Original Paper Digestion 1992;53:149-156
H. Kaneko T. Mitsuma K. Moriseb K. Uchida A. Furnsawab Y. Maedah K. Nakada K. Adachi a -b a
a -b
Effect of Serotonin on the Immunoreactive Thyrotropin-Releasing Hormone Concentrations of the Rat Stomach
a
a
Keywords Serotonin 5-HT2-receptor 5-HT3-receptor Thyrotropin-releasing hormone
Abstract The effects of serotonin and its related compounds on immu noreactive thyrotropin-releasing hormone (ir-TRH) concen trations of the rat stomach wall and gastric juice were studied. Either serotonin, cyproheptadine or GR38032F was injected intraperitoneally, and the rats were decapitated at various times after the injection. ir-TRH concentrations of the stom ach wall and gastric juice were measured by radioimmunoas say, and gastric serotonin concentrations were measured by HPLC. ir-TRH concentrations of the stomach wall decreased, and ir-TRH concentrations of gastric juice increased signifi cantly after serotonin injection. On the other hand, both cyproheptadine and GR38032F did not affect ir-TRH concen trations of the stomach. The effects of serotonin on ir-TRH concentrations of the stomach were significantly blocked by the pretreatment of cyproheptadine and GR38032F. The reciprocal changes of gastric ir-TRH and serotonin concentra tions were observed without changes in gastric juice pH. These findings suggest that serotonin stimulates ir-TRH release from the stomach wall into gastric juice, and the effects of serotonin on ir-TRH release may be partly mediated via 5-HT 2- and 5-HT3-receptors.
Thyrotropin-releasing hormone (TRH), a tripeptide originally isolated from the hypo thalamus [ 1], has subsequently been shown to be widely distributed in the mammalian body, including the stomach [2-4], TRH has
Received: May 26.1992 Received in revised form: September 10. 1992
been reported to increase gastric acid secre tion and to augment stress-induced ulcers in the rat when centrally administered [5], We have previously reported that histamine stim ulated immunoreactive-TRH (ir-TRH) re-
Hiroshi Kaneko, MD Fourth Department of Internal Medicine Aichi Medical University 21 Karimata. Yazako. Nagakutc Aichi 480-11 (Japan)
© 1992 S. Kargcr AG. Basel 0012-2823/92/ 0534-0149S2.75/0
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a Fourth Department of Internal Medicine, Aichi Medical University, Aichi, Japan, and b First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
Materials and Methods Animals Male Wistar strain rats, weighing 200-250 g, were utilized. Prior to study, the animals were housed in large cages with mesh wire bottoms to prevent coprophagia in a room maintained at constant temperature (23 ± 2°C) and humidity (60 ± 5%), with 12:12 h light-dark cycles. The rats were fed a standard diet of laboratory chow and conditioned to the room environ ment for at least 15 days prior to inclusion in the study. Drugs The following substances were used: 5-hydroxytrvptamine hydrochloride (serotonin: Sigma. St. Louis. Mo., USA), cyproheptadine hydrochloride (5-HTircccptor antagonist: Sigma) and GR38032F (5-HT?receptor antagonist: Glaxo. Ware. UK). All tested sub stances were dissolved in saline. Experimental Design All rats were deprived of food but not water for 24 h prior to the beginning of the experiments. Saline or tested substances were injected intraperitoneally. The rats were divided into five groups and were all sac
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rificed between 13:00 and 15:00 h to prevent diurnal variations of hormones. Group I, composed of 40 rats, were given sero tonin (30mg/kg). and 10 rats in each subgroup were decapitated at 0, 15. 30 and 60 min. respectively, after the injection. The rats in the 0-min subgroup were decapitated immediately after the injection. Twenty rats were used for ir-TRH concentration measure ment. and the remaining 20 for measuring serotonin concentration and gastric juice pH with a digital pH meter (CG817. Schott Geräte. Hofheim, FRG). The 20 rats of group 2 were injected with cyproheptadine (500 pg/kg), and 5 rats in each subgroup were decapi tated at 0, 15, 30 and 60 min. respectively, after the injection. In group 3, 20 rats were administered GR38032F (10 0 pg/kg). and 5 rats in each subgroup were decapitated at 0, 15, 30 and 60 min. respectively, after the injection. In group 4. serotonin (20-90 mg/ kg) was administered to the rats, and 5 rats in each subgroup were decapitated at 15 min after serotonin injection. In group 5, rats were pretreated with cypro heptadine (500 pg/kg) or GR38032F (100 pg/kg) 20 min before serotonin injection and decapitated 15 min after serotonin injection. The 20 rats of group 6 were given 1.0 ml of vehicle, and rats in each subgroup were decapitated at 0, 15, 30 and 60 min, respectively, after the injection for measuring ir-TRH concentration and gastric juice pH. Preparation o f Stomach Wall and Gastric Juice The rats were decapitated by guillotine under ether anesthesia, and the abdomen was opened by midline incision. The stomach was then removed, and 1 ml of saline was poured out of pyloric ring into the stomach, and the gastric juice was drawn as a solution contained in the saline, as previously described [6], All specimens were stored at -2 0 °C until assayed [ 12]. Extraction and Assay o f TRH TRH was extracted by the method reported pre viously [12]. In brief, the rat stomach wall was boiled with 1.0 ml of 0.1 N HC1 for 10 min at 90 °C. and then 5.0 ml of cold methanol were added. The stomach wall was homogenized, and the mixture was centrifuged. The resultant supernatants were dried under an air stream in a water bath (56 °C). Extraction of TRH from the gastric juice was essentially the same as that from the stomach wall except for the boiling proce dure. The recovery rate by these extraction methods was evaluated with the addition of l25l-TRH or a known amount ofTRH (10 ng/ml) to the homogenized stomach. By this extraction method, recovery rate was 70.1 ± 2.2% (mean ± SD; n = 8) in the stomach wall
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lease from the rat stomach wall in vivo and in vitro [6. 7] and that luminal TRH might exert a regulatory effect on gastric acid secretion [8], Changes in human gastric ir-TRH con centrations have also been demonstrated in patients with peptic ulcer disease [9], These findings suggest that gastric TRH may play a role in gastric mucosal damage in rats and humans. On the other hand, serotonin (5hydroxytryptamine: 5-HT) that induces gas tric ulcer [10] has recently attracted attention with the development of 5-HTv-reccptor an tagonists, which have a strong antiemetic ef fect [11], But no study concerning the effect of serotonin on TRH in rat stomach in vivo has been reported. Therefore, the present study was designed to investigate the effects of sero tonin and its related compounds on the con centrations of ir-TRH of the stomach wall and gastric juice in rats.
