- Email: [email protected]
Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy-d -glucose in rats
BRAIN RESEARCH ELSEVIER
Brain Research 674 (1995) 137-141
Short communication
Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy-o-glucose in rats Toshikatsu Okumura
a
Ian L. Taylor b Gordon Ohning c, Yvette Tach6 c, Theodore N. Pappas a,*
a Department of Surgery, PO Box 3479, Duke University Medical Center and Veterans Administration Medical Center, Durham, NC 27710, USA b Department of Medicine, Medical University of South Carolina, SC 29425, USA c CURE Gastroenteric Biology Center, Veterans Affairs Wadworth Medical Center and Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90073, USA
Accepted 20 December 1994
Abstract
We evaluated the effect of 2-deoxy-D-glucose (2-DG) on gastric emptying of a non nutrient solution in conscious rats using a Phenol red method. Intravenous injection of 2-deoxy-o-glucose dose-dependently increased the rate of gastric emptying. This stimulatory action of 2-DG was abolished by subdiaphragmatic vagotomy. Intracisternal injection of thyrotropin-releasing hormone (TRH) antibody blocked intracisternal TRH and intravenous 2-DG-induced enhancement of gastric emptying but not the stimulation of gastric emptying induced by intracisternal pancreatic polypeptide. The TRH antibody injected intraperitoneaily had no effect. These results suggest that endogenous TRH in the brain is involved in vagal-dependent stimulation of gastric emptying by 2-DG.
Keywords: 2-Deoxy-o-glucose; Central nervous system; Gastric emptying; Thyrotropin-releasing hormone (TRH), Vagotomy; Pancreatic polypeptide
2-Deoxy-o-glucose (2-DG) has been used as a tool for central activation of the vagal pathway. Earlier studies demonstrated that 2 - D G administered peripherally acts in the brain especially in the hypothalamus to increase vagal tone, thereby stimulating gastric acid secretion [2,7,10]. In addition to its role in stimulating the release of acid, 2 - D G induces a vagal dependent stimulation of gastric motility [16] and gastric lesion formation [17]. Although 2 - D G has been established as a central vagal stimulant to activate gut function, the mechanism by which 2 - D G increases vagal tone in the central nervous system remains to be clarified. In addition, whether 2 - D G stimulates gastric emptying is unknown. T h e r e is neuroanatomical, electrophysiological and functional evidence that medullary T R H plays a physiological stimulatory role in the vagal regulation of
* Corresponding author. Fax: (1) (919) 681-7934. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 0 0 5 - 4
gastric s e c r e t o r y and m o t o r function in rats [4,5,6,12,22,26,28,29]. In particular, recent studies demonstrated that T R H endogenously released in the D M N in response to raphe pallidus activation induced either chemically or by cold exposure mimics the effect of exogenous T R H in stimulating gastric secretory and motor function and lesion formation [1,3,15,31,32]. These results led us to speculate that T R H in the brain might be involved in the 2-DG-induced stimulation of vagal tone, which in turn enhanced gastric motor function. In support of this possibility, it has been shown that 2 - D G alters neuronal activity in the hypothalamus [2] and the hypothalamic neurons send projections to medullary raphe nuclei [8,9], the site of TRH-ergic input to the D M N [29]. Furthermore, it has been demonstrated that peripheral administration of 2 - D G decreased hypothalamic T R H content and increased serum thyrotropin and triiodothyronine [13]. These resuits suggest that T R H is released from the hypothala-
138
T. Okumura et at/Brain Research 674 (1995) 137-141
mus in response to 2-DG. Although we do not know yet whether T R H is also released in the medullary raphe, other conditions such as cold exposure or hypothyroidism increase T R H synthesis and release in both the hypothalamus and medulla [32,33]. These results further strengthen our hypothesis that endogenous T R H may be involved in 2-DG-mediated change in gastric emptying. In the present study, we investigated in conscious rats whether (1) 2 - D G stimulates gastric emptying and (2) endogenous T R H in the brain mediates the change of gastric emptying in response to 2-DG. Male S p r a g u e - D a w l e y rats weighing 300-350 g were housed under controlled light-dark conditions (lights on: 07.00-19.00 h) with room t e m p e r a t u r e regulated at 23-25°C. Rats were allowed free access to food (Solid rat chow, Purina, Richmond, IN) and tap water. All experiments were performed on animals deprived of food for 48 h but given free access to water up to the experiments. 2 - D G (Sigma Chemical Co., St Louis, MO) was dissolved in normal saline just before each experiment. Saline alone was used in control groups. Rat pancreatic polypeptide (PP) and T R H purchased from Peninsula Laboratories (Belmont, CA) were dissolved in 0.1% bovine albumin in normal saline and then stored in aliquots at - 2 0 ° C until required. The vehicle alone (0.1% bovine serum albumin in normal saline) was given to control animals. Polyclonal T R H antibody (140 /xg in 10 /xl) and control antibody (purified I g G from non-immune rabbit serum) (140 / x g / 1 0 / x l ) were raised, purified and characterized as detailed in a previous publication [31]. Briefly, a high-affinity and -specificity T R H antibody 8964 was obtained in one rabbit after the second booster immunization of T R H . T R H immune rabbit serum and non-immune rabbit serum were purified using Protein A-Sepharose affinity chromatography. The T R H antibody dose not cross react with various related and unrelated peptides [31]. Reagents needed for the m e a s u r e m e n t of Phenol red activity were purchased from Sigma (St. Louis, MO). Gastric emptying was measured using a modification of the Phenol red method [25] which has been published previously [20]. The test solution consisted of 50 mg Phenol red dissolved in 100 ml aqueous methylcellulose (1.5%, w / v ) . Four rats were sacrificed immediately after administration of the test solution. The gastric content of Phenol red was determined and values used as the zero emptying point. Stomachs were exposed by laparotomy, quickly ligated at both the pylorus and esophago-gastric junction and then removed. The stomach and its contents were homogenized in 40 ml of 0.1 M N a O H and Phenol red content determined using methods described elsewhere [20,25]. Briefly, this assay involves precipitation of proteins with 20% trichloroacetic acid, alkalization with 0.5 M N a O H , and a colorimetric assay at 560 nm. Gastric
