Acid Secretagogue Action of Structurally Gamma-Aminobutyric Acid (GABA)-Related Compounds in Rats

Katsuya YAMASAKI and Yoshiaki GOTO* Department of Pharmacology, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan

Accepted October 13, 1989

Abstract-The properties of GABA related compounds on gastric function were studied in standardized perfused rat stomach preparations. Intravenous GABA (400 mg/kg) produced a rapid increase in acid secretion. Acid secretagogue actions of 3-aminobutyric acid and 2-aminobutyric acid were less potent than that of GABA. Intravenous injection of 5-amino-n-valeric acid (400 mg/kg) stimulated gastric acid secretion, but 6-amino-n-caproic acid was inactive. Isoguvacine (40 mg/kg, s.c.) stimulated acid output, whereas guvacine, an isomer of isoguvacine, did not. Systemically administered 3-hydroxy-GABA and 8-(p-chlorophenyl) GABA (PCPGABA) showed significant secretagogue actions. Acid responses to GABA-related compounds were significantly reduced by surgical truncal vagotomy and completely antagonized by atropine. The acid responses to PCPGABA and isoguvacine were partially augmented by yohimbine and propranolol. These results suggest that the secretagogue action of GABA is mimicked by structurally GABA-related compounds, which is mediated through cholinergic receptors, with slight implication of alpha-2 and beta-adrenoceptor mechanisms.

It is well-established that the central (13, 14). GABAergic mechanisms to stimu nervous system participates in the regulation late acid secretion are, however, complicated of gastric acid secretion (1). The relationships because both types of GABAA and GABAB between neurotransmitters and gastric acid receptor agonists stimulate gastric acid secre secretion were widely studied with regard to tion responses. There is no evidence to show aspects such as inhibitory effects of norepi that GABA mechanisms in the regulation of nephrine (2), dopamine (3), calcitonin-gene gastric secretion is selectively activated by related peptide (CGRP) (4), nicotine (5), GABA or a GABA-moiety. This led us to ex bombesin (6), neurotensin (7) and somato plore the effect of structurally GABA-related statin (8) and secretagogue effects of nicotine compounds on gastric secretion. (9) and thyrotropin-releasing hormone (TRH) In the present study, we examined the ef (10). Gamma -amino-n-butyric acid (GABA) fect of various types of GABA-related com is well-established as an inhibitory transmitter pounds on gastric acid secretion by the route in the CNS. We previously reported that of intravenous administration in most cases GABA produced a dose-dependent increase because of its quick or rapid onset to cause in gastric acid secretion (11 ); and also a certain changes. We also compared the effect lipophilic derivative of GABA, 3-(p-chloro of GABA with those of other amino acids that phenyl)-GABA (PCPGABA), stimulated gas have chemical structures similar to GABA, tric acid secretion more potently at a small with special reference to structure-activity dose (2-8 mg/kg, s.c.) (12). The acid secre relationships, and also attempted to define the tagogue effects of GABA and GAB,^-A-mimetics autonomic neuronal mechanisms involved. have been also confirmed in dogs and humans Materials and Methods