Measurement ofSerotonin Concentrations o f the Stomach Wall The rat stomach wall was homogenized with 1.0 ml of 4.0 N perchloric acid (HCIO4). and serotonin was extracted. Serotonin concentrations were measured by HPLC methods previously described with a minor modification [13]. The apparatus consisted of a Mod el-590 pump (Millipore, Milford. USA). Model-460 detector (Millipore) with a glassy carbon electrode and a reverse phase chromatographic column (4.6 X 150 mm i.d.; EiCOM, Kyoto, Japan). The mobile phase was prepared from concentrated solutions of 0.1 M sodium acetate, 5% acetonitrile. 0.1 M citrate, and pH was adjusted to 3.7. The flow rate and column temperature were 1.0 ml/min and 25 °C, respectively. The recovery rate of extraction in this method was approximately 80%. Serotonin concentrations were expressed as nanograms per milligram wet weight. Stability and Recovery o f TRH in the Homogenates o f the Stomach Wall. Gastric Juice or Plasma The inactivation of TRH immunoreactivity with rat stomach was examined by the following method. One hundred nanograms of synthetic TRH were added to 1,200 mg wet weight of homogenated stomach wall, 1.0 ml of gastric juice or 1.0 ml of plasma (in 1.0 ml 0.01 M phosphate buffer. pH 7.4) at 4 °C after seroton in, cyproheptadine or GR38032F injection and then incubated at 37 °C for 30 min. The recovery of added TRH was measured by a radioimmunoassay, and the results were expressed as a percent of TRH added. Sta bility of TRH in gastric juice was examined as follows: 10 ng/ml of synthetic TRH were added to pH 2 or 7 of human gastric juice, then the gastric juice was incu-
Fig. 1. Changes in serotonin concentrations of the rat stomach wall after intraperitoneal injection of sero tonin (30 mg/kg). Values are expressed as means ± SE in each group of 5 rats.a p < 0.05 vs. the 0-min group, w.w. = Wet weight.
bated for 10, 30 or 60 min at 37 °C, and recoveries of added TRH were measured. Statistics Data are expressed as means ± SE for each group. The statistical significance of difference between the control and the experimental groups was analyzed using one-way analysis of variance, followed by the Dunnett’s test for nonpaired comparisons for ir-TRH and serotonin concentrations and by the Kruskal-Wallis test for gastric juice pH. In all the statistical tests, p values of 0.05 or less were considered to represent significant differences.
Results After intraperitoneal serotonin injection, serotonin concentration of the stomach wall increased significantly at 15 min and then rapidly decreased over 30 min (fig. 1), while pH in gastric juice tended to decrease up to 30 min, but not significantly, and increased significantly at 60 min (fig. 2). No macro scopic lesion was observed within 60 min af ter serotonin injection. 151
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and 70.4 ± 4.2% (mean ± SD; n = 8) in the gastric juice. TRH was measured by radioimmunoassay [12]. The elution profile of TRH in the acid-methanolextracted rat stomach on a Sephadex G-10 column (10 X 150 mm. Pharmacia. Uppsala, Sweden) showed a single peak and was identical to that of synthetic TRH, and the dilution curve of acid-methanol-extractcd stomach in a TRH radioimmunoassay system was parallel with the standard curve of synthetic TRH as reported previously [6], The intra-assay coefficient of variation, when assessed by five identical samples, was 6.0%, the interassav coefficient of variation, when assessed by five identical samples five times, was 9.0%. ir-TRH concentrations of the stomach wall were ex pressed as picograms per milligram wet weight, and irTRH concentrations of gastric juice were expressed as picograms per milliliter.
Gastric juice
Stomach wall
Stom ach wall
Fig. 2. Changes in pH in gastric juice after intrapcritoneal injection of serotonin (30 mg/kg) or vehicle. Values are expressed as means ± SE in each group of 5 rats. b p < 0.0! vs. the 0-min group,c p < 0.05 vs. the control group. • ----- • = Serotonin; o----- o = vehicle. Fig. 3. Changes in ir-TRH concentrations of the rat stomach wall and gastric juice after intraperitoneal injection of serotonin (30 mg/kg) or vehicle. Values are expressed as means ± SE in each group of 5 rats.a p < 0.05,b p < 0.01, vs. the 0-min group. cp < 0.05.d p < 0.01, vs. the control group. •----- • = Serotonin; o----- o = vehicle; w.w = wet weight. Fig. 4. Effects of various doses of intraperitoneally administered serotonin on ir-TRH concentrations of the rat stomach wall and gastric wall. w.w. = Wet weight. Values arc expressed as means ± SE in each group of 5 rats.a p < 0.05;b p < 0.01. vs. the control group.
