Disparate effects of intracisternal RX 77368 and ODT8-SS on gastric acid and serotonin release: role of adrenal catecholamines

Regulatory Peptides, 36 (1991) 21-28

21

© 1991 Elsevier Science Publishers B.V. All rights reserved 0167-0115/91/$03.50

REGPEP 01085

Disparate effects of intracisternal RX 77368 and ODT8-SS on gastric acid and serotonin release" role of adrenal catecholamines Robert L. Stephens, Jr. The Ohio State University,Department of Physiology, Columbus, OH (U.S.A.)

(Received 4 April 1991;revisedversionreceivedand accepted 17 June 1991) K e y words: Stomach; Thyrotropin releasing hormone; Somatostatin; Peptide;

Cisterna magna; Vagus

Summary Intracisternal injection of the thyrotropin releasing hormone (TRH) analogue RX 77368 (100 ng) or the somatostatin analogue ODT8-SS (1 #g) produced an 82~o and 101 ~o increase in gastric acid secretion in 2 h pylorus-ligated rats. In contrast, dissimilar effects were produced by intracisternal injection of these peptides on the secretion of serotonin into the gastric lumen. Intracisternal RX 77368 (100 ng) produced a 496~o increase in intraluminal serotonin release, while in contrast, intracisternal ODT8-SS (1/~g) produced a 78~o inhibition in intraluminal serotonin release. Bilateral adrenalectomy reversed the stimulatory effect of intracisternal RX 77368 (100 ng) on serotonin, but not acid release. The data reveal a difference in the ability of the two peptides, which act as gastric secretagogues, to produce intraluminal acid and serotonin release, and suggest that combined activation of the vagus and the adrenal gland are important in mediating basal and RX 77368-stimulated serotonin release into the gastric lumen. In particular, differential effects on adrenal catecholamine release are implicated in the divergent effects of the two peptides.

Correspondence: R.L. Stephens,Jr., The Ohio State University,Departmentof Physiology,4196 Graves Hall, Columbus,OH 43210, U.S.A.

22 Introduction

It is well established that centrally administered neuropeptides have marked effects on gastric acid and biogenic amine secretion and gastrointestinal motor activity [ 1-4]. Thyrotropin releasing hormone (TRH) has potent centrally mediated actions on gastrointestinal function. The effects produced by TRH on gastric acid secretion and motility are mediated by enhanced efferent vagal activation [5,6]. Recent studies have described the effect of injection of TRH and analogues into the cerebral ventricles to augment the release of the biogenic amine serotonin into the gastric lumen and portal blood [2,4]. Serotonin has pronounced effects on the stomach, which includes an inhibition of stimulated gastric acid secretion, altered gastric motility, and a depletion of gastric mucous [7-9]. 90~o of mammalian serotonin is present in the gastrointestinal tract, where it exists predominantly in endocrine cells of the gastrointestinal mucosa [ 10]. These studies suggest the existence of central mechanisms effective to stimulate gastrointestinal endocrine cell secretion. The TRH stimulatory effect on gastric luminal serotonin release has been characterized as vagal-dependent. Recently, it was shown that the cholinergic agonist bethanechol was ineffective in stimulating gastric luminal serotonin release at doses effective to stimulate gastric acid secretion [4]. This work suggests that (an)other event(s) in addition to the stimulation of muscarinic receptors distal to the nerve terminals of the vagus may be important in mediating centrally-evoked gastric serotonin release. The stimulation of adrenoreceptors has also been linked to vagaUy-stimulated serotonin release from the gastrointestinal tract [ 11 ]. Since intracerebroventricular TRH has been characterized as a stimulant of both vagal efferent outflow and sympathetic outflow to the adrenal gland [ 12], it is proposed that the catecholaminergic component of the TRH response may contribute to its effect on serotonin release. The octapeptide analogue of somatostatin Des-AA1'2'4'5"~2,13_[ D.TrpS ] somatostatin (ODT8-SS) also produces vagal-dependent stimulation in gastric acid output after intracerebroventricular injection [13 ]. However, in contrast to TRH, the somatostatin analogue produces a suppression of peripheral adrenergic neuronal and endocrine systems activated by stimuli of the central nervous system [ 14]. As a probe to assess the role of peripheral catecholamines in the centrally-mediated serotonin secretory response, the effects of intracisternal injection of the somatostatin analogue ODT8-S S and the TRH analogue RX 77368 on gastric luminal secretion of acid and serotonin will be compared.

