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Stimulatory effects of centrally injected kainate and N-methyl-d -aspartate on gastric acid secretion in anesthetized rats
Brain Research 914 (2001) 115–122 www.elsevier.com / locate / bres
Research report
Stimulatory effects of centrally injected kainate and N-methyl-Daspartate on gastric acid secretion in anesthetized rats a, a b a Shizuko Tsuchiya *, Syunji Horie , Shingo Yano , Kazuo Watanabe b
a Laboratory of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263 -8522, Japan Laboratory of Molecular Pharmacology and Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263 -8522, Japan
Accepted 3 July 2001
Abstract The effects of N-methyl-D-aspartate (NMDA), kainate and (6)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), ionotropic glutamate agonists, on gastric acid secretion were investigated in the continuously perfused stomach of anesthetized rats. The lateral ventricular (LV) injection of kainate (0.01–1 mg) or NMDA (0.3–3 mg) dose-dependently stimulated gastric acid secretion. AMPA (3–10 mg) also stimulated gastric acid secretion but the effect was very weak. Repeated injections of kainate (0.1 mg) or NMDA (1 mg), at least twice, stimulated gastric acid secretion to a similar degree. The effect of kainate (0.1 mg) was blocked by the kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (3 mg, LV) and D-g-glutamylaminomethanesulfonic acid (30 mg, LV), but not by NMDA receptor antagonists. The effect of NMDA (10 mg) was blocked by (6)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid (10 mg, LV), a competitive NMDA receptor antagonist, and (1)-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine hydrogen maleate (10 mg, LV), a non-competitive NMDA receptor antagonist, but not by kainate receptor antagonists. Moreover, the gastric acid secretion stimulated by kainate and NMDA were completely blocked by systemic atropine injection (1 mg / kg, i.v.) and vagotomy. These findings suggest that kainate and NMDA receptor mechanisms are independently involved in the central nervous system to control gastric acid secretion through vagus cholinergic activation. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Excitatory amino acids: pharmacology Keywords: Excitatory amino acid; N-Methyl-D-aspartate; Kainate; Central nervous system; Stomach; Acid secretion
1. Introduction Accumulating evidence suggests that glutamate may be the primary excitatory neurotransmitter in the mammalian central nervous system, with the majority of neurons expressing functional glutamate receptors [6]. Based on the effects of selective excitatory amino acid (EAA) receptor agonists, subtypes of ionotropic glutamate receptors have been classified as N-methyl-D-aspartate (NMDA), kainate and (6)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors [3,17]. Molecular cloning and expression studies have revealed that NMDA receptor (NMDAR) subunits consist of the mandatory subunit 1
*Corresponding author. Tel.: 181-43-290-2923; fax: 181-43-2903021. E-mail address: [email protected] (S. Tsuchiya).
(NMDAR1) and variable subunits 2A–2D (NMDAR2A– 2D), kainate (KA) receptors consist of KA1, KA2 or glutamate receptor subunits 5–7 (GluR5–7), and AMPA receptors consist of glutamate receptor subunits 1–4 (GluR1–4) [5]. There is much evidence that food intake and gastric motility are regulated by ionotropic EAA receptors. For instance, in the lateral hypothalamus (LH), glutamate acts as a physiological regulator of eating and body weight control [22–25]. In the dorsal vagal complex (DVC), activation of kainate and NMDA receptors can independently cause vagally mediated gastric motor excitation [21]. Stimulation with glutamate of the nucleus raphe pallidus (Rpa) increases gastric contractility via the Rpa– DVC pathway [7]. Likewise in cats, excitation of the medullary raphe, Rpa and the nucleus raphe obscurus (Rob), by glutamate causes an increase in gastric motility by excitation of vagal neurons in the DVC [10]. The importance of the DVC and LH in the control of gastric
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function has been previously established by electrical and chemical stimulation of the region [26,33]. Gastric acid secretion has also been proposed to be regulated by ionotropic EAAs. For instance, stimulation of Rpa by kainate or glutamate, which releases thyrotropinreleasing hormone (TRH) and stimulates the DVC, induces vagal muscarinic-dependent stimulation of gastric acid secretion [7,11,27,34]. In cats, gastric acid secretion was increased by kainate into the Rob, via the DVC pathway [32]. In these studies, however, it was not clarified whether the effects of kainate or glutamate were receptor dependent, and what subtypes of the glutamate receptor participated in the gastric acid secretion. However, Namiki et al. [18] reported that the administration of L-glutamate into LH produced no significant effect on gastric acid secretion. Thus, the effects of EAAs in the central nervous system on gastric acid secretion have not been finally established. We previously reported that centrally injected kainate strongly stimulated gastric acid secretion, and NMDA or glutamate itself also weakly stimulated acid secretion in urethane anesthetized rats [31]. In the present study, the effects of glutamate receptor antagonists, injected into the lateral ventricle (LV), on gastric acid secretion induced by NMDA, kainate and AMPA injected LV were examined. We found that kainate and NMDA receptor mechanisms regulated gastric acid secretion independently through vagus cholinergic activation.
2. Materials and methods
2.1. Animals Male Wistar rats weighing 200–300 g (Takasugi Exp. Animals, Kasukabe, Japan) were housed under conditions of controlled temperature (24628C) and a 12-h light / dark cycle (lighting on at 07:00 h), for at least 1 week before the experiment. Food and tap water were available ad libitum. All experiments were performed in rats anesthetized with urethane. The animals were fasted for 18 h before the experiment, but free access to water was maintained. Animal experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, approved by The Japanese Pharmacological Society.