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The concentrations of ir-TRH in the nor mal rat stomach wall and gastric juice were 0.88 ± 0.08 pg/rng wet weight and 42.0 ± 3.7 pg/ml, respectively. In the serotonintreated group. ir-TRH concentrations of the stomach wall decreased significantly at 15 min after injection, and ir-TRH concentra tion of gastric juice increased significantly, peaking at 15 min (fig. 3). However, no signif icant changes in ir-TRH concentrations of the stomach wall and gastric juice were found either in the cyproheptadine- or GR38032Ftreated group (data not shown). The effects of serotonin on ir-TRH con centrations of the stomach wall or gastric juice tended to be dose dependent (fig. 4). ir-TRH concentrations of the plasma showed no changes with any drug (data not shown). The effects of serotonin on ir-TRH concentrations in the stomach wall and gastric juice were significantly blocked by the pretreatment of cyproheptadine or GR38032F (fig. 5).
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Gastric juice
Stomach wall
Gastric juice
Fig. 5. Effects of serotonin on ir-TRH concentrations of the stomach wall and gastric juice in rats with cyproheptadine or GR38032F pretreatment, w.w. = Wet weight. Values are expressed as means ± SE in each group of 5 rats.h p < 0.01 vs. the group with serotonin injection alone.
TRH irnmunoreactivity was not dimin ished by the incubation with either pH 2.0 or 7.0 of gastric juice for 60 min (fig. 6). The inactivation of TRH irnmunoreactivity by the stomach wall, gastric juice or plasma in vi tro after serotonin, cyproheptadine or GR38032F injection was around 63.4 ± 1.0% (mean ± SE) by stomach wall. 99.6 ± 0.8% by gastric juice and 7.2 ± 0.5% by plas ma. and did not differ from that of control.
Evidences of widespread localization of TRH and its many actions upon the gastroin testinal tract [2, 14] indicate that this com pound probably has certain roles in the gas trointestinal tract. We previously reported the presence of TRH both in the stomach wall and in gastric juice, and ir-pro-TRH (TRHprecursor peptide) in the stomach wall [15], suggesting that TRH is synthesized in the
stomach wall and secreted directly into the gastric lumen. It is also well known that sero tonin influences gastric function [16]. On the other hand, relationships between serotonin and TRH have been supposed by a few evi dences including stimulated gastric luminal serotonin release by intracistemal TRH injec-
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Discussion
Fig. 6. Stability ofTRH in gastric juice. The inacti vation of TRI I irnmunoreactivity by gastric juice was not found up to 60 min at pH 2.0 and 7.0 of gastric juice.
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blocked by the pretreatmcnl of cyprohep tadine (5-HT2-receptor antagonist) and GR38032F (5-HT3-receptor antagonist). Mitsuma et al. [21] have earlier reported that serotonin stimulated ir-TRH release from the rat stomach in vitro, which agrees with the present study. Both endogenous and exoge nous serotonin has been proposed to be closely related to gastric mucosal damage without increase in the gastric acid output [23] , in agreement with the present study. The question can be raised as to whether the increase in ir-TRH concentrations of gastric juice might be due to contamination from the blood. However, the concentrations of gastric juice ir-TRH were much higher than those in plasma [7], and no macroscopic mucosal damage was observed within 60 min after se rotonin injection. On the other hand, the sero tonin concentration of the stomach wall in creased significantly at 15 min after serotonin injection and rapidly decreased over 30 min, then the peak change in ir-TRH concentra tion of the stomach wall was identical to that of the serotonin concentration in the stomach wall. Therefore, the present study and a pre vious report taken together suggest that sero tonin acts on the stomach and stimulates irTRH release. The importance of serotonin has been re vealed by the identification of different sero tonin receptors, 5-HT,, 5-HT3 and 5-HT3, and the development of drugs interferes selec tively with different serotonergic systems [11], We examined the effects of 5-HT 2- and 5-HT3-receptor antagonists, because both 5HT 2- and 5-HT3-receptor antagonists have been widely used for some gastrointestinal dysfunction and 5-HT 1-receptor is known to be similar to [i-adrenergic receptor. It is well known that GR38032F is effective in the pro phylaxis of emesis induced by chemotherapy [24] , while cyproheptadine, whose effects on ir-TRH concentrations of the rat stomach in
Kaneko/Milsuma/Morise/Uchida/ Furusawa/Maeda/Nakada/Adachi
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tion in rats [17] or coexistence of serotonin and TRH in medullary neurons projecting to the spinal cord [ 18]. Furthermore, histological studies revealed that both TRH and serotonin existed in endocrine cells (G and EC cells, respectively) of the gastric mucosa [ 19, 20]. Therefore, it was though that the investiga tion into the effects of serotonin on TRH release from the rat stomach wall into the gas tric lumen would presumably serve to clarify the physiological significance of TRH in the stomach. In order to study the effect of serotonin on ir-TRH concentrations of the rat stomach, one should examine experimental conditions. First, we examined several basic conditions of our TRH assay system. TRH immunoreactivity was not changed by incubation of either pH 2.0 or 7.0 of gastric juice up to 60 min. The inactivation of ir-TRH by stomach or plasma in vitro after serotonin and its related compound injection was not different from the control. From these findings, it is sug gested that the present experimental condi tions allow measurement of TRH in the rat stomach, and that changes in ir-TRH concen trations would not be dependent on the degra dations of TRH in the rat stomach or plasma. On the other hand, serotonin concentrations of the stomach in the present study were simi lar to those reported elsewhere [10]. There fore, the serotonin assay system used in the present study is also suitable to measure the serotonin concentration of the rat stomach. The doses of drugs, which were not physiolog ical but pharmacological in the present study, were similar to those in previous reports [10, 21, 22 ], The present study demonstrated that sero tonin decreased ir-TRH concentrations of the stomach wall and increased ir-TRH concen trations of gastric juice in a dose-dependent manner, and that the effects of serotonin on ir-TRH concentrations of the stomach were
vitro were previously reported [21], is presently used in patients with dumping syn drome [25]. In the present study, the effects of serotonin on ir-TRH concentrations were sig nificantly blocked by cyproheptadine or GR38032F pretreatment. Different changes in the effects on ir-TRH concentrations in the rat stomach between cyproheptadine and GR38032F administration were not observed in the present experimental conditions. The characteristic hallmark of action of serotonin has been a quite remarkable variability of effect between species and different areas of the gastrointestinal tract, among animals of the same species and in the same individual over time [ 11 ]. On the other hand, it has been estimated that selectivity of several serotonin antagonists was not so high, and some antago nists might partly act as other serotonin ago nists [11] and vice versa. From the previous and present data, it is suggested that the effects of serotonin on ir-TRH concentrations
in rat are at least partly mediated via both 5HT2- and 5-HT3-receptors. Since the dose of serotonin used in the present study is high, it cannot elucidate the physiological signifi cance of TRH in the stomach from the present study. Further studies, such as using passive immunization or TRH antagonists, will be necessary to clarify the physiological importance of the gastric TRH. In summary, the present investigation sug gests that serotonin stimulates ir-TRH release from the stomach and that the effects of sero tonin are mediated partly via both 5-HT2and 5-HT3-receptors.
