ROLE OF CALCIUM IN DRUG ACTION ON SMOOTH MUSCLE 1, 2 NORIKO YUKISADA AND FUMIKO EBASHI

Transcription

1 Jap. J. Pharmacol. 11, (1961) ROLE OF CALCIUM IN DRUG ACTION ON SMOOTH MUSCLE 1, 2 NORIKO YUKISADA AND FUMIKO EBASHI Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo Received for publication September 11, 1961 Evans and Schild (1) have shown that smooth muscle retains its ability to contract in response to drugs when suspended in Ringer solution in which sodium ions have been replaced by potassium ions. This finding together with their later observation with intracellular microelectrode (2) suggests that drugs can affect the contractile element of smooth muscle by a mechanism which is not mediated by the depolarization of the excitable membrane. We have preliminarily reported (3) that the presence of calcium ions in K-Ringer solution as well as in normal Ringer solution is the prerequisite for the stimulating action on smooth muscle of most agents except barium. The chief aim of this paper is to make clear the role of calcium ions in a mechanism which links the drug action on smooth muscle with contractile protein. Emphasis is also laid upon the unique nature of barium which behaves quite differently from other stimulating drugs. METHODS Isolated preparations of smooth muscle were obtained from rat ileum, guinea pig ileum, rabbit ileum and rat uterus. For recording muscle contraction Gohara's isotonic lever was used. Temperature of bath solutions was kept at about 26 Ž. Normal Ringer solution, designated as "Ringer" in this article, is composed of M NaCl, 1.8 mm CaCl2, 5.4 mm KCl, 2.8 mm NaHCO3 and 5.5 mm glucose. "K-Ringer" is a solution in which Na+ is replaced by K+, and K2SO4-Ringer is the one in which Na+ and Cl- are replaced by K+ and SO4-- (instead of NaCl, KCl and CaCl2, M K2SO4 and 8 mm CaSO4 were used respectively); "Ca (-)" means that the solution contains no calcium ions. Every solution contains 20 ƒêg per ml of hexamethonium bromide to eliminate the ganglion stimulating action of drugs. Figures under the name of drugs in every illustration indicate the concentration of the drug (ACh chloride, serotonin creatinine sulfate, histamine dihydrochloride, and BaCl2) in ƒêg per ml. In case of oxytocin, figures mean the numbers of unit per ml. 1. The following abbreviations are used in the text: ACh, acetylcholine; EDTA, ethylenediaminetetraacetic acid; CyDTA, cyclohexyldiaminetetraacetic acid. As for Ringer, K-Ringer and K2SO4- Ringer see METHODS in the text. 2. This paper was in part published in Japanese by Noriko Yukisada [Folia pharmacologica japonica, 56, 936 (1960)].

2 CALCIUM IN SMOOTH MUSCLE CONTRACTION 47 RESULTS I. Effect of ACh, serotonin and histamine on isolated ileum in calcium-free bath solutions As Evans and Schild (1) have shown, stimulating action of ACh, serotonin and histamine can be observed in K-Ringer. However, when we remove calcium ions from K-Ringer (Figs. 1-4), the effect of ACh, serotonin and histamine is considerably decreased. Essentially the same tendency is observed in normal Ringer solution (Fig. 5). This process is fairly reversible in case of ACh (Fig. 1), but not so obvious with serotonin (Fig. 2). Removal of calcium from bath solutions can not completely abolish the stimulating action of ACh and histamine (Figs. 1 and 4). This action is finally eliminated by adding a small amount of EDTA (Figs. 4 and 5). Since EDTA can be replaced by other chelating agent, CyDTA, the effect of EDTA is explained on the basis of the removal of trace calcium from bath solutions. FIG. 1. Effect of ACh on rat ileum in Ca (+)- and Ca (-) K-Ringer (reversible depression of ACh action in Ca (-) K-Ringer). First, the responses of muscle preparation in normal Ringer were presented. Then, at every arrow, bath solution was changed to a solution noted in the figure. Other details are described in METHODS in the text. FIG. 2. Effect of serotonin on rat ileum in Ca (+)- and Ca (-) K-Ringer.

3 48 N. YUKISADA & F. EBASHI FIG. 3. Effect of ACh, serotonin and barium on rabbit ileum in Ca (-) and Ca (+) K-Ringer FIG. 4. Effect of ACh, histamine and barium on guinea pig ileum in Ca (-) K-Ringer and Ca (-) K-Ringer containing EDTA. Concentration of EDTA, 0.1 mm.

