STUDIES ON THE ROLE OF NEUROTRANSMITTERS ...

albo thank Mn.S. Subcaaanian, Rebeatch Olticea, VCRC 604adbibtiny me in the btati6tical analybib.I wish to thank all the 6acuLty membetb and non-teaching 6ta66 06 the School 06 Li6e Sciences 604 theik helpand encounayement thtoughout the couhbe 06 thib wohk.Ispecially thank Dn.K. Stikumat, Lectuteh in Li6e Sciencebhot the help(ul di6cubbionb and bugge6tionb.I would Like to thank my colleaguebMib4. Jayadevi, Mibb. Malatuizhi and Mh. Ptakabaa and othetb60h theit encounagemen.t and help6ul company.I thank my(amity membetb and gtiendb 60t theitencoutagement and buppott thtoughout my academic cateet.Thank6 ate aldo due to M/6 Ramtec Computehb,Pondichetty dot the data ptoce66ing and typing 06 themanudctipt.This wotk wad cattied out dutiny the tenuhe 06 atedeatch Fetlowbhip 6t0m Pondichetty Univetbity and 6tom the

CONTENTSPAGE NO.CXAPTW I : Introduction and Review of Literature. 1CHAPTER I1 : Materials, Methods and ExperimentalProcedures. 23CUFTEl I11 : Opiate and GABA-ergic Interaction onhypothalamic g a m glutamyl trnnspeptidaseactivity, plasma gonadotropins,prolactin,GH, TSH and T levels and testicular lactatedehydrogenase4activity in male rats. 40CUPTER IV : Effect of Morphine and Morphine antiserumon plasma T T and hypothalamic gammaglutamyl trynsp%ptidase and testicularlactate dehydrogenase activity in male rats. 56CHBPTER VCBBPTER VI: Effects of Serotonin synthesis blockade andopioids on plasma T and T andhypothalamic gamma glutam91 transpejtidaseand testicular lactate dehydrogenaseactivity in adult male rats. 65: Effect of Naloxone, Morphine, Morphineantiserum, Glutamic acid and Aspartic acidon plasma T and T levels testicularlactate dedfrdrogekse a)nd Sorbitoldehydrogenase activity in adult male rats. 73CBAeTER VII : Effect of intratesticular injection ofDynorphin, Morphine and Morphine antiserumon testicular lactate dehydrogenase andSorhitol dehydrogenase activity in adultmale rats. 83CBAPTW VIII: General discussion and conclusion. 90References. 96

AACTHAOMARMARCB-mBSACRFCLIPDY nM)PFSHGABAT-GTGSHGnRHGHGRFGRIFhCG125 I- adrenaline: adrenocorticotropic hormone- aminooxy acetic acid- antirabbit gamma globulin- arcuate nucleus- beta-endorphin- bovine serum albumin- corticotropin releasing factor- corticotropin like immunoreactive peptide- dynorphin- endogenous opioid peptide- follicle stimulating hormone- gamma amino butyric acid- gamma glutamyl transpeptidase- glutathione- gonadotropin releasing hormone- growth hormone- growth hormone releasing factor- growth hormone release inhibiting factor- human chorionic gonadotropin- 125 iodine- intraperitonral- intravenous- lactate dehydrogenase

LHLHRHYPOAMSHM-ASngPCPAPrlFOMCRNASDHSEMTSHTRH- leucineenkephalin- lipotrophin-. luteinizing hormone- luteinizing hormone releasing hormone- medial preoptic arga- median eminence- mediobasal hypothalamus- melanocyte stimulating hormone- millicurie- morphine sulfate- morphine antiserum- methionineenkephalin- noradrenaline- nanogram- parachlorophenyl alanine- prolactin- proopiomelanocortin- ribonucleic acid- sorbitol dehydrogenase- standard error of mean- Triiodothyronine- Thyroxine- Thyroid stimulating hormone- Thyrotropin releasing hormone- total counts- 100 $ binding.

CEAPTEIL IINTBOWCFION AND REVIEW OF LITERATURE

W g RINTKWCTION AND REVIEW OF LITERATUREIThe concept of neurohumoral control of piturtaryhormone secretron is well es tablrshed (Harris, 1948,Everett 1964, McCann 1974, Everett 1988). Green and Harris(1949) demnstrated the transport of neurohumoral factorsfrom the bran to the anterior prtuitary vla the pituitaryportal system. Subsequent search for the 'factors' led tothe discovery of releasing and lnbibltint; hormones from tnehypothalamus. This led to the identification of corticotro-piu releasing factor (CHF) (Saffran g g., 1055), Luterni-zing Hormone Relenslog Hormone (LHRH) (McCann g Q., 1960,Ilarris 1361), Tbyrotropin releasing factor (TRF), growthhormone releasin& factor (GRF) and prolactin inhibitingfactor (Review: YcCann 5 g., 1974).stimulating hormone (YSH) releaseLater on melanocyteand growth hormonerelease iuhibitlng factor (GHIF) were also ldentlfied(Kruiich et G. , 1972).Characterization and elucldatlon of amino acidsequences of LIIRH, TRli nnd GRIH by Schally a,, (1969)and Guillemla g& a., (1971) and the award of Nobel Prize rn1977 ushered In a new ora in Neuroendocrinology. Ensuingsearch for factors affectin& release of hypophysiotroprc

substances resulted in the discovery of various neurotrans-mitters and neuropeptides actingsystem.at the central nervousThe presence of tuberoinfundibular dopamlnerg~ctract (Fuxo and Hokfelt, 1966), noradrenergic projections(Ungerstedt 1971) and adrenergic axons (Hokfelt 5 &.,1974) In various sltes in the hypothalamus was demonstrated.Dlrect action of dopamine on prtuitary was reported by Birge- et Q., (1969) and Macleod (1969). McCann G., (1974)provided evldence showlng presence of a peptldic prolactininnibiting factor. Gamma amino butyric acid (GABA)(Grandlson and Guldottl, 1979), acetylcholine, serotonin,histamine, noreplnephrlne and epinephrine are the othersmall molecular weight transmitters reported to be ~nfluen-clng pituitary hormone release (McCann 1980, Vljayan 1985).Endogenous ogioid peptides IS a recent addrtion to the neu-romodulators nctlng at hypothalamus to influence pituitarysecretion 01 hormones (HughesRees, 1983).g., 1975, Grossman andThere are 40 or more substances includingneurotransmitters, oeuro-hormones, amino aclds and peptldeslocalized In different regloos of central nervous system.(Table I)-et aJ.,Ttre discovery of oploid peptldes in brain by Iluglies(1975) Lnltiated intense research on the role of

these peptrdes in- pituitary hormone release. Morley (1983)classified these peptides into five main groups.1. The two pentapeptides, methionineenkeyhalin andleucineenkephalin (Hughes aJ., 1975)keg - enkephalin - Tjr-Cly-Cly-Phe-Yet&d - enkephalin - Tyr-Glj-Cly-Phe-Leu2. Peytldes that arlse or are presumed to arise from enke-pbalin precursor. This includes Met enkephalinylArg-Phe (Rossrer g g., 1980), Peptide B (Kilyatrick- et g., 1981), Met enkeyhalinyl-Arg-Gly-Leu (Gubler- et g., 1982, Noda g g., 1982), dynoryhrn (Goldstein- et aJ., 1981) d and neoendorghln (Kanagava et g.,1981).3. p - endorphin (Bradbury g Q., 1976, Li 8 Chung 1976)and the related d , 5 and 6 endorph~ns (Lln g aJ. ,1976)4. Pronase resistant pegtides present in body fluids eg:p-casomorphin -5 aud -7 (Henschen et g., 1979) andpossibly anodynin (in blood) (Pert g g., 1976a).5. Various other peptides vhose opiate-like arop?rties donot arise from dlrect lnteractlon w~th op~ate oropiate-like receptors e&. Kyotorphln (Takagi & g.,

1079) which seems to act via release of enkephalins andinhibition of enkepblin degradatron.All the known endodenous opioid peptides are orl-ginated from three precursor molecules namely, prooplomela-nocortin, pro-dynoryhin and pro-enkephalin A.Pro-oplomelanocortln is the common precursor forp-END, adrenocorticotropin (ACTH),& - Uelanocyte stimulatingLiormone (1 -USH) and related bioactive peptides.cleaved to yield ACTH, from whichIt is-#SH 1s subsequentlysplit, and p-li~troyhin (PLPH), from whlch p-END, the 31ammo acid resldue C-terminal component is generated. Thereare major inter-tissue differences in the processing, post-translational modlf lca tion, and storage of p-END and ACTHrelated species (Watson g g., 1977, Zakarian and Smith1979, Holt 1986). These are primarily found in the anteriorand intermediate lobes of pltultary. However, thesepeptides have been reported from a wide variety of tissuesrncluding the pancreas, gastrointestinal tract, plncenta,thyroid, mast cells, the male reproductive tract and theCentral nervous system (Nakai 9 a., 1978, Larsson 1977,Kricger & g., 1980, Onoll and Kendall, 1980; TsongaJ., 1982a). Pro-dynorphin or Pro-enkephalin B encodes afamily of o~iolds comprising dynoryhin A (DYN A),dynorphinB (DYN B) and & - nooendorphiu, preddminantly located in

tlie neural lobe of .the pituitary. In the brain, DYN - rela-ted peptides are found in many hypothalamic nuclei, namely,suprachiasmatic nuclei, paraventricular nuclei, supraopticnuclei, arcuate nuclel, limbic system and midbrain(Goldstein, 1984).Enkephalrn immunoreactive neurons have been loca-ted in high concentrations in areas related to pain andanalgesia Ln the mammalian nervous system (Hokfelte.,1977a, Slmontov g., 1977). They are found in hypotha-lamic and extrahypothalamic sites of endocrine regulation.The external zone of the medlan eminence is particularlyrlch in enkephallnergic lnnervatlon whlch may have multlpleorigins, the yaraventrlcular nucleus and the arcuate nucleus(Udenfrlend and Kllpatrlk 1984, Holt 1986, WatsonaJ.,1984): The adrenal glands of some specles are part~cularlyrich sources of enkephallns aud related peptldes. Numerousenkephalin-contalnlng peptides ranging in molecular welghtfrom 0.5 to 20 k~lodaltons have been isolated from bovrnechromaff~n cells (Kilpatrick g e., 1982).OPIOID IUCWKHUOn the basis of the different pharmacologicalprollles of a number of oplates in neurophyslological andbehavioral tests it was suggested that there were three

types of receptqrs, designated p, Ir and w (Gilbert andMartin, 1976, Yartin g G., 1976).These have beenreported in rat, pigeon, squirrel monkey and rhesus monkey(Adler, 1981, Cowan 1981, Herling and Woods, 1981).Opioidreceptor consists of both a recognition site, to which thedrug binds and a factor that translatesthe binding rntobiochemical events which ultimately leads to a biologicalresponse. Three subtypes of opioid binding sites P, 6 andk have been analysed (Paterson fi d., 1983). Occupation ofreceptor sites by opioid agonists and opioid peptides leadto an inbibrtion of adenosine 3:5' cyclb phosphate (cyclicAMP) production, via an inhibition of adenylate cyclaseactivity (lest and ailler, 1983).Opioid dlrectedinhibition of adenylate cyclase also requires ~ a + and GTP(Blume g g., 1979, Lichtshtein 3 d., 1979). Electro-physiological experiments suggest that p and k opioid recey-tors can cou~le to G-protein which mediates activation ofinwardly rectifying potassium channels.k-type receptorshave been shown to be coupled to inhibition of voltagedependent calcium channels in several neuronal systems(Simnds, 1988).MDXMB: WIOID ANTAGONISTOplate research got a boost with the developmentof ryecific opiate antagonists, wst notably naloxone which

was shown to have pure antagonlst activities (Folders ae., 1983). Naloxone is an N-ally1 analogue of oxymorphone.Investigations by Blumberg g g., (1961) demonstrated thatnaloxone is a potent narcotic antagonist.Naloxonesyntbesised by Lewontein and Fishman (Humberg and Dayton1071) acts as an antagonist preferentially at receptors(Holt and Herz 1978).In a variety of studies, both in vivoand in vitro test systems, naloxone has been shown toproduce effects simllar to morphide (Mc Millan g &., 1970,Sawyknok g g., 1973, Soteropoulos g a., 1973) Prom thestudies on the effect of drugs interacting with dopaminergicsystem it 1s not clear as to whether these effects arebrought about oy naloxone directly or vla interaction withendogenous oplold peptldes (Sawynok a., 1979).KNDOGEWOUS OPIOIDS AND PITUITARY FUNCTIONBarraclough and Sawyer (1955) reported theinhibitory action of morphine on the release of pituitaryovulatory hormone. It was the discovery of stereoselectiveopiate receptors (Pert and Snyder 1973; Terenius 1973) andsubsequent finding of endogenous opiold peptides (HughesQ., 1975) which resulted in an Intense research into theendocrine aspects of brain opioid system. Endogenous opioidpeptides are primarily classifled into three familiesdorived from three distinct genes (Hoelt 1983).The

distribution of eddogenous opioid peptides (Cuello, 1983)and the very high concentration of opioids in the hypo-thalamus suggested that they maybe important inneuroendocrine regulation. In the anterior pituitary thereis little or no enkephalin, while thep-endorphin ispresent only in the few cells which also stain foradrenocorticotropic hormone (Rossier g., 1980).'Anterior pituitary has few opioid receptors whereasposterior pituitary is rich in both opioid receptors andopioid peptides.Shaare., (1977) demonstrated that opiates andendogenous opioids stimulate growth hormone and prolactinsecretion.Bruni g g., (1977) using opioid antagonistnaloxone, supported this finding.Further, they showed thatnaloxone increased serum LH and FSH hut failed to alterserum TSH concentrations in male rats (Bruni g aJ., 1977).pndorphin induced increases in serum Prl and CH (Lazarius,-et g., 1976, Li and Chung,(Rivier, g al., 1977).1976) was blocked by naloxoneThe tonic inhibitory action of MIPon the release of gonadotropins was confirmed by studieswith nnloxone which could increase LH release in sexuallyiuture female rats, in mature female and male rats (Blankst g., 1979; Cicero et e., 1979; Ieri aJ., 1979). Theeffects of opioids depend on a number of factors such as theage lad sex of the animal.Naloxone could raise serum LH

levels in castrated or castrated testosterone primed ratswhereas morphine lowered serum LH levels (Van fugt andHeites, 1980, Van Vugt 3 &., 1982). A variety of opioidagonists elevate ACTH and corticosterone in the rat afteracute administration (Haracz g g., 1981).On the otherhand administration of naloxone alone leads to elevations incirculating ACTH (Volavka g &., 1979a; Blankstein 9 &.,1980) and corticosterone (Ferri g g., 1980). It ispossible that the acute stimulatory effects of opioids aredue to nonspecific stress, when the rats are subjected toacute experimental stress opioids block the release of ACTHand corticosterone (De Souza &., 1981), and chronicmorphine administration has long been known to lead tosuppression of ACTH release (Briggs and Yunson, 1955). Thephysiological significance of the opioid tone may be that itis involved ln the ACTH response to stress possibly via anoradrenergic pathway (Grossman g Q., 1982 b, Grossman andBesser 1982).The posterior pituitary hormones, axytocin andvasopressin are predominantly inhibited by Dpioids. Opioidshave powerful influence on the magnocelluler neurosecretoryoxytocia system: they act both upon the secretory nervetelrin61s in the neurobypophysis and upn the electricaldischarge activity of the oxytocin cell bodies in thehypothaluus. Wrybine and other opioids block suckling

induced milk-ejection in rats (Clarke g a., 1979, Clarkeand Wright 1984, Wright 1985) and mice- (Iialdar and -Sawyer1978, Haldar g Q., 1982). Predominant effect of opioidson oxytocin neurons at both sites is inhibitory (VanWimersmaGreidanus and Ten Haaf 1985)Vasopressin release is alsoinhibited by opioids (Kamoi g g., 1979).Stressful circumstances lead to the impairment ofreproductive functions In rats (Euker g g., 1973, Hivier,- et aJ., 1986). These effects of stress include a decreasein circulating LH levels which may be due to an alteredpituitary responsiveness of other secretagogues (Suter andSchwartz 1985) as well as.by a pDssible decrease in theactivity of GnRH neurons (Gray et g., 1378, Kawakami andlliyuche 1981, Khorram et g., 1985, Rivrer g a., 1986).Centrally acting CW (Plotsky and Vale 1984, Hivier and Vale1984, Ono et g., 1984) and endogenous oploids have beenreported to be mediating stress induced suppression of LHsecretion. (Hulse et g., 1982, Hulse and Coleman 1983,Brisky g., 1984, Gilbeau and Smith, 1985). Petraglia- et g., (1986) suggested the activation of CRF/B -ENDpathway in the stress induced inhlbi tion of reproductivefunctions.

