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MODULATION BY CENTRAL SEROTONIN OF THE GASTRIC ULCERATION INDUCED BY WATER-IMMERSION STRESS IN THE RAT
MODULATION GASTRIC
BY CENTRAL ULCERATION
IMMERSION Hiroshi Y. WATANABE,
SEROTONIN
INDUCED
STRESS
IN THE
OF THE
BY WATER RAT
Hiroshi KUNO and Kazuo WATANABE
Research Institute for Wakan-yaku, Toyama University, Toyama, Japan Accepted February 5, 1975
Reciprocal
regulation by cholinergic and noradrenergic
been previously investigated traventricular
administration
in gastric ulceration of 6-hydroxydopamine
caused
neuronal mechanisms has by stress (1-4).
An in
reportedly prevents the formation
of gastric ulcer induced by water-immersion stress in rats, and it was suggested that the catecholaminergic neuron of the brain may be involved in the formation of gastric ulcers (5). However, the role of serotonin (5HT) in response to stress has been little investigated despite the known distribution of serotonergic neurons in the CNS. To observe the changes in response to stress in the 5HT-depleted rat, we examined the incidence of gastric ulcera tion induced by water-immersion stress in rats treated with p-chlorophenylalanine and found that gastric ulcer formation was exacerbated in such animals.
(PCPA),
SHORT COMMUNICATIONS Japan. J. Pharmacol.25, 217 (1975) Male Wistar rats weighing 200 to 250 g were given i.p. 400 mg/kg of PCPA. After a 12hr fast (40 hr after PCPA), each rat was placed in a multi-perforated 20 cm long plastic cylinder, and immersed in water for 8 hr to the depth of the xiphoid process according to the method of Takagi et al. (6). The water temperature was maintained at 25°C. After water-immersion stress, the rats were decapitated, and the stomachs and brains excised. The size of the ulcer in the glandular stomach was measured under a dissection microscope and the total length was expressed as the ulcer index in nom. The forebrain was dissected for amine assay. 5HT and 5-hydroxyindoleaccticacid (5HIAA) contents were deter mined by the methods of Maickel et al. (7) and Curzon and Green (8). In the specimens from the rat which had been given L-5-hydroxytryptophan(5HTP) before water-immersion, borate wash was added twice for 5HT determination, and the 5HIAA determination was omitted. Dopamine (DA) and noradrenaline (NA) were assayed according to a modifica tion of Hogan's method as quoted in Ref. 9. Unhandled specimens from naive controls sacrificedfor amine assay were left undisturbed during course of the experiment. Student's t-test was used to determine the statistical difference between control and drug groups. Marked hemorrhage and gastric ulceration in the rat stomach were produced by water immersion for 8 hr. 5HT content of the forebrain was reduced about 9 % as compared with the naive controls. This slight reduction of the 5HT content and the remarkable in crease of the 5HIAA content in the forebrain may be due to an increased turnover of 5HT as a result of increased nerve impulse flow in the serotonergic neuron, since our present results are in parallel to those of Sheard and Aghajanian (10) that an electrical stimulation of the midbrain raphe yielded a fall in 5HT and a rise in 5HIAA in the rat forebrain. PCPA (400 mg/kg, i.p.) caused a statistically significant exacerbation of the gastric ulceration caused by water-immersion,as compared with the CMC-salinecontrol (Table 1). 5HT and 5HIAA contents of the forebrain in PCPA-treated rats were less than 27% those of the control after water-immersion (Table 2). The NA content of the forebrain decreased to TABLE 1. Effectof PCPA alone or in combinationwith an amine precursoron gastric hemorrhageand ulcerationinduced by water-immersionstress in the rat.
The rat was immersed (for 8 hr) 40 hr after 400 mg/kg of PCPA. Ro 4 4602 (50 mg, kg) was given 1.5 hr before and L-5HTP (100 mg, kg) or L dopa (100 mg/kg) was given 1 hr before water-immersion. a) Hemorrhage in stomach and intestine was roughly estimated as +, ++ or 4H. b) Size of the gastric ulcer was measured under dissection microscope and expressed as ulcer index in mm. ** P< .01 compared to rats injected with CMC-saline.