4 CALCIUM IN SMOOTH MUSCLE CONTRACTION 49 FIG. 5. Effect of ACh and serotonin on rat ileum in Ca (-) Ringer containing EDTA. Concentration of EDTA, 0.1 mm. As for details see the lengend of Fig. 1. Fic. 6. Effect of ACh on rat ileum in K2SO4-Ringer. The tendency described above is also observed in K2SO4,-Ringer (Fig. 6), in which no resting potential has been demonstrated by Evans et al. (2). Hence, there is no doubt that the effect of calcium ions is not exerted through depolarizing mechanism. II. Stimulating action of barium ions on isolated ileum in calcium-free bath solutions Fic. 7. Effect of barium on rat ileum in Ca (-) K-Ringer and Ca (+) K-Ringer.

5 50 N. YUKISADA F. EBASHI FIG. 8.. Effect of barium on rat ileum in Ca (-) Ringer and Ca (-) K-Ringer. Concentration of EDTA, 0.1 mm. Effect of barium on isolated ileum is also observed in K-Ringer (Figs. 3, 4 and 7). In contrast with the cases of ACh etc., however, removal of calcium from bath solutions, even in the presence of EDTA, does not abolish barium action (Figs. 3, 4, 7 and 8). On the contrary, barium action seems to be rather accelerated in the absence of calcium ions (Figs. 3, 4 and 7). III. Response of isolated,uterus in calcium-free solutions FIG. 9. Effect of oxytocin and barium on rat uterus in Ca (-) Ringer.

6 CALCIUM IN SMOOTH MUSCLE CONTRACTION 51 Removal of calcium from Ringer remarkably depresses ACh -contraction of uterus as in the case of ileum and abolish oxytocin action, but does not influence the barium effect (Fig. 9). In these respects the situation is essentially the same as in intestinal muscle. However, barium action is also considerably depressed in K-Ringer, though it has a.definite action, in contrast with the case of intestine (Fig. 10). Generally speaking, uterus muscle is more susceptible to the changes in the composition of Ringer solution; if once immersed in K-Ringer, uterus muscle can hardly restore its former sensitivity to drugs when it is returned to normal Ringer. DISCUSSION All the experimental results in this paper suggest that calcium is an essential factor in producing the contraction of smooth muscles with drugs except barium, even when the experiments are conducted in a solution in which sodium ions are replaced by potassium ions. Similar preliminary observations were also reported by Robertson (4), and Edman and Schild (5). In view of the observations made by Evans et al. (2), it is obvious that this calcium action is not correlated with the mechanism of depolarization in muscle membrane. FIG. 10. Effect of oxytocin and barium on rat uterus in Ca (-) K-Ringer. As for details see the legend of, Fig. 1. Calcium ions have long been believed by physiologists (6-8) to play an important role in initiating the contraction of skeletal muscle. Recently S. Ebashi {9-11), based on his biochemical studies on the relaxing factor, has presented a view that the contractionrelaxation cycle of skeletal muscle is explained by the shift of calcium ions between contractile element and a specified structure in muscle cell, probably some parts of sarcoplasmic reticulum; his concept mainly rests on two facts, namely, that calcium is essential for contraction of actomyosin systems, and that the relaxing factor, probably derived from parts of sarcoplasmic reticulum, has such a strong calcium binding capacity in the presence of ATP as is comparable with that of EDTA. Though the structure