SITE 09 OPIOID ACl'IONA clear prcture of possible site(s) of opiateaction emerged when naloxone was dlrectly applied at putativesites within the preoptic-tuberal pathway in the brainof estrogen-progesterone pretreated ovarlectomised rats(Kalra 1981). Intracranial implantation of Naloxone inextrahypothalamic regions failed to stimulate LH releasewhereas both naloxone implants as well as intracerebralinjection of naloxone, anywhere in the narrow medial zoneextending caudally from the medlal preoptic area (UPOA) tothe median eminence-arcuate nucleus (YE-ARC) readily stimulatedLH release (Kalra 1981, Kalra 1983). Further, morphinepretreatment blocked the local effect of naloxoneimplants ln eliciting LH release. This evldence suggestedthat oploid receptors lnvolved In LH release may be locatedIn the YYOA and YE-ARC area. Naloxone lnfusion In vrtroreadily stimulated GnRH release from the medial basal hypothalamus(YBH) - POA of ovarlan sterold-prlmed ovarrectomi-sed and intact and castrated male rats (Leadem g g., 1985,Kalra a g., 1986, Kalra and Kalra 1987, Kalra g.,1988a).Studies on modulation of CnRH release byendogenous opioids and opiates revealed that concomitantalth GnRH release naloxone evoked prompt release of

noradrenaline (NA) and adrenaline (A) Prom the hypothalamus(Kalra aud Leadem 1984, Leadem et G., 1985, Kalra et &. ,1986). A functional Interaction between adrenergic neuronesand opiate receptors in reguiating GnRH was supported by thefinding that Naloxone induced LH release was inhibited byprior blockade of post-synaptic adrenergic receptors or byreduction in the releasable NA and A pools in axons andnerve terminals in the preoptic tuberal pathvay (Kalra 1981,Kalra and Simpkins 1981).These studies suggested the viewthat EOP neurones may ccmunicate vith GnRH neurones viaadrenergic systems In the preoptlc-tuberal pathvay.However, Killer g g., (1985) showed that opiate receytorblockade by naloxone is able to stimulate LH secretion inNE-lesloned male rats. Further, they demonstrated thenonesse~ltiality of adrenerglc system In mediating opioidaction on LH secretion.Dopamine (Rasmussen, 1991), GABA(Yasotto g g., 1987) Serotonin (Blackford g a., 1992)are also reported to he involved In the regulation of oyioidmediated pitul tary hormone release. Likewise, the site ofactlon of morphine and endogenous opioids with regard to TSHsecretion also remains controversial.However both hypo-thalamic (iomax s g., 1970) and pltultary (Judd and Hedge,1983) site of action for the peptlde In TSH secretion hasbeen reported.

OPIOIDS IN TESTICULAR FUNCTIONMaamalian testes have two interrelated functions:to produce the male gametes, the spermatozoa, and to producehormones. The hormones are primarily required for theyroduction of the spermatozoa, in regulating behaviour anddevelopment, structure and function of the accessory sexorgans and also in controlling, by negative feedback, thesecretion of gonadotropins from pituitary.Testicularfunction is regulated by the two bormones FSH and LH.Sertoll cells have been identified as the principal targetsite for FSH and testosterone (Fritz, 1978).Leydig cellsare the endocrine component of the testis, which is underthe influence of LH secreted from the pituitary (Setchell,1978, Sharpe, 1982, Tahka, 1986). Testicular function canbe locally modulated by pcptides (Sharpe 1983, Saez, 1989).Testosterone maintalns spermatogenesis by ~ts action onSertoli cells (Ritzen g., 1989). Normal Sertoli cellactivity is an absolute requirement for germ cellmultiplication and maturation and these cells cyclicallyregulate the Sertoli cell function (Saez 1987, Griswolda., 1988). Proteins of testlcular origln, both from Leydigcells and Sertoli cells play a significant role intesticulrr function. They exert autocrine, paracrine andalso endocrine function as in the case of inhibin whichinhibits FSH secretion whereas activln entiances FSH

secretion (Nurkadli 9 al-. , 1989). The four peptidesoxytocin, vasopressin, LHRB and opioids, originally ofneuronal origln have been reported to he present in thetestls. (Sharpe and Cooper 1986).Vasopressin in thetestis has been found to inhibit gonadotroprn-inducedandrogen biosynthesis (Adashi and Hsueh 1981, Wathes 1984).GnRH like immuno and/or bioactive materials were detected inthe rat testis (Sharpe and Fraser 1980, Hedger g g., 1985,Habert & aJ., 1991).In the adult rat native GnRH and GnRHagonlsts were observed both to stlmulate and lnhihit Leydigcell steroidogenesls In vitro and In vivo (Hsueh andErickson 1979, Hunter g g., 1982).Each of the threeoploid peytide precursors POMC, proenkephalin andprodynorptrln are expressed ln the testes (Chen g g., 1984,Kllpatrick Q., 1985, Douglas G., 1987). Theproenkephalin gene 1s widely expressed wlthin the male andfemale reproductive systems of rat and hamster (Kilpatrickand Bosenthal 1986).B-endorphin like materlal wasidentifled ln Leydlg cells and the eplthelra of eyididymls,seminal vesicles, and vas deferens of the rat (Tsong g a.,1982). & , and a endorylrln along w~th ALTH-like materialwas also lbcated in these tissues (Tsongg., 1982). POMClike mRNA was also detected in testes and eyididymis whichare 150 bases Shorter than those In the pltustaryandhypothalamus but were of the same size as detected In

sllygdala (Chen. g g., 1984. Chen a. , 1986). In thehypothalamus and extrapituitary tissues the processing ofFCUC resembles to that in the intermediate lobe.FOMC isprocessed to give rise to ACTH and PLPH. ACi'H is proces-sed to give rise to .G -YSH and Corticotropine like immunorerctive peptide (CLIP) whilePLPH is processed to giverise to 7-LPH and p-endorphin (Krieger &., 1980, 1984).It ha6 been established that !-endorphinlikepeptides are directly synthesised in Leydig cells (Pabbria., 1988), rather than being transported to the testis fmmOther tissues (ValenCa and Negro Vilar 1986).It was shornthat the levels of lmmunoreactive N-acetyl endorphin wasfound to be equivalent to that of immunoreactive pndoryhin(Cheng g Q., 1985).Opiate receptors have been located onthe Sertoli cells (Fabbri Q., 1985). These observationssuggested that POYC derived oploid peptides have local para-crine and autocrine effects on testicular function such assuppression of Sertoli cell growth during the quiescentstate of testicular development and modulation of rat andro-gen binding protein (r-ABP) and testosterone secretion inthe adult (Pabhri g g., 1988; Gerendai g e., 1986). Thenubar and intensity of staining of p-endorphin containingcells in muse and hamster testes were found to increase atpuberty (Sbaha a., 1984). In tbe rat POYC mRNA and thepeptide content has been found to be elevated at puberty

(Chen and Madigad 1987; Adams and Cicero 1989). The preciserole of POMC derived opioid peptides intion of testicular function is not clear.the normal regula-Enzymes of the testis have been examined aspossible products of bormone induced protein sgntbesisoccurring during maturation of germinal epithelium. Thuslactate dehydrogenase, hexokinase, acid phosphatase,B-glucuronidase, carnitine acetyl transferase, hyaluronidase,-(-glutmy1 transpeptidase, sorbitol dehydrogenase havebeen studied as possible aarkers for spermatogenesis or forhormone induced changes In the testis (Steinberger andSteinberger 1975).NEUROTRANSMITTEBS IN THE CONTROL OF ANTERIOR PITUITARYWWCT.IONWith the discovery that neurohumoral control ofpituitary was basically peptidtc in nature, efforts were onto locate, identify and cbaracterise the factors affectingthe release of various hypopbysiotropic factors.Demonstration that tubero infundibular dopaminergic tract(Fuxe, 1964), projections from the brain stem ofnoradrenergic (Ungerstedt, 1971) and adrenergic axons(Hokfelt a,, 1974), serotoninergic projections frommedian and dorsal rapbae nuclei (Azmita and Segal, 1970)

into various sites within hypothalamus suggested theirpossible role in release/inbibition of hypophysiotropicfactors (Vijayan g e., 1978; Review Mc Cann g& g., 1979).CUMMA MINO BUTPBIC ACID (GABA)GABA is widely distributed throughout the centralnervous system and appears to be an important inhibitorysynaptic tranaaitter in many areas of the brain (Davidson,1976). GABA is found in high concentrations in thehypothalamus (Okada Q., 1971). Immunocytochemicalstudies using antibodies directed against glutamic aciddecarhoxylase have revealed high concentrations of GABAterminals In most hypothalamic areas and also very highconcentrations of terminals In the internal and externallayers of the median eminence (Tappaz g., 1981). Thusit was suggested that GABA may be interactingphysiologically within the hypothalamus with neurons whichcontrol anterior pituitary homne secretion. Specific GABAreceptors have been remrted in the anterior pituitary gland(Grandison and Guidotti, 1979) suggesting direct action ofGABA on p,itutt.ry.Intraventricular injection of GABAproduced ararked alterations in pituitary howone release,with awntation of LH and GH release, inhibition of TSHreleu18 rsd a, biphasic effect on Prl release (inhibitoryeffects at low and stimulatory effects at high doses of

GABA) (ViJayan and Mc Cann 1978 a, Locatelli g Q., 1979;.Grandison, 1980, Mc Cann 3 e., 1981).Glutathione (L-r-glutamyl- L -cysteinyl- glycine)(CSH) has a ubiquitous distribution in the living cells.The functions that have been ascribed to glutathione include(a) maintenance of SH groups of proteins and other molecules(b) destruction of hydrogen peroxide, other peroxides andfree radicals (c) catalyst for disulfide exchange reactions(d) co-enzyme for certain enzymes like glyoxylase (e)detoxification of foreign compounds (f) translocation ofamino acrds across cell membranes (Melster and Tate, 1976)Metabolic degradation of llutathrone is catalysed by T-GTand a series of enzymes via the 3-glutamyl cycle (Meister,1974). S -GT is a membrane bound enzyme and is abundant inkidney (Albert &., 1962, Glossman and Neville, 1972,George and Kenny, 1973) and epithelia of jejunal villi,choroid plexus, salivary glands, bile duct, seminalvesicles, epididymis and ciliary body (Albert Q., 1964Albert &., 1966; Hanker, 1966, Greenberg Q., 1967,Rutenberg aJ., 1969, Albert, 1970, Seligman g Q., 1970,Tate gg aJ., 1973; Ross, 1973).It has been postulated thatX-GT functions In the translocation of aminoacids acrosscall wmbranes in a process in which I-glutamyl moiety

functions as a carrier (Meister, 1973, Orlowski, andMeister, 1970, Meister, 1974). The Substrates for theenzyme are glutathione (GSH), oxidised glutathione (GSSG),S-substituted glutathione and other I-glutamyl compounds.Recently Vali Pasha and Vijayan (1989) reported an increasein the hypothalamic level of glutathione in pubertal rats.Intraventricular administration of glutathione stimulatesthe release of anterior pituitary hormones (Pasha, 1988,Pasha and Vijayan, 1989). Intraventricular administrationof LHRR significantly increased glutathione level andactivity of T-GT in various regions of the brain includinghypothalamus. A number of peptides exert synergistic actionon the anterior pituitary. It is possible that someinteraction may exist between LHRH and glutathione in thehypothalamus. (Pasha and Vijayan, 1990).LACTATE DgBBaGEWASEIn I U ~ ~ five ~ S , tetrameric lactate dehydrogenase(LDH) isozymes, U)H-1 (B 2, LDH-2 (A p 2, LDH-3 (A 2 Z), LDH-4 (AgB1) and LM1-5 (A4) are found in various proportionsmng different somatic tissues, whereas the homotetramericLDR-C4 isozyme is present only in mature testis andspermatozoa (Yarkert g g., 1975, Blanco, 1980). Duringtssticular development, the LDH-A, -B- AND -C genes areexpressed differentially. Studies examining the activity

of LDH isozymes in unfractionated mouse testis found thatthe proportion of LDH-A supunlts decrease between 2 and 10days of age, while LDH-B subunits rncrease during thisinterval, and LDH-C4 activity is not detected until thepresence of pachytene spermatocytes (14-16 days postpartum)(Goldberg and Hawtrey, 1967; Wieben, 1981). Goldberg (1973)concluded that LDH-B subunits are synthesized only Insomatic cells within the testis. In contrast, LDH-C4 isconfined to spermatogenlc cells and has been detectedinmunohistochemically In spermatocytes beginning at the midpchytenestage of melosis and In haploid cells throughoutsp?rmiogenesis (Hlntz and Goldberg, 1977). Prepubertal germcell populations containing a mlxture of spermatogonia, prepachytenespermatocytes and nongermlnal cells showed nbevidence of LDH-C synthesis (Meistrlch g& g., 1977).LDH 1s localized primarily In the cytosol,although a small proportion appears to be localized inmitochondria and on the cell surface of spermatozoa (Storeyand Kayne, 1977, Alvarez and Storey, 1984). The enzyme playsan important role as a shuttle system for NADH betweencytoylasm.and mitochondria of sperm and may serve torqul6te energy generation and mol;ili ty of spermatozoa (Vandop g g., 1977, Blanco & g., 1976, Blanco, 1980).

Study of Testicular Sorbitol dehydrogenase (SDH)activity provldes a developmental marker under theinfluence of hormones. SDH activity has been reported to belucreased after gonadotropin (FSH, LH, hCG) administrationto inunature or hypophysectomised rats. (Tang aJ., 1970).SDH activity increases rapidly along with testis weight asthe development of testis proceeds. Like other testisenzymes SDH levels also are dependent on the stages ofspermatogenesis.SCOPE OF WENT INVESTIGATIONFrom tbe d~scusslon it 1s evtdent that oploidpeptides are present in central nervous system and manyother perlpberal organs. Oplold peptldes of all the threeclasses are present in the braln. Slmllarly, receptors forthe peptides are widely dlstrlbuted In the braln, pituitary,testis and other periplieral tlssues. Studies uslng opioidpeptides, thelr agonists and antagonists have shown thelrinvolvement in the function of pltultary hormone secretlon.Though various neurotransmitters are reported to bemediatrng opioid action on hormone release from pitultary,the exact mechauism(s) and transmitters lnvolved are notclear.Immunoreactive peptides of opioid class have been

located in the gonads of rat and other species. Rowever,the physiological significance of these peptides inreproductive processes is not well understood.In this study, opioid receptor blocker naloxonewas used to evaluate role of endogenous opioids in therelease of anterior pituitary hormones and thyroid hormones.Further, role of GABA-ergic and serotoninergic systems inmediating opioid action was evaluated. Aminoacidneurotransmitters, aspartic acid and glutamic acid wereadministered to rats pretreated with morphine or itsantiserum or naloxone to study their effect on thyroidhormone secretion. Hypothalamic gamma-glutamyl transpeptidaseand testlcular enzymes lactate dehydrogenase andsorbitol dehydrogenase activity were also studied under theabove experimental conditions. Finally, effect ofintrrtesticular administration of dynorphin, morphine andits antiserum on activity of testicular lactatedebydrogen&se and sorbitol dehydrogenase were studied. Itis expected that the present studies would reveal the roleof endogenous opioids on pituitary and testicular functionand thyroid hormone release and neurotransmitters mediatingopioid action on the endocrine system.

