Role of the autonomic nervous system in the cytoprotective effect of neurotensin against gastric stress ulcers in rats

Psvchoneuroendocrmolopo~. Vol I0, No 2, pp 149- 157. 1985

0306-4530/8553.00 "4-0 00 tcj 1985 Pergamon Press Lid

Prmlcd m Great Britain

R O L E O F T H E A U T O N O M I C N E R V O U S S Y S T E M IN T H E CYTOPROTECTIVE EFFECT OF NEUROTENSIN AGAINST G A S T R I C S T R E S S U L C E R S IN R A T S * ROY C. ORLANDO,* DANIEL E. HERNANDEZ,~f ARTHUR J. PRANGE, CHARLES B. NEMEROFF|

Jr.,t$§

and

*Department of Medicine, tBiological Sciences Research Center, SDepartment of Psychiatry, and the §Neurobiology Program of the University of North Carolina School of Medicine, Chapel Hill, North Carolina, U.S.A.; and IDepartments of Psychiatry and Pharmacology and The Center for Aging and Human Development, Duke Umversity Medical Center, Durham, North Carolina, U.S.A. (Received 30 March 1983; m final form 27 August 1984)

SUMMARY Pharmacologic agents were used to study the role of the autonomic nervous system in the cytoprotection produced by intracisternal neurotensin against cold plus restraint stress-induced gastric ulcers in rats. Drugs which stimulated a- or B-adrenergic receptors or blocked muscarinic cholinergic receptors reduced the incidence of ulcers to a similar degree as intracisternal neurotensin; ct-adrenerg~c or B-adrenergic blockade as well as cholinergic stimulation prevented neurotensin's beneficial effect. However, pretreatment with indomethacin blocked only the cytoprotective effect of neurotensin or l~-adrenergic stimulation, but not that of muscarinic cholinerg~c blockade. In addition, pretreatment with reserpine or guanethidine also was effective in preventing cytoprotection by intracisternalneurotensin. These data indicate that the mechanism for cytoprotection by centrally administered neurotensin is mediated at least in part through activation of the sympatheuc nervous system. This activation by neurotensin appears to produce cytoprotection by stimulation of gastric mucosal prostaglandin synthesis. INTRODUCTION

NEUROTENSIN (NT) is a tridecapeptide isolated from bovine hypothalamus (Carraway & Leeman, 1973). Since identified in the brain and gastrointestinal tract of man and several mammalian species (Carraway & Leeman, 1976; Kitabgi et aL, 1976; Orci et al., 1976; Facer et aL, 1977), NT has been injected both into the central nervous system (CNS) and systemically to determine its actions. When injected systemically, NT produces vasodilatation, hypotension, hyperglycemia, altered pituitary hormone secretion, and inhibition of meal and pentagastrin-stimulated gastric acid secretion (Andersson et al., 1976; Carraway et al., 1976; Leeman ef al., 1977; Rivier et al., 1977). When injected centrally, NT produces hypothermia, analgesia, catalepsy, and muscle relaxation and potentiates the acute sedative effects of ethanol and barbiturates (Nemeroff et al., 1977; Osbahr et al., 1979; Luttinger et al., 198 l). Although central administration also has been reported to inhibit basal gastric acid secretion (Osumi et al., 1978) and insulin-induced gastric acid secretion (Shiraishi ef al., 1980); this effect has not been observed by others (Tache et aL, 1980; Hernandez et aL, 1982).

USend all correspondence and reprint requests to: Charles B. Nemeroff, MD, PhD, Duke University Medical Center, Box 3859, Durham, NC 27710, U.S.A. 149

150

RoY C. ORLANDO, DANIEL E. HERNANDEZ, ARTHUR J. PRANGE, Jr. and CHARLES B. NEMEROFF

We previously reported that intracisternal NT was cytoprotective against cold plus restraint stress (CRS)-induced gastric ulcers in rats (Nemeroff et ai., 1982). This effect was of particular interest because it was apparently unrelated to several of NT's other central actions including hypothermia, neuroleptic-like properties or inhibition of gastric acid secretion, but was dependent upon the integrity of the prosta#andin synthetic pathway. In addition NT does not appear to cross the blood - brain barrier (Nemeroff et ai., 1980a; 1980b) and when administered intravenously is not cytoprotective in this model (Nemeroff et al., 1982). These data imply existence of an intermediate pathway for translation of NT's central action into its peripheral effect, i.e. gastric mucosal cytoprotection. The autonomic nervous system (ANS) has long been recognized as an important intermediate between the CNS and gastric function (Brooks, 1977), and other centrally administered neuropeptides have been found to alter autonomic function (Tache & Brown, 1981). Therefore, we investigated the effect of ANS activity on the development of CRS-induced gastric ulcers in the rat and its potential for mediating gastric mucosal cytoprotection by centrally administered NT.

