) AChE.Glycyl-L-glutamine did not change the specific activity of eitherintracellular or secreted AChE. The response to glycyl-L-glutamineappeared to be relatively specific to the extent that neitherglycyl-L-glutamate nor glycyl-D-glutamine had any effect on AchEexpression. These results provide the first evidence that glycyl-L-glutamire produces trophic effects in the heart.

al 8-EndorDhin Processing in Rat Heart (Appendix III): Theobservation that glycyl-L-glutamine induces trophic effects incardiac cells supports the hypothesis that glycyl-L-glutamine actsas an autocrine factor produced locally in heart. Recent evidencethat B-endorphin immunoreactivity are localized in rat heartsuggests that glycyl-L-glutamine may be synthesized by cardiaccells (Forman et al., 1989). To obtain indirect evidence as towhether glycyl-L-glutamine may also be synthesized in cardiactissue, we analyzed the molecular forms of B-endorphin.

Sequential gel filtration and ion exchange HPLC analysesrevealed that B-endorphin-1-31 is extensively converted toendoproteolytic cleaved and N-acetylated derivatives in rat heart.Indeed, B-endorphin-1-31 comprised only 16 ± 4 % of total B-

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endorphin immunoreactivity in heart extracts (Millington et al.,1993). The single major form of cardiac S-endorphin immunoreac-tivity co-eluted with N-acetyl-B-endorphin-1-31. In addition, 8-endorphin-1-27, S-endorphin-1-26 and their N-acetylated con-generseach comprised between eight and sixteen percent of total 8-endorphin immunoreactivity. Hence, virtually all the 8-endorphinpeptides synthesized in heart are inactive as opioid receptoragonists.

These studies further revealed that B-lipotropin, ACTH and a-MSH were also localized in rat heart. Only low levels of 8-lipo-tropin and ACTH were detected, indicating that they primarily serveas precursors to 8-endorphin and a-MSH, respectively. Reverse phaseHPLC analysis further revealed that multiple forms of c-MSH werealso present, and like B-endorphin, acetylated peptides, a-MSH andN,O, diacetyl-a-NSH, redominated. These studies are the first toshow that multiple forms of a-MSH and B-endorphin are localized inheart and provide strong inferential evidence that glycyl-L-glutamine is also expressed.

It remained to be determined, however, whether the POMCpeptides found in rat heart are synthesized by cardiac myocytes orby autonomic neurons innervating the heart. To discern betweenthese two possibilities, we initiated immunohistochemicalexperiments using antisera against a-MSH, B-endorphin and, forcomparative purposes, atrial natriuretic factor (ANF). Thisrevealed that myocytes do, indeed, contain immuncreactive a-MSH andB-endorphin (Nyquist-Battie et al., 1994). Virtually every myocytewas stained, although the staining intensity was substantiallyhigher in atria than ventricles. In neonatal heart, staining wasof equivalent intensity in both regions. ANF immunoreactivity wassimilarly distributed in both adult and neonatal heart, consistentwith earlier reports (Ruskoaho, 1992). Axons were not stained byany of the antisera.

To further test the hypothesis that myocytes synthesize B-endorphin, a-MSH and related POMC-derived peptides, we examinedwhether POMC mRNA is localized in rat heart by using in situhybridization with colorimetric detection. We found that POMC mRNAis also detectable in rat atria; virtually all cells were labeled,albeit with varying intensities (Nyquist-Battie et al., 1994).Relatively intense staining was also present in cultures preparedfrom neonatal atrial or ventricular myocytes. These data providepreliminary evidence that POMC is synthesized and processed bycardiac myocytes. Experiments now in progress seek to confirmthese results by Northern blot analysis of POMC mRNA in rat heartand myocyte cultures.

3. Neuroimmune Effects: In addition to acting on peripheraltissues, a wealth of evidence indicates that B-endorphin peptidesmodulate immune function. For example, B-endorphin enhances phyto-hemagglutinin (PHA) induced T-lymphocyte proliferation, a measureof the ability of lymphocytes to respond to antigenic stimuli which

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is often used to teat immune competence (Gilmore and Weiner, 1988)Little is known about the role of glycyl-L-glutamine, however.