TABLE I1. Acetylcholine2. Biogenic aminesi) Dopamineii) Noradrenalinelii) Adrenalineiv) Serotoninv) Histaminevi) Octapaminevii) Phenyl ethylamineviii) Phenyl ethanolamine3. Amino acids1) Gamma aminobutyric acid (GABA)ii) Aspartic acldlli) Glutamlc acid4. Neuropeptidesi) LHRHli) TRHiii) SomatostatiniV) CRFV) Vasopressinvi) Oxytocinviii) endorphinvii) ! ḻipotropinix) drenocorticotroplc hormonex) 4-Yelanocyte stimulating hormonexi) Growth hormonexii) Thymtropinxiii) Prolactinxiv) Substance Pxv) Neurotensinxvi) Enkephalinsxvii) Dynorphinxviii) Bombesinxix) Neumpeptide YXX) Angiotensinsxxi) Bradykininsuii) Sleep Inducing Peptidexxiii) Chstrinxxiv) Cholecystokinin

(3uF'Tm I1MATERIALS, YgPRODS AND EXPERIYENTAL PROCEDURESAll the exprlments were performed in colony bredrats, derived from Wistar strain, purchased from the AnimalFacility of Jawaharlal Institute of Post Graduate MedicalEducation and Research (JIPMER) Pondicherry, India and KingInstitute of Preventive Medicine, Madras, India.20, 40 and 60 days old and adult male rats of 2.5-3 month old, were used in this study. They were maintainedunder controlled conditions of light and temperature (10hour dark / 14 hour light and 25 + ~OC), with free access todrinking water and standard rat pellets (Lipton India Ltd.Bangalore).Gama glutamyl para nitro anilide, Glycyl-glycine,Sodium pyruvate, 1-NADH (disodium salt), Triton X-100,Bovine serum albumin, Naloxone, Amlnooxyacetic acid,Morphine sulfate, Parachlorophenylalanine, Glutamic Acid,Aspartic Acid, Heparin, Chloramine-T were obtained fromSigma chemical Co., St. Louis, USA.

2D-AlaDynorphin was purchased from PeninsulaLaboratories, California, USA and Morphine antiserum fromOEM Concepts Inc USA.Radio Imuno Assay Kits for Thyroxine(T4) and Triiodothyronine (Tg) were obtained from BRIT,BARC, Bombay, India.RIA Kits for assay of LH, FSH, Prl, GHand TSR were gifts from the Natlonal Pituitary Agency,~ational' '~nstitute of Arthritis Metabolism and Digestive~iseases) (NIAMDD), Bethesda, USA.were of 'analytical grade and purchased locally.All other chemicals used1mplmk)ion of intravenous (iv) cannula:Indwelling silastic catheters were introduced intothe external jugular vein using the technique of Rams andOjeda (1974) as described by Vijayan and Mc cann (1979 a).Cannula wde of silastic sheets and tubing (Dow corning,USA) was placed in the superior venacava, adjacent to theheart, vra cne external jugular vein after the animal wasanaestbtized with ether.The external end of the cannulawas gui,d@da under the skin to emerge at the back of the head,and vas then sutured in place.

Estimation of G&me Glutmy1 Transpepticlase ActivityThe activity of gamma glutamyl transpeptidase wasestrmated according to the method of Tate and Ueister (1974)as standardlsed by Pasha and Sadasivudu (1984). The assaymixture (2.01~1) consisted of 80 micromoles of Tris-HC1buffer (ph 9.0), 150 micromoles of NaC1, 40 micromoles ofglycyl-glycine, 5 micromoles of gamma-glutamyl paranitroanilideand 0.4 ml 2% homogenate (in 0.25 M sucrose).After 30 min incubatlon at 37'~ the reaction was terminatedby the addition of 2 ml of 10% acetic acid. A control wasset up havlng all the above ingredients except that thehomogenate was added after the addition of 10% acetic acid,After centrifugation at 5000 rpm for 20 min the absorbanceof clear supernatant was read at 410 nm. The enzymeactivity was expressed as micromoles of paranitroanilineliberated per gram weight of tlssue per hour with referenceto a standard graph plotted uslng different concentrationsof paranitroaniline and treatlng it with acetic acid.Estirtiomof hctate Dehydrogearse ActivityLactate debydrodenase activity in the testis wasmeasured by the method of Bergmeyer (1974). Enzyme wasextracted after blunt forceps macerations (500 compressions)in isotonic saline until no solid macroscopic fragments

emained. The resultant suspension was centrifuged for 5min at 2000 g and the supernatant was used for assay. Thereaction mixture and the homogenate was brought to 25O~before the assay. To 3.5 ml of phosphate pyruvate 0.05 mlcoenzyme (11.3 mY NADH) and 0.10 ml of the supernatant wereadded. Blank tubes were run simultaneously without theaddition of NADH. The tubes were mixed and optical densityread at 60 sec intervals for 3 to 5 min at 340 urn and 360nm. Protein content was estimated according to the methodof Lmri et a1 G951)Assay of Sorbitol Dehydrogenase ActivitySorbitol dehydrogenase activity in the testis wasmeasured by the method of Bergmeyer (1974).The assaymixture consisted of 1.6 ml of Triethanolamine buffer (0.2M,pH adjusted to 7.4 with 2N NaOH) 0.1 ml of coenzyme (12 mM pNADH) and 1.0 ml of supernatant (50% W/v homogenate was madein 0.035 Y phosphate buffer pH 7.0 with a drop of Triton x100. The homogenate was centrifuged at 1000 g for 45 min.and supernatant taken as enzyme source) were mixed andincubated at 25Oc for 30 min until the extinction wasConstant.Finally 0.3 ml of D(-) fructose solution wasadded to the assay system, mixed and absorbance was read at80 second intervals for 5-8 minutes at 340. 334 or 365 nm.Blank tubes were run simultaneously using distilled water in

placeof supernatant. Proteln content was estimatedaccordrng to the method of Lowri et a1 6953.Plasma levels of LH, FSH, Prl, GH and TSH weremeasured by radioinmuno assay (RIA) using a double antibodyprocedure as standardised In our laboratory. (Babu 1982).Radioinmuno assay kits for rat LH. FSH, Prl, CH and TSH wereobtained from the NIAMDD-NIH Pituitary hormone distributionprogramme. Radioinununo assay was performed according to theguidelines provlded with tbe klt for each hormone. RadioImmuno Assay Kits for Thyroxine (T4) and Triiodothyronine(Tg) were obtained from BRIT, BARC, Bombay, India.ASSAY OF TBPROXINE (T4)Principle and Features of the TestUnlabelled endogenous T4 competes witb radiolabelledT4 for the limited binding sites on the antibodymade spec!fically for T4. At the end of incubation, the T4bound to antibody (Ag-Ab) and free T4 are separated by theaddition of polyethylene glycol (22%). The amount bound tothe 8ntiboby in the assay tube is compared with values ofknown T4 standards and the T4 concentration in the test8mpl8 is calculated. 8-anilino-1-naphthalene sulphonic

acid (ANS) is used in this Kit for displacing T4 bound tothyroxine binding globulin (TBG). This test is performedwith 10 )11 of plasma/serurn volume and covers a sample rangeof 0-200 ng/nl.1. T~ standard2. T4 antiserum3. 125~ - T44. T4 free human serum5. Control serum A and B6. Concentrated assay buffer7. Polyethylene glycolThe assay was performed as per the instructionsprovided with the kit.ASSAY OF TgPrinciple and Features of the TestUnlabelled endogenous Tg competes with radiolabelledTg for the limited blnding sites on the antibody(Ahl) made specifically for T3. The antibody is in the formOf complex with second antibody (Ab2). At the end ofincubation, the Tg (Ag) bound to antibody - second antibody

complex (Ag-Abl-Ab2) and free T3 are separated by theaddition of polyethylene glycol (12%). The amount bound tothe antibody complex in the assay tube is compared withvalues of known T3 standards and the Tg concentration in thetest sample is calculated. 8-anilino-1-naphthalenesulphonic acid (ANS) rs used in this Kit for displacing Tgbound to thyroxine binding globulin (TBC). This test isperformed with 50 p1 of plasma/serum volume and covers asample range of 0-4.8 ng/ml.Reagents :1. T3 standard2. Tg antisera complex3. 1251 - T~4. Tg free human serum5. Control serum A and B6. Concentrated assay buffer7. Polyethylene glycol.The assay was performed as per the instructionsprovided with the kit .

LH Kit consisted of1. Rat luteinizing hormone antigen highly purified foriodination NIAMDD rLH-1-5.2. flat luteinizing hormone antiserum (rabbit) NIAMDD-antir-LH-S-6.3. flat luteinizing hormone reference preparation NIAMDD-r-LH-RP-2.Preparation of the GelFive gms of sephadex C-75, was added to 100 ml ofpbosphosaline buffer (PBS) (0.01 M W4, 0.05 U NaC1, 0.1%Sodium azide, pH 7.6) and stirred for 30 min using amagnetic stirrer. It was (a) kept in a boiling water bathfor 5h (b) allowed to stand at room temperature for 72h (c)stored in a refrigerator up to 4 months (d) placed at roomtemperature for 24 hour before use.Prepantion of the Colun1. Ten a1 glass pipettes were used. The mouth piece wascut off.

2. Tubes were cleaned with chromic acid, hot water, tapwater and double distilled water and dried.3. A three-way stopcock (Pharmaseal, Puerto Rico, USA) wasattached to the tube by a 4 cm long latex tubing, glasswool was placed in the tip of the tube.4. The tube was washed twlce with phosphosaline buffer andfilled up to the 7 ml mark.5. The gel was continusouly stirred using a magneticstirrer to keep the suspension homogenous.6. The gel was pipetted from the bottom of the flask as awell mixed slurry. When settling was under way, theoutlet was opened and allowed to run freely. Theslurry was continuously added as needed. The top wasnever allowed to settle before addlng more slurry. Thecolumn was filled to a height of 15-20 cm. About 2 mlof buffer (PBS) was left at the top of the column. Onthe day of iodination (maximum 4h before use) thecolumn was equilibrated with 1 ml of 2% bovine serumalbumin (BSA) in P6S and then washed with PBS. After asingle use the column was discarded.

Iodination of Rat LA1. 125~odine, carrier free, as sodium iodide with specificactivity of 400 mCi/ml suitable for rodination ofprotein.2. 0.5 M sodium phosphate buffer, pH 7.6.3. Chloramine-T (5 mg/lOml of 0.05 M W4, pH 7.6 buffer).4. Sodium wtabisulfite (Na2S205) (25 mg/lO ml of 0.05 1PO4, pH 7.6 buffer).Chloramine-T and sodium metablsulfite wereprepared freshly just prior to use. 1 mCi 125~ was added toa small disposable glass vial used as the reaction vessel.25 p1 of 0.5M W4 buffer pH 7.6 was added. 2 pg of NIAMDDRat LH-1-5 in 20 p1 of PO4 buffer was added next. 10 pl ofchloramine T was then added. The vial was then agitated for50 sec after which 25 pl of sodium metabisfulfite was added.The entire reaction mixture was applied to the sephadex G-75column. The column was then eluted with phosphosaline (0.01POq, 0.15Y NaCl buffer, pH 7.6).Fractions of 0.5 ml werecollected in test tubes containing 50 microlitres (pl) of 2%BSA in PW buffer. These fractions were counted in a LKB-Pharmacia minigamma counter. Two peaks of radioactivity were

33detected. The first yak began at tubes 3-4 and trailed offby tube 6. A second peak containingI began at abouttube 7. The iodinated rat LH was contained in the firstpeak (Tube 4-5). The fraction high on the trailing shoulderof this peak (tube 4 and 5) contained the mostimmunoreactive and least dam2ed rat LH.This fraction wasadded to buffer in order to give 10,000 cpm per 100 ~ 1 and ,stored at -20°c, until use.Double Antibody RIA ProcedureThe following steps were performed in sequence forthe assay of plasma LH.1. 10 x 75 mm disposable test tubes were used.2. Buffer (1% BSA in 0.01 M PO4, 0.15 M NaCl, 0.1% sodlumazide, pH 7.6) was added to each tube in sufficientquantity to produce a final volume of 0.7 ml.3a. 25 t.11 plasma to be assayed was added for3b. The reference preparation (NIAMDD-Rat-LH-RP-2) wasdissolved in 1% BSA in phosphosaline and added in dosesranging from 8 ng to 0.03 ng per tube, In sufficientdetail (8.0, 4.0, 2.0, 1.0, 0.50, 0.25, 0.12, 0.06,0.03 ng) so that the entire curve can be constructedgraphically.

4. Iodinated rat LH was added such that approximately10,000 cpm were contained in 100 pl of 0.1% BSA-phosphosaline buffer.5. 200 pl of the antiserum (NIAMDD-Rat-LH-S-6) in a finaldilution of 1:40,000 in 3% normal rabbit serum. (NRS) -0.05 Y EDTA-PBS was added (at these dilutions, theantiserum was observed to bind 25% of the labelled rat.GLH (B = -- x 100, see below).TC6. In some tubes 200 pl buffer and 200 p1 3% NRS-EDTA-PBSand 100 ~1 label were added to serve as background.7. In a few tubes 200 pl buffer and 100 pl label and 200111 antiserum were added to serve as zero (100% binding= 2).8. In 2 or 3 tubes 100 ~1 label was taken to get the totalcounts (TC) .9. Tubes were agitated on vortex mixer.10. Tubes. were incubated for 24 hour at room temperature.11. At the end of this period, 200 pl of goat anti-rabbitgamma globulin (ARGG) was added to precipitateuxirmlly the antibody bound labelled rat LH.12. Tubes .ere agitated on a vortex mixer.

13. Tubes were again incubated for 24 hour at roomtemperature.14. At the end of this incubation period all tubes werecentrifuged at 1000 g for 30 min in a refrigeratedcentrifuge. The supernatant was discarded and theprecipitate was counted in a gamma counter.15. The unknown samples were compared to the percentage ofcounts precipitated with the rat LH referencepreparation, NIAMDD rat LH-RP-2. A curve wasconstructed on semilogarithmic paper, and the unknownread directly from the curve obtained with LH-RP-2.Results are expressed as nanograms (ng) of rat LH-RP-2per ml of plasma.RIA of FSEThe following were provided vith the FSH kit.1. Rat FSH antigen NIAMDD-r-FSH-1-5, highly purified foriodination.2. Rat FSH antiserum (Rabbit) NIAMDD-Anti-r-FSH-S-11.3. Rat FSH reference preparation NIAMDD-r-FSH-RP-2(calculated to RP-1).

Iodination of Rat FSBIodination was performed as for LH except that of10 mg/ml of Chloramine T was used.Double Antibody RIA ProceduresProcedure was same as for LH, except, referencepreparation (FSH-RP-2) was dissolved in 1% BSA phosphosalinein doses range of 40 to 0.62 (40, 20, 10, 5, 2.5, 1.25, 0.62ng). FSH antiserum was used at dllution of 1:2500.RIA of PrlThe RIA Kit for Prl consisted of1. Rat prolactin antlgen NlAMDD-r-Prl-1-5, highly purifiedfor iodlnatlon.2. Rat prolactin antiserum (rabbit) NIAMDD-Anti-r-Prl-S-8.3. Rat prolactin reference preparation NIAMDD-r-Prl-RP-3.(~idlo~icaal potency = 30 ~u/mg (pigeon local crop sacassay of Nlcoll).

lodination: As for LH.Double Antibody ProcedureAs per LH and PSH except the reference preparationwas diluted in PBS in a range of 0.25, 0.5, 1, 2.0, 4.0,8.0, 16.0 ng and antiserum was diluted so as to get a finaldilution of 1:12,500.As far as possible samples from a particularexperiment were run in one assay, each in duplicate, toavoid inter assay variation, In our laboratory thesensitivities for assay were 0.5 ng LH, 10 ng FSH, and 0.25ng Prl. The inter and intrassay co-efficients of variationwere 10 and 6% for LH, 9 and 5% for FSH and 10.4 and 5.5%for Prl respectively.The RIA Kit for GH consisted of1. Rat C8 antigen NIAYDD-r-CH-1-5, highly purified foriodination.2. Rat GH antiserum (rabbit) NIAMDD-Anti-r-CH-S-8.3. Rat CI1 reference preparation NIAMDPr-GH-RP-2.

lodination: As for LH.Double Antibody ProcedureAs per LH and FSH except the reference preparationwas diluted in PBS in a range of 0.25, 0.5, 1, 2.0, 4.0,8.0, 16.0 ng and antiserum was diluted so as to get a finaldilution of 1:12,500.RIA of TSEThe RIA Kit for TSH consisted of1. Rat TSH antigen NIAMDD-r-TSH-1-5, highly purif~ed foriodination.2. Rat TSH antiserum (rabbit) NIAMDD-Anti-r-TSH-S-8.3. Rat TSH reference preparation NIAMDD-r-TSH-RP-2.Iodination: As for LH.Double Antibody ProcedureAs per LH and FSH except the reference preparationwas diluted in PBS in a range of 0.18, 0.36, 0.73, 1.45,2.91, 5.82, 11.64 ng and antiserum was diluted so as to geta final dilution of 1:12,500.

STATISTICAL ARALYSISSignificance of difference between group meanswere compared by analysis of varlance (ANOVA). Significanceof difference between control and exper~mental groups at anygiven point vere calculated by Student's 't' test.