2.2. Compounds used Kainate, NMDA, AMPA hydrobromide, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX), D-gglutamylaminomethanesulfonic acid (GAMS), (6)-3-(2carboxypiperazin-4-yl)-1-propylphosphonic acid (CPP) and (1)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten5,10-imine hydrogen maleate (MK-801) were obtained from Research Biochemicals International (Natick, MA, USA). Atropine sulfate was obtained from Nacalai Tesque
(Japan). All compounds were dissolved in 0.9% saline. The volume for i.v. injection was 1 ml / kg, and that for LV injection was 5 ml.
2.3. Measurement of gastric acid secretion Gastric acid secretion was determined by the gastric perfusion method as described by Watanabe et al. [30]. Rats were anesthetized with urethane (1.35 g / kg, i.p; Tokyo Kasei, Japan), the esophagus was ligated at the cervical level, and a cannula was inserted into the trachea. After laparotomy, the pylorus was ligated and a dual cannula was inserted through a small incision into the forestomach. The lumen was continuously perfused with saline (adjusted to pH 5.0, 378C) through the inlet tube of the dual cannula at a rate of 1 ml / min. The intragastric pressure was maintained at 5 cm H 2 O. The perfusate was continuously titrated in the reservoir with 0.02 N NaOH to pH 5.0 using an automatic titrator (ABT-101, TOA Electronics, Japan) connected to a computer system.
2.4. Cannulation for lateral ventricular or intravenous injection Animals were placed in a stereotaxic instrument (SR-6, Narishige, Japan) in the mouth down position (23.3 mm). A stainless steel cannula was positioned unilaterally (right side) through a small hole in the skull made by a drill. The bregma region of the parietal skull was exposed. Stereotaxic coordinates were taken from the atlas of Paxinos and Watson [20], and were as follows: 21.0 mm anteroposterior from the bregma, 1.3 mm lateral from the bregma, 3.8 mm dorsoventral from horizontal skull surface. The cannula was secured by dental cement (GC Corporation, Japan). In some animals, the femoral vein was cannulated for injection of atropine or the bilateral vagus nerves were cut at the cervical level.
2.5. Experimental procedure The animal was left for 1 h to stabilize gastric acid secretion. After the determination of basal acid secretion for 30 min, NMDA, kainate and AMPA were injected LV. The amount of acid output was expressed in terms of mEq H 1 / 10 min. In the antagonist study, antagonist or vehicle was injected 10 min before agonist injection. In some animals, repeated injection of the agonist was applied. When the acid secretion of the first agonist injection returned to the basal level after about 120 min, the second injection of the agonist was applied.
2.6. Statistics Values are expressed as D mEq H 1 / 10 min of the basal values measured 10 min prior to injection of NMDA, kainate or AMPA. The values were expressed as the
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mean6S.E. Grouped data of gastric acid secretion were statistically analyzed by one-way ANOVA followed by the Bonferroni multiple comparisons test. In some cases in the antagonist study (Figs. 4 and 5), the first value for the vehicle was compared with the second value for the antagonist by the paired t-test. Probability values ,0.05 were considered significant.
3. Results
3.1. Effect of NMDA, kainate and AMPA on gastric acid secretion In urethane anesthetized rats, basal acid secretion was relatively low during the experimental period, and LV injection of the vehicle did not influence the basal acid secretion (D 2.9264.20 mEq H 1 / 120 min, n56). LV injection of kainate (0.01–1 mg (0.05–5 nmol)) and NMDA (0.3–3 mg (2.04–20.4 nmol)) evoked gastric acid secretion in a dose-dependent manner (Figs. 1 and 2). AMPA (3 and 10 mg (11.2 and 37.4 nmol)) also stimulated gastric acid secretion but the effect was very week (Figs. 1 and 2). The onset of the secretory response was within 10 min after the agonist injection, the response reached the peak value after 50–60 min for kainate, 20–30 min for NMDA and 20–30 min for AMPA. The response of kainate, NMDA or AMPA returned to the basal level after about 120 min, although the acid level after kainate (1 mg) at 120 min was higher than the basal level. The effect of kainate was stronger than that of NMDA and AMPA: the maximal effect obtained with 3 mg (20.4 nmol) NMDA
Fig. 2. Dose–response curves of LV injection of kainate, NMDA and AMPA on the acid output in urethane-anesthetized rats. All values represent the total acid output for 120 min and means6S.E. (n54–7).
was about one-third of that with 1 mg (5 nmol) kainate (Fig. 2). When kainate (0.3–1 mg), NMDA (0.3–3 mg) and AMPA (3 and 10 mg) were injected, hyperpnea was immediately observed. Trembling of the barba was observed after kainate (0.3–1 mg) injection. Rats began
Fig. 1. Time course of the acid output by LV injection of (a) kainate, (b) NMDA and (c) AMPA in urethane-anesthetized rats. All values represent the acid output for 10 min and means6S.E. (n54–7). Dose–response analysis is represented in Fig. 2.
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Fig. 3. Effects of repeated LV injection of kainate and NMDA on the acid output in urethane-anesthetized rats. The second injection was applied about 120 min after the first injection. All values represent the total acid output for 120 min and means6S.E. (n55).
salivating at 40 min and continued until 80–100 min after injection of AMPA (3 and 10 mg).