Acknowledgment The author wish to express their heartfelt thanks to Prof. Hidehiko Saito. MD, First Department oflnternal Medicine. Nagoya University School of Medicine. Nagoya. Japan, for his kind guidance and critical review of the manuscript.
1 Boter J. Enzmann F. Folkers K. Bowers C'Y. Schally AV: The iden tity of chemical and hormonal prop erties of the thyrotropin-releasing hormone and pyroglutamyl-histidvlproline amide. Biochem Biophys Res Commun 1969:37:705—710. 2 Morley JE: Extrahypothalamic thy rotropin-releasing hormone (TRH) - Its distribution and its function. Life Sci 1979;25:1539-1550. 3 Leppaluoto J. Koivusalo F. Kraama R: Thyrotropin-releasing factor: Distribution in neural and gastroin testinal tissues. Acta Physiol Scand 1978:104:175-179. 4 Dolva L0. Hanssen KF. Aadland E. Sand T: Thyrotropin-releasing hor mone immunoreactivity in the gas trointestinal tract of man. J Clin En docrinol Metab 1983;56:524-529.
5 Taché Y, Vale W. Brown M: Thyro tropin-releasing hormone - CNS ac tion to stimulate gastric acid secre tion. Nature 1980:287:149-151. 6 Nakada K, MitsumaT. Furusawa A. Maeda Y. Morise K: The effects of histamine on the concentrations of immunoreactive thyrotropin-releas ing hormone in the stomach and hy pothalamus in rats. Gastroenterol Jpn 1990;25:425-431. 7 Mitsuma T. Sun DH. Nogimori T. Chaya M, Ohtake K. Hirooka Y: Effects of histamine and related compounds on the release of imrnunoreactivc thyrotropin-releasing hormone from the rat stomach in vitro. Peptides 1987:8:473-475.
8 Furusawa A, Morise K. Maeda Y. Uchida K. Kaneko H. Nakada K. Mitsuma T: Effect of luminal ad ministration of thyrotropin-releas ing hormone or somatostatin on gas tric pH and interaction of these pep tides in rats. Gastroenterol Jpn 1992:27:165-171. 9 Nakada K, Mitsuma T, Furusawa A, Maeda Y. Iizuka A. Morise K: TRH. VIP and somatostatin in human gas tric juice. Biochem Res 1988; 9(supp! I): 141. 10 Furuhashi S: Experimental study of gastric ulcer - Serotonin ulcer. Jpn J Gastroenterol 1968:65:670-682. 11 Costall B. Naylor RJ: 5-Hydroxytryptamine: New receptors and novel drugs for gastrointestinal mo tor disorders. Scand J Gastroenterol 1990:25:769-787.
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References
18 Johansson O. Hökfelt T. Pemow B. Jeffcoate SL. White N. Steinbusch HWM. Verhofstad AAJ. Emson PC. Spindel E: Immunohistochemical support for three putative transmit ters in one neuron: Coexistence of 5hydroxytryptamine. substance P and thyrotropin-releasing hormone like immunoreactivity in medullary' neurons projecting to the spinal cord. Neuroscience 1981:6:18571881. 19 Tsuruo Y. Hökfelt T. Visser TJ. Kimmel JR. Brown JC, Verhofsladt A, Walsh J: TRH-like immunorcactivitv in endocrine cells and neurons in the gastro-intestinal tract of the rat and guinea pig. Cell Tissue Res 1988:253:347-356. 20 Rubin W. Schwartz B: Identifica tion of the serotonin-synthesizing endocrine cells in the rat stomach by electron microscopic radioaulography and amine fluorescence. Gas troenterology 1983:84:34-50.
21 Mitsuma T, Hirooka Y, Nakada K. Nomura A. Sun DH: Effects of sero tonin on the release of thyrotropin releasing hormone from the rat stomach in vitro. J Aichi Med Univ Assoc 1987;15:765-770. 22 Costall B. Gunning SJ. Naylor RJ. Tyers MB: The effect of GR38032F. novel 5-HTj-receptor antagonist on gastric emptying in the guinea-pig. BrJ Pharmacol 1987:91:263-264. 23 Canfield SP. Spencer JE: The inhibi tory effects of 5-hydroxytryptamine on gastric acid secretion by the rat isolated stomach. Br J Pharmacol 1983:78:123-129. 24 Stables R, Andrews PER. Bailey HE. Costall B, Gunning SJ, Haw thorn J. Naylor RJ. Tyers MB: An tiemetic properties of the 5HTweceptor antagonist. GR38032F. Can cer Treat Rev 1987; 14:333—336. 25 Johnson LP. Sloop RD. Jesseph JE. Harkins HN: Serotonin antagonists in experimental and clinical ‘dump ing’. Ann Surg 1962;156:537-549.