MATERIALS AND METHODS Adult male Sprague-Dawley rats (180-200 g, Zivic-Miller, Cincinnati, Ohio) were habituated to a controlled environmental animal facility and fed laboratory chow with water ad h b u u m for a minimum of 7 days prior to experimental use. Before each experiment rats were food-, but not water-deprived for 24 hr. Rats were injected subcutaneously (SC), intrapcritoneally (IP) or under light ether anesthesia intracisternally (IC), as previously described (Nemeroff et aL, 1977). NT in a dose (30 lag in 10 lal) previously shown to prevent CRSinduced gastric ulcers in rats (Nemeroff et al., 1982) or its vehicle (0..90/o NaCI, 10 lad was injected IC. Drugs which act on the ANS were administered by routes and m doses previously reported to selectively block or stimulate adrenergic or cholinergic receptors (Fielden & Green, 1967; Goodman & Gilman, 1975; Glavm, 1980; lnnes & Tansy, 1980). The ~x-adrenergic receptor antagonists phenoxybenzamine (5 mg/kg) and phentolamme (5 mg/kg), the 15-adrenergic receptor antagonist propranolol (12 mg/kg), the muscarinic cholinergic receptor antagonist atropine methyl bromide (1 mg/kg), the muscarimc plus nicotinic cholinergic receptor agonist carbachol (0.25 mg/kg), and saline were injected IP. The ct-adrenergic receptor agonist methoxamine (3 mg/kg), the I~-adrenergic receptor agomst isoproterenol (5 mg/kg), the muscanmc cholinergic receptor agomst bethanechol (5 mg/kg), and saline were injected SC. In experiments that required the injection of two drugs, mdomethacin (5 mg/kg SC), a potent inhibitor of prostaglandin synthesis (Vane, 1971), was administered just prior to treatment with IC NT (30 lag), IP atropine (1 mg/kg), or SC isoproterenoi (5 mg/kg), and NT (30 V.g IC) was admimstered just prmr to treatment with IP phenoxybenzamine (5 mg/kg), IP propanolol (12 mg/kg), or SC bethanechol (5 mg/kg), in experiments requiring catecholamine depletion, rats were injected with reserpine (I mg/kg IP) (Wakade, 1980), and 24 hr later these same rats received IC NT (30 lag) or saline. S~milarly, rats were reJeCted SC with guanethidine (20 mg/kg) and 2 hr later reeewed IC NT (30 lag) or saline. Immediately after IC injection, rats were restrained in wire mesh and placed supine m a cold room (4°C) for 3 hr. Thls reglmen of C R S has previously been shown to reliably produce gastric ulcers m food-dep/'wed rats (Boyd et al., 1977; Pare et aL, 1978; Vincent et aL, 1980). After CRS, the rats were killed by decapitation and the stomachs removed for inspection. Stomachs were cut along the lesser curvature, rinsed with normal saline, and pinned on a Hat surface. The presence of gastric ulcers was assessed with the m d of a dissecting m~croscope (I0 x ) by an experienced examiner who had no knowledge of the treatment regimens. W h e n present, ulcers were readily ~dentified as depicted photographically elsewhere (Nemeroff et al., 1982). In the experiments wlth reserpine the colonic temperature of the animals was measured using a model BAT-12 d~g~tal thermocouple probe (Bailey Instruments, Saddlebrook, N e w Jersey). N T was purchased from Bachem (Torrance, California); indomethacm, reserpine and all autonomic nervous system agonists and antagonists were purchased from Sigma Chemical C o m p a n y (St. Louis, Missouri), except for methoxamine which was provided by Dr P. J. Manberg {Burroughs Wellcome, Research Triangle Park, North Carolina) and phentolamine and guanethidme which were provided by Dr T. A. Slotkin (Duke University Medical Center, Durham, North Carolina).

