Effect of Sodium Propionate on the Contractile Response of the Rat Ileum in Situ

Transcription

1 Japan. J. Pharmacol. 35, (1984) Effect of Sodium Propionate on the Contractile Response of the Rat Ileum in Situ Takaji YAJI MA Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186, Japan Accepted March 30, 1984 Abstract \Effects of short-chain fatty acids (SOFA) on the contractile response of rat ileum were studied in vivo. The contractile response was estimated by means of changes in the intraluminal pressure under the isometric condition. Intravenous administration of sodium salts of propionate, butyrate, valerate or caproate produced biphasic contractions: an initial phasic contraction and a subsequent tonic contraction. The effect of propionate was studied in detail. A sigmoid dose-response curve was obtained for the phasic contraction. Atropine, hexamethonium and tetrodotoxin inhibited the phasic contraction, while neostigmine vigorously enhanced it. On the other hand, the tonic contraction was not inhibited by atropine, hexamethonium or tetrodotoxin. Repeated administration of propionate at intervals of less than 3 min led to tachyphylaxis, and this tachyphylaxis disappeared by about 10 min. These results suggest that SCFA induced the biphasic contraction of the rat ileum, probably by neurogenic and myogenic mechanisms. In nature, short-chain fatty acids (SCFA) are usually found throughout the gastrointestinal tract. They are particularly abundant in the large intestine of mammals and in the forestomach of ruminants (1-3). They are produced by the gut microbes from indigestible carbohydrates. Acetate, propionate and butyrate are the major products. They are rapidly absorbed and then transported into the portal circulation after partial metabolization in the epithelial cells (4-6). On the other hand, pharmacological drugs containing short-chain fatty acid ester are very commonly encountered. In some cases free SOFA have been generated in the plasma by enzymatic hydrolysis. Recently, large amounts of sodium butyrate were administered to humans as anticancer agents (7). We previously demonstrated that SCFA injected intravenously causes a transient increase in the transmural potential difference across the ileum (8) and the colon (9) of rats. In this study, I determined the effect of SOFA injected intravenously on the contraction of the rat ileum and tried to elucidate the mechanism of their action. Materials and Methods Animal: Male Sprague- Dawley rats, weighing g, were used. They were maintained on a diet of pellets (Type MF, Oriental Yeast Co., Tokyo, Japan) with free access to water. Measurement of ileal motility: The animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). When tetrodotoxin was administered (i.v.), the animals were artificially ventilated after tracheotomy with a respirator (Model 680, Harvard Apparatus, Ltd.). The rats were placed on a heated table to maintain rectal temperature at C. After tracheotomy and midline laparotomy, the terminal ileum, approximately 2 cm long, was isolated by a ligature at the oral end and by insertion of an L-shaped cannula at the aboral end. The loop was carefully washed with warmed 0.9% NaCI. The peritoneal cavity was kept in the form of a well and then was filled with warm 0.9% NaCI solution. Motility was measured as a change in intraluminal pressure (ILP). The amount of fluid (0.9% NaCI) supplied to the segment

2 T. Yajima was adjusted initially to give a resting intraluminal pressure ranging between 2 cm and 4 cm of water. The I LP was measured with a pressure transducer (Nihon Kohden, LPU- 0.1) under the isometric condition. Measurement of blood pressure and heart rate: After anesthetization with pentobarbital sodium (50 mg/kg, i.p.), the right carotid artery and the femoral vein were exposed and polyethylene cannulae were inserted. Heparin sodium (1000 U/kg) was injected into the femoral vein. The systemic blood pressure was measured with a pressure transducer (Nihon Kohden, MPU-0.5). Drug solutions in a volume of ml were injected into the jugular vein within 2 sec. All recordings were made on an inkwriting rectigraph (Nihon Kohden, W1-681G). Drugs: The drugs used were as follows and were obtained from the sources shown in parenthesis: sodium formate, sodium acetate, sodium propionate, sodium n- butyrate, sodium n-valerate, sodium n- caproate and sodium caprylate (Kanto Chemical Co., Inc.), sodium succinate and sodium ascorbate (Wako Pure Chemical Industries, Ltd.), atropine sulfate, hexamethonium bromide, sodium lactate, acetylcholine chloride (ACh) (Sigma Chemical Co.), tetrodotoxin (Sankyo Co., Ltd.), neostigmine bromide (ICN Pharmaceuticals, Inc.). All drugs were dissolved in 0.9% NaCl. Doses of the drugs are expressed in terms of the salt. Results Effect of SCFA on ileal motility: Intravenous injection of propionate (1.2 mg/kg), butyrate (1.4 mg/kg), valerate (1.6 mg/kg) and caproate (1.7 mg/kg) evoked a biphasic contraction: initial phasic and subsequent tonic contraction (Fig. 1). Acetate (1.0 mg/ kg) produced only a tonic contraction. Formate (0.8 mg/kg) and caprylate (2.1 mg/ kg) produced no response. The pattern of the phasic contraction produced by propionate was similar to that produced by ACh. There was a slight delay in the onset of contraction induced by propionate. The time required to start the contraction after the administration of propionate and ACh from the jugular vein was and sec (mean }S.D., n=4), respectively. Propionate slightly decreased the blood pressure and the heart rate (Fig. 2). Effects of atropine and neostigmine: Atropine (0.06 mg/kg, i.v.) inhibited the propionate-induced phasic contraction (Fig. 3). On the other hand, it did not inhibit the propionate-induced tonic contraction. In contrast, neostigmine (0.05 mg/kg, i.v.) vigorously enhanced the propionate-induced phasic contraction (Fig. 3). There was a sigmoid relationship between the logarithm of the propionate dose and the peak height of the phasic contraction (Fig. 4). Atropine (0.04 mg/kg, i.v.) shifted the dose-response curve to the right and decreased the maximum response of the phasic Fig. 1. Tracings showing the effects of acetate, propionate, butyrate, valerate and caproate on the rat ileum. The contractions were recorded as changes in intraluminal pressure (cm of water). Drugs were administered via the jugular vein. Typical for three experiments.