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12 Mitsuma T, Hirooka Y. Nihei N: Radioimmunoassay of thyrotropin releasing hormone in human serum and its clinical application. Acta En docrinol 1976;83:225-235. 13 Kumazawa T. Adachi K. Ando K: Simultaneous determination of ca techols in thalamic slices with liq uid ehromatography/elcctrochemistry. LifeSci 1986;40:41-45. 14 Hernandez DE: Neuroendocrine mechanisms of stress ulceration: Fo cus on thyrotropin-releasing hor mone (TRH). LifeSci 1986:39:279296. 15 Rhu R. Sobue G. Hirooka Y. Mit suma T: Immunohistochemical dis tribution of pro-thvrotropin-releasing hormone in the rat. J Aichi Med Univ Assoc 1989:17:665-672. 16 Bech K: Effect of serotonin on bethancchol-stimulated gastric acid se cretion and gastric antral motility in dogs. Scand J Gastroenterol 1986: 21:655-661. 17 Stephens RL, Taché Y: Intracisternal injection of a TRH analogue stimulates gastric luminal serotonin release in rats. Am J Physiol 1989: 256:G377-G383.
H. Kaneko T. Mitsuma K. Moriseb K. Uchida A. Furnsawab Y. Maedah K. Nakada K. Adachi a -b a
a -b
Effect of Serotonin on the Immunoreactive Thyrotropin-Releasing Hormone Concentrations of the Rat Stomach
a
a
Keywords Serotonin 5-HT2-receptor 5-HT3-receptor Thyrotropin-releasing hormone
Abstract The effects of serotonin and its related compounds on immu noreactive thyrotropin-releasing hormone (ir-TRH) concen trations of the rat stomach wall and gastric juice were studied. Either serotonin, cyproheptadine or GR38032F was injected intraperitoneally, and the rats were decapitated at various times after the injection. ir-TRH concentrations of the stom ach wall and gastric juice were measured by radioimmunoas say, and gastric serotonin concentrations were measured by HPLC. ir-TRH concentrations of the stomach wall decreased, and ir-TRH concentrations of gastric juice increased signifi cantly after serotonin injection. On the other hand, both cyproheptadine and GR38032F did not affect ir-TRH concen trations of the stomach. The effects of serotonin on ir-TRH concentrations of the stomach were significantly blocked by the pretreatment of cyproheptadine and GR38032F. The reciprocal changes of gastric ir-TRH and serotonin concentra tions were observed without changes in gastric juice pH. These findings suggest that serotonin stimulates ir-TRH release from the stomach wall into gastric juice, and the effects of serotonin on ir-TRH release may be partly mediated via 5-HT 2- and 5-HT3-receptors.
Thyrotropin-releasing hormone (TRH), a tripeptide originally isolated from the hypo thalamus [ 1], has subsequently been shown to be widely distributed in the mammalian body, including the stomach [2-4], TRH has
Received: May 26.1992 Received in revised form: September 10. 1992
been reported to increase gastric acid secre tion and to augment stress-induced ulcers in the rat when centrally administered [5], We have previously reported that histamine stim ulated immunoreactive-TRH (ir-TRH) re-
Hiroshi Kaneko, MD Fourth Department of Internal Medicine Aichi Medical University 21 Karimata. Yazako. Nagakutc Aichi 480-11 (Japan)
© 1992 S. Kargcr AG. Basel 0012-2823/92/ 0534-0149S2.75/0
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a Fourth Department of Internal Medicine, Aichi Medical University, Aichi, Japan, and b First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan
Materials and Methods Animals Male Wistar strain rats, weighing 200-250 g, were utilized. Prior to study, the animals were housed in large cages with mesh wire bottoms to prevent coprophagia in a room maintained at constant temperature (23 ± 2°C) and humidity (60 ± 5%), with 12:12 h light-dark cycles. The rats were fed a standard diet of laboratory chow and conditioned to the room environ ment for at least 15 days prior to inclusion in the study. Drugs The following substances were used: 5-hydroxytrvptamine hydrochloride (serotonin: Sigma. St. Louis. Mo., USA), cyproheptadine hydrochloride (5-HTircccptor antagonist: Sigma) and GR38032F (5-HT?receptor antagonist: Glaxo. Ware. UK). All tested sub stances were dissolved in saline. Experimental Design All rats were deprived of food but not water for 24 h prior to the beginning of the experiments. Saline or tested substances were injected intraperitoneally. The rats were divided into five groups and were all sac
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rificed between 13:00 and 15:00 h to prevent diurnal variations of hormones. Group I, composed of 40 rats, were given sero tonin (30mg/kg). and 10 rats in each subgroup were decapitated at 0, 15. 30 and 60 min. respectively, after the injection. The rats in the 0-min subgroup were decapitated immediately after the injection. Twenty rats were used for ir-TRH concentration measure ment. and the remaining 20 for measuring serotonin concentration and gastric juice pH with a digital pH meter (CG817. Schott Geräte. Hofheim, FRG). The 20 rats of group 2 were injected with cyproheptadine (500 pg/kg), and 5 rats in each subgroup were decapi tated at 0, 15, 30 and 60 min. respectively, after the injection. In group 3, 20 rats were administered GR38032F (10 0 pg/kg). and 5 rats in each subgroup were decapitated at 0, 15, 30 and 60 min. respectively, after the injection. In group 4. serotonin (20-90 mg/ kg) was administered to the rats, and 5 rats in each subgroup were decapitated at 15 min after serotonin injection. In group 5, rats were pretreated with cypro heptadine (500 pg/kg) or GR38032F (100 pg/kg) 20 min before serotonin injection and decapitated 15 min after serotonin injection. The 20 rats of group 6 were given 1.0 ml of vehicle, and rats in each subgroup were decapitated at 0, 15, 30 and 60 min, respectively, after the injection for measuring ir-TRH concentration and gastric juice pH. Preparation o f Stomach Wall and Gastric