NEUROTENSIN AND CYTOPROTECTION

15 i

Statistical analyses were performed using Clopper-Pearson intervals for confidence limits of 95% to compare ulcer incidence (CIopper & Pearson, 1934), and Student's t-test for unpaired samples to compare colonic temperatures (Goldstein, 1964). RESULTS

In the first series of experiments the effect of modifying sympathetic and parasympathetic nervous system activity on the incidence of CRS-induced gastric ulcers was evaluated in rats by pretreatment with an adrenergic or cholinergic receptor agonist or antagonist. For direct comparison, a group of animals pretreated with IC NT (30 ~g) was simultaneously studied. As shown in Fig. 1, NT and the u-adrenergic agonist methoxamine, the I~-adrenergic agonist isoproterenol, and the muscarinic cholinergic antagonist atropine markedly reduced the incidence of ulcers when compared to saline controls (p < 0.05). Although the u-adrenergic antagonist phenoxybenzamine also reduced the incidence of ulcers (33°/o vs "/2°/o for saline controls), statistical significance was not achieved (p > 0.05). In contrast, rats pretreated with the [~-adrenergic antagonist propranolol or the cholinergic agonists bethanechol and carbachol developed an incidence of ulcers similar to that of saline controls. 100 9O 0')

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FIG. 1. The effect of ANS agonists and antagonists on the development of CRS-inducedgastric ulcers compared with the effect of IC neurotensin(NT) the e- and B-adrenergicagonists methoxamineand isoproterenol, and the cholinergic muscarinic antagonist atropine reduced the incidence of ulcers (*p < 0.05 compared to saline controls. Since the incidence of ulcers for all saline control groups (IC, IP and SC) was similar, only the combined total is shown.

Figure I also shows that the stimulation of ~ or ~-adrenergic receptors by methoxamine or isoproterenol, respectively or inhibition of muscarinic cholinergic receptors by atropine reduced the incidence of CRS ulcers to the same degree as 1C NT. Thus, it was

152

Roy C. ORLANDO, DANIEL E. HERNANDEZ, ARTHUR J. PRANGE, Jr. and CHARLES B NEMEROFF

hypothesized that the cytoprotection by IC NT could be mediated by activation of one or more of these ANS pathways. To further evaluate this possibility a second set of experiments was performed in which IC NT was injected into rats which also were pretreated with the a-adrenergic antagonist phenoxybenzamine, the 13-adrenergic antagonist propranolol, or the muscarinic cholinergic agonist bethanechol. As shown in Fig. 2, pretreatment with propranolol or bethanechol completely blocked the ability of IC NT to reduce the incidence of CRS ulcers, while pretreatment with phenoxybenzamine •only partially blocked it. Because the results obtained with phenoxybenzamine were less clear, the effects of another a-adrenergic antagonist, phentolamine, were assessed. As illustrated in Table I, phentolamine blocked the cytoprotective effect of IC NT. In a third group of experiments an attempt was made to further link B-adrenergic stimulation or muscarinic cholinergic inhibition with the ability of IC NT to prevent the development of CRS ulcers. This was done by pretreating rats with indomethacin before treatment with NT, isoproterenol, or atropine, three agents which had been shown to reduce the incidence of CRS ulcers (Fig. 1). As shown in Fig. 3, indomethacin completely blocked the ability of NT and isoproterenol to prevent the development of CRS ulcers, while failing to have any effect on the ability of atropine to prevent ulcers.

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FIG. 2. The effect of a- or B-adrenerglc blockade by phenoxybenzamine or propranolol respectwely and muscanmc cholinerglc stimulation by bethanechol on the ability of IC N T to prevent CRS-induced gastric ulcers. B-Adrenergic blockade or stimulation of muscarinic cholinergic receptors blocked the abilityof IC N T to reduce the incidence of ulcers (*p < 0.05 compared to saline controls). Since the incidence of ulcers for all saline control groups (IC, IP, and SC) was similar, only the combined total is shown.