To determine whether glycyl-L-glutamine is also involved inneuroimmune regulation, we examined the effect of S-endorphin andglycyl-L-glutamine on lymphocyte proliferation in a human T-lymphocyte cell line, Jurkat E6-1. Mitogenic stimulation inducesa rapid increase in c-myc mRNA levels, both in normal lymphocytesand in Jurkat E6-1 cells (Hough et al., 1990). We found thatglycyl-L-glutamine (30 nM) produced a 2.5-fold increase inconcanavalin A (50 ng/ml) stimulated c-myc mRNA expression when co-incubated with sub-effective amounts of S-endorphin; when added tothe cell cultures alone, glycyl-L-glutamine had no significanteffect. Interestingly, these effects did not appear to be mediatedby interleukin-2 (IL-2) because B-endorphin had no effect on IL-2release from Jurkat cells. While yet preliminary, these resultssuggest that glycyl-L-glutamine potentiates S-endorphin induced c-myc expression.

al B-Endorphin Processing in the Human Pituitary Gland(Appendix IV): The finding that glycyl-L-glutamine modulates theeffect of B-endorphin on a human lymphocyte cell line raised thequestion as to the source of glycyl-L-glutamine which normallyregulates immune cell function. One source may be the lymphocytesthemselves, which are known to express B-endorphin and other POMCderived peptides (Morley et al., 1987). Alternatively, glycyl-L-glutamine may be released from the intermediate pituitary lobewhich, in the rat, produces large amounts of glycyl-L-glutamine(Plishka et al., 1985). To determine whether the human pituitaryalso synthesizes glycyl-L-glutamine, we analyzed the molecularforms of S-endorphin, extending our previous studies of B-endorphinprocessing in human brain (Millington and Smith, 1991). Thisrevealed that only small amounts of C-terminally shortened 8-endorphin peptides are localized in the human pituitary (Evans etal., 1993). B-Endorphin-(1-31) was the predominate 8-endorphinpeptide, comprising 85% of total 8-endorphin immunoreactivity.This indicates that the human pituitary does not producesignificant amounts of glycyl-L-glutamine; lymphocytes may be thesource of the glycyl-L-glutamine which modulates immune function.

Correlative immunohistochemical studies further revealed twounexpected findings regarding pituitary 8-endorphin processing. Weused several antisera to identified pituitary cells that synthesizeS-endorphin, including one which specifically recognizes N-acetyl-S-endorphin peptides. As expected, this antiserum stained a smallnumber of cells along the border between the anterior and neurallobes where the remnants of the fetal intermediate lobe arelocalized. Unexpectedly, however, N-acetyl-8-endorphin, as wellas a-MSH, immunoreactive cells were also dispersed throughout theanterior lobe. This indicates that cells resembling melanotrophs,biochemically, are distributed throughout the human pituitary,unlike virtually every other mammalian species in which N-acetyl-B-endorphin and a-MSH are localized in the intermediate, but notthe anterior lobe.

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These studies also revealed that S-endorphin immunoreactiveaxons were present within the human neural lobe (Manning et al.,1993). ACTH immunoreactivity has previously been reported in ratneural lobe axons (Knigge and Joseph, 1982) but innervation of thehuman neural lobe by POMC neurons has not been previously reported.We showed that human neural lobe axons were also labeled by anti-sera to ACTH and a-MSH, confirming their identity as POMC axons.Unexpectedly, a smaller number of axons were intensely stained bythe N-acetyl-B-endorphin antisera, suggesting that a subpopulationof neural lobe POMC axons N-acetylate S-endorphin peptides.

C. Analytical Methods:

An additional broad objective of our research has been todevelop analytical methods for isolating and measuring glycyl-L-glutamine concentrations in order to study its distribution inbrain and release in vitro. We also plan to use radiolabeledglycyl-L-glutamine to investigate glycyl-L-glutamine binding sitesand metabolism in brain, and for these applications, it is essen-tial to develop methods for separating glycyl-L-glutamine from itsconstituent amino acids, glycine and glutamine. We have had goodsuccess in developing HPLC methods for separating and quantifyingglycyl-L-glutamine although our efforts to develop highly sensitiveimmunoassays has been less successful.