emptying was calculated according to the following formula (PR, Phenol red): Gastric emptying (%) ( amount of PR recovered from a test stomach ) = laverage amount of PR recovered from standard stomachs × 100 First, we examined the dose-response effect of 2-DG on gastric emptying of a non caloric solution. Under brief isoflurane anesthesia, rats received intravenous injection of either vehicle or 2 - D G (75, 100 or 125 m g / k g ) into the tail vein. Immediately after the injection, the Phenol red methylcellulose solution was given by oral intubation in an amount of 1.5 ml. Rats were killed by cervical dislocation 25 min later. To investigate if the vagus nerve mediates the 2DG-induced change of gastric emptying, 2-DG was administered intravenously to vagotomized rats. Subdiaphragmatic vagotomy or sham operation (skin incision) was performed under isoflurane anesthesia 24 h prior to 2 - D G administration in 24-h fasted rats as described previously [21]. U n d e r brief isoflurane anesthesia, rats received intravenous injection of 2-DG (100 m g / k g ) into the tail vein. Immediately after the injection, the acaloric solution was given by oral intubation in an amount of 1.5 ml and rats were killed by cervical dislocation 25 min later. To test the hypothesis that endogenous T R H in the brain may mediate 2-DG-induced stimulation of gastric emptying, a specific T R H antibody was used. Rats received intracisternal injection of T R H antibody 8964 (140 /xg/10 /xl) or control antibody (140 /xg/10 /xl). Intracisternal injection was performed under brief isoflurane anesthesia with a 10 /xl-Hamilton microsyringe after the rats were mounted on ear bars of a stereotaxic apparatus (David Kopf Instruments, Tijunga, CA). To exclude the possibility that T R H antibody acts peripherally to influence gastric emptying, intraperitoneal injection of either T R H antibody (140 txg/0.5 ml) or control antibody (140 /zg/0.5 ml) was performed as a control. Immediately after central or peripheral injection of T R H antibody or control antibody, 2 - D G in a dose of 100 m g / k g was administrated intravenously and the acaloric solution was given by oral intubation in an amount of 1.5 ml. Rats were killed by cervical dislocation 25 min later. To evaluate the specificity of T R H antibody action, we examined the effects of intracisternal injection of T R H antibody on central T R H or pancreatic polypeptide (non-TRH)-stimulated gastric emptying of a liquid solution. First, we confirmed the stimulatory action of intracisternal injection of T R H (1 p~g) or PP (1 Fzg) on gastric emptying in conscious rats which has been reported in previous reports [12,19]. Next, rats received intracisternal injection of T R H antibody 8964 (140 / x g / 1 0 / x l ) or control antibody (140 p~g/10/xl). Immediately after the central injection, intracisternal injec-
T. Okumura et al. /Brain Research 674 (1995) 137-141
100
139
90 /kg)
so
6o
i
60 40 ,~
.wl
.,.h
30
20
0 0
75 2-DG
100
0
125
(mg/kg)
Fig. 1. Dose-related effect of intravenous administration of 2-deoxyD-glucose (2-DG) on gastric emptying of a liquid meal in conscious rats. 2-DG was injected just before the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the mean _+S.E.M. of 4 or 5 animals. * P < 0.05, when compared with saline (2-DG, 0).
tion of T R H (1 /xg/10 /zl) or PP (1 /zg/10 /~1) was performed. Gastric emptying was measured 25 min after the injection as described above. Values were expressed as the means _+ S.E.M. Statistical analysis was performed by one way analysis of variance and subsequent Duncan's new multiple range tests. A P value < 0.05 was considered to be significant. Fig. 1 illustrates the effect of intravenous injection of 2-DG on gastric emptying of a non-caloric solution in conscious rats. A N O V A detected a significant effect of 2-DG on gastric emptying (F3,14 = 11.87, P < 0.01). Although 2-DG in a dose of 75 m g / k g did not alter gastric emptying rate, two higher doses of 2-DG (100 and 125 m g / k g ) significantly stimulated gastric emptying. Gastric emptying rate (%) increased from 60.3 + 5.9 to 93.9 _+ 2.7 (mean _+ S.E.M.) after intravenous administration of saline or 2-DG in a dose of 125 m g / k g . We investigated the role of the vagus in mediating the effects of 2-DG on gastric emptying. As shown in Fig. 2, vagotomy did not change the rate of gastric emptying in saline administered rats but blocked the stimulatory action of gastric emptying in response to intravenous administration of 2-DG at 100 m g / k g (F3,16 = 30.7, P < 0.01). Intracisternal injection of T R H antibody 8964 in a dose of 1 4 0 / z g / 1 0 / z l completely abolished the stimulation of gastric emptying induced by 2-DG (100 mg/kg), but did not change the basal rate of gastric emptying in saline-treated rats. In contrast, intraperitoneal injection of T R H antibody 8964 in a dose of 140 /~g/0.5 ml failed to inhibit gastric emptying stimulated by 2-DG (Fig. 3, F7,27 = 19.8, P < 0.01).
Sham
Vagotomy
Fig. 2. Effect of vagotomy on 2-deoxy-o-glucose (2-DG)-induced stimulation of gastric emptying of a liquid meal in conscious rats. Vagotomy or sham operation was performed 24 h before 2-DG injection. 2-DG was injected just before the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the mean + S.E.M. of 5 animals. * P < 0.05, when compared with saline in sham operation.
Table 1 represents the effect of intracisternal injection of T R H antibody on intracisternal T R H - or PPstimulated gastric emptying. Gastric emptying was significantly stimulated by either intracisternal T R H or PP. Control antibody did not alter the rate of gastric emptying stimulated by intracisternal administration of T R H or PP. Pretreatment with the T R H antibody (140
[] saline 100
•
2-DG (100 m2/k2)
eo
'~
6o
40 ~
2o 0
control TRH Ab [ Intracisternal I
control TRH Ab I Intraperitoneal ]
Fig. 3. Effect of pretreatment with intracisternal injection of T R H antibody on gastric emptying stimulated by 2-DG. Rats received intracisternal injection of T R H antibody or control antibody in a dose of 140 / z g / 1 0 / z l under brief isoflurane anesthesia. Then 2-DG was injected intravenously followed by the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the m e a n + S.E.M. of 4 or 5 animals. * P < 0.05, when compared with saline (control). * * P < 0.05, when compared with 2-DG (intracisternal control).