3.2. Effects of antagonists on the kainate or NMDAinduced response Fig. 3 shows the effects of repeated injections of kainate
(0.1 mg) or NMDA (1 mg). The response after the second injection of kainate was similar to that after the first injection. The effect of NMDA was also repeatable. In the antagonist study of kainate, vehicle was injected 10 min before the first kainate injection, and then the antagonist was injected 10 min before the second agonist injection. The response to the first agonist stimulation was used as a control for the following second antagonist response (Figs. 4 and 5). Injection of CNQX (3 mg, LV), an antagonist of AMPA / kainate receptor [9], blocked the kainate (0.1 mg)-stimulated gastric acid secretion (Fig. 4). The inhibitory effect of CNQX was significant from 60 min after the kainate injection, but not significant at 0–50 min. The reason may be because CNQX (3 mg) itself stimulated gastric acid secretion slightly (D 9.8864.90 mEq H 1 / 120 min, n55, P50.3059 compared with vehicle), and / or CNQX at 3 mg was not sufficient as an antagonist of the AMPA / kainate receptor. We did not use CNQX at doses above 3 mg because of its own stimulatory effect. CNQX (3 mg, LV) also slightly inhibited the effect of AMPA (3 mg, LV), but not significantly because of the stimulatory effect of CNQX and the weak effect of AMPA. GAMS (30 mg, LV), another type of antagonist of the AMPA / kainate receptor [4,28], also blocked kainate (0.1 mg)-stimulated gastric acid secretion (Fig. 5). Injection of GAMS alone had no effect on gastric acid secretion and the inhibition of kainate-stimulated gastric acid secretion was observed after 20 min. Injection of GAMS did not inhibit the NMDA (1 mg)-stimulated gastric acid secretion (data not shown). CPP (10 mg, LV) and MK-801 (10 mg, LV), antagonists for NMDA receptors, blocked the NMDA (10 mg, LV)stimulated gastric acid secretion almost completely (Fig. 6). Similar doses of CPP and MK-801 had no effect alone and did not influence the kainate effect (data not shown).
Fig. 4. Effects of CNQX on gastric acid secretion by kainate in urethane-anesthetized rats. CNQX (3 mg) was injected LV 10 min before LV injection of kainate (KA, 0.1 mg). (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n57). *P,0.05, **P,0.01 and ***P,0.001 compared with kainate alone (paired t-test).
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Fig. 5. Effects of GAMS on gastric acid secretion by kainate in urethane-anesthetized rats. GAMS (30 mg) was injected in LV 10 min before LV injection of kainate (KA, 0.1 mg). (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n56). **P,0.01 and ***P,0.001 compared with kainate alone (paired t-test).
3.3. Effect of atropine or vagotomy on kainate- and NMDA-induced gastric acid secretion Injection of atropine (1 mg / kg, i.v.) blocked the NMDA-stimulated gastric acid secretion (Fig. 7). Atropine also blocked the kainate-stimulated gastric acid secretion (kainate, D 227.4665.1 mEq H 1 / 120 min; kainate1 atropine, D 2.85615.43 mEq H 1 / 120 min, P,0.01). Vagotomy blocked the NMDA-stimulated gastric acid secretion (Fig. 7). Vagotomy also blocked the kainatestimulated gastric acid secretion (kainate, D 244.5662.6 mEq H 1 / 120 min; kainate1vagotomy, D 3.663.6 mEq
H 1 / 120 min, P,0.01). These findings suggest activation of kainate or NMDA receptors in the central nervous system stimulates gastric acid secretion through vagus cholinergic nerves.
4. Discussion
4.1. Stimulatory effect of kainate and NMDA on gastric acid secretion In the central nervous system, there are studies showing
Fig. 6. Effects of CPP (10 mg, LV) and MK801 (10 mg, LV) on gastric acid secretion by NMDA (10 mg, LV) in urethane anesthetized rats. (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n53–5). *P,0.05, **P,0.01 and ***P,0.001 compared with NMDA alone.
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Fig. 7. Effects of atropine (Atr., 1 mg / kg, i.v.) and vagotomy (Vago.) on gastric acid secretion by NMDA (10 mg, LV) in urethane anesthetized rats. (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n53–5). *P,0.05, **P,0.01 and ***P,0.001 compared with NMDA alone.
that glutamate receptors regulate gastric functions, such as eating, gastric motility or intragastric pressure. For example, LH injection of kainate, NMDA and AMPA elicited a dose-dependent eating response [22–25]. Stanley et al. [24] suggested that glutamate may act through several subtypes of its receptors on LH neurons to elicit eating, and showed that LH injection of the NMDA receptor antagonist ( D-AP5) suppressed eating elicited by LH injection of NMDA, but not kainate or AMPA. DVC injection of kainate and NMDA increased intragastric pressure and gastric motility, which were inhibited by selective antagonists for each receptor subtype [21]. Intracerebral injection of kainate or glutamate stimulated gastric acid secretion, as mentioned in Section 1 [7,11,32,34]. However, it has not been demonstrated whether kainate or glutamate stimulates its receptor, or what subtypes of receptors are concerned with gastric acid secretion. In the present study, it was confirmed that centrally injected kainate (0.01–1 mg; LV), NMDA (0.3–3 mg, LV) stimulated gastric acid secretion in a dose-dependent manner (Figs. 1 and 2). AMPA (3 and 10 mg, LV) also stimulated gastric acid secretion but the effect was weak (Figs. 1 and 2). GAMS (30 mg, LV), an AMPA / kainate receptor antagonist, blocked the stimulatory effect of kainate, but not NMDA (Fig. 5). CNQX (3 mg), an AMPA / kainate receptor antagonist, blocked the stimulatory effect of kainate (Fig. 4). The effect of AMPA (10 mg) was significantly less than that of kainate (0.1 mg). This finding appeared to indicate that the effect of kainate is mediated by a kainate receptor, not by an AMPA receptor. However, kainate is not a specific agonist [17]. Some kainate responses may be mediated by an AMPA receptor. In addition, CNQX (IC 50
of tritiated ligands to rat cortical membranes: AMPA, 0.3 mM; kainate, 1.5 mM [9]) and GAMS (relative potency ratios by comparison of CD 50 values for the presence of respected antagonist versus agonist alone in the convulsant action of EAAs in mice: kainate, 90.7; quisqualate, 36.7; NMDA, 29.3 [28]) are not so selective for kainate receptor. Moreover, three types of ionotropic EAA receptors distributed throughout the brain, including the hypothalamus [5,29] or DVC [1,2,14,15]. Thus, further experiments will be necessary to distinguish the kainate receptor from the AMPA receptors. NMDA receptor antagonists, CPP and MK801, inhibited the stimulation of gastric acid secretion by NMDA, but not by kainate (Fig. 6). From these findings, it was suggested that glutamate receptor mechanisms, especially kainate and NMDA receptors, are independently involved in the central nervous system control of gastric acid secretion. EAAs, administered into the LH, elicited eating responses and the order was kainate.AMPA>NMDA.glutamate [22,23]. Kainate and NMDA affected intragastric pressure and motility, and the order was kainate.NMDA [21]. In the present experiments, the stimulatory responses of agonists of glutamate receptors on gastric acid secretion were in the order kainate.NMDA.AMPA, in potency and in affinity. It appeared that both kainate and NMDA receptors took part in stimulation of gastric acid secretion.