1. HPLC Analysis: The development of HPLC methods addressed twoobjectives. First, to provide a method for separating radiolabeledglycyl-L-glutamine from glycine and glutamine. This is essentialfor receptor binding studies with 3H-glycyl-L-glutamine, for whicha necessary criterion is to demonstrate that the radioactivitybound to membrane receptors is 3H-glycyl-L-glutamine and not iH-glycine generated by enzymatic hydrolysis of the radioligand. Itmay also be necessary to purify 3H-glycyl-L-glutamine to be usedfor this purpose. A second objective is to develop a method formeasuring tissue glycyl-L-glutamine concentrations.

a) HPLC Separation of Glvcyl-L-glutamine, Glycine andGlutamine: To develop a method for separating radiolabeled glycyl-L-glutamine from glycine and glutamine, we initially used reversephase HPLC, a standard method for isolating neuropeptides. Wefound, however, that glycyl-L-glutamine was poorly retained byreverse phase columns, presumably due to its weak hydrophobicproperties. Conversely, we found that glycyl-L-glutamine was notsufficiently polar to be retained on standard anion or cationexchange resins. We therefore tested columns designed to analyzeamino acids and found that glycyl-L-glutamine could be completelyseparated from glycine and glutamine using a Beckman Spherosil Acolumn eluted with 10 mM ammonium acetate containing 50 mM sodiumchloride with retention times of 56, 22 and 12 min, respectively.This analytical HPLC method has enabled us to initiate receptorbinding experiments because it provides the capability ofmonitoring the purity of radiolabeled glycyl-L-glutamine.

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b) HPLC analysis with NDA derivatization: Subsequently, wedeveloped an HPLC assay, using fluorescent derivatization,initially using ortho-phthalaldehyde (OPA) as a derivatizing agentand separating the glycyl-L-glutamine/OPA derivative by reversedphase HPLC, a standard approach. This was marginally successfulbut its sensitivity (10 pmol) and resolving power were limited.To overcome these problems, we coupled glycyl-L-glutamine tonaphthalene-2,3-dicarboxaldehyde (NDA), a newly developed, moresensitive fluorescent derivatizing agent (Lunte and Wong, 1989)and used a modified RP-HPLC column (VyDac 201HS54). This methodsubstantially improved the sensitivity, linearity (100 fmol to 100pmol) and resolving capability of the assay. With it, we have beenable to detect endogenous glycyl-L-glutamine concentrations in acidextracts of the intermediate lobe of the pituitary. The assaysensitivity is too low to measure brain glycyl-L-glutamine concen-trations, although ongoing experiments seek to develop a tissueextraction method which will sufficiently improve its sensitivity.The assay, in its present form is clearly adequate for testingwhether glycyl-L-glutamine undergoes metabolism when incubated withimmune cells or brain homogenates in lymphocyte proliferation andradioreceptor binding assays, a critically important control.Hence, these experiments succeeded in establishing a relativelysensitive and rapid method for measuring glycyl-L-glutamine concen-trations in pituitary extracts and in vitro incubation medium.

2. Glycyl-L-Qlutamine Assay: Efforts to develop analyticalmethods for measuring endogenous glycyl-L-glutamine concentrationshave been unexpectedly problematic and, despite extensive timeinvestment, only partially successful. Three alternative methodsof analysis have been evaluated thus far: RIA, ELISA and HPLC withfluorescent detection. We first attempted to develop an RIA forglycyl-L-glutamine using an antisera generated by Plishka et al.(1985). We found that, like many antisera raised against smallmolecules coupled to larger proteins (Buijs et al., 1989) theantiserum did not recognize unconjugated C 6I]Bolton-Hunter- or3H-glycyl-L-glutamine and, hence, RIA analysis was not feasible.