NEUROTENSIN AND CYTOPROTECTION

I ~3

TABI_F [. EFFECT OF G-RECEPTOR BLOCKADE ON NEUROTENSIN-INDUCED GASTRIC ¢ YTOPROTECTION AGAINST STRESS-INDUCED GASTRIC ULCERS IN RATS

Treatment group

n

Gastric ulcers

Saline (IC + IP) Neurotensin (30 gg, IC) Phentolamine (5 mg/kg IP) Phentolamine (5 mg/kg IP) and Neurotensin (30 gg,) IC)

8 8 9 9

8 2 8 8

% 100 25* 88 88

*p < 0.05 when compared wRh saline controls. IC: intracisternal; IP: mtraperitoneal.

To further discern whether the prevention of CRS ulcers by IC NT requires adrenergic activation, rats were treated with either reserpine or guanethidine (to deplete catecholamines) before receiving IC NT or IC saline. Because catecholamine depletion is accompanied by hypothermia (Askew, 1963), prior to CRS the colonic temperatures were measured and shown to be lower for the reserpinized rats (36.2 _+0.2°C, n = 19, vs 37.5 + 0.3°C, n = 20 for non-reserpinized rats [mean _ S.E.M., p < 0.05]). Following CRS, IOO -

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reserpinized rats given IC NT developed a similar incidence of ulcers to that of reserpinized rats given saline (p > 0.05) (Table II). To ensure that reserpinization alone did not produce ulcers unrelated to CRS in NT-treated rats, a group of reserpinized rats (n =

154

Roy C ORLANDO, DANIEL E. HERNANDEZ, ARTHUR J. PRANGE, Jr. and CHARLES B. NEMEROFF TABLE 11. EFFECT OF RESERPINE PRETREATMENT ON CYTOPROTECTION BY IC NEUROTENSINAGAINSTCRS-INDUCED ULCERS Treatment group

Number of rats

Reserpine (I m g / k g IP) + IC saline ( 10 IJ.l)

Number with lesions (070)

9

6 (67)

10

7 (70)

Reserpine (1 mg/kg IP)

+ 'k IC neurotensm(30 ~tgin I0 lad

TABLE Ill. EFFECT OF GUANETHIDINEON NEUROTENSIN-INDUCEDCYTOPROTECTION AGAINSTSTRESS-INDUCEDGASTRICULCERS IN RATS

Saline (IC + SC) Neurotensm (30 lag [C) Guanethtdine (20 m g / k g SC) Guanethtdtne (20 mg/kg SC)

n

Gastric ulcers

°70

9 10 10 9

6 0 7 6

67 0* 70 67

and Neurotensln (30 lag IC) *p < 0 05 compared to saline controls

4) were not stressed; at sacrifice all were free of ulcers (data not shown). Similar results were obtained with guanethidine (Table III). The cytoprotective effect of IC NT was abolished in rats pretreated with this drug. DISCUSSION

This investigation confirms our previous report that IC NT is cytoprotective against CRS-induced gastric ulcers in rats (Nemeroff et al., 1982). In addition, to determine whether the ANS mediates this central action of NT, the effect of ANS agonists and antagonists on the development of CRS-induced ulcers was assessed. Stimulation of either a or I~-adrenergic receptors and blockade of muscarinic cholinergic receptors were shown to be cytoprotective against CRS-induced gastric ulcers. Although blockade of ct-adrenergic receptors with phenoxybenzamine reduced the incidence of ulcers, this effect was small and failed to achieve statistical significance. Moreover, phentolamine, another a-adrenergic antagonist, clearly exerted no cytoprotective effect. Moreover, neither blockade of IB-adrenergic receptors nor stimulation of muscarinic or muscarinic plus nicotinic cholinergic receptors altered the incidence of ulcers. These findings are consistent with the concept that stress ulceration results from the depletion of catecholamines from inhibitory sympathetic fibers, thus permitting unopposed and excessive parasympathetic (cholinergic) stimulation to the stomach (Brodie & Hook, 1976; Kasuya et al., 1978; Benedict et al., 1979; Taylor & Nabi Mir, 1982). Therefore, in our