140
T. Okumura et al. / B r a i n Research 674 (1995) 137-141
Table 1 Effect of intracisternal injection of TRH antibody 8964 on gastric emptying stimulated by intracisternal pancreatic polypeptide (PP) or TRH Treatment n Gastric emptying(%) Saline PP Control Ab + PP TRH Ab + PP
5 5 4 4
59.2_+3.0 84.8 ± 2.9 * 81.5 _+3.8 * 81.0 _+2.8 *
TRH Control Ab + TRH TRH Ab + TRH
5 4 5
94.0 ± 1.5 * 92.5 _+2.4 * 57.6 _+3.8 * *
Rats received intracisternal injection (10 ~1) of either saline, PP (1 ~g) or TRH (1 #g) with or without pretreatment with intracisternal injection of control or TRH antibody (Ab) in a dose of 140 ~g/10 pA. Rats were sacrificed and the stomach was removed 25 min after Phenol red methylcellulose ingestion to determine gastric emptying. Each data represents the mean_+S.E.M. of the indicated number of animals. ANOVA, F6.25 = 26.1, P < 0.01. * P < 0.05, when compared with saline alone. * * P < 0.05, when compared with control antibody plus TRH.
/.~g) abolished the stimulation of gastric emptying induced by intracisternal T R H but not by PP. The present study demonstrated that intravenous injection of 2-DG increased the rate of gastric emptying of a non-caloric solution in a dose-dependent manner. Although vagotomy did not modify the emptying of liquid, which is in agreement with other reports in rats [12,19,23,27], it did prevent the stimulatory effect of 2-DG on gastric emptying. These results suggest that 2-DG stimulates gastric emptying through vagal dependent pathways as established previously for acid secretion [2,7,10]. A number of findings reported previously suggest that 2-DG acts in the brain to increase vagal tone to stimulate gastric function [2,10]. These results led us to speculate that changes in neuronal activity triggered by neurotransmitter(s) must occur at the D M N which contains preganglionic neurons contributing vagal innervation of the stomach [11,18]. A m o n g many chemical neurotransmitters or peptides tested, T R H is a well-established stimulant of D M N neurons and increases gastric function through a vagal dependent pathway [29]. In particular, intracisternal injection of T R H induces a vagal-mediated stimulation of gastric emptying of a Phenol red methylcellulose solution [12], suggesting that 2-DG-induced stimulation of gastric emptying might result from the action of T R H on the DMN. The present data obtained using the specific T R H antibody 8964 demonstrated that intracisternal, but not intraperitoneal injection of T R H antibody abolished 2-DG-induced enhancement of gastric emptying. These findings support an involvement of medullary T R H in mediating the vagal dependent stimulation of gastric emptying in response to 2-DG. These results also indicate that endogenous T R H in the brain,
probably in the DMN, plays an important role in stimulating vagally-mediated gastric motility as previously reported [3]. In contrast, intracisternal injection of T R H antibody did not inhibit basal (unstimulated)gastric emptying. This might suggest that vagal tone is not involved in regulating basal gastric emptying of liquid as already indicated by the unchanged gastric emptying rate after vagotomy [12,19,23,27]. One may speculate that the T R H antibody has some non-specific effects which disrupt vagally mediated gastric emptying. However, this possibility is unlikely because (1) the T R H antibody prevented the stimulation of gastric emptying elicited by exogenous administration of T R H and (2) central PP-stimulated gastric emptying was not changed by pretreatment with the T R H antibody. These data further confirm in vivo the specificity of T R H antibody to T R H and show that it dose not react with related or unrelated peptides [31]. It also demonstrated that PP action in the brain is not mediated by T R H release. These data support our present conclusion that endogenous T R H may be involved in 2-DG-induced stimulation of gastric emptying. We recently reported that pretreatment with intracisternal injection of T R H antibody significantly reduced the severity of gastric mucosal lesions induced by 2-DG [17]. The present study revealed that immunoneutrtalization of T R H in the brain prevents gastric emptying stimulated by 2-DG. Since the rate of gastric emptying is linearly correlated with intragastric pressure [14], the present results suggest that the T R H antibody might block the vagally mediated increase in gastric contractility. It is therefore speculated that the abolition of gastric contractile response might contribute to the inhibition of 2-DG-induced gastric lesion formation by centrally injected T R H antibody because stimulated gastric contractility is one of the aggressive factors in gastric ulcer formation [30,34]. The present study suggest that 2-DG may activate T R H release in the brain to stimulate gastric emptying in a vagal dependent fashion. It has been demonstrated that intraperitoneal injection of 2-DG resulted in a significant reduction of hypothalamic T R H content followed by an increase in serum thyrotropin and triiodothyronine, suggesting a release of T R H from the hypothalamus in response to 2-DG [13]. Our results suggest that 2-DG also stimulates medullary caudal raphe ( T R H containing neurons)-DMN pathways which are involved in the stimulation of the vagal outflow to the stomach [29]. In summary, our data demonstrated that peripheral administration of 2-DG stimulates gastric emptying of a liquid meal through vagal dependent pathways. Endogenous T R H in the brain may be involved in the stimulation of gastric emptying by 2-DG. We speculate that 2-DG, a well-established central vagal stimulant,
T. Okumura et al. /Brain Research 674 (1995) 137-141 e x e r t s its s t i m u l a t o r y a c t i o n o n g u t f u n c t i o n t h r o u g h activating medullary TRH release.
T h i s s t u d y w a s s u p p o r t e d by V e t e r a n s A d m i n i s t r a t i o n a n d N I H G r a n t s D K 44072, D K 38216, D K 4 0 7 9 0 - 0 5 , D K 41301 ( G a s t r o e n t e r i c B i o l o g y C e n t e r , A n t i b o d y c o r e ) a n d M H 00663.