We then attempted to establish an ELISA assay by couplingglycyl-L-glutamine to bovine serum albumin (BSA), using eithercarbodiimide or glutaraldehyde as coupling agents. We found thatthe antiserum recognized the glycyl-L-glutamine-BSA conjugate bySDS PAGE and Western blotting or by simply applying the conjugateto nitrocellulose filters. Dilution studies indicated that glycyl-L-glutamine immunoreactivity was concentration dependent withrespect to both antigen and antiserum. The method was specific forglycyl-L-glutamine to the extent that the antiserum did not recog-nize closely related dipeptides or B-endorphin conjugated to BSA.Consistent with our RIA experience, the antiserum did not recognizefree glycyl-L-glutamine applied to the nitrocellulose filter,confirming that protein coupling is required for the antiserum torecognize glycyl-L-glutamine.

Further development of a useful ELISA assay proved to beproblematic, however. In immunohistochemical studies we had found

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4 9 ..

that glycyl-L-glutamine displaced antisera labeling of pituitarytissue in which, presumably, glycyl-L-glutamine is conjugated totissue proteins by glutaraldehyde fixation. We therefore reasonedthat, as in other ELISA assays, unconjugated glycyl-L-glutamineshould displace antibody binding to the glycyl-L-glutamine-BSAconjugate. This turned out to be the case, but unfortunately, theassay sensitivity was in the micromole range, too low to measureendogenous glycyl-L-glutamine levels. Efforts to increase thesensitivity of the assay were unsuccessful.

3. Immunohistochemical Studies: Immunohistochemical studiesdesigned to map the distribution of glycyl-L-glutamine in thepituitary and brain have also been only partially successful. Wewere highly successful in labeling intermediate lobe melanotrophswhich, from studies of B-endorphin processing, are known to containglycyl-L-glutamine (O'Donohue and Dorsa, 1982; Plishka et al.,1985). As expected, anterior lobe corticotrophs, which do notfurther process B-endorphin, were not immunoreactive. Immunohi- -chemical analysis glycyl-L-glutamine's distribution in brevealed immunoreactive axons in the supraoptic, and ot.hypothalamic nuclei but the staining intensity was too low to becompletely convincing and did not label glycyl-L-glutamine immuno-reactive processes in other brain regions. We further evaluatedseveral alternative methodologies but none has substantiallyimproved the staining intensity. These studies were, nonethelessvaluable, because no neuronal processes were identified in regionsother than those known to be innervated by B-endorphin releasingneurons, consistent with our hypothesis that glycyl-L-glutamine issynthesized only in POMC neurons. However, further studies willrequire developing additional antisera. This remains a longer termobjective although other avenues of investigation currently holda higher priority status.

D. Receptor Binding Studies:

We have recently initiated experiments to determine whethersaturable and stereospecific glycyl-L-glutamine binding sites arepresent in brain membrane preparations. To accomplish thisobjective, we used a standard homogenate receptor binding method(Bennett and Yamamura, 1985) using 3H-glycyl-L-glutamine (5Ci/mmol). Non-specific binding was determined with 10 AM unlabeledglycyl-L-glutamine. Initially, we used a centrifugation method forseparating free and bound H-glycyl-L-glutamine, rather thanfiltration to facilitate testing whether 3H-glycyl-L-glutamine washydrolyzed during incubation.

In preliminary studies, we found that 3H-glycyl-L-glutaminespecifically bound to rat brain membrane preparations. Specificbinding was approximately 60% of total binding and was linear overa broad range of protein concentrations. Initial saturationanalysis indicated that the binding Kd for 3H-glycyl-L-glutaminebinding is 20 nM. HPLC analysis indicates that 3H-glycyl-L-glutamine binding is attributable to the intact dipeptide, rather

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than hydrolysis to glycine and glutamine. Glycine displaced 3H-glycyl-L-glutanine binding at very high concentrations; the IC. wasapproximately %mM. These results, while yet preliminary, indrcatethat specific "H-glycyl-L-glutamine binding sites are present inrat brain althoug4 additional characterization is necessary todetermine whether ýH-glycyl-L-glutamine binds to a unique glycyl-L-glutamine binding site or to a previously identified receptor.