NEUROTENSIN AND CYTOPROTECTION

]~

experiments cytoprotection by blockade of cholinergic receptors or stimulation of adrenergic receptors can be explained by reduced acetylcholine release and/or blockade of its end-organ effects. To determine if the cytoprotective effect of IC NT is mediated by stimulation or blockade of a pathway in the ANS, experiments were carried out focusing on those pathways in which activation produced cytoprotection similar to that of NT. This requirement was satisfied by stimulation of ¢t- or ~-adrenergic receptors or blockade of muscarinic cholinergic receptors. For this reason, rats were pretreated with a-adrenergic blockers, a [~-adrenergic blocker, or a muscarinic cholinergic agonist proir to the injection of IC NT. Because c~-adrenergic or [~-adrenergic blockade as well as muscarinic cholinergic stimulation completely prevented NT-induced cytoprotection, stimulation of adrenergic receptors or blockade of muscarinic cholinergic receptors retained the potential to act as possible mediators of NT's effect. The former possibility was of particular interest since adrenergic agonists had previously been reported to prevent CRSulcers in rats and through mechanisms like NT, unrelated to inhibition of gastric acid secretion (Esplugues et ai., 1982). In order to further establish a link between stimulation of 13-adrenergic receptors and/or blockade of muscarinic cholinergic receptors with the cytoprotection of IC NT, indomethacin was administered to rats prior to treatment with NT, isoproterenol, or atropine. This study was undertaken because it had been demonstrated previously that pretreatment with indomethacin, an inhibitor of prostaglandin synthesis (Vane, 1971), could prevent cytoprotection by IC NT in this model (Nemeroff et al., 1982). Confirming this finding, indomethacin blocked the ability of IC NT to prevent CRS-induced gastric ulcers. In addition, it also blocked the cytoprotective effect of isoproterenol, the [3-adrenergic agonist, while it had no effect on the cytoprotective effect of atropine, the cholinergic antagonist. The ability of indomethacin to block the cytoprotective effect of isoproterenol can be explained by the finding that other stimulants of adrenergic receptors (i.e. epinephrine and norepinephrine) have been reported to increase prostaglandin synthetase activity in the fundic mucosa of rat stomachs (Pace-Asciak, 1972). This suggests that the mechanism for cytoprotection by adrenergic stimulation in this model may be more of a positive effect by stimulation of gastric mucosal prostaglandin synthesis than a negative effect through inhibition of acetylcholine release. Of further importance, the results with indomethacin pretreatment indicate that IC NT and stimulation of 13-adrenergic receptors as with isoproterenol share in common a final pathway for gastric mucosal cytoprotection and that this pathway requires the synthesis of prostaglandins. It is unclear at this time whether the cytoprotection induced by activation of ~-adrenergic receptors is mediated via prostaglandin synthesis. Finally, using another approach to support an adrenergic pathway as the mediator of cytoprotection by NT, rats were catecholamine-depleted by pretreatment with reserpine or guanethidine. Because we did not confirm the catecholamine depletion after guanethidine, the notion must be entertained that this drug acted in this dose and treatment regime as an adrenergic neuron blocking agent. Because NT was no longer cytoprotective when injected 1C into these animals, it reaffirmed the requirement for an intact sympathetic nervous system for its action. The similarity in ulcer prevention by drugs which stimulate 13-adrenergic blockade receptors and by IC NT, the ability to block

156

RoY C. ORLANDO,DANIELE. HERNANDEZ,ARTHURJ. PRANGE,Jr and CHARLESB. NEMEROFF

N T ' s c y t o p r o t e c t i o n by ~-adrenergic b l o c k a d e or p r e t r e a t m e n t with reserpine a n d g u a n e t h i d i n e , a n d the ability of i n d o m e t h a c i n to p r e v e n t c y t o p r o t e c t i o n by N T a n d [~-adrenergic agonists s u p p o r t the c o n c l u s i o n that s t i m u l a t i o n o f adrenergic receptors is a n i m p o r t a n t i n t e r m e d i a t e p a t h w a y for the expression o f IC N T ' s cytoprotective effect. I n s u m m a r y , i n j e c t i o n o f N T into the CNS a p p e a r s to p r o d u c e its peripheral effect, gastric m u c o s a l c y t o p r o t e c t i o n , by s t i m u l a t i o n o f s y m p a t h e t i c o u t f l o w to the s t o m a c h . T h e s t i m u l a t i o n o f adrenergic receptors in t u r n activates gastric m u c o s a i p r o s t a g l a n d i n synthesis, ultimately leading to c y t o p r o t e c t i o n against CRS ulcers in rats. A l t h o u g h a definite m e c h a n i s m by which p r o s t a g l a n d i n s protect the gastric m u c o s a from ulceration has n o t been defined, the possibilities include increases in gastric m u c o s a l b l o o d flow, ion t r a n s p o r t , m u c u s secretion a n d a d e n y l a t e cyclase activity ( R o b e r t , 1979). T h e last m e c h a n i s m is o f special interest because in m a n y tissues m e m b r a n e a d e n y l a t e cyclase activity has been linked to a c t i v a t i o n o f ~-adrenergic receptors ( R o b i s o n et al. 1971). However, the ability o f i n d o m e t h a c i n to block the cytoprotective effect o f the ~-adrenergic agonist, i s o p r o t e r e n o l , suggests that direct s t i m u l a t i o n o f a d e n y l a t e cyclase activity a l o n e (resulting in increased cellular cyclic A M P ) is unlikely to m e d i a t e this effect. Acknowledgements--This research was supported by NIMH MH-32316, MH-22536, MH-33127, MH-39415