[1] Bonaz, B. and Tach6, Y. Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion, Brain Res., 652 (1994) 56-64. [2] Colin-Jones, D.G. and Himsworth, R.L., The location of the chemoreceptor controlling gastric acid secretion during hypoglycaemia, J. Physiol., 206 (1970) 397-409. [3] Garrick, T., Prince, M., Yang, H., Ohning, G. and Tach6, Y. Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats, Brain Res., 636 (1994) 343-347. [4] Garrick, T., Stephens, R., Ishikawa, T., Sierra, A., Avidan, A., Weiner, H., and TachS, Y., Medullary sites for TRH analogue stimulation of gastric contractility in the rat, Am. J. Physiol., 259 (Gastroinetst. Liver Physiol. 19) (1989) G1011-G1015. [5] Goto, Y. and TachS, Y., Gastric erosions induced by intracisternal thyrotropin-releasing hormone (TRH) in rats, Peptides, 6 (1985) 153-156. [6] Hernandez, D.E. and Emerick, S.G., Thyrotropin-releasing hormone: medullary site of action to induce gastric ulcers and stimulate acid secretion, Brain Res., 459 (1988) 148-152. [7l Hirschowitz, B.I. and Sacks, G., Vagal gastric secretory stimulation by 2-deoxy-o-glucose, Am. J. Physiol., 209 (1965) 452-460. [8] Holstege, G., Some anatomical observations on the projections from hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the rat, J. Comp. Neurol., 260 (1987) 98-126. [9] Hosoya, Y., Hypothalamic projections to the ventral medulla oblongata in the rat, with special reference to the nucleus raphe pallidus: a study using autoradiographic and HRP techniques, Brain Res., 344 (1985) 338-350. [10] Kadekaro, M., Timo-Iaria, C. and Valle, L.E.R., Neural systems responsible for the gastric secretion provoked by 2-deoxy-D-glucose cytoglucopenia, J. Physiol., 252 (1975) 565-584. [11] Kalia, M. and Sullivan, J.M., Brainstem projection of sensory and motor components of the vagus nerve in the rat, J. Cornp. NeuroL, 211 (1982) 248-264. [12] Maeda-Hagiwara, M. and TachS, Y., Central nervous system action of TRH to stimulate gastric emptying in rats, Regul. Pept., 17 (1987) 199-207. [13] Martino, E., Bambini, G., Aghini-Lombardi, F., Breccia, M. and Baschiere, L., Effect of 2-deoxy-o-glucose on hypothalamicpituitary-thyroid axis in rats, Life Sci., 35 (1984) 1569-1574. [14] Minami, H. and McCallum, R.W., The physiology and pathophysiology of gastric emptying in humans, Gastroenterology, 86 (1984) 1592-1610. [15] Niida, H., Takeuchi, K., Ueshima, K. and Okabe, S., Vagally mediated acid hypersecretion and lesion formation in anesthetized rat under hypothermic conditions, Dig. Dis. Sci., 36 (1991) 441-448. [16] Okada, M., Niida, H., Takeuchi, K. and Okabe, S., Role of prostaglandin deficiency in pathogenetic mechanism of gastric lesions induced by indomethacin in rats, Dig. Dis. Sci., 34 (1989) 694-702.
141
[17] Okumura, T., Grant, A.P., Taylor, I.L., Ohning, G., Tach6, Y. and Pappas, T.N., Gastric mucosal damage induced by 2-deoxyD-glucose involves medullary TRH in the rat, Regul. Pept., in press. [18] Okumura, T. and Namiki, M., Vagal motor neurons innervating the stomach are site-specifically organized in the dorsal motor nucleus of the vagus nerve in rats, J. Auton. Nerv. Syst., 29 (1990) 157-162. [19] Okumura, T., Pappas, T.N. and Taylor, I.L., Intracisternal injection of pancreatic polypeptide stimulates gastric emptying in rats, Neurosci. Lett., 178 (1994) 167-170. [20] Okumura, T., Uehara, A., Kitamori, S., Watanabe, Y., Taniguchi, Y., Tsuji, K. and Namiki, M., Basic fibroblast growth factor (bFGF) acts centrally in the brain to inhibit gastric emptying in rats, Neurosci. Lett., 137 (1992) 53-55. [21] Okumura, T., Uehara, A., Taniguchi, Y., Watanabe, Y., Tsuji, K., Kitamori, S. and Namiki, M., Kainic acid injection into medullary raphe produces gastric lesions through the vagal system in rats, Am. J. PhysioL, 264 (Gastrointest. Liver Physiol. 27) (1993) G655-G658. [22] Okumura, T., Uehara, A., Watanabe, Y., Taniguchi, Y., Kitamori, S. and Namiki, M., Site-specific formation of thyrotropinreleasing hormone-induced gastric ulcers through the vagal system, Scand. J. Physiol., 29 (1994) 226-231. [23] Porreca, F. and Burks, T.F., Centrally administered bombesin affects gastric emptying and small and large transit in the rat, Gastroenterology, 85 (1983) 313-317. [24] Rogers, R.C. and Hermann, G.E., Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility, Peptides, 8 (1987) 505-513. [25] Scarpignato, C., Capovilla, T. and Bertacini, G., Action of caerulein on gastric emptying of the conscious rats, Arch. Int. Pharmacodyn. Then, 246 (1980) 286-294. [26] Stephens, R.L., Ishikawa, T., Weiner, H., Novin, D. and TachS, Y., TRH analogue, RX77368, injected into the dorsal vagal complex stimulates gastric secretion in rats, Am. J. Physiol., 254 (Gastrointest. Liver Physiol. 17) (1988) G639-G643. [27] Tach6, Y., Maeda-Hagiwara, M. and Turkelson, C.M., Central nervous system action of corticotropin-releasing factor to inhibit gastric emptying in rats, Am. J. Physiol., 253 (1987) G241-G245. [28] TachS, Y., Vale, W. and Brown, M., Thyrotropin-releasing hormone: CNS action to stimulate gastric acid secretion, Nature, 287 (1980) 149-151. [29] Tach6, Y., Yang, H. and Yoneda, M., Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex, Digestion, 54 (1993) 65-72. [30] Takeuchi, K., Ueki, S. and Okabe, S., Importance of gastric motility in the pathogenesis of indomethacin-induced gastric lesions in rats, Dig. Dis. Sci., 31 (1986) 1114-1122. [31] Yang, H., Ohning, G. and Tach6, Y., TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats, Am. J. Physiol., 265 (Gastrointest. Liver Physiol. 28) (1993) G880-G886. [32] Yang, H., Wu, S.V., Ishikawa, T. and Tach6, Y., Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei; relationship with vagally mediated gastric erosions, Neuroscience, 61 (1994) 655-663. [33] Yang, H., Wu, V. and Tach6, Y., Medullary thyrotropin-releasing hormone (TRH) mRNA expression is increased after thyroidectomy, Gastroenterology, 102 (1992) A 767. [34] Yano, S., Akahane, M. and Harada, M., Role of gastric motility in development of stress-induced gastric lesions of rats, Jpn. J. Pharmacol., 28 (1978) 607-615.