SHORT
COMMUNICATIONS
Japan. J . Pharmacol. 25, 218 (1975)
TABLE2. CDncentratiDns of 5HT and 5HIAA* in rat forebrain after water immersion stress.
Rats were sacrificed after water-immersion stress. The concentration of amine was expressed as <100 ng,ig when a fluorescence peak the same wavelength as that obtained with 5HT was not obtained in the brain extract. * ng'g wet tissue , mean+S.E.M. ** P< .01 compared to unhandled naive control.
77 % that of the naive control, while the DA content did not decrease in the CMC-saline group. DA and NA contents of the forebrain in the PCPA group were lowered to 60 and 72 % respectively of the naive control. Concomitant administration of 100mg/kg of L dihydroxyphenylalanine (L-dopa) and a decarboxylase inhibitor, seryl-2,3,4-trihydroxyl benzylhydrazinehydrochloride (Ro 4-4602), did not exacerbate further the gastric hemor rhage and ulceration. Formation of gastric ulcers due to various stresses has been reported to be affected by peripheral and central catecholaminergicmechanisms (2-5). The present results, how ever, imply that the exacerbation of gastric ulceration due to water-immersion stress in PCPA-treated rats may not be mediated by catecholaminergic mechanisms, but may be explained by peripheral and/or central mechanisms related to 5HT. Administrations of Ro 4-4602 50 mg/kg and 5HTP 100 mg/kg reversed the exacerbation of gastric hemor rhage and ulceration in PCPA-treated rats. 5HT content of the forebrain recovered to the level of the control after concomitant Ro 4-4602 and 5HTP administrations. These results support the theory that increased 5HT synthesis within the brain, as a consequence the drastic stimuli of water-immersion stress, inhibits the formation of gastric hemorrhage and ulceration, although the peripheral mechanism cannot be altogether excluded. Fur ther studies are required to clarify the following: 1) the area in the brain in which 5HT plays a modulatory role in stress-inducedgastric ulceration, and 2) through which system, i.e. cholinergic, noradrenergic or pituitary-adrenal systems, the response of gastric mucosa to stress is modified after depletion of brain 5HT. Acknowledgements: Thanks are due to Mrs. Carla Utech for assistance with the English script. Wistar rats were provided by the Biological Research Inst. of Eisai Co., Ltd., Gifu and L-5HTP by the Ajinomoto Co., Inc., Tokyo. REFERENCES 1) WATANABE, K., Chem. Pharm. Bull., 14,101 (1966): 2) DOTEUCHI,M., Japan. J. Pharmacol., 17, 638 (1967): 3) KIDO, R., Brain & Nerve, 19, 467 (1967) (in Japanese): 4) FUJIWARA, M. AND MORI, J., Saishin Igaku, 25, 2058 (1970) (in Japanese): 5) IKEDA, H., NARUMI, S.,
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 219 (1975)
NAGAWA,Y., SHIMAMOTO, K. AND TANAKA,C., Amine Fluorescence Histochemistry, ed. by Fuji WARA, M. AND TANAKA,C., p. 82, Igaku Shoin, Tokyo (1974): 6) TAKAGI, K., KASUYA,Y. AND WATANABE,K., Chem. Pharm. Bull., 12, 465 (1964): 7) MAICKEL,R.P., Cox, Jr., R.H., SAILLANT,J. AND MILLER, F.P., Int. J. Neuropharmacol., 7, 275 (1968): 8) CURZON, G. AND GREEN, A.R., Brit. J. Pharmacol., 39, 653 (1970): 9) JACOBOWITZ,D.M., COOPER, T. AND BARNER,H.B., Circulation Res., 20, 289 (1967): 10) SHEARD,M.H. AND AGHAJANIAN,G.K., J. Pharmacol. exp. Ther., 163, 425 (1968)
BY CENTRAL ULCERATION
IMMERSION Hiroshi Y. WATANABE,
SEROTONIN
INDUCED
STRESS
IN THE
OF THE
BY WATER RAT
Hiroshi KUNO and Kazuo WATANABE
Research Institute for Wakan-yaku, Toyama University, Toyama, Japan Accepted February 5, 1975
Reciprocal
regulation by cholinergic and noradrenergic
been previously investigated traventricular
administration
in gastric ulceration of 6-hydroxydopamine
caused
neuronal mechanisms has by stress (1-4).