3 SCFA on Ileal Contraction contraction. Neostigmine (0.04 mg/kg, i.v.) increased the maximum response of the phasic contraction. Effects of hexamethonium and tetrodotoxin: Figure 5 shows that both hexamethonium (0.1 mg/kg, i.v.) and tetrodotoxin (0.009 Fig. 2. Effects of propionate on the blood pressure and the heart rate. Drugs were administered via the jugular vein. Typical for three experiments. Fig. 3. Effects of atropine (A) and neostigmine (B) on the contractile response of the rat ileum to propionate. The contractions were recorded as changes in intraluminal pressure (cm of water). Drugs were administered via the jugular vein. Typical for five or six experiments.

4 T. Yajima mg/kg, i.v.) abolished the propionateinduced phasic contraction without inhibiting the ACh-induced phasic contraction. Neither drug, however, inhibited the tonic contraction. Tachyphylaxis of ileal contractile response: The repeated administration of the same dose of propionate (1.2 mg/kg, i.v.) at approximately 3-min intervals elicited rapid tachy- Fig. 4. Effects of atropine and neostigmine on the dose-dependent contraction of rat ileum in response to propionate. The height of the phasic contraction is expressed as the percentage of the maximal contraction evoked by propionate (3 mg/kg) in the control. Drugs were administered via the jugular vein. Each value represents the mean±s.e. for six experiments. *P<0.05 and **P<0.01 vs. control (Student's t-test). Fig. 5. Effects of hexamethonium (A) and tetrodotoxin (B) on the contractile response of the rat ileum to propionate. The contractions were recorded as changes in intraluminal pressure (cm of water). Drugs were administered via the jugular vein. Typical for three or five experiments.

5 SCFA on lleal Contraction Fig. 6. Effects of repeated administration of propionate on the contractile response of the rat ileum to propionate (A) and the effect of propionate and ACh on the contractile response of the rat ileum during propinate infusion from the jugular vein (B). The contractions were recorded as changes in intraluminal pressure (cm of water). Drugs were administered via the jugular vein. Typical for three experiments. phylaxis (Fig. 6). The responsiveness of the ileal segment to propionate was restored after approximately 10 min. On the other hand, there was no cross tachyphylaxis between propionate and ACh since the ACh-induced contraction was not altered during the tachyphylaxis developed by the i.v. infusion of propionate (4.8 mg/kg/min) (Fig. 6). Discussion The present study shows that the intravenous injection of propionate, butyrate, valerate and caproate induces the biphasic contraction of the rat ileum. The partial inhibition by atropine and the potentiation by neostigmine suggest that the first phasic contraction produced by propionate is probably mediated by ACh. Moreover, the inhibitory effects of hexamethonium and tetrodotoxin suggest that the first phasic contractile response of the rat ileum to propionate involves a cholinergic component which mediates in part the contractile effect of propionate. However, we do not know at present whether propionate exerts its effect on the cholinergic mechanism directly or indirectly. If propionate stimulates release of enough ACh from cholinergic nerve endings to cause full contraction of the ileal smooth muscle to occur, an appropriate amount of atropine or neostigmine must parallelly shift the doseresponse curve to the right or left, respectively. In the present study, however, atropine shifted the dose-response curve for propionate to the right, but decreased the maximum response (Fig. 4); and on the other hand, neostigmine increased the maximum response (Fig. 4). The results can be interpreted as follows: the maximum response produced by propionate is dependent on saturation of ACh-release from the nerve endings, but not the saturation of the contractile response of the ileal smooth muscle;