4.2. Dose-related dual roles of stimulants on gastric functions It was reported that NMDA (68 or 136 nmol / ml) destroyed LH cells [16]. Kainate (80 nmol / ml) was also reported as a cell-specific neurotoxin [8]. In the present
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study, the response to kainate (0.5 nmol / 5 ml, LV) or NMDA (6.8 and 20.4 nmol / 5 ml, LV) returned to the basal levels after about 120 min (Fig. 1). The effects of kainate and NMDA injection on gastric acid secretion were observed repeatedly to similar degrees. These findings suggest that the glutamate agonists did not destroy cells in this experimental procedure, and played a role in gastric acid secretion. Namiki et al. [18] reported that the administration of L-glutamate (1.1 nmol / ml) into LH produced no significant effect on gastric acid secretion, although it led to an increase in gastric mucosal blood flow. In addition, injection of kainate into the Rpa at a low dose (0.1 pmol / 30 nl) increased gastric mucosal blood flow and protected against ethanol-induced gastric damage, but did not alter gastric acid secretion [13]. However, injection of kainate at 56.3 pmol / 30 nl into Rpa and 216 pmol / 50 nl into Rob stimulated gastric acid secretion and induced gastric erosion [11,19]. The eating-stimulatory effect of glutamate (300–900 nmol) was weaker than kainate (0.1– 1.0 nmol) [22,23]. Kainate injected into the brain at a low dose acted in a gastric defensive manner, but at higher doses acted as a gastric acid stimulator [11–13,34]. These findings suggested that the dose of the glutamate agonist is very important for the response of gastric acid secretion. In the present conditions, kainate (0.1–1 mg / 5 ml (0.5–5 nmol / 5 ml)) administered into the LV stimulated gastric acid secretion.
4.3. Brain regions regulating gastric acid secretion The importance of DVC and LH in the control of gastric function has previously been established by electrical and chemical stimulation of the region [22–26,33]. For instance, in the lateral hypothalamus (LH), glutamate acts as a physiological regulator of eating and body weight control [22–25]. In addition, electrical stimulation of the dorsal motor nucleus of the vagus (DMN) of the DVC or LH stimulated gastric acid secretion [26,33]. Neurons in the DMN are the main source of vagal efferent nerve fibers innervating the stomach [27]. Furthermore, three types of ionotropic EAA receptors are distributed throughout the brain, including the hypothalamus [5,29] or DVC [1,2,14,15]. In the present study, vagotomy inhibited the kainate or NMDA stimulated gastric acid secretion (Fig. 7), so perhaps at least DMN-vagal activation to the stomach was included. EAAs were injected into the LV, so we did not examine the actual nucleus for a stimulatory effect of gastric acid secretion by EAAs. In addition, it was suggested that endogenous transmitters such as TRH [7,10–12,34] might be released by EAAs injections, which stimulate the gastric acid secretion. Further experiments will be necessary to clarify the actual locus of these effects and the final endogenous transmitters. In summary, the present study showed that kainate and
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NMDA stimulated gastric acid secretion through separate receptors, and these effects were via activation of the central nervous system and DMN-vagal activation to the stomach.
Acknowledgements We thank Professor Toshihiko Murayama for critical reading of the manuscript.