Brain Research 674 (1995) 137-141
Short communication
Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy-o-glucose in rats Toshikatsu Okumura
a
Ian L. Taylor b Gordon Ohning c, Yvette Tach6 c, Theodore N. Pappas a,*
a Department of Surgery, PO Box 3479, Duke University Medical Center and Veterans Administration Medical Center, Durham, NC 27710, USA b Department of Medicine, Medical University of South Carolina, SC 29425, USA c CURE Gastroenteric Biology Center, Veterans Affairs Wadworth Medical Center and Department of Medicine and Brain Research Institute, UCLA, Los Angeles, CA 90073, USA
Accepted 20 December 1994
Abstract
We evaluated the effect of 2-deoxy-D-glucose (2-DG) on gastric emptying of a non nutrient solution in conscious rats using a Phenol red method. Intravenous injection of 2-deoxy-o-glucose dose-dependently increased the rate of gastric emptying. This stimulatory action of 2-DG was abolished by subdiaphragmatic vagotomy. Intracisternal injection of thyrotropin-releasing hormone (TRH) antibody blocked intracisternal TRH and intravenous 2-DG-induced enhancement of gastric emptying but not the stimulation of gastric emptying induced by intracisternal pancreatic polypeptide. The TRH antibody injected intraperitoneaily had no effect. These results suggest that endogenous TRH in the brain is involved in vagal-dependent stimulation of gastric emptying by 2-DG.
Keywords: 2-Deoxy-o-glucose; Central nervous system; Gastric emptying; Thyrotropin-releasing hormone (TRH), Vagotomy; Pancreatic polypeptide
2-Deoxy-o-glucose (2-DG) has been used as a tool for central activation of the vagal pathway. Earlier studies demonstrated that 2 - D G administered peripherally acts in the brain especially in the hypothalamus to increase vagal tone, thereby stimulating gastric acid secretion [2,7,10]. In addition to its role in stimulating the release of acid, 2 - D G induces a vagal dependent stimulation of gastric motility [16] and gastric lesion formation [17]. Although 2 - D G has been established as a central vagal stimulant to activate gut function, the mechanism by which 2 - D G increases vagal tone in the central nervous system remains to be clarified. In addition, whether 2 - D G stimulates gastric emptying is unknown. T h e r e is neuroanatomical, electrophysiological and functional evidence that medullary T R H plays a physiological stimulatory role in the vagal regulation of
* Corresponding author. Fax: (1) (919) 681-7934. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 0 0 5 - 4
gastric s e c r e t o r y and m o t o r function in rats [4,5,6,12,22,26,28,29]. In particular, recent studies demonstrated that T R H endogenously released in the D M N in response to raphe pallidus activation induced either chemically or by cold exposure mimics the effect of exogenous T R H in stimulating gastric secretory and motor function and lesion formation [1,3,15,31,32]. These results led us to speculate that T R H in the brain might be involved in the 2-DG-induced stimulation of vagal tone, which in turn enhanced gastric motor function. In support of this possibility, it has been shown that 2 - D G alters neuronal activity in the hypothalamus [2] and the hypothalamic neurons send projections to medullary raphe nuclei [8,9], the site of TRH-ergic input to the D M N [29]. Furthermore, it has been demonstrated that peripheral administration of 2 - D G decreased hypothalamic T R H content and increased serum thyrotropin and triiodothyronine [13]. These resuits suggest that T R H is released from the hypothala-
138
T. Okumura et at/Brain Research 674 (1995) 137-141
mus in response to 2-DG. Although we do not know yet whether T R H is also released in the medullary raphe, other conditions such as cold exposure or hypothyroidism increase T R H synthesis and release in both the hypothalamus and medulla [32,33]. These results further strengthen our hypothesis that endogenous T R H may be involved in 2-DG-mediated change in gastric emptying. In the present study, we investigated in conscious rats whether (1) 2 - D G stimulates gastric emptying and (2) endogenous T R H in the brain mediates the change of gastric emptying in response to 2-DG. Male S p r a g u e - D a w l e y rats weighing 300-350 g were housed under controlled light-dark conditions (lights on: 07.00-19.00 h) with room t e m p e r a t u r e regulated at 23-25°C. Rats were allowed free access to food (Solid rat chow, Purina, Richmond, IN) and tap water. All experiments were performed on animals deprived of food for 48 h but given free access to water up to the experiments. 2 - D G (Sigma Chemical Co., St Louis, MO) was dissolved in normal saline just before each experiment. Saline alone was used in control groups. Rat pancreatic polypeptide (PP) and T R H purchased from Peninsula Laboratories (Belmont, CA) were dissolved in 0.1% bovine albumin in normal saline and then stored in aliquots at - 2 0 ° C until required. The vehicle alone (0.1% bovine serum albumin in normal saline) was given to control animals. Polyclonal T R H antibody (140 /xg in 10 /xl) and control antibody (purified I g G from non-immune rabbit serum) (140 / x g / 1 0 / x l ) were raised, purified and characterized as detailed in a previous publication [31]. Briefly, a high-affinity and -specificity T R H antibody 8964 was obtained in one rabbit after the second booster immunization of T R H . T R H immune rabbit serum and non-immune rabbit serum were purified using Protein A-Sepharose affinity chromatography. The T R H antibody dose not cross react with various related and unrelated peptides [31]. Reagents needed for the m e a s u r e m e n t of Phenol red activity were purchased from Sigma (St. Louis, MO). Gastric emptying was measured using a modification of the Phenol red method [25] which has been published previously [20]. The test solution consisted of 50 mg Phenol red dissolved in 100 ml aqueous methylcellulose (1.5%, w / v ) . Four rats were sacrificed immediately after administration of the test solution. The gastric content of Phenol red was determined and values used as the zero emptying point. Stomachs were exposed by laparotomy, quickly ligated at both the pylorus and esophago-gastric junction and then removed. The stomach and its contents were homogenized in 40 ml of 0.1 M N a O H and Phenol red content determined using methods described elsewhere [20,25]. Briefly, this assay involves precipitation of proteins with 20% trichloroacetic acid, alkalization with 0.5 M N a O H , and a colorimetric assay at 560 nm. Gastric