6 T. Yajima therefore, the response of the ileal smooth muscle to ACh released by stimulation of the maximum dose of propionate decreases and increases in the presence of atropine and neostigmine, respectively. Neostigmine stretched upward the dose-response curve of propionate in the upper plot, but did not shift it to the left (Fig. 4). A similar doseresponse pattern for ACh in the presence of eserine has been demonstrated with the rabbit intestine (10). Since the tonic contraction evoked by propionate was not inhibited by atropine, hexamethonium or tetrodotoxin, this contraction is not attributed to a cholinergic mechanism. It may have resulted from the direct action of propionate on smooth muscle cells. However, there is no study so far on the direct contractile effect of SCFA on smooth muscle. The precise mechanism of this possible direct action of propionate on the smooth muscle remains to be investigated. The changes in the blood pressure and the heart rate produced by propionate were very weak compared with those produced by ACh. Therefore, the ileal contractile response to propionate may be not indirectly elicited by the cardiovascular changes. The present study showed that in the propionate-induced phasic contraction, tachyphylaxis appeared rapidly and was reversible. This tachyphylaxis resembles that developed in the changes in the potential difference produced by propionate (i.v.) in the rat ileum (8). The tachyphylaxis of the contraction was not dependent on the residual concentration of propionate in the blood preceding subsequent injection since the propionate concentration rapidly declines within 90 sec (8). The propionate-induced tachyphylaxis of the contraction may not be due to the ACh-induced tachyphylaxis in the muscarinic receptor since the phasic contraction was elicited by ACh administered even during the development of propionateinduced tachyphylaxis. The mechanism of propionate-induced unclear. tachyphylaxis remains The ability to contract the ileum differed among the acids. Propionate, butyrate, valerate and caproate showed similar effects on the ileal contraction. However, formate, acetate and caprylate were different from them and had no such effect. The reason for this difference is yet unknown. SCFA injected intravenously suppresses the food intake via the hepatic nerve plexus i the ruminant (11) and increases the transmural potential difference across the ileum (8) and the colon (9), possibly via mediation of ACh release in the rat. However, the precise mechanism of SCFA action on the nerve is still obscure. This study showed the involvement of the intramural cholinergic nerve system in the contractile response to SCFA. Acknowledgments: I would like to thank Dr. H. Ono, Hatano Institute, and Drs. Y. Umesaki and T. Sakata for their helpful discussion and advice during the preparation of this manuscript. References 1 Wrong, 0.M., Edmonds, C.J. and Chadwick, V.S.: The Large Intestine: Its Role in Mammalian Nutrition and Homeostasis. MTP Press, Lancaster (1981) 2 Wolin, M.J.: Fermentation in the rumen and human large intestine. Science 213, (1981) 3 Cummings, J.H.: Short chain fatty acids in the human colon. Gut 22, (1981) 4 Dobson, A. and Phillipson A.T.: Absorption from ruminant forestomach. In Handbook of Physiology, Section 6, Alimentary Canal, Edited by Code C.F., Vol. 5, p , American Physiological Society, Washington, D.C. (1968) 5 Umesaki, Y., Yajima, T. and Mutai, M.: Effect of organic acid absorption on bicarbonate transport in rat colon. Pfluegers Arch. 379, (1979) 6 Roediger, W.E.W.: The effect of bacterial metabolites on nutrition and function of the colonic mucosa. Symbiosis between man and bacteria. In Colon and Nutrition, Edited by Kasper. H. and Goebell, H., p , MTP Press, Lancaster, (1982) 7 Prasad, K.N.: Butyric acid: A small fatty acid with diverse biological functions. Life Sci. 27, (1980) 8 Yajima, T., Kojima, K., Tohyama, K. and Mutai, M.: Effect of short-chain fatty acids on electrical activity of the small intestinal mucosa of rat. Life Sci. 28, (1981) 9 Yajima, T., Kojima, K., Tohyama, K. and Mutai, M.: Alteration in sensitivity of transmural electrical response to propionate in rat colon after chronic luminal infusion of short-chain

7 SCFA on lleal Contraction fatty acids. Life Sci. 32, (1983) 10 Matumoto, H. and Chihara, M.: Types of drug synergism classified by the concentrationaction curve. Japan. J. Pharmacol. 8, (1958) 11 Anil, M.H. and Forbes, J.M.: Feeding in sheep intraportal infusions of short-chain fatty acids and the effect of liver denervation. J. Physiol. (Lond.) 298, (1980)