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coagonist glycine site: evidence for a role in lateral hypothalamic stimulation of feeding, Am. J. Physiol. 273 (1997) R790–R796. ´ Central nervous system regulation of gastric acid secreY. Tache, tion, in: L.R. Johnson (Ed.), Physiology of the Gastrointestinal Tract, Vol. 2, Raven Press, New York, 1987, pp. 911–930. ´ H. Yang, M. Yoneda, Vagal regulation of gastric function Y. Tache, involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex, Digestion 54 (1993) 65–72. L. Turski, B.S. Meldrum, A.W. Jones, J.C. Watkins, Anticonvulsant action of stereoisomers of g-glutamylaminomethylsulphonic acid in mice, Eur. J. Pharmacol. 111 (1985) 279–283. A.N. Van Den Pol, I. Hermans-Borgmeyer, M. Hofer, P. Ghosh, S. Heinemann, Ionotropic glutamate-receptor gene expression in hypothalamus: localization of AMPA, kainate, and NMDA receptor RNA with in situ hybridization, J. Comp. Neurol. 343 (1994) 428–444. K. Watanabe, S. Yano, Y. Minakawa, Morphine inhibits the gastric acid secretion stimulated by 2-deoxy-D-glucose via a central mechanism in anesthetized rats, Eur. J. Pharmacol. 143 (1987) 293–298. K. Watanabe, S. Yano, M. Mizuno, Stimulatory effect of intracerebroventricularly injected kainic acid and related compounds on gastric acid secretion in anesthetized rats, Eur. J. Pharmacol. 183 (1990) 2092. R.L. White Jr., C.D. Rossiter, P.J. Hornby, J.W. Harmon, D.K. Kasbekar, R.A. Gillis, Excitation of neurons in the medullary raphe increases gastric acid and pepsin production in cats, Am. J. Physiol. 260 (1991) G91–G96. W. Wyrwicka, R. Garcia, Effect of electrical stimulation of the dorsal nucleus of the vagus nerve on gastric acid secretion in cats, Exp. Neurol. 65 (1979) 315–325. ´ TRH in dorsal vagal complex H. Yang, G. Ohning, Y. Tache, mediates acid response to excitation of raphe pallidus in rats, Am. J. Physiol. 265 (1993) G880–G886.
Research report
Stimulatory effects of centrally injected kainate and N-methyl-Daspartate on gastric acid secretion in anesthetized rats a, a b a Shizuko Tsuchiya *, Syunji Horie , Shingo Yano , Kazuo Watanabe b
a Laboratory of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263 -8522, Japan Laboratory of Molecular Pharmacology and Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263 -8522, Japan
Accepted 3 July 2001
Abstract The effects of N-methyl-D-aspartate (NMDA), kainate and (6)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), ionotropic glutamate agonists, on gastric acid secretion were investigated in the continuously perfused stomach of anesthetized rats. The lateral ventricular (LV) injection of kainate (0.01–1 mg) or NMDA (0.3–3 mg) dose-dependently stimulated gastric acid secretion. AMPA (3–10 mg) also stimulated gastric acid secretion but the effect was very weak. Repeated injections of kainate (0.1 mg) or NMDA (1 mg), at least twice, stimulated gastric acid secretion to a similar degree. The effect of kainate (0.1 mg) was blocked by the kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (3 mg, LV) and D-g-glutamylaminomethanesulfonic acid (30 mg, LV), but not by NMDA receptor antagonists. The effect of NMDA (10 mg) was blocked by (6)-3-(2-carboxypiperazin-4-yl)-1-propylphosphonic acid (10 mg, LV), a competitive NMDA receptor antagonist, and (1)-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine hydrogen maleate (10 mg, LV), a non-competitive NMDA receptor antagonist, but not by kainate receptor antagonists. Moreover, the gastric acid secretion stimulated by kainate and NMDA were completely blocked by systemic atropine injection (1 mg / kg, i.v.) and vagotomy. These findings suggest that kainate and NMDA receptor mechanisms are independently involved in the central nervous system to control gastric acid secretion through vagus cholinergic activation. 2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Excitatory amino acids: pharmacology Keywords: Excitatory amino acid; N-Methyl-D-aspartate; Kainate; Central nervous system; Stomach; Acid secretion
1. Introduction Accumulating evidence suggests that glutamate may be the primary excitatory neurotransmitter in the mammalian central nervous system, with the majority of neurons expressing functional glutamate receptors [6]. Based on the effects of selective excitatory amino acid (EAA) receptor agonists, subtypes of ionotropic glutamate receptors have been classified as N-methyl-D-aspartate (NMDA), kainate and (6)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors [3,17]. Molecular cloning and expression studies have revealed that NMDA receptor (NMDAR) subunits consist of the mandatory subunit 1
*Corresponding author. Tel.: 181-43-290-2923; fax: 181-43-2903021. E-mail address: [email protected] (S. Tsuchiya).
(NMDAR1) and variable subunits 2A–2D (NMDAR2A– 2D), kainate (KA) receptors consist of KA1, KA2 or glutamate receptor subunits 5–7 (GluR5–7), and AMPA receptors consist of glutamate receptor subunits 1–4 (GluR1–4) [5]. There is much evidence that food intake and gastric motility are regulated by ionotropic EAA receptors. For instance, in the lateral hypothalamus (LH), glutamate acts as a physiological regulator of eating and body weight control [22–25]. In the dorsal vagal complex (DVC), activation of kainate and NMDA receptors can independently cause vagally mediated gastric motor excitation [21]. Stimulation with glutamate of the nucleus raphe pallidus (Rpa) increases gastric contractility via the Rpa– DVC pathway [7]. Likewise in cats, excitation of the medullary raphe, Rpa and the nucleus raphe obscurus (Rob), by glutamate causes an increase in gastric motility by excitation of vagal neurons in the DVC [10]. The importance of the DVC and LH in the control of gastric
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02784-6
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function has been previously established by electrical and chemical stimulation of the region [26,33]. Gastric acid secretion has also been proposed to be regulated by ionotropic EAAs. For instance, stimulation of Rpa by kainate or glutamate, which releases thyrotropinreleasing hormone (TRH) and stimulates the DVC, induces vagal muscarinic-dependent stimulation of gastric acid secretion [7,11,27,34]. In cats, gastric acid secretion was increased by kainate into the Rob, via the DVC pathway [32]. In these studies, however, it was not clarified whether the effects of kainate or glutamate were receptor dependent, and what subtypes of the glutamate receptor participated in the gastric acid secretion. However, Namiki et al. [18] reported that the administration of L-glutamate into LH produced no significant effect on gastric acid secretion. Thus, the effects of EAAs in the central nervous system on gastric acid secretion have not been finally established. We previously reported that centrally injected kainate strongly stimulated gastric acid secretion, and NMDA or glutamate itself also weakly stimulated acid secretion in urethane anesthetized rats [31]. In the present study, the effects of glutamate receptor antagonists, injected into the lateral ventricle (LV), on gastric acid secretion induced by NMDA, kainate and AMPA injected LV were examined. We found that kainate and NMDA receptor mechanisms regulated gastric acid secretion independently through vagus cholinergic activation.