An in
reportedly prevents the formation
of gastric ulcer induced by water-immersion stress in rats, and it was suggested that the catecholaminergic neuron of the brain may be involved in the formation of gastric ulcers (5). However, the role of serotonin (5HT) in response to stress has been little investigated despite the known distribution of serotonergic neurons in the CNS. To observe the changes in response to stress in the 5HT-depleted rat, we examined the incidence of gastric ulcera tion induced by water-immersion stress in rats treated with p-chlorophenylalanine and found that gastric ulcer formation was exacerbated in such animals.
(PCPA),
SHORT COMMUNICATIONS Japan. J. Pharmacol.25, 217 (1975) Male Wistar rats weighing 200 to 250 g were given i.p. 400 mg/kg of PCPA. After a 12hr fast (40 hr after PCPA), each rat was placed in a multi-perforated 20 cm long plastic cylinder, and immersed in water for 8 hr to the depth of the xiphoid process according to the method of Takagi et al. (6). The water temperature was maintained at 25°C. After water-immersion stress, the rats were decapitated, and the stomachs and brains excised. The size of the ulcer in the glandular stomach was measured under a dissection microscope and the total length was expressed as the ulcer index in nom. The forebrain was dissected for amine assay. 5HT and 5-hydroxyindoleaccticacid (5HIAA) contents were deter mined by the methods of Maickel et al. (7) and Curzon and Green (8). In the specimens from the rat which had been given L-5-hydroxytryptophan(5HTP) before water-immersion, borate wash was added twice for 5HT determination, and the 5HIAA determination was omitted. Dopamine (DA) and noradrenaline (NA) were assayed according to a modifica tion of Hogan's method as quoted in Ref. 9. Unhandled specimens from naive controls sacrificedfor amine assay were left undisturbed during course of the experiment. Student's t-test was used to determine the statistical difference between control and drug groups. Marked hemorrhage and gastric ulceration in the rat stomach were produced by water immersion for 8 hr. 5HT content of the forebrain was reduced about 9 % as compared with the naive controls. This slight reduction of the 5HT content and the remarkable in crease of the 5HIAA content in the forebrain may be due to an increased turnover of 5HT as a result of increased nerve impulse flow in the serotonergic neuron, since our present results are in parallel to those of Sheard and Aghajanian (10) that an electrical stimulation of the midbrain raphe yielded a fall in 5HT and a rise in 5HIAA in the rat forebrain. PCPA (400 mg/kg, i.p.) caused a statistically significant exacerbation of the gastric ulceration caused by water-immersion,as compared with the CMC-salinecontrol (Table 1). 5HT and 5HIAA contents of the forebrain in PCPA-treated rats were less than 27% those of the control after water-immersion (Table 2). The NA content of the forebrain decreased to TABLE 1. Effectof PCPA alone or in combinationwith an amine precursoron gastric hemorrhageand ulcerationinduced by water-immersionstress in the rat.
The rat was immersed (for 8 hr) 40 hr after 400 mg/kg of PCPA. Ro 4 4602 (50 mg, kg) was given 1.5 hr before and L-5HTP (100 mg, kg) or L dopa (100 mg/kg) was given 1 hr before water-immersion. a) Hemorrhage in stomach and intestine was roughly estimated as +, ++ or 4H. b) Size of the gastric ulcer was measured under dissection microscope and expressed as ulcer index in mm. ** P< .01 compared to rats injected with CMC-saline.
