with a mechanism coupling excitation to contraction and involving calcium. ... Saxby (1958) was used but only longitudinal isotonic contractions were recorded.
THE EFFECT OF BILE SALTS ON CONTRACTION OF VISCERAL SMOOTH MUSCLE^ by B. H. LAURENCE AND W . J. SIMMONDS (From tbe Department of Physiology, tbe University of Western Australia, Nedlands, Western Australia). (Accepted for publication 18th March, 1963.) SUMMARY. Sodium taurocholatc, 0-125 to 2-0 mg./ml. bath fluid, revcrsibly inhibited isometric contractions of rat'.s ileum in response to Ach, 1()~** to 10-*. The degree of inhibition depended on the relative proportions of the two substances. In depolarizing solution, K^SO^Ringer, taurocholate inhibited the Acb response of rat's ileum, rat's utems and circular musele of toad's stomach. In calcium-free K^SO^-Ringer taurocholate inhibited calcium eontractures in these tbrce preparations. It is suggested tbat tauroebolate may interfere with a mechanism coupling excitation to contraction and involving calcium.
INTRODUCTION. Deficiency of bile salts impairs tbe propulsion of a marker through the small intestine of unanaesthctizcd rats (Morgan and Simmonds, 1962). In contrast to their stimulant effect in vivo, bile salts have frequently been reported to inhibit intestinal motility in vitro. It bas been shown that such inhibition is manifest only wben bile salts bave access to the serosal aspect of the gut and does not occur if tbey are confined in physiological concentrations within the lumen (Emmelin, 1941; Meyer and McEwen, 1948). From tbe serosal aspect, quite low concentrations inhibit not only spontaneous motility but also the response to a variety of stimulants including acetylcholine, histamine, substance P and barium chloride (Emmelin, 1943). This paper reports experiments which suggest tbat tbe non-specific inhibitory effect of bile salts might be due to interference with a mechanism coupling excitation to contraction and involving calcium. METHODS. Heal segments were obtained from male rats weighing about 200 gm., and uterine homs from virgin female rats of about the same weight, after the animals were killed by a blow on the head. Tbe preparations were flushed with saline and kept in Ringer's solution at 5° C. for several hours until used. Circular muscle was obtained from stomachs freshly excised
• This work was carried out with the aid of grants from the National Health and Medical Research Couneil, Canberra, and tbe Medical Research Grants Committee, the University of Western Australia.
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from pithed toads (Bufo marinus) by stripping off thin layers after removal of the serosa, the stomach being held between grooves on a glass rod of suitable diameter. Smooth muscle preparations were mounted vertically in an organ bath containing 40 ml. of fluid for rat tissues and 10 ml. for toad's stomach muscle. Usually, isometric tension was recorded by an R.C.A. 5734 transducer valve, D.C. amplifier and inkwriter. For testing serosal against mucosal applications in rat's ileum, a preparation similar to that of Bulbring, Crema and Saxby (1958) was used but only longitudinal isotonic contractions were recorded. The solutions used had the following composition (millimolar): Mammalian Ringer-NaCl 147, KCl 2 7, CaCla 2 0, MgCl^ 1 0 , NaH2PO4 4-2, NaHCOg 11-9, dextrose 12. KjSO^-Ringer-KaSO^ 126, KCl 5-5, MgCU M 5 , CaCla 2-0, KHCOg 3-6, dextrose 5-5. Amphibian K^SO4-Ringer-K2SO4 80, K C r i - 9 , CaCIg 2 0, KHCO3 4 4, dextrose 5 0. In calcium-free solutions, calcium was omitted and disodium EDTA added (5 x 10"*) to cover impurities in analytical grade chemicals. In addition, short periods of exposure to moderately concentrated EDTA (10--^) were often used to accelerate the process of calcium depletion. All distilled water was passed through a deionizing column before use. All solutions were equilibrated with 5 p.c. CO.,, 95 p.c. O2 for 1 hr. before use and gassed continuously in the organ bath with the same mixture. The pH of the solutions used varied from 6 7 to 7-2, being lower in sulphate-Ringer than in normal Ringer. Drugs and other materials were added directly to the organ bath. Tbree minutes were allowed between additions in the ca.se of mammalian tissue and 10 minutes for toad's stomach muscle. The bath was flushed twice after peak tension bad developed and the tissue then rested until tlie next injection. Preparation and sources of test materials were as follows: acetylcholine chloride (Rocbe) and ntropine sulphate solutions were freshly made On the day of experiment; recrystallized diethyl stilboestrol was dissolved in propane diol, diluted in aqueous solution just prior to use and injected as a fine suspension; sodium taurocholate solutions were freshly made from a purified preparation (Light Chemical Co.) which nevertheless included some bile pigment; CaClg was made up from stock solution (IM) kept at 5° C. in a polythene container. The volume added in all cases was one hundredth of the fluid in the bath.