* To whoa, correspondence should be addressed . Measurement for gastric acid secretion: Male Wistar rats weighing 180-240 g were The cervical ligation of the esophagus was fasted in meta! cages with mesh bottoms for followed by resections of the cervical truncal 18-24 hr prior to the experiment but were vagi. Vagotomy was performed 30 min prior allowed free access to water ad libitum. Rats toa dministration of GABA or GABA-related were anesthetized with urethane (1.25 g/kg, compounds. i.p.). A cannula was inserted into the trachea Drugs: The drugs used were as follows: 4 followed by the ligation of the esophagus to amino-n-butyric acid (GABA), DL-2-amino avoid the reflex of the perfusate. A laparotomy n-butyric acid (2A-BA), DL-3-amino-n was performed, and the pylorus was ligated, butyric acid (3A-BA), 5-amino-n-valeric acid care being taken not to interfere with the (5A-VA), 6-amino-n-caproic acid (6A-CA), vascular supply and neural connections to the DL-4-amino -3-hydroxy-n-butyric acid stomach. A small incision on the forestomach (GABOB), atropine sulfate, yohimbine hy was made, and the gastrib lumen was gently drochloride, and DL-propranolol hydro rinsed with saline to remove the solid gastric chloride, which were all purchased from contents. A dual polyethylene cannula was Nacalai Tesque Ltd., Japan. Isoguvacine and introduced into the gastric lumen through the guvacine were from Funacoshi Pharm. Co., forestomach. The stomach was perfused with Japan; and DL-4-amino-3-(p-chloro saline, pH adjusted to 5.5 by HCI, using a phenyl)-butyric acid (PCPGABA) was a peristaltic pump at a flow rate of 5 ml/min. generous gift from Ciba-Geigy, U.S.A. The Acid output was determined by titrating the chemical structures of these GABA-related perfusate with 0.02 N NaOH to pH 5.5 using compounds are shown in Fig. 1. The com an automatic titrator (Toa Electronic Co. Ltd, pounds dissolved in saline or distilled water Japan. TSB-10A) and continuously recorded were administered through the femoral vein or in terms of every 2 min response via a zero administered subcutaneously. Pretreatment suppression adaptor (Toa Electronic Co. Ltd, with atropine, yohimbine or propranolol was Japan, C96667T). Acid output was expressed performed 20 min before each secretagogue in terms of /i equivalents of hydrogen ions was administered. (freq.). After stabilization of basal acid secre Statistical analysis: Results are expressed tion for usually 30 min after the initiation of as the mean±S.E. Basal acid output was very the gastric lumen perfusion, each compound low and usually less than 2.0 aeq./10 min. was intravenously or subcutaneously given at For evaluation of drug effects, the net in a volume of 1 or 2 ml/kg. crease in gastric acid output was calculated

Fig. 1. Chemical structures of GABA-related compounds used in this study. by subtracting the basal response from the little or no secretagogue actions. Intravenous acid response to each compound. Statistical injection of 5A-VA (400 mg/kg) and GABOB significances were determined by analysis of (200 mg/kg) markedly stimulated gastric variance followed by Dunnett's test for multi secretion, reaching maximal responses of group comparison of parametric data. 12.1±2.0 ueq./10 min (70 min after, N=6) and 7.2±1.5 ueq./10 min (60 min after, N=6), Results respectively. Isoguvacine also showed a Effect of GABA-related compounds on potent secretagogue effect at the dose of 40 gastric acid secretion: Typical responses of mg/kg, s.c. (maximal response: 15.7±3.2 GABA-related compounds on basal acid ueq./10 min at 90 min after the medication, secretion are shown in Fig. 2. GABA (400 N=6), while guvacine had no influence on mg/kg) given intravenously significantly in gastric acid secretion. Dose-response rela creased the gastric acid production. The tions of 5A-VA, GABOB and isoguvacine secretagogure effect was observed with 15 were also confirmed at the dose ranges of min latency and maintained for more than 120 100-400 mg/kg, i.v.; 50-200 mg/kg, i.v.; and min. The maximal response to GABA was 10-40 mg/kg, s.c., respectively. PC PGABA observed 60 min after the administration and was more than 100-fold potent than GABA was 8.4+1.2 ueq./10 min (N=7). The acid on a molecular basis. PCPGABA (4 mg/kg, secretagogue action of GABA was dose s.c.) exhibited secretagogue action and its dependent (100-400 mg/kg, i.v.). The effect peak response was obtained within 90 min of GABA in anesthetized rats was mimicked (25.6±4.3 ueq./10 min (N=10)). Beth by 5A-VA, GABOB, isoguvacine and PCP anechol (1 mg/kg) and histamine (10 mg/kg) GABA. The results obtained with structurally given subcutaneously caused potent acid related compounds are summarized in Fig. 3. increments, and the peak responses were 3A-BA, 2A-BA and 6A-CA, however, had 16.6±1.8 (30 min, N=10) and 17.7±2.9 (70

Fig. 2. Typical secretory responses to GABA, 5-aminovalleric acid (5A-VA), 4-amino-3-hydroxybutyric acid (GABOB) and isoguvacine in stornach perfused rats. Fig. 3. Secretagogue action of GABA-related compounds in rats . Each column represents the ne increase in gastric acid output for 60 min. GABA (400 mg/kg, i.v.), 3A-BA (400 mg/kg , i.v.), 2A-BA (400 mg/kg, i.v.), 5A-VA (400 mg/kg, i.v.), 6A-CA (400 mg/kg, i.v.), GABOB (200 mg/kg, i.v.), iso guvacine (40 mg/kg, s.c.), guvacine (40 mg/kg, s.c.), PCPGABA (4 mg/kg, s.c.), bethanechol (1 mg, kg, s.c.) and histamine (10 mg/kg , s.c.).