SHORT
COMMUNICATIONS
Japan. J . Pharmacol. 25, 218 (1975)
TABLE2. CDncentratiDns of 5HT and 5HIAA* in rat forebrain after water immersion stress.
Rats were sacrificed after water-immersion stress. The concentration of amine was expressed as <100 ng,ig when a fluorescence peak the same wavelength as that obtained with 5HT was not obtained in the brain extract. * ng'g wet tissue , mean+S.E.M. ** P< .01 compared to unhandled naive control.
77 % that of the naive control, while the DA content did not decrease in the CMC-saline group. DA and NA contents of the forebrain in the PCPA group were lowered to 60 and 72 % respectively of the naive control. Concomitant administration of 100mg/kg of L dihydroxyphenylalanine (L-dopa) and a decarboxylase inhibitor, seryl-2,3,4-trihydroxyl benzylhydrazinehydrochloride (Ro 4-4602), did not exacerbate further the gastric hemor rhage and ulceration. Formation of gastric ulcers due to various stresses has been reported to be affected by peripheral and central catecholaminergicmechanisms (2-5). The present results, how ever, imply that the exacerbation of gastric ulceration due to water-immersion stress in PCPA-treated rats may not be mediated by catecholaminergic mechanisms, but may be explained by peripheral and/or central mechanisms related to 5HT. Administrations of Ro 4-4602 50 mg/kg and 5HTP 100 mg/kg reversed the exacerbation of gastric hemor rhage and ulceration in PCPA-treated rats. 5HT content of the forebrain recovered to the level of the control after concomitant Ro 4-4602 and 5HTP administrations. These results support the theory that increased 5HT synthesis within the brain, as a consequence the drastic stimuli of water-immersion stress, inhibits the formation of gastric hemorrhage and ulceration, although the peripheral mechanism cannot be altogether excluded. Fur ther studies are required to clarify the following: 1) the area in the brain in which 5HT plays a modulatory role in stress-inducedgastric ulceration, and 2) through which system, i.e. cholinergic, noradrenergic or pituitary-adrenal systems, the response of gastric mucosa to stress is modified after depletion of brain 5HT. Acknowledgements: Thanks are due to Mrs. Carla Utech for assistance with the English script. Wistar rats were provided by the Biological Research Inst. of Eisai Co., Ltd., Gifu and L-5HTP by the Ajinomoto Co., Inc., Tokyo. REFERENCES 1) WATANABE, K., Chem. Pharm. Bull., 14,101 (1966): 2) DOTEUCHI,M., Japan. J. Pharmacol., 17, 638 (1967): 3) KIDO, R., Brain & Nerve, 19, 467 (1967) (in Japanese): 4) FUJIWARA, M. AND MORI, J., Saishin Igaku, 25, 2058 (1970) (in Japanese): 5) IKEDA, H., NARUMI, S.,
SHORT
COMMUNICATIONS
Japan. J. Pharmacol. 25, 219 (1975)
NAGAWA,Y., SHIMAMOTO, K. AND TANAKA,C., Amine Fluorescence Histochemistry, ed. by Fuji WARA, M. AND TANAKA,C., p. 82, Igaku Shoin, Tokyo (1974): 6) TAKAGI, K., KASUYA,Y. AND WATANABE,K., Chem. Pharm. Bull., 12, 465 (1964): 7) MAICKEL,R.P., Cox, Jr., R.H., SAILLANT,J. AND MILLER, F.P., Int. J. Neuropharmacol., 7, 275 (1968): 8) CURZON, G. AND GREEN, A.R., Brit. J. Pharmacol., 39, 653 (1970): 9) JACOBOWITZ,D.M., COOPER, T. AND BARNER,H.B., Circulation Res., 20, 289 (1967): 10) SHEARD,M.H. AND AGHAJANIAN,G.K., J. Pharmacol. exp. Ther., 163, 425 (1968)