RESULTS. Confirming the results of previous workers, it was shown that sodium taurocholate, 10-2 0 mg./ml. Ringer solution, reversibly inhibited peristalsis, evoked by raised intraluminal pressure, and rhythmical contractions of the small intestine of the rat in vitro, when applied to tbe serosal aspect. When restricted to tbe fluid bathing the lumen taurocholate had no inhibitory effect. Taurocholate and acetylcholine contractions. Tliese experiments were conducted in mammalian Ringer on segments of rat's ileum. The temperature was 34 ° C, spontaneous contractions being minimal or absent. In eight segments the response to different doses of acetylcholine was measured without taurocholate and with taurocholate 1 mg./ml. in tbe bath (see Fig. 1). In six segments (Fig. 2) the steady response to Ach (10~*) was measured and the segment then exposed to increasing or decreasing concentrations of taurocholate between 0-125 and 2-0 mg./ml. of Ringer in the bath. Tbe segment was then repeatedly washed and, after a rest, the steady response to Ach again measured.
TAUROCHOLATE AND SMOOTH MUSCLE
4-0 -
3O •
^ 20
-9
-7 -6 -5 LOG. CONC. ACETYLCHOLINE
-4
Fig. I. Effect of tatirocholate on longitudinal isometric tension response of segments of rat ileum to varying concentrations of acetylcholine. Average results for eight experiments in mammalian Ringer. O O No taurocholate. X X Same segments, tatirocholate 1 mg./ml. Tlie linear regression of tension on log. dose of Ach in the presence of taurocholate, 1 mg./ml. was highly significant (p < 0-001).
0^125 025
a 5^
10
TAUROCHOLATE IN MG/HL.
Fig. 2. Effect of varying concentrations of taurocholate on longitudinal isometric tension response of segments of rat ileum to acetylcholine, 10-5. Average results for six experiments in mammalian Ringer. Left hand anow: Steady response to Ach 10 —>* before exposure to taurocholate = 4-3 ± 0-27 gm. (mean ± s.e.m.). Right hand arrow: Steady response after recovery from series of taurocholate doses = 4-1 ± 0-23 gm. (mean ± s.e.m.). The linear regression of tension on log. dose of taurocholate was highly significant (p < O-OOI).
Figs. 1 and 2 show that taurocholate inhibited acetylcholine contractions, the degree of inhibition depending on the relative concentrations. The effect was reversible. Similar experiments were carried out on ileal segments from bile-fistula rats in which all the bile had been drained to the exterior for 7-8 days. The quantitative results did not differ significantly from those in normal segments. In five experiments strips were cut, some in a circular and some in a longitudinal direction, from adjacent segments of ileum. A steady response to Aeh 10~* was established by repeated injection at three-minute intervals first in normal Ringer, then in Ringer containing 1 mg. tauroeholate per ml., and then, after 10 min. washing, in normal Ringer again. Contractions were weaker with strips cut in a circular direction, but the effect of bile was reversible and the average percentage inhibition (36 p.c.) was the same for both types of strip. There was no evidence, therefore, that taurocholate affected the two layers of the muscularis differently.
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Acetylcholine contractions in depolarizing solution. As was shown by previous workers, acetylcholine elicited contractions in K2SO4-Ringer, which is said to depolarize the cell membrane almost completely (Evans, Schild and Theslcff, 1958). In four segments of rat's ileum steady responses to Ach 10~^ were established by repeated injection in normal and in K2SO4-Ringcr. In K:.SO4 solution contractions were weaker, developing about 50 p.e. of the tension shown in normal Ringer. Taurocholate, 0 5 mg./ml., had an inhibitory effect in both solutions, reducing tension by 65 p.c. in normal Ringer and by 16 p.c. in K2SO4 solution. Taurocholate, 1 mg./ml., reduced tension by 50 p.c. in K2SO4 solution (three experiments). Calcium eontractures in calcium-free K^SO>,-Ringcr depolarizing solution. When CaCl;! was omitted from the depolarizing solution, responses to Ach became progressively weaker, as previous workers have shown (Robertson, 1960; Duibin and Jenkinson, 1961a and b). Evidently acetylcholine requires at least traces of calcium for its effect to become manifest. Small amounts must be sufficient, since prolonged soaking was required to produce a large reduction in response. Disodium EDTA, a cbelating agent, accelerated the process. Addition of CaCI^ to the caleium-free depolarizing solution gave a prolonged contracture (Edman and Schild, 1962). A concentration of 5 to 7 mM, calculated from the amount added to the bath, was usually sufficient to produee a maximal effect. The aetual concentration of ionized calcium in the bath was not measured. Taurocholate, 1 mg./ml., greatly reduced or abolished the response to added caleium in all of 20 experiments with rat ileum (7), rat uterus (6), and circular muscle of toad's stomach (7). The effect was reversible but tlic period of washing required was much longer than in normal Ringer. Atropine, 10~^, had no effect on the calcium contracture but, as was expected, antagonized the increase in tension produced by injection of acetylcholine during a calcium eontracture. There was no evidence, then, that "endogenous" Ach was involved in the calcium contracture. The effect of stilboestrol. Bass and Setliff (1960) found that a variety of steroids, regardless of their endocrine specificity, inhibited the response of smooth muscle in normal Ringer to a variety of chemical stimulants, including acetylcholine. Stilboestrol, although it shows only some steric resemblance to the steroid configuration, was also a very potent inhibitor. This was confirmed in nine experiments in which stilboestrol, 27 /xg./m]., diminished by an average of 35 p.c. the response of rat's ileum to Ach 10~^. In six experiments with taurocholate, 0-5 mg./ml., tbe tension response was diminished by about 45 p.e. Propane diol alone (2 x 10 ~^) had no effeet. In preliminary experiments stilboestrol in the same concentration also inhibited the response to Ach in depolarizing solution (3/4) and calcium eontractures in calcium-free depolarizing solution (3/3).