Juice The rats were decapitated by guillotine under ether anesthesia, and the abdomen was opened by midline incision. The stomach was then removed, and 1 ml of saline was poured out of pyloric ring into the stomach, and the gastric juice was drawn as a solution contained in the saline, as previously described [6], All specimens were stored at -2 0 °C until assayed [ 12]. Extraction and Assay o f TRH TRH was extracted by the method reported pre viously [12]. In brief, the rat stomach wall was boiled with 1.0 ml of 0.1 N HC1 for 10 min at 90 °C. and then 5.0 ml of cold methanol were added. The stomach wall was homogenized, and the mixture was centrifuged. The resultant supernatants were dried under an air stream in a water bath (56 °C). Extraction of TRH from the gastric juice was essentially the same as that from the stomach wall except for the boiling proce dure. The recovery rate by these extraction methods was evaluated with the addition of l25l-TRH or a known amount ofTRH (10 ng/ml) to the homogenized stomach. By this extraction method, recovery rate was 70.1 ± 2.2% (mean ± SD; n = 8) in the stomach wall
Kaneko/Mitsuma/Morise/Uchida/ Furusawa/Maeda/Nakada/Adachi
Serotonin and TRH of the Rat Stomach
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lease from the rat stomach wall in vivo and in vitro [6. 7] and that luminal TRH might exert a regulatory effect on gastric acid secretion [8], Changes in human gastric ir-TRH con centrations have also been demonstrated in patients with peptic ulcer disease [9], These findings suggest that gastric TRH may play a role in gastric mucosal damage in rats and humans. On the other hand, serotonin (5hydroxytryptamine: 5-HT) that induces gas tric ulcer [10] has recently attracted attention with the development of 5-HTv-reccptor an tagonists, which have a strong antiemetic ef fect [11], But no study concerning the effect of serotonin on TRH in rat stomach in vivo has been reported. Therefore, the present study was designed to investigate the effects of sero tonin and its related compounds on the con centrations of ir-TRH of the stomach wall and gastric juice in rats.
Measurement ofSerotonin Concentrations o f the Stomach Wall The rat stomach wall was homogenized with 1.0 ml of 4.0 N perchloric acid (HCIO4). and serotonin was extracted. Serotonin concentrations were measured by HPLC methods previously described with a minor modification [13]. The apparatus consisted of a Mod el-590 pump (Millipore, Milford. USA). Model-460 detector (Millipore) with a glassy carbon electrode and a reverse phase chromatographic column (4.6 X 150 mm i.d.; EiCOM, Kyoto, Japan). The mobile phase was prepared from concentrated solutions of 0.1 M sodium acetate, 5% acetonitrile. 0.1 M citrate, and pH was adjusted to 3.7. The flow rate and column temperature were 1.0 ml/min and 25 °C, respectively. The recovery rate of extraction in this method was approximately 80%. Serotonin concentrations were expressed as nanograms per milligram wet weight. Stability and Recovery o f TRH in the Homogenates o f the Stomach Wall. Gastric Juice or Plasma The inactivation of TRH immunoreactivity with rat stomach was examined by the following method. One hundred nanograms of synthetic TRH were added to 1,200 mg wet weight of homogenated stomach wall, 1.0 ml of gastric juice or 1.0 ml of plasma (in 1.0 ml 0.01 M phosphate buffer. pH 7.4) at 4 °C after seroton in, cyproheptadine or GR38032F injection and then incubated at 37 °C for 30 min. The recovery of added TRH was measured by a radioimmunoassay, and the results were expressed as a percent of TRH added. Sta bility of TRH in gastric juice was examined as follows: 10 ng/ml of synthetic TRH were added to pH 2 or 7 of human gastric juice, then the gastric juice was incu-
Fig. 1. Changes in serotonin concentrations of the rat stomach wall after intraperitoneal injection of sero tonin (30 mg/kg). Values are expressed as means ± SE in each group of 5 rats.a p < 0.05 vs. the 0-min group, w.w. = Wet weight.
bated for 10, 30 or 60 min at 37 °C, and recoveries of added TRH were measured. Statistics Data are expressed as means ± SE for each group. The statistical significance of difference between the control and the experimental groups was analyzed using one-way analysis of variance, followed by the Dunnett’s test for nonpaired comparisons for ir-TRH and serotonin concentrations and by the Kruskal-Wallis test for gastric juice pH. In all the statistical tests, p values of 0.05 or less were considered to represent significant differences.
Results After intraperitoneal serotonin injection, serotonin concentration of the stomach wall increased significantly at 15 min and then rapidly decreased over 30 min (fig. 1), while pH in gastric juice tended to decrease up to 30 min, but not significantly, and increased significantly at 60 min (fig. 2). No macro scopic lesion was observed within 60 min af ter serotonin injection. 151
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and 70.4 ± 4.2% (mean ± SD; n = 8) in the gastric juice. TRH was measured by radioimmunoassay [12]. The elution profile of TRH in the acid-methanolextracted rat stomach on a Sephadex G-10 column (10 X 150 mm. Pharmacia. Uppsala, Sweden) showed a single peak and was identical to that of synthetic TRH, and the dilution curve of acid-methanol-extractcd stomach in a TRH radioimmunoassay system was parallel with the standard curve of synthetic TRH as reported previously [6], The intra-assay coefficient of variation, when assessed by five identical samples, was 6.0%, the interassav coefficient of variation, when assessed by five identical samples five times, was 9.0%. ir-TRH concentrations of the stomach wall were ex pressed as picograms per milligram wet weight, and irTRH concentrations of gastric juice were expressed as picograms per milliliter.