Materials and Methods Animals. Male Sprague-Dawley rats (200-240 g, Harlan Industries, Indianapolis, IN) were maintained ad libitum on Purina Laboratory Chow and tap water. They were housed under controlled conditions of temperature (22 + 1 ° C) and illumination (6 a.m. to 6 p.m.). All experiments were performed in animals deprived of food but not water 18-24 h before the beginning of the experiment.

23

Measurement of gastric acid secretion. Gastric acid secretion was measured in conscious 2 h pylorus ligated rats. The animals were injected intracisternally with 10 #1 of vehicle or the peptides of interest under light diethyl ether anesthesia utilizing a stereotaxic instrument. Laparotomy and pylorus ligation were performed immediately after intracisternal injection. The animals regained the righting reflex 3-5 min after these procedures and were returned to the home cage. Conscious animals were decapitated 2 h later and the stomachs were removed, the gastric contents collected and centrifuged. A small aliquot of the gastric juice was separated for the measurement of serotonin content (0.1-0.5 ml). The volume and pH of the remaining gastric juice were measured, the quantity of acid determined by titration with 0.1 M NaOH to pH 7 utilizing an automatic titrator (Radiometer, Copenhagen, Denmark). The final quantity of acid was determined after correction for the removed volume. In some animals, bilateral adrenalectomy was performed 72 h before the experiment through dorsal incisions in rats under light diethyl ether anesthesia. Measurement of serotonin secretion. The centrifuged aliquots of gastric secretion from pylorus-ligated or gastric fistula rats were centrifuged at 15,000 g at 4 ° C for 20 min and a volume of 5-25 #1 was injected into a high-performance liquid chromatograph. The liquid chromatograph (Shimadzu Corp., Kyoto Japan) was equipped with a L-ECD-6A electrochemical detector, a LC-6A solvent delivery system, a SIL-6A autoinjector system and a C-R5A integrator. The system was connected to an ODS, 5/~m C-18 Bondipak column. The details of the mobile phase composition and operating conditions have been described previously [4]. The operating potential of the HPLC system was + 700 inV. Standard curves were made by injecting 0.5-2.0 ng of serotonin dissolved in the gastric sample. Peptides. The stable TRH analogue, RX 77368, (Reckitt and Colman, Kingston upon Hull, U.K.) obtained in powder form, was dissolved in 0.5~o bovine serum albumin and 0.9~/o saline at an initial concentration of 20/~g/ml and kept at - 2 0 °C. The somatostatin analogue ODT8-SS (Catherine Rivier, Peptide Biology Laboratory, San Diego, CA) was obtained in powder form, dissolved in 0.5~o BSA and 0.9~o normal saline in a 1 mg/ml concentration and frozen in 20/~1 aliquots at - 70 °C until use in this study. Statistics. The data were analyzed utilizing analysis of variance with post hoc Newman-Keuls test. Differences between groups was considered significant ifP < 0.05.

Results

Effect of intracisternal peptides on gastric acid and serotonin release in 2-h pylorus-ligated rats In conscious, 2 h pylorus-ligated rats, intracisternal doses of the TRH and somatostatin analogue were chosen to give similar magnitudes of elevated gastric acid secretion. Thus, intracisternal injection of RX 77368 (100 ng) or ODT8-SS (1/~g) produced a 82~o and 101 ~o increase, respectively, in gastric acid secretion as compared to vehicle treated controls (Table I). The enhanced gastric response was expressed as an augmented secretory volume; RX 77368 and ODT8-SS stimulated gastric fluid secretion 73 ~o and

24 TABLE I Effect of intracisternal injection of RX 77368 (100 ng) or ODT8-S S (1 #g) on gastric acid volume, output and concentration in 2 h pylorus-ligated rats Treatment

n

Volume (ml/2 h)

Acid output (#Eq/2 h)

Acid concentration (#Eq/ml)

Vehicle RX 77368 ODT8-SS

10 16 13

3.0 + 0.6 5.2 + 0.6* 5.6 + 0.5*

220 + 44 401 + 45* 442 + 51"

74 +_ 6 78 + 5 78 + 4

Values are means + SEM; n = number of diethyl ether anesthesia with artificial CSF was then ligated and the rats returned to secretion collected and titrated. * P < 0.05

rats. Rats fasted for 24 h were injected intracisternally under (vehicle), RX 77368 (100 ng) or ODT8-SS (1 #g). The pylorus their home cage. 2 h later, rats were decapitated and gastric as compared to the vehicle-treated group.