OPIATE AHD GABA-IIILGIC mCTIORBPPOTBhLhYIC GAWAQarm TwmPwrIDAsB ACTIVITY, PLASMA GONaDJWDPIIS,WL, a, TSB AmI T4 LWElS AND TESTIC[IWLB LACTATEDmmxmG[(RAsE ACrIVITY In YaLB RATS

OPIATE AND GABA-EGIC INTERACTION ON -ICCAYYAUDTAHYL TRAUSPKPTIDASE ACTIVITY, PLASMA GONADOTROPINS,PRL, GB, TSB AND T4 LEVW AND TESTI-CULAR LACTATEDIPIYDROGENASE ACTIVITY IN llllLg RBTSOpiates and endogenous opioid peptides have beenreported to influence the release of hormones from thepituitary gland (Howlett and Rees 1985, Bruni g., 1977,Meites g., 1979). Primarily the site of action is thehypophysiotroplc areas of hypothalamus where they alter therelease of various neurotransmitters (see Chapter I). Gammaaminobutyric acid (CABA) 1s an amino acid neurotransmitterpresent in the central nervous system. GABA has beenreported to play a role in the anterior pituitary function(Review Mc Cann g., 1984). With regard to gonadotropinrelease, the precise role of CABA is not clear since bothstimulatory (Vijayan and Mc Cann, 1978, Ondo, 1974) andinhibitory (Adler and Crowley, 1986; Donoso, 1988) effectsof GABA have been reported. Various neurotransmitters suchas norepinephrine, epinephrine, dopamine, serotonin and GABAhave been implicated in the mediation of opioid action onanterior pituitary function (Chapter I). Recently, Masottoand Negro Vilar (1987) demonstrated in male rats that

activation of GABAB (not GABAA) receptor abolishes naloxonestimulated LH release, suggesting that GABA-opioidinteractions may be important in the control of LHsecretion.The present chapter describes results on theeffect of naloxone and Aminooxyacetic acid on pituitaryhormone release and the interactions between opioidergic andGABA ergic systems in male rats.Aminoaxy acetic acid whichelevates brain GABA levels by way of inhibiting GABAdegradation and naloxone, specific opioid receptorantagonist are used in the experiments. Activity ofhypothalamic Y-glutamyl transpeptidase and testicularlactate dehydrogenase and assay of plasma LH, FSH, Prl, GH,TSH and T4 levels were measured under various experimentalconditions.MATgBIIUS AND YgTBODSAnimals and chemicals used in this study wereobtained as described in chapter 11. Rats of Wistar strainof 20, 40 and 60 day old were used. Naloxone (2 mg/kg bw)and Amino oxyacetic acid (15 mg/kg bv) were dissolved in0.9% saline and administered subcutaneously. Treated ratsWen, sacrificed at specified time intervals. Trunk blood wasCollected in beparinized test tubes and plasma was

separated and stored for later assay of hormones.Radioimrnunoassay of LH, FSH, Prl, GH, TSH and T4 werecarried out as described in chapter 11.Activity ofhypothalamic 7 -glutamyl transpeptidase and testicularlactate dehydrogenase werermeasured as described above.Ew5lInmnAL DESIGN1. Administration of RaloxoaeRats of three different age groups (20, 40 and 60days old) were given subcutaneous injection of Naloxone (2.mg/kg body weight dissolved rn 0.9% saline). One group ofrats in each age group were sacrificed one hour after andthe other group, 4 hours after injection.Blood wascollected in heparinised tubes, plasma was separated andstored frozen for later hormone assay.Hypothalamus andtestes were dissected out and processed for measurement ofactivity ofY-GT and LDH respectively (as described inChapter 11).2. Afhinistrrtion of Amiwoxy Acetic Acid (AOM)20, 40 and 60 day old rats were administered with6Ein00xy acetic acid (15 mg/kg bw dissolved in 0.9% saline)subcutaneously (sc).Treated rats were divided into twogroups: one group was sacrificed 1 hour after and the other

group at 4 hour after AOAA injection.Trunk blood wascollected, plasma was separated and stored for assay ofhormones. Hypothalamus and testes were dissected out andprocessed for measurement of activity of y-GT and LDHrespectively.3. Administration of Naloxone to AOAA Treated RatsRats of 20, 40 and 60 day age were administered,sc, with AOAA (15 &/kg bw). After 1 hour of AOAA injectionNaloxone (2 mg/kg bw) was administered. These rats weredivided into two groups: one group was sacrificed at 1 hourand the other group at 4 hour after naloxone administration.Trunk blood was collected, plasma separated and stored forlater hormone assays. Hypothalamus and testes weredissected out and processed for measurement of activity ofY-GT and LDH respectively.RESULTS1. Effect of hloxone AdministrationRypothalamicy-GT activity varies from 33.2 to44.1 M d 52.9 moles/gm tissue/hr in control rats ofdifferent rge groups studied. The enzyme activity waseigaifiantly reduced by naloxone treatment in 20 day oldrats at 4 hours after injection while no change in enzyme

actlvlty was observed in the other two age groups studied(Fig.1-3).Plasma LH levels ln control rats of different agegroups studied varies from 0.7 to 1.2 and 3.1 ng/ml. Thehormone levels were raised at 1 hr after naloxoneadministration while In 60 days old rats a slight decreasein hormone level was observed at 4 hours afteradministration (Pig.3-6).Plasma FSH level in control rats vanes from 2483ng/ml to 2234.6 ng/ml and 2681.8 ng/ml in rats of 20, 40 and60 days old. The hormone levels did not register anysignificant dification after naloxone treatment (Fig.7-9).In control rats of 20, 40 and 60 days age thebasal level of plasma prolactin is 4.4 ng/ml, 5.6 ng/ml and5.8 ng/ml respectively. The plasma prolactln levels weresignificantly reduced by administration of naloxone in ratsof all age groups (Flg.10-12).Plasma CH levels varies from 8.7 ng/ml to 10.8nglml in control rats of different age groups. Naloxoneadministration caused significant increase in plasma CHlevel in rats of 20 and 60 day of age at 1 and 4 hours afterinjection, while it remained unmodified in 40 day old rats

45Plasma TSH levels varies from 68.2 ng/ml to 169.9ng/ml in control rats of different age groups. The homnelevels were significantly diminished by naloxone treatmentat 4 hours after injection in 20 and 60 days old ratswhereas a Significant Increase in the hormone level was seenin 40 day old rats (Fiy.16-18).Plasma thyroxine varies from 44 to 73 ng/ml incontrol rats of different age groups. The plasma T4 levelsremained unmodified after naloxone administration in rats ofall age groups studied (Fig.19-21).Testicular lactate dehydrogenase activity variesfrom 49.1 to 88.4 units/mg proteln in 20, 40 and 60 daycontrol rats. Administration of naloxone significantlyreduced the enzyme activity in 20 day old rats. However in40 and 60 day old rats lt produced an increase in LDHactivity at 4 hours after treatment (Fig.22-24).2. Effect of AOM Administration.Hypothalamic 7-GT varies from 33.2 to 52.9moles/p tissue/hr In control rats of different age groupsstudied.Administration of AOAA caused a significantreduction in tbe enzyme activity in 20 day old rats.TheY-GT actlvity was increased in 60 day old rats at 1 hourafter administration of AOAA. The enzyme activity wasU~aodified in 40 day old rats (Fig. 1-3).

Plasma LH levels varies from 0.7 to 1.2 and 3.1ng/ml in 20, 40 and 60 day old control rats. The hormonelevels were reduced after AOAA treatment in rats of all agegroups (Fig.4-6).Plasma FSH level varies from 2483.4 to 2234.6 and2681.8 ng/ml in control rats of 20, 40 and 60 day old rats.The plasma FSH levels remained unmod~fied in 20 day old ratstreated with AOAA while it caused a significant reduction inthe hormone level in 40 and 60 days old rats (Fig. 7-9).Plasma prolaction level vanes from 4.4 ng/ml to5.8 ng/ml in 20, 40 and 60 day old control rats. AOAAtreatment caused significant reduction in plasma prolactinlevel rn 20 and 40 day old rats while it was reduced at 1hour and remained unmodified at 4 hours after injection in60 day old rats (Flg.10-12).Plasma GH levels varies from 8.7 to 10.7 ng/ml in20, 40 and 60 day old control rats. Administration of AOAAcaused a significant increase in plasma GH in 40 day oldrats at .1 bour whereas the hormone levelremalnedunmodified in 20 and 60 day old rats (Fig.13-15).inPlhsma TSH levels varies from 68.2 to 169.9 ng/ml20, 40 and 60 day old control rats. Administration ofin 20 b y old rats led to reduction of plasma TSH both

at 1 and 4 hour after injection (Fig.16). However in 40 dayold rats AOAA caused a significant increase in TSH level at1 hour while in 60 day old rats no significant change in thehormone level was observed (Fig.17, 18).Plasma thyroxine level varies from 44.0 to 73.0ng/ml in 20, 40 and 60 day old control rats. Administrationof AOAA resulted in changes in T4 level corresponding tochanges in plasma TSH levels. In 20 and 60 day old ratsAOAA reduced plasma T4 significantly at 4 hours and 1 hourrespectively after injection. In 40 day old rats AOAAcaused an increase in plasma T4 level at 1 hour afterinjection (Fig.19-21).Testlcular lactate dehydrogenase activity variesfrom 49.1 unitslmg proteln to 88.4 unlts/mg protein in 20,40 and 60 day old control rats. AOAA induced a decrease intesticular LDH activity in 20 and 60 day old rats.However, it enhanced the enzyme actrvlty at 1 hour afterinjection, in 40 day old rats with no significant change at4 hours (Yig.22-24).3. Effect of mloxone on AOM pretreated ratsHypothalamic Y-CT activity varies from 33.2mles to 52.9 u mles/gm tissuelhr In 20, 40 and 60 day oldcontrol rats. Naloxone administration to rats pretreatedP

with AOAA caused an increase in hypothalamic y-GT activltyand decreased the enzyme activity in 60 day old rats. Nomodification in the enzyme activity was observed ln 40 dayold rats (Flg.1-3).Plasma LH levels varles from 0.7 ng/ml to 1.2 and3.1 og/ml in 20, 40 and 60 day old control rats. Plasma LHlevels were comparable to that of control values in rats ofall age groups treated wlth naloxone (Flg.4-6).Plasma FSH level 10 control rats varies from2234.6 to 2681.8 ng/ml in control rats of 20, 40 and 60 day.Naloxone treatment caused enhanced FSH secretion in 20 daysand decreased FSH level in 60 days old rats whlle ~t had noeffect on 40 days old rats (F1g.7-9).Plasma prolactin level varles from 4.4 ng/ml to5.6 ng/ml in 20, 40 and 60 day old control rats. Naloxoneadministration had a pronounced effect on the inhibitoryaction of AOAA in 20 day old rats with the hormone levelremaining subdued in 40 and 60 day old rats at 1 hour and 4hours respctively (Fig.10-12).Plasma GHlevel varies from 8.7 to 10.8 ng/ml incontrol rats. Prior treatment with AOAA blocked the naloxonestimulated rise in GH level in 20 and 60 day old rats whilein 40 day old rats naloxone caused inhibition of GH release

at 1 hour after injection (Fig.13-15). Plasma TSH levelvaries from 68.2 to 169.9 ng/ml in 20, 40 and 60 day oldcontrol rats. Pretreatment with AOAA did not modify theeffect of naloxone on plasma TSH levels (Fig.16-18).Plasma thyroxine level vanes from 44.0 to 73.0ng/ml in 20, 40 and 60 day old control rats. Pretreatmentwith AOAA blocked the effect of naloxone on plasma T4 level(Fig.19-21).Testicular lactate dehydrogenase activity variesfrom 49.1 to 88.4 units/mg protein in 20, 40 and 60 day oldcontrol rats. Testicular LDH activity was enhanced in 40day old rats at 4 hours after injection while in the otherage groups naloxone blocked the inhibitory effect of AOAA onthe enzyme activity (Fig.22-24).DISCUSSIONThe results of thls study indicate that endogenousopioids have an inhibitory effect on gonadotropin release.Administration of opioid receptor antagonist naloxone raisedplasma LH and to a lesser extent plasma FSH In rats of allage groups studied.. This finding supports some of theearlier observations under comparable experimentalconditions (Meites g nJ., 1979, Blank g g., 1980,Grossman and Rees 1983, Yasotto and Negro vilar 1987).

Effect of naloxone on gonadotropin release is mediatedthrough an enhanced release of GnRR from the hypothalamuswhich acts upon the pituitary gonadotropes (Blank g g.,1985; Negro-vilar g., 1987). Aminaoxy acetic acid whichelevates internal levels of GABA in the brain (Camnag., 1980, Wood and Peesker, 1973) inhibited gonadotropinsecretion. Inhibitory action of GABA on gonadotropinrelease is reported to be mediated by the B-type ofreceptors (Yasoto and Negro vilar 1987; Yasottofi.,1989). This type of receptor is selectively activated byhigh concentrations of GABA or by the specific GABA-Bagonist baclofen (Nicoll 1988, Bower), g G., 1980).It wasobserved in the present study that pretreatment wlth AOAAabolishes naloxone effect on gonadotropins, particularly onLH release. Yasotto and Negrovilar (1987) have alsoreported that GABA-B agonist, baclofen could also blocknaloxone induced LH release.These findings suggest thatinhibitory actlon of opioid peptides on LH release aremediated by GABA-B receptors.This is supported by thereport that baclofen blocked naloxone stimulated LRRHrelease in vitro from arcuate nucleus-median eminence (AN-ME)(Masotto* a,, 1989). Further, inhibitory effect ofbaclofen or naloxone stimulated LHRH release was completelyblocked by %aminovalarate (Yasotto & a., 1989, Muhyaddin& &. , 1983). Mndrenergic system Is reported to exert a

stimulatory effect on gonadotropin release (Ojeda g &.,1979, Negrovilar a Q., 1979; Vijayan and tdcCann 1978)Thus the emerging picture is that opioid peptides activateGABA-8 receQtors which In turn inhibit stimulatorynoradrenergic input resultlpg in inhibition of GnRH release.Naloxone treatment had no effect on plasma FSHlevels suggesting absence of role for endogenous opioids onYSH release. However, pretreatment with AOAA modified theeffect of naloxone on plasma FSH level. AOAA alone produceda reduction in the hormone level in 40 and 60 day old rats.Recently, Yoguilevsky et a1 (1991) showed that GABAfacilitates gonadotropin secretion in 16 day old andrnhiblts the hormone secretion in 30 days old female rats.This dual control is by a mechanism of activation ofdifferent types of GABA receptors.Administration of naloxone led to reduction inplasma prolactin level suggesting oyiold control of basalsecretion of prolactin.This has been reported to bemediated at the hypothalamic level by influencind doyaminerelease (Van Loon g& a., 1980). Dopamine has an inhibitorystimulus on prolactin release (McCann g g., 1979, Vijayan,1982, Nagesh Babu and Vi~ayan 1983). Similarly reducedlevels of plasma prolactin following AOAA administration isin agreement with earlier reports that high doses of GABA or

GABA agonists inhibit Prl release from pituitary (Schally gf&., 1977; Grandison and Guidotti 1979). No additive effectof AOAA and naloxone was observed on the prolactin secretionexcept in 20 day old rats indicating different mechanisms ofaction of opioidergic and GABA-ergic modulation of prolactinsecretion.Response of plasma growth hormone to naloxonetreatment was varied with respect to the age of rats.Whilein 20 and 60 day old rats it raised plasma GH level,naloxone had no effect in 40 days old rats.Whereasmorphine and opioids are known to increase GH secretion(Bruni g., 1977; Mikl g., 1984) effect of naloxoneon plasma GH level is controversial.Earlier reportssuggest that naloxone suppressed growtb hormone release(Meites g g., 1979; Bruni g., 1977). Bruni et a1(1977) showed absence of effect of naloxone at a dose of 2-/kg b.w. Naloxone failed to modify plasma GH level in 10day old male and, female rat pups (Arce g c., 1991).Considering these findings, the effect of opioids on basallevels of. CH is not well understood.The results of thisstudy show that GH release in response to activation ofGABA-ergic system is age dependent.While in 40 days oldrats AOAA caused an increase in GH release, it had no effectin the other two age groups.In contrast, intraventricularapplication of CABA raised GA levels (Mc Cann g &., 1984).