SUMMARY AND CONCLUSIONS

A. Pharmacoloqy: CNOZ ffects

A principal objective of this research, was to determinewhether glycyl-L-glutamine is centrally active and if its actionsin brain are consistent with its biosynthesis with other PONCpeptides. The results demonstrate that glycyl-L-glutaminemodulates the cardiorespiratory effects of B-endorphin but producesno consistent effects on S-endorphin-induced antinociceptionsuggesting that glycyl-L-glutamine's effects are functionally, andperhaps, anatomically specific. Glycyl-L-glutamine also inhibiteda-MSH-elicited hyperthermia, indicating that its modulatory actionis not specific to B-endorphin from which it is synthesized.Glycyl-L-glutamine did not influence these physiological parameterswhen administered alone, however, suggesting that it functions asan inhibitory modulator but does not act independently. These dataprovide firm evidence that glycyl-L-glutamine is centrally active.

A second objective, was to explore the possibility thatglycyl-L-glutanine's central effects might be replicated byperipherally active analogs. We identified one lead compound,cyclo-glycyl-L-glutamine, which is essentially equipotent toglycyl-L-glutamine when centrally injected and which reproduces theeffects of the linear peptide when administered intra-arterially.These data suggest that cyclo-glycyl-L-glutamine permeates theblood-brain barrier by virtue of its lipid solubility, as shownfor other cyclic dipeptides using direct assays for blood-brainbarrier transport (Hoffman et al., 1977).

In the longer term, the specific effects produced by glycyl-L-glutamine and cyclo-glycyl-L-glutamine may also be of interestfrom a therapeutic standpoint. Respiratory depression, forexample, continues to be a limiting side effect of certain typesof therapy with opiate drugs. Although respiratory depression canbe controlled with opioid receptor antagonists, such as naloxone,antagonists also inhibit morphine's therapeutic effects. Incontrast, glycyl-L-glutamine does not act by blocking opioidreceptors and does not appear to inhibit opioid induced anti-nociception, raising the possibility that glycyl-L-glutamineanalogs may inhibit the side effects, but not the therapeuticactions of opioids. The finding that glycyl-L-glutamine's effectsare anatomically and/or functionally specific is not unprecedented;S-endorphin-l-27, for example, is a potent antagonist of a-endorphin-induced analgesia (Nicolas and Li, 1985) but acts as an

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agonist in tests of cardiovascular function (Hirsch and Millington,1991). Nevertheless, we have not, as yet, tested whether glycyl-L-glutamine and cyclo-glycyl-L-glutauine influence morphineanalgesia and so further study will be necessary before concludingdefinitively that glycyl-L-glutamine selectively inhibits thedeleterious, but not the therapeutic actions of opioids.

Studies on the thermoregulatory effects of glycyl-L-qlutaminealso led to unanticipated results. Initial experiments showed thatglycyl-L-glutamine inhibits a-MSH-induced hyperthermia when the twopeptides are injected into highly specific sites in the mPOA knownto contain thermoregulatory neurons as well as relatively highconcentrations of POMC-derived peptides. PGE injection into theseidentical sites also elicits thermogenesis a;though PGE2 does notmediate the effects of a-MSH (personal communication, Dr. G.Resch). These observations ultimately led to the finding thatglycyl-L-glutamine also blocked the fever induced by PGE and IL-l8 injection into the mPOA. These findings suggest ihat thefunction of glycyl-L-glutamine is not limited to the modulation ofother co-released POMC peptides. Nevertheless, glycyl-L-glutaminewas inactive when injected alone, and thus appears to opposepyrogenic increases in body temperature without changing normaltemperature homeostasis. Hence, glycyl-L-glutamine appears toinhibit perturbations in both cardiovascular and thermoregulatoryfunction without altering the homeostatic regulation of eitherblood pressure or body temperature when given alone.

a. Pharmacology: Peripheral Effects

Glycyl-L-glutamine also produces trophic and neuroimmuneeffects in peripheral tissues. We found, in some of the firstexperiments of the project, that glycyl-L-glutamine induces theexpression of the A,2 form of AChE in neonatal rat ventricularmyocyte cultures (Nyquist-Battie, 1993), suggesting the possibilitythat the dipeptide may be involved in the normal maintenance ofAChE expression. This observation was predicated on similarresults in skeletal muscle (Lotwick et al., 1990) and sympatheticganglia (Koelle et al., 1988); together, these finding suggest thatglycyl-L-glutamine's trophic action on AChE expression is wide-spread and not limited to cardiac tissue.