87%, respectively, over the control group. The acid concentration, however, was not altered by intracisternal injection of either peptide. In contrast to the similar effect of the two peptides on acid secretion, there was a disparity in the effect of the peptides on the secretion of serotonin into the gastric lumen. Intracisternal injection of RX 77368 (100 ng) produced a 5-fold enhancement in gastric luminal serotonin response in this study, whereas intracisternal ODT8-SS (1 #g) produced a 78% inhibition in gastric luminal serotonin release as compared to the control group (Fig. 1).

E Z 8OO

~ Z ~ W K 0 W W ~ I

800 400 3OO 200 N

100

=

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E W


0

VEHICLE

RX 77366 (lO0ng)

ODT8-SS (1 Jug)

Fig. 1. Effect of intracisternal injection of RX 77368 (100 ng) or ODT8-SS (1 #g) on gastric intraluminal serotonin release in 2 h pylorus-ligated rats. Rats under light diethyl ether anesthesia were injected intracisternaUy with vehicle (artificial CSF), RX 77368 or ODT8-SS. The pylorus was then ligated and rats returned to home cage. 2 h later, the rats were killed, the gastric contents centrifuged, and 25 #1 injected directly into the HPLC. * P < 0.05 as compared to the vehicle-treated groups.

25 Effect of bilateral adrenalectomy on RX 77368 and basal acid and serotonin release in 2-h pylorus-ligated rats Bilateral adrenalectomy or sham surgery was performed 72 h before the experiment. In this study, sham + intracisternal RX 77368 (100 ng) produced a 432%, 252% and 354% elevation in gastric acid, fluid and serotonin secretion, respectively, as compared to sham + vehicle-treated animals. In comparing RX 77368-treated groups, adrenalectomy produced an 84% reduction in luminal serotonin release, while the gastric acid or volume response was not altered. In addition, in comparing the intracisternal vehicle treated groups, adrenalectomy produced a 88~/o reduction in gastric luminal serotonin release without changing the acid response.

Discussion

The major findings of this study were the disparity of the effects of intracisternal RX 77368 and ODT8-SS on acid and serotonin secretion into the gastric lumen, and the selective effect of adrenalectomy on serotonin but not acid secretory response. The data suggest a dissociation between central mechanisms effective to stimulate gastric acid and intraluminal serotonin secretion. Both RX 77368 and ODT8-SS injection into the rat CSF stimulate gastric acid secretion via a vagal-dependent pathway [5,13]. However, examination of other effects of central injection of the tested peptides on the activity of the autonomic nervous system reveal important differences. TRH produces an enhancement in sympathetic outflow to the adrenal gland, and an elevation of plasma catecholamine levels [15,16]. ODT8-SS, however, produces an inhibition in adrenal epinephrine secretion by a centrally mediated mechanism [ 14]. Thus, the disparity in the effects of RX 77368 and ODT8-SS on intraluminal serotonin release might be explained by their dissimilar effects on adrenal catecholamine release. The mediatory role of plasma catecholamines is supported by the finding that RX 77368 failed to stimulate in adrenalectomized animals. While a role for mineralocorticoid and/or glucocorticoid depletion cannot be ruled out, this finding taken together with the opposite effects of intracisternal RX 77368 and ODT8-SS on serotonin release supports the notion that stimulated adrenergic and vagal systems work in concert to augment gastric luminal serotonin release. Adrenergic mechanisms have been shown to contribute to the intraluminal release of gastrointestinal serotonin. Serosal application of the fl-agonist isoproterenol produces significant mucosal serotonin release into the duodenal lumen in vitro [ 11 ]. Splanchnic nerve stimulation produces a significant depletion in the serotonin content of enterochromaffin cells of the small intestine [ 17]. However, it appears that adrenerglc mechanisms alone are insufficient to stimulate serotonin release because the effect of intracisternal RX 77368 is reversed by vagotomy or atropine pretreatment [4]. The pylorus-ligation rat model produces a stomach under an enhanced vagal tone, resulting in enhanced basal acid and serotonin levels in this model [ 18]. The inhibitory effect of adrenalectomy on intraluminal serotonin release in vehicle treated animals supports the role of combined vagal cholinergic and adrenal adrenergic influence mediating this response.