emptying was calculated according to the following formula (PR, Phenol red): Gastric emptying (%) ( amount of PR recovered from a test stomach ) = laverage amount of PR recovered from standard stomachs × 100 First, we examined the dose-response effect of 2-DG on gastric emptying of a non caloric solution. Under brief isoflurane anesthesia, rats received intravenous injection of either vehicle or 2 - D G (75, 100 or 125 m g / k g ) into the tail vein. Immediately after the injection, the Phenol red methylcellulose solution was given by oral intubation in an amount of 1.5 ml. Rats were killed by cervical dislocation 25 min later. To investigate if the vagus nerve mediates the 2DG-induced change of gastric emptying, 2-DG was administered intravenously to vagotomized rats. Subdiaphragmatic vagotomy or sham operation (skin incision) was performed under isoflurane anesthesia 24 h prior to 2 - D G administration in 24-h fasted rats as described previously [21]. U n d e r brief isoflurane anesthesia, rats received intravenous injection of 2-DG (100 m g / k g ) into the tail vein. Immediately after the injection, the acaloric solution was given by oral intubation in an amount of 1.5 ml and rats were killed by cervical dislocation 25 min later. To test the hypothesis that endogenous T R H in the brain may mediate 2-DG-induced stimulation of gastric emptying, a specific T R H antibody was used. Rats received intracisternal injection of T R H antibody 8964 (140 /xg/10 /xl) or control antibody (140 /xg/10 /xl). Intracisternal injection was performed under brief isoflurane anesthesia with a 10 /xl-Hamilton microsyringe after the rats were mounted on ear bars of a stereotaxic apparatus (David Kopf Instruments, Tijunga, CA). To exclude the possibility that T R H antibody acts peripherally to influence gastric emptying, intraperitoneal injection of either T R H antibody (140 txg/0.5 ml) or control antibody (140 /zg/0.5 ml) was performed as a control. Immediately after central or peripheral injection of T R H antibody or control antibody, 2 - D G in a dose of 100 m g / k g was administrated intravenously and the acaloric solution was given by oral intubation in an amount of 1.5 ml. Rats were killed by cervical dislocation 25 min later. To evaluate the specificity of T R H antibody action, we examined the effects of intracisternal injection of T R H antibody on central T R H or pancreatic polypeptide (non-TRH)-stimulated gastric emptying of a liquid solution. First, we confirmed the stimulatory action of intracisternal injection of T R H (1 p~g) or PP (1 Fzg) on gastric emptying in conscious rats which has been reported in previous reports [12,19]. Next, rats received intracisternal injection of T R H antibody 8964 (140 / x g / 1 0 / x l ) or control antibody (140 p~g/10/xl). Immediately after the central injection, intracisternal injec-
T. Okumura et al. /Brain Research 674 (1995) 137-141
100
139
90 /kg)
so
6o
i
60 40 ,~
.wl
.,.h
30
20
0 0
75 2-DG
100
0
125
(mg/kg)
Fig. 1. Dose-related effect of intravenous administration of 2-deoxyD-glucose (2-DG) on gastric emptying of a liquid meal in conscious rats. 2-DG was injected just before the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the mean _+S.E.M. of 4 or 5 animals. * P < 0.05, when compared with saline (2-DG, 0).
tion of T R H (1 /xg/10 /zl) or PP (1 /zg/10 /~1) was performed. Gastric emptying was measured 25 min after the injection as described above. Values were expressed as the means _+ S.E.M. Statistical analysis was performed by one way analysis of variance and subsequent Duncan's new multiple range tests. A P value < 0.05 was considered to be significant. Fig. 1 illustrates the effect of intravenous injection of 2-DG on gastric emptying of a non-caloric solution in conscious rats. A N O V A detected a significant effect of 2-DG on gastric emptying (F3,14 = 11.87, P < 0.01). Although 2-DG in a dose of 75 m g / k g did not alter gastric emptying rate, two higher doses of 2-DG (100 and 125 m g / k g ) significantly stimulated gastric emptying. Gastric emptying rate (%) increased from 60.3 + 5.9 to 93.9 _+ 2.7 (mean _+ S.E.M.) after intravenous administration of saline or 2-DG in a dose of 125 m g / k g . We investigated the role of the vagus in mediating the effects of 2-DG on gastric emptying. As shown in Fig. 2, vagotomy did not change the rate of gastric emptying in saline administered rats but blocked the stimulatory action of gastric emptying in response to intravenous administration of 2-DG at 100 m g / k g (F3,16 = 30.7, P < 0.01). Intracisternal injection of T R H antibody 8964 in a dose of 1 4 0 / z g / 1 0 / z l completely abolished the stimulation of gastric emptying induced by 2-DG (100 mg/kg), but did not change the basal rate of gastric emptying in saline-treated rats. In contrast, intraperitoneal injection of T R H antibody 8964 in a dose of 140 /~g/0.5 ml failed to inhibit gastric emptying stimulated by 2-DG (Fig. 3, F7,27 = 19.8, P < 0.01).
Sham
Vagotomy
Fig. 2. Effect of vagotomy on 2-deoxy-o-glucose (2-DG)-induced stimulation of gastric emptying of a liquid meal in conscious rats. Vagotomy or sham operation was performed 24 h before 2-DG injection. 2-DG was injected just before the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the mean + S.E.M. of 5 animals. * P < 0.05, when compared with saline in sham operation.
Table 1 represents the effect of intracisternal injection of T R H antibody on intracisternal T R H - or PPstimulated gastric emptying. Gastric emptying was significantly stimulated by either intracisternal T R H or PP. Control antibody did not alter the rate of gastric emptying stimulated by intracisternal administration of T R H or PP. Pretreatment with the T R H antibody (140
[] saline 100
•
2-DG (100 m2/k2)
eo
'~
6o
40 ~
2o 0
control TRH Ab [ Intracisternal I
control TRH Ab I Intraperitoneal ]
Fig. 3. Effect of pretreatment with intracisternal injection of T R H antibody on gastric emptying stimulated by 2-DG. Rats received intracisternal injection of T R H antibody or control antibody in a dose of 140 / z g / 1 0 / z l under brief isoflurane anesthesia. Then 2-DG was injected intravenously followed by the administration of a test liquid meal. Rats were sacrificed and the stomach was removed 25 min after meal ingestion to determine gastric emptying. Each column represents the m e a n + S.E.M. of 4 or 5 animals. * P < 0.05, when compared with saline (control). * * P < 0.05, when compared with 2-DG (intracisternal control).