7 52 N. YUKISADA & F. EBASHI and function of smooth muscle* are distinctly different from those of skeletal muscle, these findings and concepts led us to postulate the following picture for explaining our observation: Calcium itself is the trigger substance for contraction also in smooth muscle. Drugs may release the calcium bound to cell membrane and/or some specified structure, or increase the permeability of cell membrane to make calcium ions penetrate into the interior of cells. Thus released or permeated calcium ions induce the contraction of smooth muscle. As is well known, the important role of calcium in the contraction of heart muscle has long been discussed and investigated. All the evidences, especially of Niedergerke (12, 13), Weidmann (14) and Otsuka (15), indicate that calcium plays. an essential part in the mechanism of linking excitation and contraction. Indispensable nature of calcium has also been noticed in the contraction of frog tonus fibers (16). Thus calcium now appears to be a common key substance to different kinds of muscles for initiating the response of contractile protein. Among the various smooth muscle stimulants barium is entirely different from other drugs in its mode of action, i.e., barium does not need calcium for its stimulatory action. It has been demonstrated by Fujita (17) that barium behaves in a similar way to calcium in reversing the relaxed glycerinated muscle to contraction. Accordin to the recent view of S. Ebashi (11) this may be explained by the direct action of barium on actomyosin systems like calcium. Indeed, the action of barium on actomyosin syneresis is essentially the same as calcium (unpublished data). et al. (18) with the actomyosin extracted from smooth muscle. Similar observation was also made by Takeya Therefore, if the action of calcium on smooth muscle contraction with drugs is explained by its direct action on contractile protein, there will be not a few possibilities that the action of barium is also produced by the same mechanism as that of calcium, though so far no evidence has been obtained that barium penetrates the cell membrane of muscle in such an amount as is sufficient to induce the contraction of actomyosin system. The facts that barium stimulates all kinds of muscle cells and does not have a remarkable and consistent effect on membrane potential inspite of its drastic action may substantiate the above explanation. The hypothesis described above, however, is largely speculative and not yet proved by definite evidences. Among other possible mechanisms which can explain the observation in this paper, the most plausible one is: every drug except barium needs calcium for its binding to the site of action, or receptor; then, the binding will induce the contraction through unknown mechanism. At present one cannot decide between these two alternatives. is particularly noteworthy that the endoplasmic reticulum is poorly developed in smooth muscle (17). This is probably reflected in the fact that no one has so far obtained relaxing factor from smooth muscles, though some success is achieved in cardiac muscle (18, 19).

8 CALCIUM IN SMOOTH MUSCLE CONTRACTION 53 SUMMARY 1. Calcium is essential for the stimulating action of ACh, serotonin, histamine and oxytocin on smooth muscle, both in normal Ringer and K-Ringer. 2. In contrast with the above drugs, barium does not require the presence of calcium in Ringer or K-Ringer for its stimulatory action. Calcium rather depresses the barium effect. 3. These phenomena are discussed in relation to a concept that calcium and barium act directly on contractile protein of smooth muscle. REFERENCES 1) EVANS, D.H.L. AND SCHILD, H.O.: Nature 180, 341 (1957) 2) EVANS, D.H.L., SCHILD, H.O. AND THESLEFF, S.: J. Physiol. 143, 474 (1958) 3) YUKISADA, N., EBASHI, F. AND EBASHI, S. Folia pharmacol. japon. 55, 124 (1959) 4) ROBERTSON, P.A.: Nature 186, 316 (1960) 5) EDMAN, K.A. AND SCHILD, H.O.: J. Physiol. 155, 10 P (1961) 6) HEILBRUN, L.V. AND WIERCINSKI, F. J.: J. cell. comp. Physiol. 29, 15 (1947) 7) SHANES, A.M.: Pharmacol. Rev. 10, 165 (1958) 8) HODGKIN, A.L. AND HOROWICZ, P.: J. Physiol. 153, 386 (1960) 9) EBASHI, S.: J. Biochem. 48, 150 (1960) 10) EBASHI, S.: Progress of Theoretical Physics, Suppl. No. 17, p. 35 (1961) 11) EBASHI, S.: J. Biochem. 50, 236 (1961) 12) NIEDERGERKE, R.: J. Physiol. 134, 584 (1956) 13) NIEDERGERKE, R.: Ibid. 138, 506 (1957) 14) WEIDMANN, S.: Experientia 15, 128 (1959) 15) OTSUKA, M.: J. Physiol. Soc. Japan 22, 525 (1960) 16) DENTON, E.J.: J. Physiol. 107, 32 P (1948) 17) FUJITA, K.: Folia pharmacol. japon. 50, 183 (1951) 18) TAKEYA, K., KANDA, Z. AND SEKIYA, J.: Presented at the 34 th Annual Meeting of Pharmacological Society of Japan (1961) 19) CAESER, R., EDWARDS, G.A. AND RUSKA, H.: J. Biophys. and Biochem. Cytol. 3, 867 (1957) 20) BRIGGS, F.N.: Fed. Proc. 18, 372 (1959) 21) EBASHI, F. AND EBASHI, S.: to be published.