26 Previous work has shown that activation of peripheral histamine, gastrin or muscarinic receptors alone are ineffective to stimulate gastric intraluminal serotonin release in the rat [4]. The effect ofintracisternal ODT8-S S to inhibit serotonin release provides further evidence in support of a role of adrenal catecholamines in mediating intraluminal serotonin release. Serotonin release in response to intracerebral TRH may modulate gastrointestinal function. Recent work suggests that released serotonin may produce an inhibitory influence on TRH analogue-stimulated gastric acid secretion and contractility [19]. However the present data showed no change in the gastric acid secretory response stimulated by intracisternal RX 77368 after a marked reduction in luminal serotonin caused by adrenalectomy (Fig. 2). It has been demonstrated that vagal stimulation can

acid output IO0

6OO

SO0

0

i=1 gastric volume

8

ni

6

Jl q~l

4

i

2 0

N

luminal serotonin lOOO

t

'l~ml

aoo

Jm

eoo 400 200

I

N

10 Preb'estment: Trestment:

,N,

,

I

5

11

5

8ham

Adx

8hsm

Adx

Veh

Veh

RX77368

RX77368

Fig. 2. Effect of bilateral adrenalectomy on acid output, gastric volume and luminal serotonin release in animals treated with intracisternal vehicle or RX 77368. Adrenalectomy was performed 72 h before the experiment. Rats under light diethyl ether anesthesia were injected intracisternally with vehicle (artificial CSF) or RX 77368. The pylorus was then ligated and the rats returned to the home cage. 2 h later, the rats were killed, the gastric contents centrifuged, and analyzed for acid secretion and serotonin release as described under Materials and Methods. * P < 0.05.

27 release serotonin into both the lumen and the portal blood [11]. It remains to be determined whether catecholamine depletion can attenuate basilar serotonin release, and whether this is the locus of its inhibitory effect on gastric secretory function. In addition, the role of released serotonin, which produced gastric lesions when given exogenously, to modulate intracerebral T R H - i n d u c e d gastric erosions needs to be investigated. The results of this study suggest a disparity in the effects o f the two centrally active gastric secretagogues to stimulate gastric acid and biogenic amine secretion into the gastric lumen. An important role for adrenal catecholamines to mediate centrally stimulated intraluminal serotonin release but not gastric acid secretion is proposed. Thus, there appear to be differences in the central control mechanisms of gastric endocrine and parietal cell secretion.

Acknowledgements The work was funded by N I H DK42880-01. The technical assistance of Ms. Kathy Lepard and Mr. Ronald Shockley is gratefully acknowledged. The author also thanks Dr. Catherine Rivier for the supply of O D T 8 - S S and Dr. J. G. Lloyd-Jones (Rickitt & Colman, Kingston upon Hull, U.K.) for the supply o f RX 77368.

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28 13 Tache, Y., Rivier, J., Vale, W. and Brown, M., Is somatostatin or a somatostatin-like peptide involved in central nervous system control of gastric secretion, Regul. Pept., 1 (1981) 307-315. 14 Fisher, D.A. and Brown, M.R., Somatostatin analog: plasma catecholamine suppression mediated by the central nervous system, Endocrinology, 107 (1980) 714-718. 15 Somiya, H. and Tonoue, T., Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves, Regul. Pept., 9 (1984) 47-52. 16 Brown, M. R., Thyrotropin releasing factor: a putative CNS regulator of the autonomic nervous system, Life Sci., 28 (1981) 1789-1795. 17 Larsson, I., Dahlstrom, A., Pettersson, G., Larsson, P.A., Kewenter, J. and Ahlman, H., The effects of adrenergie antagonists on the serotonin levels of feline enterochromatfin cells after splanchnic nerve stimulation, J. Neural Transm., 47 (1980) 89-98. 18 Brodie, D.A.,Themechanism ofgastric hyperacidity produced by pylorus ligationin the rat, Am. J. Dig. Dis., 11 (1966) 231-241. 19 Stephens, R.L., Garrick, T., Weiner H. and Tache, Y., Serotonin depletion potentiates gastric secretory and motor responses to vagal but not peripheral gastric stimulants, J. Pharmacol. Exp. Ther., 251 (1989) 524-530.