Gastric juice
Stomach wall
Stom ach wall
Fig. 2. Changes in pH in gastric juice after intrapcritoneal injection of serotonin (30 mg/kg) or vehicle. Values are expressed as means ± SE in each group of 5 rats. b p < 0.0! vs. the 0-min group,c p < 0.05 vs. the control group. • ----- • = Serotonin; o----- o = vehicle. Fig. 3. Changes in ir-TRH concentrations of the rat stomach wall and gastric juice after intraperitoneal injection of serotonin (30 mg/kg) or vehicle. Values are expressed as means ± SE in each group of 5 rats.a p < 0.05,b p < 0.01, vs. the 0-min group. cp < 0.05.d p < 0.01, vs. the control group. •----- • = Serotonin; o----- o = vehicle; w.w = wet weight. Fig. 4. Effects of various doses of intraperitoneally administered serotonin on ir-TRH concentrations of the rat stomach wall and gastric wall. w.w. = Wet weight. Values arc expressed as means ± SE in each group of 5 rats.a p < 0.05;b p < 0.01. vs. the control group.
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The concentrations of ir-TRH in the nor mal rat stomach wall and gastric juice were 0.88 ± 0.08 pg/rng wet weight and 42.0 ± 3.7 pg/ml, respectively. In the serotonintreated group. ir-TRH concentrations of the stomach wall decreased significantly at 15 min after injection, and ir-TRH concentra tion of gastric juice increased significantly, peaking at 15 min (fig. 3). However, no signif icant changes in ir-TRH concentrations of the stomach wall and gastric juice were found either in the cyproheptadine- or GR38032Ftreated group (data not shown). The effects of serotonin on ir-TRH con centrations of the stomach wall or gastric juice tended to be dose dependent (fig. 4). ir-TRH concentrations of the plasma showed no changes with any drug (data not shown). The effects of serotonin on ir-TRH concentrations in the stomach wall and gastric juice were significantly blocked by the pretreatment of cyproheptadine or GR38032F (fig. 5).
Kaneko/Mitsuma/Morise/Uchida/ Furusawa/Maeda/Nakada/Adachi
Serotonin and TRH of the Rat Stomach
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Gastric juice
Stomach wall
Gastric juice
Fig. 5. Effects of serotonin on ir-TRH concentrations of the stomach wall and gastric juice in rats with cyproheptadine or GR38032F pretreatment, w.w. = Wet weight. Values are expressed as means ± SE in each group of 5 rats.h p < 0.01 vs. the group with serotonin injection alone.
TRH irnmunoreactivity was not dimin ished by the incubation with either pH 2.0 or 7.0 of gastric juice for 60 min (fig. 6). The inactivation of TRH irnmunoreactivity by the stomach wall, gastric juice or plasma in vi tro after serotonin, cyproheptadine or GR38032F injection was around 63.4 ± 1.0% (mean ± SE) by stomach wall. 99.6 ± 0.8% by gastric juice and 7.2 ± 0.5% by plas ma. and did not differ from that of control.
Evidences of widespread localization of TRH and its many actions upon the gastroin testinal tract [2, 14] indicate that this com pound probably has certain roles in the gas trointestinal tract. We previously reported the presence of TRH both in the stomach wall and in gastric juice, and ir-pro-TRH (TRHprecursor peptide) in the stomach wall [15], suggesting that TRH is synthesized in the
stomach wall and secreted directly into the gastric lumen. It is also well known that sero tonin influences gastric function [16]. On the other hand, relationships between serotonin and TRH have been supposed by a few evi dences including stimulated gastric luminal serotonin release by intracistemal TRH injec-
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Discussion
Fig. 6. Stability ofTRH in gastric juice. The inacti vation of TRI I irnmunoreactivity by gastric juice was not found up to 60 min at pH 2.0 and 7.0 of gastric juice.