Studies using GABA receptor blocker bicuculline in male ratsshowed a delayed rise in plasma CH levels (Yc Cann g.,1984). It is not clear whether involvement of differenttypes of GABA receptors is responsible for such variedresponse. Activation of GABA-ergic system by administeringAOAA, abolished naloxone induced rise in plasma GH levels in20 and 60 day old rats while in 40 day old rats thiscombination inhibited CH release. Tbis observationindicates that opioid action may be mediated through GABAergicsystem.An age-dependent change in response to plasma TSHlevels after naloxone administration was observed suggestinginvolvement of endogenous opioids In TSH secretion.Someother reports suggest the absence of naloxone action onbasal levels of TSH (Bruni fi &., 1977, Meites g.,1979). Opiates and cold stress are known to suppress TSHsecretion. In contrast, plasma T4 level remained unchangedafter naloxone treatment indicating absence of effect ofendogenous opioids on thyroid gland. The fact that TSHresponse .to naloxone administration develops by a durationof 4 brs only, may be responsible for the apparent absenceof response in T4 levels. It was reported that morphine andopioids inhibit TSH by dopamine mediated inhibition oftbyrotropin releasing hormone (TRH) (Sharp fi g., 1981).AOAA treatment, did not evoke a uniform response in rats of

various age groups studied. McCann et a1 (1984) reportedthat intracerebroventricular injection of GABA inovariectomised rats declined TSH levels while intravenousinfusion of bicuculline, a GABA antagonist into either malesor ovariectomised females resulted in inhibition of TSHsecretion. The results obtained in the present studysuggest that GABA may stimulate (40 day), inhibit (20 day)or has no role (60 days old) in TSH secretion. Plasma ~4levels reflected changes in TSH secretion following AOAAtreatment. Pretreatment with AOAA, had no effect onnaloxone induced TSH or T4 levels indicating absence ofinteractions between opioiderglc and GABA-ergic systems onTSH release.Changes in the hypothalamic y-GT activity suggestthat the tripeptide glutathione may be involved inmodulating hormone secretion under the influence ofneurotransmitters. Naloxone or AOAA reduced Y -GT activityin 20 day old rats and AOAA had a stimulatory effect onenzyme activity in 60 day old rats. In rats pretreatedwith AOAA; naloxone reversed both these actions. These datasuggest that activation of GABA-ergic system reducesglutathione utilisation.Further, the results suggestexistence of interaction between GABA-ergic and opioidergicsystems through glutathione. Its relevance on pituitaryborne release is to be explored as glutathione is reported

to De inrluencing pituitary function (Pasha and Vijayan1989; 1990). The enhanced (20 day old) and reduced (60 dayold) levels of plasma FSH rn rats treated with AOAA followedby naloxone may be due to the effect of glutaihione onrelease of FSH (Pasha 1988; Pasha and Vijayan 1989).Naloxone, AOAA or the sequential administration ofboth agents brings about significant changes in testicularlactate dehydrogenase activity. LDH has been studied as amarker enzyme in relation to hormone treatments. Thecbangea in gonadotropin levels brought about by treatmentswith naloxone and/or aminooxyacetic acid may be responsiblefor the observed changes in lactate dehydrogenase activity.A direct action of opioid receptor antagonist naloxone ontestis is supported by the presence of opioid receptors onSertoli cells (Fabbri G., 1985). Sertoli cells Intest16 is the major site of synthesis of pyruvate andlactate which are the substrates for LDH action (Means1980).In conclusion, the data presented in this chapterindicates that endogenous opioids exert regulatory influenceon hormone secretion by the pituitary. Effects of opioidson gonadotropin secretion was found to be medlated vla theGABA-ergic system. Developmental changes in theneuroendocrine axis reflects in the response to these

Pig.1: Effect of administration of naloxone, AOAA and/orNaloxone on hypotha1nmic~-CT activity in 20 day oldmlo rats at 1 and 4 hours after treatment. Valuesare Yean + SEY for 5 rats in each group. The enzymeactivity is expressed as p moles p-nitroanilinereleased/gr~ wt. tissue/ hour. In this and subsequentfigures the P value is with respect to control.

Pig.2:Effe'ct of Naloxone, AOAA andlor Naloxone onhypothalamic Y-GT activity in 40 day old male ratsat 1 md 4 hours after treatment. Values are Mean 2SEM for 4 - 6 rats in each group. The enzymeactivity is expressed as p moles p-nitroanilinerele&sed/gm wt. tissue/hour.

sfFie.3: Effect,Ualoxone. AOAA and/or Naloxone on hypothalamicS T activty in 60 day old male rats at 1 and 4 hoursafter treatment. Values are Mean 2 SEM for 4-6 ratsin each group. The enzyme activity is expressed asmoles p-nitroaniline releasedl gm wt. tissue/hour.

Pig.4: Effect of Naloxone, AOM and/or Naloxone on plasma LHlevels in 20 day old male rats at 1 and 4 hours aftertreatment. Values are Mean 2 SEM for 4-6 rats ineach group.

Pig.5: Effect of Naloxone, AOAA kndjor Naloxone on plasma LHlevels in 40 day old male rats at 1 and 4 hours aftertreatment. Values are Mean 2 SEM for 4-6 rats ineach group.

Fig.6: Effect of Naloxone, AOAA and/or Naloxone on plasma LHlevels in 60 day old male rats at 1 and 4 hours aftertreatment. Values are Mean 2 SEY for 4-6 rats inacb group.

Pig.7: Effect of Naloxone, AOAA and/or Naloxone on plasmaFSH levels in 20 day old male rats at 1 and 4 hoursafter treatment. Values are Mean 2 SEY for 4-6 ratsin eacb group.

Pig.8: Effect of Naloxone, AOAA and/or Naloxone on plasmaFSH levels in 40 day old male rats at 1 and 4 hoursafter treatment. Values are Mean 2 SEM for 4-6 ratsin each group.

Control Nb! Ww Na4 4ke MX4*MA 4hra~MAIP(~I ltw llll K)MI)(OI Ah18Fig.9: Effect of Naloxone, AOAA and/or Naloxone on plasmaFSH levels in 60 day old male rats at 1 and 4 hoursafter treatment. Values are Mean 2 SEM for 4-6 ratsin each group.

Pig.10: Effect of Naloxone, AOAA and/or Naloxone on plasmaProlactin levels in 20 day old male rats at 1 and 4hours after treatment. Values are Llean + SEM for 4-6 rats in each group.

Fig.11: Effect of Naloxone, AOM and/or Naloxone on plasmaPmlactin levels in 40 day old male rats at 1 and 4hours after treatment. Values are kan 2 SEN for 4-6 rats in each group.

Pig.12: Effect of Naloxone, AOAA and/or Naloxone an plasPmlactin levels in 60 day old male rats at 1 and 4bours after treatment. Values are Yem 2 SM for 4-6 rats in each group.

Fig.13: Effect of Naloxone, AOAA and/or Naloxone on plasmGR levels in 20 day old male rats at 1 and 4 hourafter treatment. Values are Mean 2 SEN for 4-6 ratsin each group.

Fig.14: Effect of Nalorone, AOM and/or Naloxone on plasGH levels in 40 day old male rats at 1 and 4 hourafter treatment. Values are Uean + SEM for 4-8 ratsIn emch group.

Pig.15: Effect of Naloxone, AOAA and/or Naloxone on plasmaGB levels in 64l day old male rats at 1 and 4 hourafter treatment. Values are Mean 2 SEN for 4-6rats.

Pig.16: Effect of Naloxone, AOAA and/or Naloxone on plasmaTSH levels in 20 day old male rats at 1 and 4 hourafter treatment. Values are Mean 2 SEM for 4-6rats.

Pig.17: Effect of Naloxone, AOAA and/or Naloxone on plasmaTSB levels in 40 day old male rats at 1 and 4 hourafter treatment. Values are Mean + SM for 4-6rats.

Pig.18: Effect of Naloxone, AOAA and/or Naloxone on plasmaTSB levels in 60 day old male rats at 1 and 4 hourafter treatment. Values are Mean + SEY for 4-6rats.

Control NU ?u W 4h8 MX4WwM 4 N 8 m~~1~44dh=1~~~4hf8Pig.19: Effect of Naloxone, AOM and/or Naloxone on plasmaT levels in 20 day old male rats at 1 and 4 hourafter treatment. Values are Mean + SEM for 4-6 ratsin each group.

Pig.20: Effect of Naloxone, AOAA and/or Naloxone on plasmaT levels in 40 day old male rats at I and 4 hourafter treatment. Values are Mean + SEM for 4-6 ratsin each group.

Pig.21: Effect of Naloxone, AOAA and/or Naloxone on plasmaT levels in 60 day old male rats at 1 and 4 hourafter treatment. Values are Mean + SEM for 4-6 ratsin each group.

Fig.22: Effect of Naloxone, AOAA and/or Naloxone ontesticular LDH activity in 20 day old rats at 1 and4 hour after treatment. Values are Yean 2 SEN for5-8 rats in each group. The enzyme activity isexpressed as unitslmg protein.

Pig.23: Effect of Naloxone, AOAA and/or Naloxone ontesticular LDE activity in 40 day old rats at 1 and4 hours after treatamit. Values are Mean 2 SEM for6 rats in each group. The enzyme activity isexpressed as units/- protein.

Pig.=:Effect of Naloxone, AOAA and/or Naloxone ontesticular LDR. activity in 60 day old rats at 1 and4 hour after treatment. Values are Mean + SEM for6 rats in each group. The enzyme activity isexpressed as units/mg protein.

OF aaPBIW AND ITS AHTISERUM ON PLASMA T3 AND T4Am EYmmUaIC GMUA GlQTAML TRANSPBPTIDASE ANDTESTICDLAR L4CTATE DEEYDRCCENASEACTIVITY IN YdLE RATS

EFFECT OF MORPHINE AND ITS ANTISEBM MPUSMA Tg AND T4 AND HYPOTAALBYIC GAMMA GUPTAMYLTRANSPEPTIDUE AND TESTICULaR LACTATE DEEYMXENASEACTIVITY IN MALE BBTSOpioid peptldes and opiates have been shown toinfluence thyroid stimulating hormone (TSH) secretion.While some reports suggest an inhibitory role for opioidsand opiates (George and Lomax 1975, Bruni 5 g,, 1977,Meites g g., 1979, Van Vugt and Meites 1980),some otherstudies showed stimulation of TSH secretion by opioids (Juddand Hedge 1982, 1983). On the other hand, Sharp e.,(1981) reported that opiate agonist morphine has no effecton basal or TRH stimulated release of TSH.In the present study the effect of morphine andits antiserum on plasma Tg and T4 levels in male rats ofvarious age groups has been evaluated. Effect of theseagents on the activity of hypothalamic y-glutamyltranspeptidase and testicular lactate dehydrogenase has alsobeen evaluated.

Animals and chemicals were used as described(Chapter 11). Plasma Tg and T4 were assayed using RadioImmuno Assay Kits purchased from BRIT, BARC Bombay, Indiaand Activity of hypothalamic Y-glutamyl transpeptidaseN-GT) and testicular lactate dehydrogenase were measured asdescribed (Chapter 11).EXPERIMENTAL LLIGN1. Administration of Morphine SulfateMale rats of 20, 40 and 60 days of age wereinjected with morphine sulfate (2 mg/kg b.w. dissolved in0.9% saline) subcutaneously (sc). The treated rats weredivided into two groups: one group was sacrificed at onehour and the other group was sacrificed at 4 hours aftermorphine administration. Trunk blood was collected inheparinized tubes and plasma was seperated and stored frozenfor later assay of thyroid hormones by RIA. Hypothalamusand testes were dissected out and used for measurement ofY-GT and LDH activity, respectively as described before.2. Administration of Yorphine Antiserum (M-As)Antiserum to morphine was administered8ubcutaneously to 20, 40 and 60 day old (0.2 rnl, 100 X

dilution). The treated rats were drvided into two groups:one group of . rats were sacrificed at 1 hour and the othergroup at 4 hour after treatment with M-As. Trunk blood wascollected rn beparinized tubes, plasma was separated andstored frozen for assay of thyroid hormones. Hypothalamusand testes were dissected out and used for measurement ofactivity of T-GT and LDH respectively.1. Effect of Morphine Sulfate and Morphine Antiserum onIiypothalaricY-Clutamyl TranspeptidaseHypothalamic Y-glutamyl transpeptidase activityvaries from 33.2 to 52.9 t~ moles/g tissuelhr in 20, 40 and60 day old control rats. Admlnlstration of morphine sulfatesignificantly reduced the enzyme activity both at one and 4hour after injectron In 20 day old rats. Administration ofM-As, however, did not modify the inhibitory effect ofmorphine sulfate and the enzyme levels remainedsignificantly reduced comparable to those values as obtainedby US administration (Fig.1). In 40 day old rats the enzymeactivity was significantly (p < 0.01) suppressed only at 4hours after injection. M-As administration to 40 day oldrats further reduced (p(0.1 vs MS) the enzyme activity atone hour, while there was no change at 4 hours. However,

oth at one and 4 hours after H-As injection, the enzymeactivity was lower when compared to saline control (Fig.2).The enzyme activity was significantly lowered both at oneand 4 hour after MS administration in 60 day old rats. M-Asadministration, however, induced a slight increase in theenzyme activity at 1 hour while there was a decrease in theenzyme activity at 4 hours after M-As (Fig.3).2. Effect of Morphine Sulfate and Morphine Antiserum onTesticular Lactate DehydrogenaseTesticular lactate dehydrogenase activlty incontrol rats of varlous age groups varied from 49.1 to 88.4unitslmg protein. Administration of morphine sulfateproduced a significant (~(0.01) and age related reductionin the enzyme activity both at one and 4 hour in rats of allage groups studied (Fig.4-6). Adrnlnlstration of M-As didnot modify the enzyme activlty in 20 day old rats butproduced significant increase at 1 hour when compared tomorphine sulfate injected values in 40 days old rats(Fig.4 8.5). M-As injection further elevated the enzymeactivity at 4 hour both in 20 and 40 day old rats (Fig.4 86). However Y-As did not cause any Increase in the enzymeactivity and the levels remained significantly suppressed,comparable to the values with morphine sulfate, both at oneand 4 hour in the 60 day old rats (Fig.6).

3. Effect of Morphine Sulfate and Morphine Antiserum onPlasm T4 LevelPlasma thyroxine level varies from 67.3 ng/ml to73 ng/ml in rats of different age groups studied.Administration of morphine sulfate significantly reducedplasma T4 level only at 4 hour in 20 day old rats.Interestingly M-AS also further reduced T4 levels in 20 dayold rats both at one and 4 hour after administration(Fig.'7).In contrast T4 level was significantly reduced at1 hour while there was no effect at 4 hour in 40 day oldrats (Fig.8).However M-As treatment produced similareffects both at one and 4 hour, in 20 and 40 days old rats(F1g.7 8 8).T4 levels were not modlfied by morphinesulfate both at one and 4 hour after administration in 60day old rats.Administration of M-As, on the other hand,significantly (y(O.OO1)decreased T4 levels both at 1 and4 hours (Fig.9).4. Effect of Morphine Sulfate and Morphine Antiserum onP1- Tg LevelPlasma T3 levels varies from 0.7 to 0.8 ng/ml incontrol rats of various age groups studied. Administrationof morphine sulfate significantly reduced plasma Tg levelsboth at one and 4 hour in 20 and 40 day old rats

(Fig.10 & 11). There was no change in the Tg levels at 1hour after morphine sulfate, while the hormone level wassignificantly reduced at 4 hours in 60 day old rats(Pig.12). Administration of M-As elevated Tg levels both in20 and 40 day old rats when compared to the values obtainedafter morphine sulfate, though the hormone values were stillsignificantly l m r with respect to saline control valuesFig10 & 1 1 In contrast M-As induced increase in T3level was evident only at 4 hours in 60 day old rats(Pig. 12).The results of the present study show thatmorphine exerts an inhibitory effect on plasma thyroidhormone levels.Opioid peptides and opiates is reported tosuppress TSH secretion from pituitary (Bruni g., 1977).This effect is thought to be primarily mediated via thehypothaluaus (George 1973; Muraki 9 g., 1980; Lomax andGeorge 1966; Loursg., 1970) although there is someevidence that the opioids may act directly at the pituitaryto decrease TSH release due to TRH administration (Yay 5s., 1079). In this study, plasma levels of both thyroxineUId triiodothyroniae were iound to be diminished followingmorphine rdainistration.Morphine did not change T4 levelin 80 day old rits while there was an inhibitory effect in

the other two age groups. This age dependent change inresponse may be due to developmental changes in hypothalamopituitary-thyroidaxis and related differential sensitivityto morphine treatment. Also significant is the observationthat morphine could alter plasma T3 levels - an increase at1 hour and reduction at 4 hours after treatment.Administration of M-As antiserum significantlyreduced both plasma T3 and T4 levels. The fall in thyroidhormone levels following morphine antiserum administrationmay be due to a direct effect on the pituitary.This issupported by the report that opioid peptides have been knownto exert a stlmulatory effect on TSH release by acting onpituitaryand Hedge 1982, 1983).which effect was not blocked by naloxone (JuddThe opioid peptides &endorphin,Yet-enkephalin, Leu-enkephalin, dynorphin and Y -endorphincould significantly increase TSH secretion (Judd and Hedge1983).Both morphine sad its antiserum significantlyreduced the activity of hypothalamic'f-glutamyltranspept'idase. As glutathione is reported to be involvedin the regulation of secretion of hormones from thepituitary (Pasha and Vijayan, 1989) and morphine was foundto have an effect on hormone secretions from the pituitary(Bruni G., 1977; Meltes a., 1979), this finding

appears to be significant.Whether the tripeptideglutathione is involved rn morphine induced changes inhormone release from pltultary rs not clear.Testicular lactate dehydrogenase activlty wasreduced by morphine treatment.Morphine and oploidpeptides have been reported to rnhiblt gonadotropinsecretion (Bruni g g., 1977; Meltes g a., 1979, seechapters I and 111).Further oploid peptide p-endorphin hasbeen reported to be synthesised by Leydig cells and theirreceptors were localrzed on Sertoli cells in rat, mice andother mammalian specles (Tsong g a,, 1982, Fabbrl g.,1988, 1985). Testlcular enzyme lactate dehydrogenase is amarker enzyme of testlcular function.The actlvlty of theenzyme 1s dependent on the age of the animal as well asstage of spermatogenesis (Mills and Means, 1972, Steinbergerand Steinberger, 1975).The reduction In testicular LDHactivlty may be due to changed level of hormones from thepituitary particularly gonadotropins (Chapter 111) or due todirect actlon on testls.The latter possibility issubstantiated by the report that opiate receptors arelocated in the testis (Fabbrl g g., 1985). Similarlyantiserum to morphine also has an inhibitory effect ontesticular LDH.