Table 1. Influence of atropine and vagotomy on gastric acid response to GABA-related compounds in rats

Atropine was subcutaneously pretreated 20 min before administration of GABA-related compounds . Each value represents the mean+S .E. of the net increase of acid output (,Ueq./60 min). 5A-VA: 5 aminovaleric acid, PCPGABA: fi-(p-chlorophenyl)-GABA . A significant difference was obtained from the secretagogue alone at the level of *: P 0.05 by Dunnett's test. The number of rats is indicated in parentheses. min, N=10) /eeq./10 min, respectively. compared to vagi-intact rats. When GABA Influence of cholinergic blockades on (400 mg/kg, i.v.), 5A-VA (400 mg/kg, i.v.), gastric acid response to GABA-related com isoguvacine (40 mg/kg, s.c.) and PCPGABA pounds: The results of the effectiveness of (4 mg/kg, s.c.) were given to vagotomized surgical and chemical vagotomy on the acid rats, the secretagogue actions of these com secretion in response to GABA-related com pounds were significantly attenuated, while pounds are shown in Table 1. Bilateral truncal bethanechol and histamine given subcutane vagotomy reduced the basal acid secretion ously still exhibited marked effects to increase Table 2. Influence of adrenergic blockers on gastric acid response to GABA-related compounds in rats