140
T. Okumura et al. / B r a i n Research 674 (1995) 137-141
Table 1 Effect of intracisternal injection of TRH antibody 8964 on gastric emptying stimulated by intracisternal pancreatic polypeptide (PP) or TRH Treatment n Gastric emptying(%) Saline PP Control Ab + PP TRH Ab + PP
5 5 4 4
59.2_+3.0 84.8 ± 2.9 * 81.5 _+3.8 * 81.0 _+2.8 *
TRH Control Ab + TRH TRH Ab + TRH
5 4 5
94.0 ± 1.5 * 92.5 _+2.4 * 57.6 _+3.8 * *
Rats received intracisternal injection (10 ~1) of either saline, PP (1 ~g) or TRH (1 #g) with or without pretreatment with intracisternal injection of control or TRH antibody (Ab) in a dose of 140 ~g/10 pA. Rats were sacrificed and the stomach was removed 25 min after Phenol red methylcellulose ingestion to determine gastric emptying. Each data represents the mean_+S.E.M. of the indicated number of animals. ANOVA, F6.25 = 26.1, P < 0.01. * P < 0.05, when compared with saline alone. * * P < 0.05, when compared with control antibody plus TRH.
/.~g) abolished the stimulation of gastric emptying induced by intracisternal T R H but not by PP. The present study demonstrated that intravenous injection of 2-DG increased the rate of gastric emptying of a non-caloric solution in a dose-dependent manner. Although vagotomy did not modify the emptying of liquid, which is in agreement with other reports in rats [12,19,23,27], it did prevent the stimulatory effect of 2-DG on gastric emptying. These results suggest that 2-DG stimulates gastric emptying through vagal dependent pathways as established previously for acid secretion [2,7,10]. A number of findings reported previously suggest that 2-DG acts in the brain to increase vagal tone to stimulate gastric function [2,10]. These results led us to speculate that changes in neuronal activity triggered by neurotransmitter(s) must occur at the D M N which contains preganglionic neurons contributing vagal innervation of the stomach [11,18]. A m o n g many chemical neurotransmitters or peptides tested, T R H is a well-established stimulant of D M N neurons and increases gastric function through a vagal dependent pathway [29]. In particular, intracisternal injection of T R H induces a vagal-mediated stimulation of gastric emptying of a Phenol red methylcellulose solution [12], suggesting that 2-DG-induced stimulation of gastric emptying might result from the action of T R H on the DMN. The present data obtained using the specific T R H antibody 8964 demonstrated that intracisternal, but not intraperitoneal injection of T R H antibody abolished 2-DG-induced enhancement of gastric emptying. These findings support an involvement of medullary T R H in mediating the vagal dependent stimulation of gastric emptying in response to 2-DG. These results also indicate that endogenous T R H in the brain,
probably in the DMN, plays an important role in stimulating vagally-mediated gastric motility as previously reported [3]. In contrast, intracisternal injection of T R H antibody did not inhibit basal (unstimulated)gastric emptying. This might suggest that vagal tone is not involved in regulating basal gastric emptying of liquid as already indicated by the unchanged gastric emptying rate after vagotomy [12,19,23,27]. One may speculate that the T R H antibody has some non-specific effects which disrupt vagally mediated gastric emptying. However, this possibility is unlikely because (1) the T R H antibody prevented the stimulation of gastric emptying elicited by exogenous administration of T R H and (2) central PP-stimulated gastric emptying was not changed by pretreatment with the T R H antibody. These data further confirm in vivo the specificity of T R H antibody to T R H and show that it dose not react with related or unrelated peptides [31]. It also demonstrated that PP action in the brain is not mediated by T R H release. These data support our present conclusion that endogenous T R H may be involved in 2-DG-induced stimulation of gastric emptying. We recently reported that pretreatment with intracisternal injection of T R H antibody significantly reduced the severity of gastric mucosal lesions induced by 2-DG [17]. The present study revealed that immunoneutrtalization of T R H in the brain prevents gastric emptying stimulated by 2-DG. Since the rate of gastric emptying is linearly correlated with intragastric pressure [14], the present results suggest that the T R H antibody might block the vagally mediated increase in gastric contractility. It is therefore speculated that the abolition of gastric contractile response might contribute to the inhibition of 2-DG-induced gastric lesion formation by centrally injected T R H antibody because stimulated gastric contractility is one of the aggressive factors in gastric ulcer formation [30,34]. The present study suggest that 2-DG may activate T R H release in the brain to stimulate gastric emptying in a vagal dependent fashion. It has been demonstrated that intraperitoneal injection of 2-DG resulted in a significant reduction of hypothalamic T R H content followed by an increase in serum thyrotropin and triiodothyronine, suggesting a release of T R H from the hypothalamus in response to 2-DG [13]. Our results suggest that 2-DG also stimulates medullary caudal raphe ( T R H containing neurons)-DMN pathways which are involved in the stimulation of the vagal outflow to the stomach [29]. In summary, our data demonstrated that peripheral administration of 2-DG stimulates gastric emptying of a liquid meal through vagal dependent pathways. Endogenous T R H in the brain may be involved in the stimulation of gastric emptying by 2-DG. We speculate that 2-DG, a well-established central vagal stimulant,
T. Okumura et al. /Brain Research 674 (1995) 137-141 e x e r t s its s t i m u l a t o r y a c t i o n o n g u t f u n c t i o n t h r o u g h activating medullary TRH release.
T h i s s t u d y w a s s u p p o r t e d by V e t e r a n s A d m i n i s t r a t i o n a n d N I H G r a n t s D K 44072, D K 38216, D K 4 0 7 9 0 - 0 5 , D K 41301 ( G a s t r o e n t e r i c B i o l o g y C e n t e r , A n t i b o d y c o r e ) a n d M H 00663.