This observation prompted us to consider whether glycyl-L-glutamine may be released locally within the heart and serve anautocrine or paracrine function. Previous studies had localized8-endorphin immunoreactivity in the heart (Forman et al., 1989) butwhether POMC-peptides are actually synthesized by cardiac myocyteswas unknown. Moreover, B-endorphin processing had not been charac-terized in heart tissue and it was uncertain whether B-endorphinwas converted to 8-endorphin-1-27 and glycyl-L-glutamine. We foundthat 8-endorphin is almost entirely converted to C-terminallyshortened forms, providing strong evidence that glycyl-L-glutamineis also a major product of S-endorphin processing in the heart.Moreover, in situ hybridization and immunohistochemical studies

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provided prelininary evidence that POMC gene is expressed withincardiac myocytes. Myocytes also synthesize and release pro-enkephalin-derived peptides (Springhorn and Claycomb, 1989). Thus,the extensive processing of B-endorphin to non-opioid forms mayfunction as an opioid inactivating mechanism, promoting the actionof a-MSH or other POHC-derived peptides. Ongoing studies willinvestigate the regulation of POMC mRNA in cardiac myocytes.

C. Analytical Studies

A third objective of this research - to map the distributionof glycyl-L-glutamine in brain using both immunohistochemical andquantitative analyses - proved to be more problematic thananticipated and delayed our progress. Glycyl-L-glutamine proveddifficult to analyze chromatographically, because it is not suffi-ciently hydrophobic to be retained by hydrophobic resins, such asreverse phase HPLC, and is not sufficiently charged to be retainedby standard ion exchange chromatography. Ultimately, we used aweak cation exchange amino acid column to separate glycyl-L-glutamine from its constituent amino acids and developed a quanti-tative HPLC method by using NDA derivatization and fluorescentdetection with sufficient sensitivity to detect pituitary glycyl-L-glutamine.

These analytical techniques are essential for characterizingglycyl-L-glutamine receptors using 3H-glycyl-L-glutamine. We havenow demonstrated that -H-glycyl-L-glutamine binds to synapticmembrane preparations prepared from rat brain and that binding isdisplaced by unlabeled glycyl-L-glutamine, linear with respect toprotein concentrations and saturable with a Kd of approximately 20nM. An important prerequisite for these experiments, is theability to monitor the purity of the 3H-glycyl-L-glut~mine and toconfirm that binding is wholly attributable to H-glycyl-L-glutamine, and not to radiolabeled glycine formed by enzymatichydrolysis. Experiments now in progress fully characterize -H-glycyl-L-glutamine binding.

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PUBLICATION8

Fifteen manuscripts were published during the project period,several of which were represent work initiated during a priorUSAMRDC funding (86PP6813). In addition, three manuscripts arecurrently in preparation and we anticipate that at least two otherpapers well be generated from research initiated under this grant.

A. Journal Articles%

1. Hirsch, M.D. and Millington, W.R. Endoproteolytic conversionof S-endorphin-l-31 to B-endorphin-l-27 potentiates its centralcardioregulatory activity. Brain Res. 550:61-68, 1991.

2. Millington, W.R. and Smith, D.L. The post-translationalprocessing of 8-endorphin in human hypothalamus. J. Neurochem.57:775-781, 1991.

3. Millington, W.R., Dybdal, N.O., Mueller, G.P. and Chronwall,B.M. N-acetylation and C-terminal proteolysis of S-endorphinin the anterior lobe of the horse pituitary. Gen. Comp.Endocrinology 85:297-307, 1992.

4. Millington, W.R., Mueller, G.P. and Lavigne, G.L. Cholecysto-kinin Type A and Type B receptor antagonists produce opposingeffects on cholecystokinin stimulated Z-endorphin secretionfrom the rat pituitary. J. Pharm. Exp. Ther. 261:454-461,1992.

5. Lavigne, G.L., Millington, W.R. and Mueller, G.P. The CCK-Aand CCK-B receptor antagonists, devazepide and L-365,260,enhance morphine antinociception only in non-acclimated ratsexposed to a novel environment. Neuropeptides 21:119-129,1992.