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DISCUSSION. There is now considerable evidence that ionized calcium is involved in the mechanism linking events at the cell membrane with contraction in both smooth and striated muscle (Shanes, 1958; Robertson, 1960; Edman and Schild, 1962; Daniel, Sehdev and Robinson, 1962). The possibility that taurocholate may interfere with such a funetion of calcium seems to warrant further investigation. This is suggested by the inhibitory effect of bile salts on responses to a variety of stimulants (Emmelin, 1943), by the ability of taurocholate to block calcium contracture in calcium-depleted, depolarized smooth musele and by the dependence of acetylcholine and probably other stimulants on calcium for their effect. It will be interesting to see whether taurocholate inhibits the response of striated and cardiac muscle. The inhibitory effect of taurocholate might be due to a reduction of membrane permeability to calcium. However, tuuroeholate inhibits promptly. Prolonged exposure to calcium-free solution, even in the presence of chelators, is necessary for loss of the acetylcholine response in the absenee of taurocholate. This, with other evidenee (Shanes, 1958; Edman and Schild, 1962; Daniel et al., 1962), suggests that displacement of calcium from cellular binding sites into the cytoplasm is important in coupling excitation to contraction. It remains to be seen whether taurocholate has any effect on the binding of calcium by cell membrane or cytoplasm. A direct effect of taurocholate on the contractile process has not yet been excluded. Disorganization of enzymes or contractile proteins seems unlikely, since the inhibition was reversible and since the response varied with tbe proportion of acetylcholine to taurocholate. However, there was evidence of a cumulative effect of taurocholate with repeated small doses and recovery time was prolonged after higher doses, e.g. 2 mg./ml. Finally, it sbould be emphasized that the inhibitory effect of tauroebolate appears to be of interest only as a possible tool for studying the coupling of contraction to excitation. No evidence was obtained to suggest tbat tbe very low coneentrations of bile salts present in tissue fluids under physiological conditions would significantly affect the performance of smooth muscle. Ackmnvledgments. We wish to thank Dr. A. Beck, Department of Agriculture, Western Australia, for a supply of recrystallized diethylstilboestrol, Mr. Clive Boundy of the Commonwealth Scientific and Industrial Research Organisation for statistical advice, and Mr. J. Finnan for his enthusiastic technical assistance. REFERENCES. Bass, A. D., and Setliff, J. A. (1960): J. Pharmacol., 130, p. 469. Bulbring, E., Crema, A., and Saxby, O. B. (1958); Brit. J. Pharmacol., 13, p. 440. Daniel, E. E., Sehdev, H., and Robinson, K. (1962): Physiol. Rev., 42, siippl. 5, p. 228. Durbin, R. P., and Jenkinson, D. H. (1961a): J. Physiol., 157, p. 74. Durbin, R. P., and Jenkinson, D. H. (1961b): Jbiii, 157, p. 90.
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Edman, K. A. P., and Schild, H. O. (1962): Ibid., 161, p. 424. Emmelin, N. (1941): Acta physiol. Scand., 3. p. 91. EmmeUn, N. (1943): Ibid., 5, p. 372. Evans, D. H. L., Schild, H. O., and Thesleff, S. (1958): J. Physiol. 143, p. 474. Meyer, A. E., and McEwen, J. P. (1948): Amer. J. Physiol, 153, p. 386. Morgan, R. G. H., and Simmonds, W. J. (1962): Quart, j . exp. Physiol, 47, p. 352. Robertson, P. A. (1960): Nature, 186, p. 316. Shanes, A. M. (1958): Pharmacol Rev., 10, p. 165.