2. Materials and methods
2.1. Animals Male Wistar rats weighing 200–300 g (Takasugi Exp. Animals, Kasukabe, Japan) were housed under conditions of controlled temperature (24628C) and a 12-h light / dark cycle (lighting on at 07:00 h), for at least 1 week before the experiment. Food and tap water were available ad libitum. All experiments were performed in rats anesthetized with urethane. The animals were fasted for 18 h before the experiment, but free access to water was maintained. Animal experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, approved by The Japanese Pharmacological Society.
2.2. Compounds used Kainate, NMDA, AMPA hydrobromide, 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX), D-gglutamylaminomethanesulfonic acid (GAMS), (6)-3-(2carboxypiperazin-4-yl)-1-propylphosphonic acid (CPP) and (1)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten5,10-imine hydrogen maleate (MK-801) were obtained from Research Biochemicals International (Natick, MA, USA). Atropine sulfate was obtained from Nacalai Tesque
(Japan). All compounds were dissolved in 0.9% saline. The volume for i.v. injection was 1 ml / kg, and that for LV injection was 5 ml.
2.3. Measurement of gastric acid secretion Gastric acid secretion was determined by the gastric perfusion method as described by Watanabe et al. [30]. Rats were anesthetized with urethane (1.35 g / kg, i.p; Tokyo Kasei, Japan), the esophagus was ligated at the cervical level, and a cannula was inserted into the trachea. After laparotomy, the pylorus was ligated and a dual cannula was inserted through a small incision into the forestomach. The lumen was continuously perfused with saline (adjusted to pH 5.0, 378C) through the inlet tube of the dual cannula at a rate of 1 ml / min. The intragastric pressure was maintained at 5 cm H 2 O. The perfusate was continuously titrated in the reservoir with 0.02 N NaOH to pH 5.0 using an automatic titrator (ABT-101, TOA Electronics, Japan) connected to a computer system.
2.4. Cannulation for lateral ventricular or intravenous injection Animals were placed in a stereotaxic instrument (SR-6, Narishige, Japan) in the mouth down position (23.3 mm). A stainless steel cannula was positioned unilaterally (right side) through a small hole in the skull made by a drill. The bregma region of the parietal skull was exposed. Stereotaxic coordinates were taken from the atlas of Paxinos and Watson [20], and were as follows: 21.0 mm anteroposterior from the bregma, 1.3 mm lateral from the bregma, 3.8 mm dorsoventral from horizontal skull surface. The cannula was secured by dental cement (GC Corporation, Japan). In some animals, the femoral vein was cannulated for injection of atropine or the bilateral vagus nerves were cut at the cervical level.
2.5. Experimental procedure The animal was left for 1 h to stabilize gastric acid secretion. After the determination of basal acid secretion for 30 min, NMDA, kainate and AMPA were injected LV. The amount of acid output was expressed in terms of mEq H 1 / 10 min. In the antagonist study, antagonist or vehicle was injected 10 min before agonist injection. In some animals, repeated injection of the agonist was applied. When the acid secretion of the first agonist injection returned to the basal level after about 120 min, the second injection of the agonist was applied.
2.6. Statistics Values are expressed as D mEq H 1 / 10 min of the basal values measured 10 min prior to injection of NMDA, kainate or AMPA. The values were expressed as the
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mean6S.E. Grouped data of gastric acid secretion were statistically analyzed by one-way ANOVA followed by the Bonferroni multiple comparisons test. In some cases in the antagonist study (Figs. 4 and 5), the first value for the vehicle was compared with the second value for the antagonist by the paired t-test. Probability values ,0.05 were considered significant.
3. Results
3.1. Effect of NMDA, kainate and AMPA on gastric acid secretion In urethane anesthetized rats, basal acid secretion was relatively low during the experimental period, and LV injection of the vehicle did not influence the basal acid secretion (D 2.9264.20 mEq H 1 / 120 min, n56). LV injection of kainate (0.01–1 mg (0.05–5 nmol)) and NMDA (0.3–3 mg (2.04–20.4 nmol)) evoked gastric acid secretion in a dose-dependent manner (Figs. 1 and 2). AMPA (3 and 10 mg (11.2 and 37.4 nmol)) also stimulated gastric acid secretion but the effect was very week (Figs. 1 and 2). The onset of the secretory response was within 10 min after the agonist injection, the response reached the peak value after 50–60 min for kainate, 20–30 min for NMDA and 20–30 min for AMPA. The response of kainate, NMDA or AMPA returned to the basal level after about 120 min, although the acid level after kainate (1 mg) at 120 min was higher than the basal level. The effect of kainate was stronger than that of NMDA and AMPA: the maximal effect obtained with 3 mg (20.4 nmol) NMDA
Fig. 2. Dose–response curves of LV injection of kainate, NMDA and AMPA on the acid output in urethane-anesthetized rats. All values represent the total acid output for 120 min and means6S.E. (n54–7).
was about one-third of that with 1 mg (5 nmol) kainate (Fig. 2). When kainate (0.3–1 mg), NMDA (0.3–3 mg) and AMPA (3 and 10 mg) were injected, hyperpnea was immediately observed. Trembling of the barba was observed after kainate (0.3–1 mg) injection. Rats began
Fig. 1. Time course of the acid output by LV injection of (a) kainate, (b) NMDA and (c) AMPA in urethane-anesthetized rats. All values represent the acid output for 10 min and means6S.E. (n54–7). Dose–response analysis is represented in Fig. 2.