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blocked by the pretreatmcnl of cyprohep tadine (5-HT2-receptor antagonist) and GR38032F (5-HT3-receptor antagonist). Mitsuma et al. [21] have earlier reported that serotonin stimulated ir-TRH release from the rat stomach in vitro, which agrees with the present study. Both endogenous and exoge nous serotonin has been proposed to be closely related to gastric mucosal damage without increase in the gastric acid output [23] , in agreement with the present study. The question can be raised as to whether the increase in ir-TRH concentrations of gastric juice might be due to contamination from the blood. However, the concentrations of gastric juice ir-TRH were much higher than those in plasma [7], and no macroscopic mucosal damage was observed within 60 min after se rotonin injection. On the other hand, the sero tonin concentration of the stomach wall in creased significantly at 15 min after serotonin injection and rapidly decreased over 30 min, then the peak change in ir-TRH concentra tion of the stomach wall was identical to that of the serotonin concentration in the stomach wall. Therefore, the present study and a pre vious report taken together suggest that sero tonin acts on the stomach and stimulates irTRH release. The importance of serotonin has been re vealed by the identification of different sero tonin receptors, 5-HT,, 5-HT3 and 5-HT3, and the development of drugs interferes selec tively with different serotonergic systems [11], We examined the effects of 5-HT 2- and 5-HT3-receptor antagonists, because both 5HT 2- and 5-HT3-receptor antagonists have been widely used for some gastrointestinal dysfunction and 5-HT 1-receptor is known to be similar to [i-adrenergic receptor. It is well known that GR38032F is effective in the pro phylaxis of emesis induced by chemotherapy [24] , while cyproheptadine, whose effects on ir-TRH concentrations of the rat stomach in
Kaneko/Milsuma/Morise/Uchida/ Furusawa/Maeda/Nakada/Adachi
Serotonin and TRH of the Rat Stomach
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tion in rats [17] or coexistence of serotonin and TRH in medullary neurons projecting to the spinal cord [ 18]. Furthermore, histological studies revealed that both TRH and serotonin existed in endocrine cells (G and EC cells, respectively) of the gastric mucosa [ 19, 20]. Therefore, it was though that the investiga tion into the effects of serotonin on TRH release from the rat stomach wall into the gas tric lumen would presumably serve to clarify the physiological significance of TRH in the stomach. In order to study the effect of serotonin on ir-TRH concentrations of the rat stomach, one should examine experimental conditions. First, we examined several basic conditions of our TRH assay system. TRH immunoreactivity was not changed by incubation of either pH 2.0 or 7.0 of gastric juice up to 60 min. The inactivation of ir-TRH by stomach or plasma in vitro after serotonin and its related compound injection was not different from the control. From these findings, it is sug gested that the present experimental condi tions allow measurement of TRH in the rat stomach, and that changes in ir-TRH concen trations would not be dependent on the degra dations of TRH in the rat stomach or plasma. On the other hand, serotonin concentrations of the stomach in the present study were simi lar to those reported elsewhere [10]. There fore, the serotonin assay system used in the present study is also suitable to measure the serotonin concentration of the rat stomach. The doses of drugs, which were not physiolog ical but pharmacological in the present study, were similar to those in previous reports [10, 21, 22 ], The present study demonstrated that sero tonin decreased ir-TRH concentrations of the stomach wall and increased ir-TRH concen trations of gastric juice in a dose-dependent manner, and that the effects of serotonin on ir-TRH concentrations of the stomach were
vitro were previously reported [21], is presently used in patients with dumping syn drome [25]. In the present study, the effects of serotonin on ir-TRH concentrations were sig nificantly blocked by cyproheptadine or GR38032F pretreatment. Different changes in the effects on ir-TRH concentrations in the rat stomach between cyproheptadine and GR38032F administration were not observed in the present experimental conditions. The characteristic hallmark of action of serotonin has been a quite remarkable variability of effect between species and different areas of the gastrointestinal tract, among animals of the same species and in the same individual over time [ 11 ]. On the other hand, it has been estimated that selectivity of several serotonin antagonists was not so high, and some antago nists might partly act as other serotonin ago nists [11] and vice versa. From the previous and present data, it is suggested that the effects of serotonin on ir-TRH concentrations
in rat are at least partly mediated via both 5HT2- and 5-HT3-receptors. Since the dose of serotonin used in the present study is high, it cannot elucidate the physiological signifi cance of TRH in the stomach from the present study. Further studies, such as using passive immunization or TRH antagonists, will be necessary to clarify the physiological importance of the gastric TRH. In summary, the present investigation sug gests that serotonin stimulates ir-TRH release from the stomach and that the effects of sero tonin are mediated partly via both 5-HT2and 5-HT3-receptors.
Acknowledgment The author wish to express their heartfelt thanks to Prof. Hidehiko Saito. MD, First Department oflnternal Medicine. Nagoya University School of Medicine. Nagoya. Japan, for his kind guidance and critical review of the manuscript.
1 Boter J. Enzmann F. Folkers K. Bowers C'Y. Schally AV: The iden tity of chemical and hormonal prop erties of the thyrotropin-releasing hormone and pyroglutamyl-histidvlproline amide. Biochem Biophys Res Commun 1969:37:705—710. 2 Morley JE: Extrahypothalamic thy rotropin-releasing hormone (TRH) - Its distribution and its function. Life Sci 1979;25:1539-1550. 3 Leppaluoto J. Koivusalo F. Kraama R: Thyrotropin-releasing factor: Distribution in neural and gastroin testinal tissues. Acta Physiol Scand 1978:104:175-179. 4 Dolva L0. Hanssen KF. Aadland E. Sand T: Thyrotropin-releasing hor mone immunoreactivity in the gas trointestinal tract of man. J Clin En docrinol Metab 1983;56:524-529.
5 Taché Y, Vale W. Brown M: Thyro tropin-releasing hormone - CNS ac tion to stimulate gastric acid secre tion. Nature 1980:287:149-151. 6 Nakada K, MitsumaT. Furusawa A. Maeda Y. Morise K: The effects of histamine on the concentrations of immunoreactive thyrotropin-releas ing hormone in the stomach and hy pothalamus in rats. Gastroenterol Jpn 1990;25:425-431. 7 Mitsuma T. Sun DH. Nogimori T. Chaya M, Ohtake K. Hirooka Y: Effects of histamine and related compounds on the release of imrnunoreactivc thyrotropin-releasing hormone from the rat stomach in vitro. Peptides 1987:8:473-475.
8 Furusawa A, Morise K. Maeda Y. Uchida K. Kaneko H. Nakada K. Mitsuma T: Effect of luminal ad ministration of thyrotropin-releas ing hormone or somatostatin on gas tric pH and interaction of these pep tides in rats. Gastroenterol Jpn 1992:27:165-171. 9 Nakada K, Mitsuma T, Furusawa A, Maeda Y. Iizuka A. Morise K: TRH. VIP and somatostatin in human gas tric juice. Biochem Res 1988; 9(supp! I): 141. 10 Furuhashi S: Experimental study of gastric ulcer - Serotonin ulcer. Jpn J Gastroenterol 1968:65:670-682. 11 Costall B. Naylor RJ: 5-Hydroxytryptamine: New receptors and novel drugs for gastrointestinal mo tor disorders. Scand J Gastroenterol 1990:25:769-787.
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