The present results demonstrate that morphlneexerts an rnhibitory action on thyroid hormone secretion.Earlier reports also suggested that this action of morphinemay be via hypothalamic level. In contrast, antiserum tomorphine presumably acts dlrectly on the pituitary, inhibitsTSH secretion and thereby suppresses thyroid function (Judd8 Hedge, 1982; 1983).

Pig.1: Effect of morphine and morphine antiserum onhypothalamic r-GT activity in 20 day old male ratsat 1 and 4 hour after treatment. Values are Mean 2SBI for 4-6 rats in each group.P < 0.05 vs Control

Pig.2: Effect of morphine and morphine antiserum onhypothalamic r-GT activity in 40 day old male ratsat 1 and 4 bour after treatment. Values are Mean 2SD1 for 4-6 rats in each group.* P < 0.05 vs Control , ** P < 0.001 vs Control

Pig.3: Effect of morphine and morphine antiserum onhypothalamic r-GT activity in 60 day old male ratsat 1 and 4 hour after treatment. Values are Mean 2SE# for 4-6 rats in each group.* P 0.05 vs Control , ** P< 0.001 vs Control

Fig.4: Effect of Morphine and M-As on testicular lactatedehydrogenase activity in 20 day old male rats at 1and 4 hour after treatment values are Mean 2 SEM for4-6 rats in each group.* P < 0.001 vs Control , ** P< 0.01 vs Control

Fig.5: Effect of Morphlne and M-As on testlcular lactatedehydrogenase activity in 40 day old male rats at 1and 4 hour after treatment values are Mean 2 SEM for4-6 rats In each group.* P C 0.01 vs Control , ** P < 0.05 vs Control

Pig.6: Effect of Morphine and M-As testicular lactatedehydrogenase actlvlty ln 60 day old male rats at 1and 4 hour after treatment. Values are Mean 2 SEMfor 4-6 rats in each group.* P < 0,001 vs Control

Ssllne Control a MmphSulI. - 1 hr MOrphSull - 4 hrsMorphAntlwr -1 IWI ~otph.~ntlaer-l t~rsFig.7: Effect of Morphine and M-As on plasma T level In 20day old male rats at 1 and 4 hour afte* treatment.Values are Mean 2 SEM for 4-6 rats in each group.* P< 0.001 vs Control

Fig.8: Effect of Morphine and M-As on plasma T level rn 40day old male rats at 1 and 4 hour afte* treatment.Values are Mean 2 SEM for 4-6 rats in each group.* P< 0.05 vs Control , ** P < 0.001 vs Control

Fig.9: Effect of Morphine and M-As on plasma T level in 60day old male rats at 1 and 4 hour aft& treatment.Values are Mean 2 SEM for 4-6 rats in each group.* P( 0.001 vs Control

SS11ne Cantrd a ~orph.8ull. - 1 hr M~tphSulI. - 4 htsEl uorph~~~rat-i hr wph.~tlaa-4 hraIFig.10: Effect of Morphlne and M-As on plasma T level ln 20day old male rats at 1 and 4 hour afte? treatment.Values are Mean + SEM for 4-6 rats in each group.* P < 0.001 vs Control , ** P < 0.05 vs Control

Sallne Control a Uorph.6ull. - 1 hr UDrQh.Sull. - 4 hrsWphMtaec-1 hrUOlph.Anti881-4 hr8Pig.11: Effect of Morphlne and M-As on plasma T level In 40day old male rats at 1 and 4 hour afte? treatment.Values are Mean 2 SEM for 4-6 rats ln each group.* P < 0.02 vs Control , ** P < 0.05 vs Control

Sa:lne Conttol MorphSull - 1 hr I Morph.Sull. - 4 hrsMmph Antlrer -1 hrM0rph.Ant188f -4 h18Fig.12: Effect of Morphlne and M-As on plasma T level in 60day old male rats at 1 and 4 hour afte3 treatment.Values are Mean + SEM for 4-6 rats in each group.* P < 0.05 vs Control

SBFKTIWIN SYNTHESIS BuCK&DE AND OPIOIDS OW PLASMA Tg AND T4AWD EYHXMUMIC GAMMA GLDTAMYL TRANSPEPTIDASE ANDTESTICXLM LACTATE DEBYDBOGENASEACTIVITY IN dDULT MALE RATS

SEROTONIN SYNTHESIS BLOCKADE AND OPIOIDS ON PLASMA Tg AND T4AND HYPOTHALAMIC GAMMA GLUTAYYL TRANSPEPTIDASE ANDTESTICULAR LACTATE DERPDROGENASE ACTIVITY IN ADULT W RATSRole of central serotoninergic system on TRH-TSHsecretion is controversial. Injection of 5-HT into thelateral ventricle or intrahypothalamic implants of 5HT werereported to deplete bioassayable hypothalamic TRH andinhibit TSH release (Mess and Peter, 1975). Jordan et a1(1978) reported that intraventrlcular injections of 5HTcaused a transient activation of TSH secretion. Krulich eta1 (1979) on the other hand provided evidence thatactivation of central serotoninergic system inhibits releaseof hypothalamic TRH which results in subsequent decrease insecretion of TSH from the pituitary. Opioid peptides on theother hand, have been reported to be inhibitory (Meites- al., 1979), stimulatory (Judd and Hedge 1983) or having norole (Sharp g g., 1981) on TSH secrctlon and thereby onthyroid function. Morphine was found to inhibit plasmalevels of thyroid hormones (see Chapter IV).The present study aims to explore the inter-actions, if any, between opioidergic andserotoninergic

s~sta on thyroid function. Para chlorophenyl alanine, aserotonin synthesis inhibitor is reported to deplete brainSerotonin levels (noe and Weissrnan, 1966; Engelman g.,1967; Cremata and Koe, 1966; Weitzman &. , 1968).Plasma levels of thyroid hormones were evaluated in PCPAalone and/or with opioid receptor antagonist naloxone,morphine and antiserum to morphine injected rats. Morphineand opioid peptides have been reported to inhibitgonadotropin secretion (Bruni g g., 1977; Meites & g.,1979; see Chapters I and 111). Further, opioid peptidePndorphin has been reported to be synthesised by Leydigcells and their receptors were localized on Sertoli cells inrat, mice and other mammalian species (Tsong g Q., 1982,Fabbri g., 1988, 1985). Testicular enzyme lactatedehydrogenase is a marker enzyme of testicular function.The activity of the enzyme is dependent on the age of theanimal as well as stage of spermatogenesis (Mills and Means,1972; Steinberger and Steinberger, 1975). Glutathione, atripeptide located in hypothalamus has recently been shownto be involved ln regulation of hormone release fromPitui~r); (Pasha and Vi~ayan, 1989). Y-GT is the key enzymeinr-glutamyl cycle through which glutathione is utilised.Hypothalamic gamma-glutamyl trasnspeptldase, (v-GT) andtesticular lactate dehydrogenase activity is also measuredin the above, experimental groups.

YbTEBIhL8 AND UETmDsAnimals and chemicals used in this study wereobtained as described In chapter 11. Parachloropbenylalanine (PCPA) was suspended in 0.9% saline and administeredintraperitonially (i.p. 3OOmg/kg b.w.). Naloxone andmorphine sulfate was dissolved in 0.9% saline and injectedsubcutaneously (sc). Antiserum to morphine was administeredsc (100X dilution with phosphate buffer pH 7.0).Treated rats were sacrificed at required timeintervals. Trunk blood was collected in heparinized testtubes, plasma was separated and stored frozen for laterassay of thyroid hormones. Hypothalamus and testis werequickly dissected out and.processed for measurement ofenzyme activity as described below.I Administration of PBA and Naloxone(A) FCPA (300 mg/kg bw) were administered intraperitonially(ip) in adult male rats. The treated rats were divided intotwo groups:Group I were sacrificed 24 hours affer PCPA injection.Group I1 were sncrificed .72 hours after PCPA injection.

68(B) Ualoxone (2mg/kg bw) was given sc by single injectionto adult male rats. Treated rats were divided into twogroups :Croup I were sacrificed 24 hours after nafoneadministration.Group I1 were sacrificed 72 hours after naloxone treatemnt.(C) Naloxpne (2mg/kg bw) sc, was administered daily toadult male rats for 3 days and sacrificed 24 hours afterlast injection.I1Administration of Naloxone, Morphine and Yorphineantiserum (Morphine-As) to rats pretreated with PCPAAdult male rats were administered ip a single doseof PCPA (300mg/kg b,w in 0.9% saline). 72 hours afterinjection rats were given the following treatments.Croup I and 11: Yorphine sulfate (2mg/kg b.w) sc, andsacrificed at 1 and 4 hour after injection.Croups It1 and IV: Naloxone was administred sc (2mg/kg bwdissolved in 0.9% NaC1) and sacrificed at 1 and 4 hour afterinjection.Group V and VI: Antiserum to morphine (0.2~11 100 Xdillution) sc, and sacrificed after 1 and 4 hours afterinjection.

1. Effect of Administration of PCPB and NaloxonePlasma thyroxine level in control rats treatedwith saline was 73 !: 5.3 ng/ml. Administration of eitherPCPA or naloxone had no effect on plasma thyroxine level.(Fig 1).Plasma Trilodothyronine (T3) level in salinetreated control rats was 0.8 + 0.2 ng/ml. Administration ofPCPA or naloxone did not alter the hormone level. (Fig 2).Hypothalamic -glutamyl transpeptidase ( Y -GT)activity in control rats was 52.9 + 1.9 t~ moles/gtissuelhr. FCPA or Naloxone had no effect on the enzymeactivity under any of the treatment regimens. (Fig 3).Testicular lactate dehdrogenase activity in salinetreated control rats was 88.4 2 4.5 units/mg protein. Theenzyme activity was significantly Increased by PCPA at 24hours after injection. The enzyme activity did not exhibitany change at 72 hour after treatment (Fig 4). Naloxonetreatment caused reduction in LDH activity at 24 and 72houre after a single injection and also at 3 days afterdaily injections (Fig 4).

2. Effect of Morphine, Naloxone and morphine-Antisem onPBA - treated ratsPlasma thyroxine level in control rats (PCPA +Saline) was 77.8 + 5.6 ng/ml. Administration of naloxone toPCPA pretreated rats reduced thyroxine levels at 1 hourafter injection. Administration of morphine sulfate alsoreduced thyroxine level at 4 hours after injection.Administration of antiserum to morphine in rats pretreatedwith PCPA also reduced plasma T4 at both 1 and 4 hours afterinjection (Fig 5).Plasma Triiodothyronine level in control rats(PCPA + Saline) was 0.8 + 0.3 ng/ml. Naloxoneadministration had no effect on plasma thyroxine levels.Injection of morphine sulfate to PCPA treated ratssignificantly reduced plasma Tg levels while administrationof morphine-As had no effect on the hormone level (Fig 6).Hypothalamic Y -glutamyl transpeptidase activityin control rats (PCPA + Saline) was 51.4 + 0.9 t~ moles/gprotein/hr. Naloxone treatment induced reduction in enzymeactivity at 4 hrs. after injection. Administration ofmorphine caused an increase in 7-GT activity at 1 hourafter injection. Administration of morphine-As in PCPApretreated rats reduced Y-GT activity both at 1 and 4 hoursafter injection (Fig 7).

Testicular lactate dehydrogenase activity incontrol rats (PCPA + Saline) was 84.8 5 5.3 units/mgprotein. Administration of morphine. naloxone or antiserumto morphine to PCPA treated rats caused significantreduction in testicular LDH activity.Adminsitration of FCPA and the results indicatethe absence of serotoninergic involvement in the thyroidfunction. Depletion of brain serotonin by administration ofPCPA had no effect on plasma thyroid hormones. Earlierstudies by Krulich et a1 (1979) had demonstrated aninhibitory role for serotonin on the TSH secretion. Asthere is no enhanced secretion of thyroid hormones in thepresent study, it may be that serotonin is not involved inthe basal secretion of TSH from pituitary. TRH releare fromhypothalamus is mainly controlled by dopamine (McCann ge., 1984).OPioid receptor blockade using rialoxone in p ~ p ~treated rats had an inhibitory effect on plasma Tq levelwhereas it had no effect on Tg level. Naloxone alone doesnot alter thyroxine levels (see Chapter 111).As naloxonecould reduce plasma TSH (Chapter 111), the reduced plasmathyroxine may he due to decreased TSH release caused bynaloxone activation of hypersensitive serotonin receptors.

The data indicate that blockade of serotoninsynthesis accelerates morphine induced suppression of TSHsecretion and subsequently thyroid hormone levels. It hasbeen observed that pharmacological activation of centralserotoninergic system inhibits release of hypothalamic TRHwhich subseqently reflects in reduced plasma TSH levels(Krulich g., 1979). Plasma, T3 level was inhibited bymorphine in PCPA treated rats at both intervals. Thisfinding is different from results obtained with morphineinjection alone (chapter IV). However, administration ofmorphine-As in PCPA pretreated rats had an inhibitory effecton all the parameters studied. These effects are similar tothose observed in rats treated with morphine-As alone.Depletion of central serotonin level with PCPA donot modify the effect of naloxone, morphine or morphine-Ason testicular LDH activity. However, all these drugs whenadministered alone significantly reduced LDH activity.Pretreatment with PCPA on the otherhand modified the effectof naloxone morphine and morphine-As on hypothalamic T-GTactivity. This indicate that serotoninergic system may beinvolved in the utilisation of glutathione in thehypothalamus. The physiological significance and exact roleon pituitary hormone secretion of such an interaction is notclear.

Pig.1: Effect of FCPA and Naloxone on plasma T level in 60day old male rats at 24 and 72 hour aft=# treatment.Values are Hean + SEY for 4-6 rats in each group.

Pig.2: Effect of PCPA and Naloxone on plasma T level in 80day old male rats at 24 and 72 hour afteg treatment.Values are Yean + SFU for 4-6 rats in each group.

Pig.3: Effect of PCPA and Naloxone on hypothalamic'Y-GTactivity in 60 day old male rats at 24 and 72 hourafter treatment. Values are Mean 2 SEM for 4-6 ratsin each group.