[1] Bonaz, B. and Tach6, Y. Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion, Brain Res., 652 (1994) 56-64. [2] Colin-Jones, D.G. and Himsworth, R.L., The location of the chemoreceptor controlling gastric acid secretion during hypoglycaemia, J. Physiol., 206 (1970) 397-409. [3] Garrick, T., Prince, M., Yang, H., Ohning, G. and Tach6, Y. Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats, Brain Res., 636 (1994) 343-347. [4] Garrick, T., Stephens, R., Ishikawa, T., Sierra, A., Avidan, A., Weiner, H., and TachS, Y., Medullary sites for TRH analogue stimulation of gastric contractility in the rat, Am. J. Physiol., 259 (Gastroinetst. Liver Physiol. 19) (1989) G1011-G1015. [5] Goto, Y. and TachS, Y., Gastric erosions induced by intracisternal thyrotropin-releasing hormone (TRH) in rats, Peptides, 6 (1985) 153-156. [6] Hernandez, D.E. and Emerick, S.G., Thyrotropin-releasing hormone: medullary site of action to induce gastric ulcers and stimulate acid secretion, Brain Res., 459 (1988) 148-152. [7l Hirschowitz, B.I. and Sacks, G., Vagal gastric secretory stimulation by 2-deoxy-o-glucose, Am. J. Physiol., 209 (1965) 452-460. [8] Holstege, G., Some anatomical observations on the projections from hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the rat, J. Comp. Neurol., 260 (1987) 98-126. [9] Hosoya, Y., Hypothalamic projections to the ventral medulla oblongata in the rat, with special reference to the nucleus raphe pallidus: a study using autoradiographic and HRP techniques, Brain Res., 344 (1985) 338-350. [10] Kadekaro, M., Timo-Iaria, C. and Valle, L.E.R., Neural systems responsible for the gastric secretion provoked by 2-deoxy-D-glucose cytoglucopenia, J. Physiol., 252 (1975) 565-584. [11] Kalia, M. and Sullivan, J.M., Brainstem projection of sensory and motor components of the vagus nerve in the rat, J. Cornp. NeuroL, 211 (1982) 248-264. [12] Maeda-Hagiwara, M. and TachS, Y., Central nervous system action of TRH to stimulate gastric emptying in rats, Regul. Pept., 17 (1987) 199-207. [13] Martino, E., Bambini, G., Aghini-Lombardi, F., Breccia, M. and Baschiere, L., Effect of 2-deoxy-o-glucose on hypothalamicpituitary-thyroid axis in rats, Life Sci., 35 (1984) 1569-1574. [14] Minami, H. and McCallum, R.W., The physiology and pathophysiology of gastric emptying in humans, Gastroenterology, 86 (1984) 1592-1610. [15] Niida, H., Takeuchi, K., Ueshima, K. and Okabe, S., Vagally mediated acid hypersecretion and lesion formation in anesthetized rat under hypothermic conditions, Dig. Dis. Sci., 36 (1991) 441-448. [16] Okada, M., Niida, H., Takeuchi, K. and Okabe, S., Role of prostaglandin deficiency in pathogenetic mechanism of gastric lesions induced by indomethacin in rats, Dig. Dis. Sci., 34 (1989) 694-702.
141
[17] Okumura, T., Grant, A.P., Taylor, I.L., Ohning, G., Tach6, Y. and Pappas, T.N., Gastric mucosal damage induced by 2-deoxyD-glucose involves medullary TRH in the rat, Regul. Pept., in press. [18] Okumura, T. and Namiki, M., Vagal motor neurons innervating the stomach are site-specifically organized in the dorsal motor nucleus of the vagus nerve in rats, J. Auton. Nerv. Syst., 29 (1990) 157-162. [19] Okumura, T., Pappas, T.N. and Taylor, I.L., Intracisternal injection of pancreatic polypeptide stimulates gastric emptying in rats, Neurosci. Lett., 178 (1994) 167-170. [20] Okumura, T., Uehara, A., Kitamori, S., Watanabe, Y., Taniguchi, Y., Tsuji, K. and Namiki, M., Basic fibroblast growth factor (bFGF) acts centrally in the brain to inhibit gastric emptying in rats, Neurosci. Lett., 137 (1992) 53-55. [21] Okumura, T., Uehara, A., Taniguchi, Y., Watanabe, Y., Tsuji, K., Kitamori, S. and Namiki, M., Kainic acid injection into medullary raphe produces gastric lesions through the vagal system in rats, Am. J. PhysioL, 264 (Gastrointest. Liver Physiol. 27) (1993) G655-G658. [22] Okumura, T., Uehara, A., Watanabe, Y., Taniguchi, Y., Kitamori, S. and Namiki, M., Site-specific formation of thyrotropinreleasing hormone-induced gastric ulcers through the vagal system, Scand. J. Physiol., 29 (1994) 226-231. [23] Porreca, F. and Burks, T.F., Centrally administered bombesin affects gastric emptying and small and large transit in the rat, Gastroenterology, 85 (1983) 313-317. [24] Rogers, R.C. and Hermann, G.E., Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility, Peptides, 8 (1987) 505-513. [25] Scarpignato, C., Capovilla, T. and Bertacini, G., Action of caerulein on gastric emptying of the conscious rats, Arch. Int. Pharmacodyn. Then, 246 (1980) 286-294. [26] Stephens, R.L., Ishikawa, T., Weiner, H., Novin, D. and TachS, Y., TRH analogue, RX77368, injected into the dorsal vagal complex stimulates gastric secretion in rats, Am. J. Physiol., 254 (Gastrointest. Liver Physiol. 17) (1988) G639-G643. [27] Tach6, Y., Maeda-Hagiwara, M. and Turkelson, C.M., Central nervous system action of corticotropin-releasing factor to inhibit gastric emptying in rats, Am. J. Physiol., 253 (1987) G241-G245. [28] TachS, Y., Vale, W. and Brown, M., Thyrotropin-releasing hormone: CNS action to stimulate gastric acid secretion, Nature, 287 (1980) 149-151. [29] Tach6, Y., Yang, H. and Yoneda, M., Vagal regulation of gastric function involves thyrotropin-releasing hormone in the medullary raphe nuclei and dorsal vagal complex, Digestion, 54 (1993) 65-72. [30] Takeuchi, K., Ueki, S. and Okabe, S., Importance of gastric motility in the pathogenesis of indomethacin-induced gastric lesions in rats, Dig. Dis. Sci., 31 (1986) 1114-1122. [31] Yang, H., Ohning, G. and Tach6, Y., TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats, Am. J. Physiol., 265 (Gastrointest. Liver Physiol. 28) (1993) G880-G886. [32] Yang, H., Wu, S.V., Ishikawa, T. and Tach6, Y., Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei; relationship with vagally mediated gastric erosions, Neuroscience, 61 (1994) 655-663. [33] Yang, H., Wu, V. and Tach6, Y., Medullary thyrotropin-releasing hormone (TRH) mRNA expression is increased after thyroidectomy, Gastroenterology, 102 (1992) A 767. [34] Yano, S., Akahane, M. and Harada, M., Role of gastric motility in development of stress-induced gastric lesions of rats, Jpn. J. Pharmacol., 28 (1978) 607-615.