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Fig. 3. Effects of repeated LV injection of kainate and NMDA on the acid output in urethane-anesthetized rats. The second injection was applied about 120 min after the first injection. All values represent the total acid output for 120 min and means6S.E. (n55).
salivating at 40 min and continued until 80–100 min after injection of AMPA (3 and 10 mg).
3.2. Effects of antagonists on the kainate or NMDAinduced response Fig. 3 shows the effects of repeated injections of kainate
(0.1 mg) or NMDA (1 mg). The response after the second injection of kainate was similar to that after the first injection. The effect of NMDA was also repeatable. In the antagonist study of kainate, vehicle was injected 10 min before the first kainate injection, and then the antagonist was injected 10 min before the second agonist injection. The response to the first agonist stimulation was used as a control for the following second antagonist response (Figs. 4 and 5). Injection of CNQX (3 mg, LV), an antagonist of AMPA / kainate receptor [9], blocked the kainate (0.1 mg)-stimulated gastric acid secretion (Fig. 4). The inhibitory effect of CNQX was significant from 60 min after the kainate injection, but not significant at 0–50 min. The reason may be because CNQX (3 mg) itself stimulated gastric acid secretion slightly (D 9.8864.90 mEq H 1 / 120 min, n55, P50.3059 compared with vehicle), and / or CNQX at 3 mg was not sufficient as an antagonist of the AMPA / kainate receptor. We did not use CNQX at doses above 3 mg because of its own stimulatory effect. CNQX (3 mg, LV) also slightly inhibited the effect of AMPA (3 mg, LV), but not significantly because of the stimulatory effect of CNQX and the weak effect of AMPA. GAMS (30 mg, LV), another type of antagonist of the AMPA / kainate receptor [4,28], also blocked kainate (0.1 mg)-stimulated gastric acid secretion (Fig. 5). Injection of GAMS alone had no effect on gastric acid secretion and the inhibition of kainate-stimulated gastric acid secretion was observed after 20 min. Injection of GAMS did not inhibit the NMDA (1 mg)-stimulated gastric acid secretion (data not shown). CPP (10 mg, LV) and MK-801 (10 mg, LV), antagonists for NMDA receptors, blocked the NMDA (10 mg, LV)stimulated gastric acid secretion almost completely (Fig. 6). Similar doses of CPP and MK-801 had no effect alone and did not influence the kainate effect (data not shown).
Fig. 4. Effects of CNQX on gastric acid secretion by kainate in urethane-anesthetized rats. CNQX (3 mg) was injected LV 10 min before LV injection of kainate (KA, 0.1 mg). (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n57). *P,0.05, **P,0.01 and ***P,0.001 compared with kainate alone (paired t-test).
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Fig. 5. Effects of GAMS on gastric acid secretion by kainate in urethane-anesthetized rats. GAMS (30 mg) was injected in LV 10 min before LV injection of kainate (KA, 0.1 mg). (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n56). **P,0.01 and ***P,0.001 compared with kainate alone (paired t-test).
3.3. Effect of atropine or vagotomy on kainate- and NMDA-induced gastric acid secretion Injection of atropine (1 mg / kg, i.v.) blocked the NMDA-stimulated gastric acid secretion (Fig. 7). Atropine also blocked the kainate-stimulated gastric acid secretion (kainate, D 227.4665.1 mEq H 1 / 120 min; kainate1 atropine, D 2.85615.43 mEq H 1 / 120 min, P,0.01). Vagotomy blocked the NMDA-stimulated gastric acid secretion (Fig. 7). Vagotomy also blocked the kainatestimulated gastric acid secretion (kainate, D 244.5662.6 mEq H 1 / 120 min; kainate1vagotomy, D 3.663.6 mEq
H 1 / 120 min, P,0.01). These findings suggest activation of kainate or NMDA receptors in the central nervous system stimulates gastric acid secretion through vagus cholinergic nerves.
4. Discussion
4.1. Stimulatory effect of kainate and NMDA on gastric acid secretion In the central nervous system, there are studies showing
Fig. 6. Effects of CPP (10 mg, LV) and MK801 (10 mg, LV) on gastric acid secretion by NMDA (10 mg, LV) in urethane anesthetized rats. (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n53–5). *P,0.05, **P,0.01 and ***P,0.001 compared with NMDA alone.
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Fig. 7. Effects of atropine (Atr., 1 mg / kg, i.v.) and vagotomy (Vago.) on gastric acid secretion by NMDA (10 mg, LV) in urethane anesthetized rats. (a) Time course of the acid output for 10 min. (b) The total acid output for 120 min. All values represent means6S.E. (n53–5). *P,0.05, **P,0.01 and ***P,0.001 compared with NMDA alone.