Ptg.4: Effect of PCPA and Naloxone on testicular LDAactivity in 60 day old male rats at 24 and 72 hourafter treatment. Values are Mean 2 SEH for 4-6 ratsin each group.P(O.OO1 vs Control

Pig.5: Effect of PCPA + Nal, FCPA + Morphine and PCPA + M-Ason plasma T level in 60 day old male rats at 1 and 4hour after treatment. Values are Mean 2 SKU for 4-6rats in each group.* P< 0.05 vs Control , ** P< 0.001 vs Control

Fig.6: Effect of PCPA + Nal, PAPA + Morphine and PCPA + Y-Ason plasma T level in 60 day old male rats at 1 and 4hour after ?,reatment. Values are Mean 2 S M for 4-6rats in each group.P < 0.05 vs Control

Pig.7: Effect of administration of PCPA + Nal, PCPA +Morphine and PCPA + Y-As on hypothalamic 7-GTactivity in 60 day old male rats at 1 and 4 hourafter treatment. Values are Mean 2 SEN for 4-6 ratsin each group.* P< 0.05 vs Control

Pig.8: Effect of PCPA + Nal, PCPA + Morphine and F WA + Y-Ason testicular LDH activity in 60 day old male rats at1 and 4 hour after treatment. Values are Mean 2 SEMfor 4-6 rats in each group.* P(O.OO1 vs Control

EFFECT OP NAmxom, YOBPBIrn, YOBPEIrn ANTISBBIIY mEXCITATORY NIUO ACIDS, CLUTAHIC ACID AND ASPARTICACID ON PLASMA Tg AND Tq AND ON TESTICULAE LACTATEDKEYDNJGENASE AND SORBITOL DKAlDROGENASEAClYVITY IN ALWLT YBLg RATS

EPFgCT OP NALOXWE, YOBPBINE, YOBPHINE ANTISERLM ANDEXCITATORY AMINO ACIDS, GLUTAMIC ACID AND ASPARTIC ACIDON PLaSYIL T3 AND Tq AND ON TESTICIRdR LACTATE DEHPDBOCENASEAND SORBITOL DKWDROGENASE ACTIVITY IN ADILT MALE RATSAmino acid neurotransmitters, by an action on thecentral nervous system has been found to regulate pituitaryhormone secretion and thereby target gland functions.Excitatory aminoacids such as aspartate (Asp), glutamate(Glu) and homocystelc acid (HCA) as well as relatedsubstances (eg. N-methyl D-aspartate, NMDA) stimulateluteinizing hormone secretion (Olney g G., 1976; Price1978, Wilson and Knobil, 1982, Gay and Plant 1987; Tal gg., 1983, Gay and Plant, 1988). Systemlc administration ofGlu to adult rats caused an immediate long lastingsuppression of growth hormone secretion due to an increasein release of somatostatin (SRIF) (Terry 5 g., 1981,Benyassi g Q., 1991), Administration of N-Methyl D, L-aspartate (NMA), specific excitatory amino acid receptoragonist, caused an increase in plasma prolactin levels(Arslan g G., 1992).The present experiments were designed to study theeffects of intravenous administration of naloxone, morphine

sulfate and antiserum to morphine on plasma level of thyroidhomes and also whether infusion of excitatory amino acidsmodulates the response to above treatments.ANIMALSChapter 11.Rats were procured and maintained as described inas described (Chapter 11).Chemicals were purchased from various sourcesTesticular LDH and SDH activitywere measured and RIA of T4 and T were carried out as3described In Chapter 11.bperimental DesignAll the experlments were conducted using adultmale rats. Rats were implanted with intrajugular cannula asdescribed In Chapter 11. On the day of the experiment, theanimals were transferred in their individual cages to aquiet laboratory. A polyethylene extension tube filled witha solution of heparin in 0.9%. Nacl was attached to thedistal end of the jugular cannula and the animals were leftundisturbed for 30-60 sec.Experiment I: A preinjection blood sample was withdrawnover a period of 60 sec. Naloxone (200 pg in 0.2 ml 0.9%

NaC1) was administered through an indwelling catheter andblood sample was withdrawn after 1 hour. Following this,aspartic acid (150 mg/kg b/w in 0.2 ml 0.9% NaC1) wasinfused over a period of 60 seconds. Blood samples weretaken at 30 minutes and 1 hour intervals. The volume ofeach sample was replaced after each bleeding by an equalvolume of 0.9% NaC1. Plasma was separated by centrifugationat 4'~ and stored frozen till the hormone assays. Glutamicacid (150 mg/kg bw) was administered following naloxonetreatment and blood samples were collected at intervals asdescribed above.Experiment 11: A preinjection blood sample was withdrawnover a period of 60 sec. Morphine sulfate (100 pg in 0.2 ml0.9% Nacl) was administered iv followed by a blood samplingat 1 hour duration. Aspartic acid (150 mg/kg bw in 0.9%Nacl) was administered by iv and blood was collected at 30'and 1 hour after injection. The volume of each sample wasreplaced after each bleeding by an equal volume of 0.9%NaC1. Plasma was separated by centrifugation at 4'~ andstored frozen till the assay of hormones. Rats weresacrificed after the last bleeding, testes were dissectedout and used for measurement of the activity of LDH and SDH.Glutamic acid (150 mg/kg bw in 0.9% NaCl) wasadministered after treatment with morphine and blood samples

were collected at the intervals described above. Rats weresacrificed after the last bleedlng and activity oftesticular LDH and SDH were measured.Experiment 111: Rats unplanted with jugular vein cannulawere used in this study. A preinjection blood sample waswithdrawn over a period of 60 Sec. Antiserum to morphlne(0.1 ml, 100 X dilution) was administered, iv, and bloodsample was withdrawn after lhr. Following this asparticacid (150 mg/kg hw in 0.9% Nacl) was administered and bloodsamples were collected at 30 minutes and 1 hour intervals.The volume of each sample was replaced after each bleedlngby an equal volume of 0.9% NaC1. Plasma was separated bycentrifugation at 4'~ and stored frozen till the assay ofhormones. Rats were sacrificed after the last bleeding andtestes were dissected out and used for measurement ofactivity of testicular LDH and SDH.Glutamic acid (150 mg/kg bw in 0.9% saline) wasadministered following treatment with antiserum to morphineand blood samples were collected at specified Intervals asdescribed above. Rats were sacrificed and activity oftesticular lactate dehydrogenase and sorbitol dehydrogenasewere measured.

1. Effect of naloxone, aspartic acid and glutamic acid onplasma T3 and T4The preinjection level of plasma T4 ranged from6.1 ng/rnl to 12.5 ng/ml. Administration of naloxone led tosignificant decrease in plasma thyroxine level within onehour of treatment. Aspartic acid injection to Nalpretreated rats produced a further reduction in plasmathyroxine levels both at 30 mlnutes and 1 hour afteradministration (Fig.1). Glutamic acid administration to Nalpretreated rats also signrficantly reduced plasma T4 levelat 30 minutes. Whereas glutamic acid treatment of Nalpretreated rats produced a marginal increase in the hormonelevel at one hour (Fig.2).Prein~ection level of plasma T3 was 0.37 ng/ml.Administration of naloxone did not modify plasma T3 levels.Treatment of Nal pretreated rats with aspartic acidsignificantly reduced the hormone level at 30 minutes afterinJection. Administration of aspartic acid to Nal pretreatedrats modified plasma T3 levels significantly at one hourafter Asp injection (Fig.3). Intravenous administration ofglutamic acid to naloxone pretreated rats significantlyreduced plasma T3 level at 30 minutes whereas the hormonelevel was significantly increased at 1 hour after injection(Fig.4).

2. Effect of Morphine Sulfate, Aspartic Acid and GlutamicAcid on Plasma T3, T4 Testicular LDB and SDBThe preinjection level of plasma T4 varies from4.38 to 5.1 ng/ml. Administration of morphine sulfate alonesignificantly raised T4 levels. Subsequent administrationof-aspartic acid and/or glutamic acid to MS pretreated ratssignificantly reduced plasma T4 level both at 30 minutes and1 hour after injection. Injection of aspartic acid produceda greater degree of suppression of T4 than those produced byClutamic acid (Fig.5 8 6).Preinjection level of plasma T3 ranges from 0.29to 0.43 ng/ml. Morphine sulfate treatment significantlyreduced plasma T3 level. Administration of Aspartic acid toMS pretreated rats induced a further reduction in thehormone level at 30 minutes while it bad no effect on thehormone level at 1 hour after injection (Fig.7).Administration of glutamic acid to morphine pretreated ratscaused an increase in plasma Tg at 30 minutes and at hourafter in~ection. The rise in hormone levels observed at 30minutes after glutamic acid was comparable to those of thepreinjection value (Fig.8).Testicular enzyme lactate dehydrogenaseshowed anactivity of 83.1units/mg protein when treated withmorphine sulfate followed by administration of saline which

served as controls. Intravenous administration of asparticacid to rats pretreated with morphine and sacrificed after 1hour had no effect on testicular lactate dehydrogenase andsorbitol dehydrogenase activity. Glutamic acid treatment torats pretreated with morphine on the other hand causedsignificant reduction in testicular LDH activity while ithad no effect on SDH activity (Fig.9810).3. Effect of Morphine Antiserum, Aspartic Acid andGlutanic Acid on Plasma T3, T4 and Testicular LDR andSDBThe preinjection level of plasma T4 varied from3.6 to 11.7 ng/ml. Administration of antiserum to morphineproduced significant reduction in T4 level at 1 hour afterintravenous injection. Administration of aspartic acid tohi-As pretreated rats caused a further reduction both at 30minutes and 1 hour after injection (Fig.11). Glutamic acidinjection to morphine antiserum pretreated rats caused asignificant (p < 0.001) rise in plasma T4 at 30 minuteswhile at hour after injection ~t diminished the hormonelevel (Fig.12).The preinjection level of plasma Tg varies from0.2 to 0.25 ng/ml. Administration of antiserum to morphinehad no effec.t on the plasma T3 level at 1 hour after

injection. Injection of aspartic acid M-As pretreated ratsproduced a significant (p < 0.002) reduction in plasma T3level at 30 minutes. The Tg level was significantly elevatedat 1 hour in M-As pretreated rats. The increase was alsosignificant when compared to those valuues obtained in M-Asinjected rats (Flg. 13). Glutamic acid injection to morphinepretreated rats produced a rise in plasma T3 level both at30 minutes and one hour after lnjectlon (Fig.14).In control rats treated with antiserum to morphineand saline and sacrificed after one hour, lactatedehydrogenase (LDH) and sorbitol dehydrogenase (SDH)activity was 60.6 and 0.89 units/mg proteln respectively.Treatment with aspartlc acld to rats pretreated withmorphine antiserum reduced only marginally LDH activlty(Flg.15) while it had no effect on testicular SDH (Flg.16).Glutamlc acid injection, on the otherhand led to increase intesticular LDH activity while it had an inhlb~tory effect onSDH activity.The present data show that the opioid antagonistnaloxone, morphine sulfate and antiserum to morphine haveregulating effect on plasma T and T levels. Intravenousinfusion of naloxone evoked an lnhibltory response of plasma

T4 level while it had no effect on Tg levels. Naloxoneadministered subcutaneously inhibited plasma TSH levelwithout modifying plasma T4 level (Chapter 111).Administration of naloxone through intravenous route appearsto have accelerated the inhibitory response of naloxone onTSH release and subsequent reduction In plasma T4 levels.Thus it appears that endogenous opioids exert regulatoryeffects on pituitary thyrotropln release.Inhibitory effect of both glutamic acid oraspartic acid on plasma thyroid hormone indicates that theseneuronally active aminoacids may be involved in TSH release.A centrally mediated action of amino acid neurotransmitterson pituitary hormone release has been reported (Davidson,1976). Amino acid neurotransmitters such as glutamic acid,aspartic acid, and homocysteic acid, as well as relatedsubstances (eg. N-methyl-D-aspartate, NMDA) stimulateluteinizing hormone secretion(~i1sonand Knobil, 1982; Gayand Plant 1987; Gay and Plant, 1988) NMDA receptor agonistmediated release of prolactin (Arslang., 1992) andsystemic .administration of glutamic acid to adult ratscauses an immediate long-lasting suppression of growthhormone secretion (Benyassi g., 1991). The inhibitionby the administration of glutamic acid and aspartic acid onplasma T and T levels is possibly via a hypothalamus4 3mediated inhibition of thyrotropin release from the

pituitary. The exact mechanisms of action of such aresponse is not clear.Morphine, administered intravenously produced arise in plasma T4 and reduction in plasma Tg levels. Incontrast, antiserum to morphine administered iv reducedplasma T4 level without modifying plasma Tg level. Thetransient increase in plasma T4 level observed is incontrast to earlier observations (Chapters IV 8 V) whereadministration of morphine led to reduction in plasma T4levels.The change in activity of testicular markerenzymes lactate dehydrogenase and sorbltol debydrogenaseunder the experimental conditions may be due to change incirculating levels of hormones (Chapter 111) as well as adirect action of morphlne and its antiserum on testis.The data presented in this chapter indicates thatamino acid neurotransmitters glutamic acid and aspartic acidexert an inhibitory effect on plasma thyroid hormone levelpossibly mediated by Inhibition of thyrotropin secretion.

Prelnj lhr Nel 30' Asp lhr AetIPig.1: Effect of intravenous administration of Naloxone +Aspartic acid on plasma T level in adult male rats.Values are Mean + SEM for 4 rats in each group.* P (0.01 ; vs Preinj ** P< 0.01 vs Nal*** P< 0.b1 vs Nal

Prdnj lhr Nal 30' Glu lhr QluPig.2: Effect of intravenous administration of Naloxone +Glutamic acid on plasma T level in adult male rats.Values are Mean + SEM for4 4 rats in each group.* P< 0.001 vs Preinj , ** P(0.05 vs Nal

Prelnl 0 lhr Nal 30' ASP ii%ii lhr ASPFig.3: Effect of intravenous administration of Naloxone +Aspartic acid on plasma T level in adult male rats.Values are Mean 2 SEM for 34 rats in each group.* P< 0.05 vs Nal

Pig.4: Effect of intravenous adainistration of Naloxone +Glutrnic acid on plama T3 level in adult mle rats.Values are Mean 2 SEY for 4 rats in each group.P(0.05Vs No1 ; ** P< 0.001 vs Nal

Pig.5: Effect of intravenoua administration of morphine +asputic acid on plam T4 level in adult ale rats.Values ua Maan 2 SEY for 4 rats in each group.* P< 0.001 vs Prein~ , ** P< 0.001 vs w

Pig.6: Effect of intravenous administration of morphine tgluta~lic acid an plasma T4 level in adult male rats.Values Ire Mean 2 SEM for 4 rats in each group.* P< 0.05 vs Prein~ , ** P< 0.001 vs NS

Pig.7: Effect of intravenous administration of morphinesulfate + aspartic acid on plasma Tg level in adultmale rats. Values are Mean 2 SEM for 4 rats in eachgroup.

Pig.8: Effect of intravenous administration of morphinesulfate + glutamic acid on plasma Tg level in adultmale rats. Values are Mean 2 S M for 4 rats in eachgroup.

Pig.@:Effect of Morphine + Asp; Morphine t Glu ontesticular lactate dehydrogenase activity in adultrats (Morphine t saline served as control). Valuesare Mean + SEY for 4 rats in each group.P< 0.001 vs Control

Fig.10: Effect of Morphine + Asp and Morphine + Glu ontesticular Sorhitol dehgdrogensse activity in adultrats (Morphine + saline served as control). Valuesare Mean + SM for 4 rats in each group.

Fig.11: Effect of intravenous administration of morphineantiserum + aspartic acid (M-As + Asp) on plasma Tlevel in adult mle rats. Values are Mean + SEM for44 rats in each group.

Fig.12: Effect of intravenous administration of morphineantiserum + glutamic acid (M-As + Glu) on plasma Tlevel in adult male rats. Values are Mean + SEU for44 rats in each group.

Fig.13: Effect of intravenous administration of morphineantiserum + nspartic acid (M-As + Asp) on plasma Tlevel in adult male rats. Values are Mean 2 SM for34 rats in each group.

Fig.14: Effect of intravenous administration of morphineantiserum + glutanic acid (Y-As + Glu) on plasma Tlevel in adult male rats. Values are Yean + SEM for34 rats in each group.