Propranolol and yohimbine were subcutaneously pretreated 20 min before the administration of GABA related compounds. Each value represents the mean±S.E. of the net increase of acid output (ueq./60 min). 5A-VA: 5-aminovaleric acid, PCPGABA: jS-(p-chlorophenyl)-GABA. A significant difference was obtained from the secretagogue alone at the level of *: P<0.05 by Dunnett's test. The number of rats is indicated in parentheses. acid output. Atropine (1 mg/kg) given sub dose-dependent increase in gastric acid secre cutaneously had little influence on basal tion (15). In contrast, Bhargava et al. (16) gastric acid secretion, but completely an observed that intracerebroventricular adminis tagonized the secretagogue action of GABA tration of GABA reduced gastric secretion in related compounds and bethanechol, and it Shay rats. This discrepancy may be at least in had little influence on histamine stimulation. part due to differences in experimental con Influence of adrenergic blockers on gastric ditions. A recent finding indicated that several acid response to GABA-related compounds: neurotransmitters administered centrally The effects of yohimbine (5 mg/kg, s.c.) and stimulated the gastric acid secretion at very propranolol (5 mg/kg, s.c.) on the acid re small doses (1 ). In this study, a large amount sponse to GABA-related compounds are of these compounds was required to produce shown in Table 2. The acid responses to the secretagogue actions by intravenous ad PCPGABA and isoguvacine were significantly ministrations. Little penetration a cross the potentiated in both yohimbine or propranolol blood-brain barrier occurs since these com treated animals. The responses to the other pounds are amino acids. Indeed, PCPGABA, GABA-re!ated compounds, GABA and 5A a lipoahilic derivative of GABA, can cross the VA, were slightly augmented in yohimbine blood-brain barrier (17) and vigorously and propranolol-pretreated groups, and the stimulated gastric acid secretion when it was responses were not significantly different given in small doses by the subcutaneous from the control values. route. The acid responses to hethanechol and In this experiment, only small numbers of histamine have, however, not been modified GABA-related compounds were available for by both adrenergic receptor blockers. studying the quantitative structure-activity relationships. The acid stimulatory effect of Discussion 3A-BA, in which the amino group is situated The present study shows that peripherally at the 3-position, was markedly less potent administered GABA and GABA-related com than that of GABA. 2A-BA, in which the pounds potently increased gastric acid secre amino group is at 2-position, and 6A-CA, tion in anesthetized rat perfused stomach prep which has a 2 carbon longer chain than GABA, aration. The maximal activity seemed nearly had little or no secretagogue effect. However, equal to those of bethanechol and histamine, 5A-VA whose chemical structure contains 5 and the durations lasted for more than 120 carbon atoms exerted an approximately equiv min. This is coincident to the findings that ad alent effect to GABA. These results and effects ministration of the GABA-related compound of GABA taken together suggest that the muscimo!, a GABAA-agonist, resulted in a terminal amino group and 3-4 carbon atoms between the amino and carbonic acid moieties the yohimbine-treated animals. The adrener are necessary for the exhibition of secre gic blocking agents used in this study tended tagogue actions. Isoguvacine had a potent to modulate the secretagogue action of GABA secretagogue effect, while guvacine, an and 5A-VA, although these effects were not isomer to isoguvacine, had no influence on significant. This postulates that sympathetic gastric acid secretion. Krogsgaard-Larsen et inhibitory mechanisms may be mediated by al. (18) have been reported that the con the adrenergic receptors in gastric acid formational!y restricted analogue of GABA, responses to these compounds. There is no isoguvacine, is a potent agonist at GABA evidence to explain the potentiation by receptors, and its activity was comparable alpha2 or beta-blockers of the acid response with that of another analogue, muscimol. to GABA-related compounds. It may be These compounds are potent as displacement assumed that this synergism is related to the ligands in receptor binding studies with 3H inhibition of acid inhibitory mechanisms. GABA. There is, however, no evidence that GABA itself and 5A-VA did not stimulate acid guvacine acts on GABA-receptors, except for output as potently as PCP-GABA and the inhibitory effect on GABA uptake (19). isoguvacine, suggesting the latter compounds These relative potencies may reflect different are more active in GABA-mechanisms that molecular mechanisms of receptor interac seem to be affected by adrenergic inhibition. tions of GABA and suggest that steric factors It has been reported that GABA receptor are important for studying structure-activity agonists, including muscimol and baclofen, relationships of GABA. stimulate gastric secretion in the anesthetized Although the mechanisms for how GABA rat (1, 11-15). The amino acids used in this stimulates acid secretion have not yet been study such as GABOB, one of the GABA completely understood, it seems that a central metabolites (27, 28); isoguvacine, a GABAA cholinergic pathway is involved in the secre agonist (29); PCPGABA, a GABAB agonist tagogue mechanisms, since these responses (30); and 5A-VA, a GABAB partial agonist were antagonized by pretreatment with atro (31), also stimulated gastric secretion with a pine and by truncal vagotomy. It is, however, potency similar to or greater than that of still unclear whether this effect is accom GABA. According to our previous finding, plished by activating GABA receptors directly both GABAA and GABAB-antagonists, how or by other mechanisms. In addition, the ever, failed to suppress the acid response to adrenergic pathways have been postulated to baclofen (K. Yamasaki et al., unpublished play a role in the inhibitory mechanisms data). Most studies on the subtypes of GABA involved in the regulation of gastric acid receptors have been based on in vitro re secretion (20, 21). Some researchers have ceptor binding assays. In the gastric acid obtained data showing the acid inhibitory secretion response in vivo, either an agonist effects of the a!pha2-receptor agonist clon of GABAA-receptors or an agonist of GABAB idine (22-24) or showing a contradictory receptors increased acid secretion through effect to stimulate acid secretion in the dog central cholinergic activation mechanisms (25). A beta2-stimulant also inhibited the (15). We have recently found that phaclofen, gastric acid response to histamine (26). In the a GABAB-receptor antagonist, had no signifi present study, the adrenergic neural activity cant effect on baclofen-induced acid re was attenuated by pretreatment with ad sponse (32). Furthermore, a specific GA.BAA renoceptor antagonists, yohimbine and pro receptor antagonist, bicuculline, had no pranolol. The acid responses to PCPGABA influence on the acid response to muscimol, and isoguvacine were significantly augmented whereas bicuculline antagonized the secre in the yohimbine-treated animals. Further tagogue action of GABA in isolated guinea more, significant potentiations of acid re pig stomach preparation (33). This suggests sponses to GABA-related compounds were that the acid stimulatory effect of GABA also observed in propranolol-treated animals mimetics might be mediated through non as compared with the saline control, but the GABAA and non-GABAB-receptors. The magnitude was smaller than that observed in question that GABAergic mechanisms could be activated by GABA or structurally restricted ventricularly applied neurotensin in rats. Life Sci. 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