that glutamate receptors regulate gastric functions, such as eating, gastric motility or intragastric pressure. For example, LH injection of kainate, NMDA and AMPA elicited a dose-dependent eating response [22–25]. Stanley et al. [24] suggested that glutamate may act through several subtypes of its receptors on LH neurons to elicit eating, and showed that LH injection of the NMDA receptor antagonist ( D-AP5) suppressed eating elicited by LH injection of NMDA, but not kainate or AMPA. DVC injection of kainate and NMDA increased intragastric pressure and gastric motility, which were inhibited by selective antagonists for each receptor subtype [21]. Intracerebral injection of kainate or glutamate stimulated gastric acid secretion, as mentioned in Section 1 [7,11,32,34]. However, it has not been demonstrated whether kainate or glutamate stimulates its receptor, or what subtypes of receptors are concerned with gastric acid secretion. In the present study, it was confirmed that centrally injected kainate (0.01–1 mg; LV), NMDA (0.3–3 mg, LV) stimulated gastric acid secretion in a dose-dependent manner (Figs. 1 and 2). AMPA (3 and 10 mg, LV) also stimulated gastric acid secretion but the effect was weak (Figs. 1 and 2). GAMS (30 mg, LV), an AMPA / kainate receptor antagonist, blocked the stimulatory effect of kainate, but not NMDA (Fig. 5). CNQX (3 mg), an AMPA / kainate receptor antagonist, blocked the stimulatory effect of kainate (Fig. 4). The effect of AMPA (10 mg) was significantly less than that of kainate (0.1 mg). This finding appeared to indicate that the effect of kainate is mediated by a kainate receptor, not by an AMPA receptor. However, kainate is not a specific agonist [17]. Some kainate responses may be mediated by an AMPA receptor. In addition, CNQX (IC 50
of tritiated ligands to rat cortical membranes: AMPA, 0.3 mM; kainate, 1.5 mM [9]) and GAMS (relative potency ratios by comparison of CD 50 values for the presence of respected antagonist versus agonist alone in the convulsant action of EAAs in mice: kainate, 90.7; quisqualate, 36.7; NMDA, 29.3 [28]) are not so selective for kainate receptor. Moreover, three types of ionotropic EAA receptors distributed throughout the brain, including the hypothalamus [5,29] or DVC [1,2,14,15]. Thus, further experiments will be necessary to distinguish the kainate receptor from the AMPA receptors. NMDA receptor antagonists, CPP and MK801, inhibited the stimulation of gastric acid secretion by NMDA, but not by kainate (Fig. 6). From these findings, it was suggested that glutamate receptor mechanisms, especially kainate and NMDA receptors, are independently involved in the central nervous system control of gastric acid secretion. EAAs, administered into the LH, elicited eating responses and the order was kainate.AMPA>NMDA.glutamate [22,23]. Kainate and NMDA affected intragastric pressure and motility, and the order was kainate.NMDA [21]. In the present experiments, the stimulatory responses of agonists of glutamate receptors on gastric acid secretion were in the order kainate.NMDA.AMPA, in potency and in affinity. It appeared that both kainate and NMDA receptors took part in stimulation of gastric acid secretion.
4.2. Dose-related dual roles of stimulants on gastric functions It was reported that NMDA (68 or 136 nmol / ml) destroyed LH cells [16]. Kainate (80 nmol / ml) was also reported as a cell-specific neurotoxin [8]. In the present
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study, the response to kainate (0.5 nmol / 5 ml, LV) or NMDA (6.8 and 20.4 nmol / 5 ml, LV) returned to the basal levels after about 120 min (Fig. 1). The effects of kainate and NMDA injection on gastric acid secretion were observed repeatedly to similar degrees. These findings suggest that the glutamate agonists did not destroy cells in this experimental procedure, and played a role in gastric acid secretion. Namiki et al. [18] reported that the administration of L-glutamate (1.1 nmol / ml) into LH produced no significant effect on gastric acid secretion, although it led to an increase in gastric mucosal blood flow. In addition, injection of kainate into the Rpa at a low dose (0.1 pmol / 30 nl) increased gastric mucosal blood flow and protected against ethanol-induced gastric damage, but did not alter gastric acid secretion [13]. However, injection of kainate at 56.3 pmol / 30 nl into Rpa and 216 pmol / 50 nl into Rob stimulated gastric acid secretion and induced gastric erosion [11,19]. The eating-stimulatory effect of glutamate (300–900 nmol) was weaker than kainate (0.1– 1.0 nmol) [22,23]. Kainate injected into the brain at a low dose acted in a gastric defensive manner, but at higher doses acted as a gastric acid stimulator [11–13,34]. These findings suggested that the dose of the glutamate agonist is very important for the response of gastric acid secretion. In the present conditions, kainate (0.1–1 mg / 5 ml (0.5–5 nmol / 5 ml)) administered into the LV stimulated gastric acid secretion.
4.3. Brain regions regulating gastric acid secretion The importance of DVC and LH in the control of gastric function has previously been established by electrical and chemical stimulation of the region [22–26,33]. For instance, in the lateral hypothalamus (LH), glutamate acts as a physiological regulator of eating and body weight control [22–25]. In addition, electrical stimulation of the dorsal motor nucleus of the vagus (DMN) of the DVC or LH stimulated gastric acid secretion [26,33]. Neurons in the DMN are the main source of vagal efferent nerve fibers innervating the stomach [27]. Furthermore, three types of ionotropic EAA receptors are distributed throughout the brain, including the hypothalamus [5,29] or DVC [1,2,14,15]. In the present study, vagotomy inhibited the kainate or NMDA stimulated gastric acid secretion (Fig. 7), so perhaps at least DMN-vagal activation to the stomach was included. EAAs were injected into the LV, so we did not examine the actual nucleus for a stimulatory effect of gastric acid secretion by EAAs. In addition, it was suggested that endogenous transmitters such as TRH [7,10–12,34] might be released by EAAs injections, which stimulate the gastric acid secretion. Further experiments will be necessary to clarify the actual locus of these effects and the final endogenous transmitters. In summary, the present study showed that kainate and
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NMDA stimulated gastric acid secretion through separate receptors, and these effects were via activation of the central nervous system and DMN-vagal activation to the stomach.
Acknowledgements We thank Professor Toshihiko Murayama for critical reading of the manuscript.
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