Pig.15: Effect of M-As + Asp and Y-As + Glu on testicularlactate dehydrogenase activity in adult male rats.Morphine antiseru t saline treated rats served ascontrol.Values are Mean + SEN for 4 rats in eachgroup.* P< 0.2 vs Control , ** P< 0.001 vs Control

Fig.16: Effect of M-As + Asp and M-As + Glu on testicularSorbitol dehydrogenase activity in male ratsMorphine antiserum t saline treated rats served ascontrol. Values are Yean + SEM for 4 rats in eachgroup.* P< 0.05 vs Control

cEummt VIIEFFECT OF I!iTRATggTIaRdB INJgCTIOW OF DYNORPEIN, YOBPEINEA?Kl WEPEINE AHTISEBM ON TESTICIRdR LACTATEDEEYDROGMASB AND SORBITOL D ~ E N A S EACl'IVITY INADULT YALE RATS

CRbPPEB VIIEFFECT OF IWTBBTBSTICUIda INJECTION OF DYNOWBIN,MOBPHIRE AND MORPHINE ANTISERUM ONTESTICULaa LbcrATE DEmDmxDEHPD AND ~ I T O LDEAPDBOGENASE AnIVITY IN ADULT YALg RATSOpioid peptides of the three different classes arepresent in pituitary and brain (Krieger et g., 1980; Saito-et aJ., 1983).Opioid peptides have also been localized ina variety of peripheral tissues like thyroid (Chengg.,1986), pancreas (Bruni & Q., 1979), gastrointestinal tract(Smyth, 1983), placenta (Genazzani & g., 1974), in thereproductive tract of male and female (Sharp g g., 1980),Lolait et aJ., 1984) and in the testes of rats (Margioris 9g., 1983; Kilpatrick g., 1985).The number and intensity of staining of p-endorphin containing cells in mouse and hamster testes werefound to increase at puberty (Shaha & Q., 1984). Chen andMadigan (1987) reported that WMC mRNA levels found to beincreased from days 15-40 in the rat testis. Theconcentration of inununo reactive p -endorphin in rat testiswas very high at 5 days of age but then decreased andremained constant throughout development and sexualmaturation (Adams and Cicero, 1989).

It has been suggested that p-endorphin has localeffects within the testes on Sertoli cell growth, androgenbinding protein and steroidogenesis (Fabbri g., 1988,Gerendai g &., 1986). Fabbri et a1 (1985) demonstrated thepresence of opioid receptors on Sertoli cells of rat.Kilpatrick et a1 (1985, 1986) suggested that proenkephalin -derived peptides may be involved in gametogenesis and mayfunction as germ cell associated hormones or paracrinefactors. A precise role for p-endorphin in testicularfunction is not yet known. In the present study, effect ofintratesticular injection of Dynorphin, morphine sulfate andantiserum to morphine on testicular lactate dehydrogenase,and sorbitol dehydrogenase, activity has been evaluated.MATERIALS AND YFPBODSSource of animals, chemicals and procedures are asdescribed in Chapter 11. Adult male rats of 2.5 to 3 monthold were used in the experiments described below:Experiment I: Morphine sulfate 25 u g and 50 u g dissolvedin 0.9% NaCl and microln~ected intratesticularly in a volumeof 50 p1. Rats were sacrificed after an interval of 1 hourand 4 hours and testicular lactate dehydrogenase andsorbitol dehydrogenase activity was measured as describedearlier.

85Experiment 11:la') Dynorphin 5 u g and 25 u grespectively, dissolved in 0.9% NaCl and microinjectedintratesticularly in a volume of 50 p1. Rats were sacrificedafter an interval of 1 hour and 4 hour and testicular LDHand SDH activity was measured.Experiment 111: Antiserum to Morphine 0.1 ml and 0.2 mlrespectively, was injected intratesticularly. Groups ofrats were sacrificed after an interval of 1 hour and 4 hoursand testicular LDH and SDH activity were measured asdescribed before.1. Effect of Morphine Sulfate on LDB and SDATesticular lactate dehydrogenase activity wasfound to be 89.1 + 3.3 units/mg protein in saline treatedcontrol rats. Both doses of morphine significantly reducedthe enzyme activity at lhr after injection whereas,thehigher dose evoked no response on LDH activity at 4 hoursafter injection (Flg 1).Sorbitol dehydrogenase activity in control ratstreated with saline was 0.9 2 0.01 units/mg protein.Administration of both doses of morphine led to increase inSDH activity at 1 hour while a significant reduction in theenzyme activity was observed at 4 hours after injection(Fig. 2).

2. Effect of Dynorphin on LDE and SDBTesticular lactate dehydrogenase activity was 89.1- + 3.3 unitslmg proteln in control rats. Dynorphin at bothdoses significantly reduced the enzyme activity at 1 and 4hour after injection. (Fig.3).Sorbitol dehydrogenase activity was 0.9 2 0.01unitslmg protein in control rats.Administration of bothdoses of dynorphin significantly reduced the enzyme activityboth at 1 and 4 hour after injection. (Fig.4).3. Effect of Morphine Antiserum on LDB and SDRLactate dehydrogenase activity in control rats was89.1 2 3.3 unitslmg protein. Both doses of antiserum tomorphine significantly reduced lactate dehydrogenaseactivity at 1 hour after treatment while the enzyme activityremained unaffected at 4 hour after injection. (Fig. 5).Sorbitol dehydrogenase activity in saline treatedcontrol rats were 0.9 2 0.01 unitslmg protein. Antiserum tomorphine caused an increase in activity of sorbitoldehydrogenase at 1 hour after treatment. The higher dosesignificantly reduced enzyme activity at 4 hour afterinjection. (Fig. 6).

The results obtained in this study indicate thatopiates and opioid peptides influence testicular metabolism.Morphine sulfate reduces lactate dehydrogenase activity,which is a key enzyme in metabolic process.Lactate andpyruvate are two metabolites secreted by the Sertoli cellsand utilised by maturing spermatocytes and spermatids (Mitaand Hall 1982).Pyruvate and lactate help to maintain highrates of protein synthesis in isolated spermatocytes andspermatids (Jutte g e., 1983). Thus ~t is suggested thatthese two secretory products of Sertoli cells are essentialfor spermatogenesis In vivo (Jutte g aJ.,g., 1975; Mita and Hall 1982).and -C1983, Markert &During testicular development, the LDH-A, -Bgenes are expressed differentially. Previous studiesexamining the activity of LDH isozymes in unfractionatedmouse testes found that the proportion of LDH-A subunitsdecreases between 2 and 10 days of age, while LDH-B subunitsincrease during this Interval and LDH-C4 is not detecteduntil the'presence of pachytene spermatocytes (Wieben 1981).LDH-C4 is confined to spermatogenic cells and has beendetected immunohistochemi~ally in mouse spermatocytesbeginning at the mid-pachytene stage of meiosis and inhaploid cells throughout spermiogenesis (Hintz and Goldberg1977). Prepubertal germ cell populations containing a

mixture of spermatogonia, pre-pachytene spermatocytes andnongerminal cells showed no evidence of LDH-C synthesis.The reduction in the actlvity of testicularlactate dehydrogenase by morphine as well as dynorphin maybe either due to reduction in lactatejpyruvate secretion bySertoli cells or by a direct action on spermatogenic cycle.The former conclusion 1s supported by the presence of opioidreceptors on Sertolr cells (Fabbrl et al., 1985).Proenkephalin gene is expressed in germ cells and somaticcells of testes (Kilpatrick g g., 1985; Kilpatrick andRosenthal, 1986).Products derived from the transcripts ofthe gene may be having inhibitory effect on the function ofgerm cells or may be inhibitory via affecting the functionof somatic cells.The paracrine regulation of testicularsteroidogenesis by opioids was demonstrated by theexperiments in which lntratesticular injection of naloxonedecreased testosterone secretion from the testicular tissueincubated in vitro (Anandalakshmi 1989; Gerendai g g.,1989). B-endorphin produced within the Leydig cells maybehave as a paracrine inhibitor of Sertoli cell function(Fabbri g g., 1985; 1988). Thus the reduction in theactivity of lactate dehydrogenase following intratesticularadministration of morphine and dynorphin may be due tocomplex interactions between the somatic and germ cellcomponents of the testis. Administration of antiserum to

89morphine caused a transient reduction in testicular LDH at 1hour. after injection while the enzyme activity remainedunaffected by the treatment at 4 hours after injection.Administration of morphine caused an increase insorbitol dehydrogenase activity at 1 hour while reducedenzyme activity was observed at 4 hours duration. Dynorphintreatment on the otherhand caused significant reduction atboth intervals and doses employed. This data suggests thatopiates and opioids exert lnhlbitory action on SDH activityand thereby the metabolic processes. Sorbitol dehydrogenasehas been reported as an important marker enzyme of thetestis (Bishop G., 1967; Bishop 1967) Mills and Means(1972) reported that sorbitol dehydrogenase activitycorrelates well with the state of germinal epithelium bothduring development and following hypophysectomy. A dose andtime dependent response in SDH activity to morphineantiserum treatment was observed. Both the doses enhancedenzyme activity at 1 hour duration while the higher dosecaused reduction in SDH activity at 4 hours duration.The data presented in this study clearly suggeststhat opiates and opioid peptides exert an inhibitory actionon the intratesticular metabolism. Available evidencessuggest that the action may be mediated by Sertoli cells asalso germ cells. The exact mechanism(s) Of action andcomplex paracrine regulatory role of opioid peptides intesticular tissue is yet to be revealed.

h: tooPig.1: Effect of intratesticular administration of morphine(25 ug, 50 ug) on testicular Lactate dehydrogenase(LDH) activity in adult rats at 1 and 4 hour aftertreatment. Values are Mean + SEY for 6 rats in eachgroup. .* P < 0.001 vs Control

Fig.2: Effect of intratesticular administration of morphine(25 ug, 50 up) on Sorbitol dehydrogenase activity inadult rats at 1 and 4 hour after treatment. Valuesare Mean + SEM for 6 rats in each group.* P< 0.001 vs Control

Fig.3: Effect of intratesticular administration of dynorphin(5 ug, 25 ug) on testicular Lactate dehydrogenase(LDH) activity in adult rats at 1 and 4 hour aftertreatment. Values are Mean 2 SEM for 6 rats in eachgroup.

Pig.4: Effect of intratesticular administration of dynorphin(5 ug, 25 ug) on testicular Sorbitol dehydrogeaaseactivity in adult rats at 1 and 4 hour aftertreataent. Values are Mean 2 SEM for 6 rate in eachgroup.

Pig.5: Effect of intratesticular administration of wrphineantiserum (M-As) (100 ul, 200 ul) on testicularLactate dehydrogenase (LDH) activity in adult rats at1 and 4 hour after treatment. Values are Mean + SQIfor 6 rats in each group.* P < 0.001 vs Control

Fig.6: Effect of intratesticular administration of morphineantiserum (M-As) (100 ul, 200 ul) on testicularSorhitol dehydrogenase (SDH) activity in adult ratsat 1 and 4 hour after treatment. Values are Mean 2SQ( for 6 rats in each group.* P< 0.05 vs Control , ** P< 0.001 vs Control

a?APTm VIIIGEHERaL DISCUSSION AND CONCLUSION

m mVIIIGEWERAL DISCUSSION AND CONCLUSIONThe pituitary gland is no more considered as the'master gland' or 'the conductor--of .endocrine orchestra' .Experimental evidence accrued during the past 3-4 decadeshave clearly established the brain, particularly thebypotbalamus, as the master gland in controlling thesecretion of pituitary hormones. Neurotransmitters andneuromodulators like norepinephrine, epinephrine, dopamine,serotonin, endogenous opioids and neuronally active aminoacids such as glutamate, asparatate, gamma amino butyricacid (GABA) are also involved in the co-ordination andrelease of hypothalamic releasing and inhibitory hormones(McCann 1980; Vijayan, 1985).The localization of opioid receptors andendogenous opioid peptides in various regions of the centralnervous system led to investigations on their role inneuroendocrine regulation (Hughes & g., 1975; Morley,1983; Gross4un and Rees, 1983; Howlett and Rees, 1985). Inthe present investigation, attempts were made to examine therole of opioid system on pituitary hormone release byadministration of ~loxone, and opioid receptor antagonist,

in 20, 40 and 60 day old male rats. Results presented inthe thesis clearly indicate an opioid induced suppression ofLH release.Involvement of neurotransmitter Interactionwith opioids at the hypothalamic level in mediating opioidaction on LH release have been suggested (Karlag.,1988). CABA-ergic system involvement in the opioid inducedsuppression of LH release was examined in the presentstudies. Administration of aminooxyacetic acid (AOAA)diminished LH levels.Administration of naloxone to ratspretreated with AOAA, abolished the suppressive effect ofAOAA on LH release indicating an inhibitory action of GABA-ergic system on LH release.The oploid systea may alsopossibly be acting through this inhibitory pathway.GABAmay play a dual regulatory influence on LH release dependingon the type of receptor that is activated (Vijayan and McCann, 1978, Donoso, 1988). High concentrations of GABAresulting from AOAA treatment lead to activation of GABABreceptors resulting in inhibition of LH release (Masotto gc., 1989). Further, opiate receptor blockade by naloxoneis able to stimulate LH secretion in NE-lesioned male rats(Miller & a,, 1985). The present observation and otherrecent findings suggest that opioid inhibition of LH releaseis possibly mediated by the CABA-ergic system.However,naloxone treatment had no effect on plasma FSH levelsindicating either absence of endogenous opioid effect or for

the presence of a separate FSH releasing factor.Administration of AOAA produced a reduction in plasma FSHlevel in 40 and 60 day old rats while no effect in 20 daysold rats. Sexual maturation has been reported to modify thestimulatory effect of GABA on LH 'and FSH release in femalerats (Moguilevsky g g., 1991).AOAA pretreatment followedby naloxone, however produced increase in FSH in 20 days oldrats, decrease in the hormone level in 60 day and no changein 40 day old rats. This suggest that activation of GABAergicsystem modifies opioid effect in 20 and 60 day oldrats and existence of possible interactions between the GABAergic and opioidergic systems.Prolactin secretion 1s facilitated by endogenousopioids (Blank g Q., 1980; Anand Lakshmi, 1989). Naloxonecaused a reduction in plasma prolactin levels. AOAAtreatment suppressed prolactin release. High doses of GABAis known to suppress prolactin release (Grandison andGuidotti, 1979) from pituitaries incubated in vitro.Further, specific GABA receptors have been localized in thepituitary.gland (Grandison and Guidotti, 1979). It appearsthat GABA-ergic and opioid system function independently toregulate prolactin release.Exogenous administration of morphine and opioidpeptides stimulate GH release.However, the role of

endogenous opioids on GH release remains controversial (Miki- -et al., 1984).Studies using naloxone has producedequivocal results. In the present study, naloxoneadministration raised plasma GH level in immature (20 days)and sexually mature (60 days) with no change in pubertal (40days) rats. Results indicate that age dependent change inresponse to AOAA and naloxone treatment prevails with regardto GH release. Pretreatment with AOAA abolished stimulatoryeffect of naloxone on GH release, indicating interactionsbetween GABA-ergic and opioidergic systems.Naloxone administration inhibited plasma TStirelease in 20 and 60 day old rats but enhanced the hormonelevel in 40 day old rats.Morphine sulfate on the otherhand reduced plasma Tg and T4 levels in rats of all the agegroups studied.Surprisingly morphine antiserum alsoproduced inhibitory effect on thyroid hormone levels.Pretreatment with AOAA or PCPA (serotonin synthesis blocker)did not modify the effect of naloxone or morphine sulfate onthyroid hormone levels. Thus, the present results indicatenon-involvement of GABA-ergic and serotoninergic systems inthe mediation of opioid action on TSH release. Excitatoryamino acids, glutamic acid and aspartic acid reduced plasmaT and T level. This action is presumably mediated at the3 4hypothalamic level.

Glutathione utilisation has been implicated inpituitary hormone release (Pasha and Vijayan, 1989). Thechanges in they-GT activity under the present experimentalconditions suggest possible interactions between glutathioneutilisation and pituitary hormone release. The results withAOAA and PCPA on Y-GT activity indicates that GABA-ergicand serotoninergic system may be utilising glutathione toInfluence pituitary hormone release.Opioids and opiate antagonist naloxone exertdirect action on the testicular LDH and SDH activity.Intratesticular administration of dynorphrn and morphinesulfate reduced LDH and SDH activity. Generally the effectof opioids on testicular metabolism is inhibitory in nature.Interestingly morphine antiserum also exerted an inhibitoryaction on testicular LDH activity.Opioid peptides are classified into three distinctclasses originated from three prezarsor molecules namely,proopiomelanocortin, prodynorphin and proenkephalin-A andare widely distributed in the CNS and other peripheralorgans. 'At the hypothalamic and pituitary levels thesepeptides are involved in the regulation of secretion ofhormones. Structural diversity of the peptides,heterogeneity of the opioid receptors and the mediation ofvarious neurotransmitters at the hypothalamic level

contribute to the complexity of opioid peptide regulation ofend~crlne system. only further investigations will re.fealthe class of opioid peptide and the receptors involved inthe activation of GABA-ergic system. .Experiments are alsonecessary to establish the precise interactions betweenglutathione and neurotransmitters and its role on hormonesecretion from the pituitary. Paracrine regulation of atleast some, if not all testicular metabolic events couldpossibly be mediated by opioid peptides since presence ofintratesticular 0~i0ids are well established.

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