and NICHHD HD-031 I0. We would also like to express our thanks to Sandra F. Woody and Jane A. Atkms for expert secretarial help in the preparation of this manuscript REFERENCES ANDERSSON, D., CHANt.,, D. & Fol kegs, K (1976) Inhibition of gastric aod secretion m dogs by neurotensm. Life Scl. 19, 367 - 370. ASKEW, B. M. (1963) A simple screemng procedure for imlpramme-like antidepressant agents. Ltfe Scl 10, 725 - 730. BENEDICT,C. R., FILLENZ,M. & STANFORD,C. (1979) Noradrenahne release m rats durmg prolonged cold-stress and repeated swim stress Br. J. Pharmacol. 66, 521 -524. BOVD, S. C., CAUL, W. F. & BOWEN,B. K. (1977) Use of cold-restraint to examine psychological factors m gastric ulceration. Phystol Behav. Ig, 865 - 870. BRODIE, D. A. & Hook, K. (1976) The effect of vasoactmveagents on gastric secretion and stress-reduced gastric hemorrhage m the rat. Fed. Proc. 29. 722 Brooks, F P (1977) Nervous control of gastrointestinal function neurophys~olog~calconsideration In Progress tn Gastroenterology, Glass, G. B. J. (Ed.), pp 373- 394 Grune and Stratton, New York. CARRAWAY,R. E., DEMERS,L. M & LEEMAn,S. E. (1976) Hyperglycemtceffect of neurotensm, a hypothalamlc peptlde. Endocrinology 99, 1452- 1462. CARRAWAY,R. ,I~ LEEMAN,S. E. (1973) The Isolation ol a new hypotenswe peptlde, neurotensm, from bovine hypothalamus. J. Biol Chem. 248, 6854-6861. CARRAWAY,R. t~¢ LEEMAN, S E (1976) Characterization ol rad~o~mmunoassayableneurotensin m the rat: ~ts differential distribution m the central nervous system, small intestine and stomach J Biol Chem. 251. 7045- 7052. CLOPPEr, C J & PEARSOn,E. S (1934) The use of conhdence ot hduoal hm~ts dlustrated m the case of the binomial. Btometrtka 26, 404 - 413 EsPI UGUES,J., Leorls, J. M., EZEQUEL, M. et al. (1982) Effects 13-adrenoceptor drug stlmulauon on various models of gastric ulcer in rats. Br. J. Pharmacol. 76, 587 - 594 FACEt, M., BROWn,M R & PEArSE,A. G. E. (1977) Specific Iocahzallon ol neurotensm to lhe N cell m human intestine by radlolmmunoassay and tmmunocytochemlstry Nature, Lond. 270, 183- 184 FIELDEN, R. & GREEN, A. L. (1967) A comparative study of the noradrenahne-depletmg and sympathetic blocking actions of guanelhldine and (-)-13-hydroxyphenethyl-guamdine. Br J Pharmacol. Chemother. 30, 155- 165 GLAVJN, G. B. 0980) Restraint ulcer: history, current research and future ~mphcatlons. Brain Res. Bull (5 Suppl.) I, 51 -58. Got DSTEIN,A. (1964) Btostatlsttcs: An Introductory Text, pp. 51 -55. MacMillan, New York.

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