6. Nyquist-Battie, C.N., Hagler, K. and Millington, W.R. Glycyl-L-glutamine regulates the expression of asymmetric acetyl-cholinesterase molecular forms in cultured cardiac postnatalmyocytes. J. Mol. Cell. Cardiol. 25:1111-1118, 1993.

7. Millington, W.R., Evans, V.R., Battie, C.N., Bagasra, 0. andForman, L.J. Pro-opiomelanocortin derived peptides and mRNAare expressed in rat heart. Ann. N.Y. Acad. Sci. 680: 575-578,1993.

8. Resch, G.E. and Millington, W.R. Glycyl-L-glutamine antago-nizes a-MSH-elicited thermogenesis. Peptides 14:971-975, 1993.

9. Millington, W.R., Evans, V.R., Forman, L.J. and Battie, C.N.Characterization of S-endorphin- and a-MSH-related peptides inrat heart. Peptides 14:1141-1147, 1993.

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10. Manning, A.B., Chronwall, B.M. and Millington, W.R. POC-derived peptide immunoreactivity in neural lobe axons of thehuman pituitary. Peptides 14:857-860, 1993.

11. Gehlert, D.R., Gackenheimer, S.L., Millington, W.R., Manning,A.B. and Chronwall, 1M. Localization of neuropeptide Yimmunoreactivity and C I]-peptide YY binding sites in thehuman pituitary. Peptides 15:651-656, 1994.

12. Evans, V.R., Manning, A.B., Bernard, L.H., Chronwall, B.M. andMillington, W.R. a-Melanocyte-stimulating hormone and N-acetyl-B-endorphin immunoreactivities are localized in thehuman pituitary but are not restricted to the zona intermedia.Endocrinology 134:97-106, 1994.

13. Chronwall, B.M., Dickerson, D.S., Huerter, B.S., Sibley, D.R.and Millington, W.R. Regulation of heterogeneity in D2 dopaminereceptor gene expression among individual melanotropes in therat pituitary intermediate lobe. Mol. Cell Neurobiol. 5:35-45,1994.

14. Millington, W.R. and Hirsch, M.D. Selective post-translationalprocessing of opioid peptides in cardioregulatory mechanismsof the dorsal medulla. In: Barraco, R.A., ed. Nucleus of theSolitary Tract. CRC Press, Boca Raton, FL, pp 315-326, 1994.

15. Unal, C.B., Owen-Kummer, M.D. and Millington, W.R. S-Endor-phin-induced cardiorespiratory depression is inhibited byglycyl-L-glutamine, a dipeptide derived from 8-endorphinprocessing. J. Pharmacol. Exp. Ther. (Submitted).

B. Publications In Preparation:

16. Resch, G.E. and Millington, W.R. Interleukin 1 and PGE2induced fever are blocked by glycyl-L-glutamine. (To besubmitted to Brain Research).

17. Unal, C.B., Owen-Kummer, M.D. and Millington, W.R.Inhibition of morphine induced hypotension and respiratorydepression by glycyl-L-glutamine and cyclo-glycyl-L-glutamine. (To be submitted to J. Pharmacol. Exp. Ther.).

18. Millington, W.R. and Manning, A.B. Opioid peptides:Analytical methods for the simultaneous measurement ofmultiple peptides derived from common prohormones. (To besubmitted to Methods in Toxicology).

C. Abstracts:

1. Hirsch, M.D., Villavicencio, A.E., McKenzie, J.E. andMillington, W.R. C-terminal proteolysis modifies cardio-regulation by B-endorphin. Society for Neuroscience, 1990.

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2. Lavigne, G.L. and Millington, W.R. Antinociceptive andpituitary B-endorphin studies with a novel cholecystokinin-Bantagonist, L-340-718. VIth World Congress on Pain, 1990.

3. Chronwall, B.M., Farah, J.M., Morris, S.J., Sibley, D.R. andMillington, W.R. Temporal characteristics of dopaminergicregulation of the rat intermediate pituitary: Secretion,POMC and D2 receptor gene expressions and cell prolifera-tion. Society for Neuroscience, 1990.

4. Lavigne, G.J. and Millington, W.R. The CCK-A and -Bantagonists, devazepide and L-365,260, potentiate morphineantinociception, but only in non-acclimated rats. ThirdIBRO World Congress of Neuroscience, 1991.

5. Dickerson, D.S., Pratt, B.S., Millington, W.R. andChronwall, B.M. D. dopamine receptor regulation in theintermediate lobe of the rat pituitary. Society forNeuroscience, 1991.

6. Bernard, L.H., Evans, V.R., Chronwall, B.M. and Millington,W.R. Beta-endorphin processing in human hypothalamus andpituitary. The Endocrine Society, 1991.

7. Bernard, L.H., Chronwall, B.M., Evans, V.R. and Millington,W.R. Post-translational processing of B-endorphin and ACTHin the human pituitary. The Midwest Anesthesiology Resi-dents Conference, 1991.

8. Evans, V.R., Forman, L.J. and Millington, W.R. Pro-opio-melanocortin-derived peptides in rat heart. Society forNeuroscience, 1991.

9. Battie, C.N., Hagler, K. and Millington, W.R. Glycyl-L-glutamine regulates the expression of acetylcholinesteraseasymmetric forms in cultured fetal cardiac myocytes. Societyfor Neuroscience, 1991.

10. Bernard, L.H., Chronwall, B.M., Evans, V.R. and Millington,W.R. Post-translational processing of 8-endorphin and ACTHin the human pituitary. American Society for Anesthesiol-ogy, 1991.

11. McQuillan, R., Lavigne, G.L., Fibuch, E. and Millington,W.R. B-Endorphin-induced antinociception is modulated byproducts of its post-translational processing. MidwestAnesthesiology Residents Conference, 1992.

12. McQuillan, R., Lavigne, G.L., Fibuch, E. and Millington,W.R. The role of B-endorphin processing in pain modulation.John J. Bonnica Pain Conference, Maui, Hawai, 1992.

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4

13. Killington, W., Evans, V., Battie, C., Bagasra, o. andForman, L. Proopiomelanocortin-derived peptides and mRNAare expressed in rat heart. Conference on The MelanotropicPeptides, Rouen, France, 1992.

14. Resch, G.E. and Millington, W.R. Glycyl-L-glutamine antago-nizes a-MSH elicited theruogenesis in PGE 2-sensitive mPOAsites. 14th Annual Winter Neuropeptide Conference,Breckenridge, CO, 1993.

15. Owen-Kummer, M.D., Unal, C.B., Fibuch, E.E. and Millington,W.R. Glycyl-L-glutamine (B-endorphin-30-31) inhibits thecentral cardioregulatory effects of B-endorphin-1-31.Midwest Anesthesiology Residents Conference, 1993.

16. McQuillan, R., Unal, C., Owen-Kummer, M., Lavigne, G.,Fibuch, E. and Millington, W. Glycyl-L-glutamine antago-nizes the antinociceptive and hemodynamic effects of B-endorphin. 7th World Congress on Pain, Paris, France, 1993.

17. Owen-Kummer, M.D., Unal, C.B., Fibuch, E.E. and Millington,W.R. Glycyl-L-glutamine inhibits the central hypotensiveeffects of B-endorphin-1-31. American Society for Anes-thesiology, 1993.

18. Unal, C.B., Owen-Kummer, M.D. and Millington, W.R. Inhibi-tion of S-endorphin-1-31-induced hypotension by glycyl-L-glutamine. Society for Neuroscience, 1993.

19. Resch, G.E. and Millington, W.R. Glycyl-L-glutamine antago-nism of a-MSH: Involvement of PGE2. Society for Neuro-science, 1993.

20. Manning, A.M., Chronwall, B.M., Evans, V.R., Millington,W.R. Localization of N-acetyl-S-endorphin immunoreactivityin anterior lobe corticotrophs and neural lobe axons of thehuman pituitary. Society for Neuroscience, 1993.

21. Pendergrass, D. and Millington, W.R. S-Endorphin inhibitionof T-lymphocyte proliferation is attenuated by glycyl-L-glutamine. Missouri Academy of Sciences, 1994.

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