Calcium

Channel

Blocker-Like in Smooth

Takumi

Satoh,

Takahiro

Moriyama,

Action

of Reserpine

Muscle Hiroyuki

Kuriki

and Hideaki

Karaki *

Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received

August

19, 1992

Accepted

September

11, 1992

ABSTRACT-The effect of reserpine on vascular and intestinal smooth muscles, reserpine inhibited the high K+-induced contraction, and this by the increase in external Ca 2+ concentration and also by a Ca 2+ rabbit aorta, increases in cytosolic Ca 2+ level and muscle tension due allel by reserpine. These results suggest that reserpine inhibits L-type muscle contraction. Keywords:

Reserpine,

Fura-2-Ca

2+ signal,

Smooth

The compounds termed as Ca2+ channel blockers have diverse chemical structures but still show a com mon mechanism of action to inhibit L-type Ca 2+ chan nels (for review see 1). Since the contractions of smooth muscle are due largely to the Ca 2+ influx through L-type Ca2+ channels, these blockers strongly inhibit smooth muscle contractions (2, 3). Reserpine has a chemical structure that is different from any group of the above Ca 2+ channel blockers. However, Casteels and Login (4) showed that reserpine inhibits the contraction and increase in Ca2+ efflux due to high K+ in rabbit ear artery and suggested that reserpine has a Ca2+ channel blocker-like action. To further characterize the effect of reserpine, we examined the effect of reserpine on contraction and cytosolic Ca2+ levels ([Ca2+]i) in smooth muscle. Four muscle preparations were used. Male New Zea land white rabbits (2.0-3.0 kg) were killed by rapid in fusion of sodium pentobarbital (50 mg/kg) and air into the ear vein. Male Wistar rats (200 300 g) were killed by a blow on the neck and exanguination. The thoracic aorta was rapidly removed and cut into spiral strips of 2 3 mm width. In rabbit aorta, the adventitial layer was removed from the medial (smooth muscle) layer in order to avoid the possible involvement of endogenous catecholamines (5), and muscle strips (10-15 mm long) were prepared. The endothelium was removed from rabbit and rat aortas by gently rubbing the intimal sur

muscles was examined. In these inhibitory effect was antagonized channel activator, Bay k8644. In to high K+ were inhibited in par Ca 2+ channels to inhibit smooth

muscle

face with a moistened finger. In a preliminary experi ment, it was confirmed that the inhibitory effect of re serpine in the aortas was not modified by vascular en dothelium. Albino male guinea pigs (250 300 g) were killed by a blow on the neck and a section of taenia, 5 10 mm in length, was dissected from the caecum. The terminal ileum was also removed from guinea pigs, and a longitudinal muscle preparation was made. The normal physiological salt solution contained 136.9 mM NaCl, 5.4 mM KCI, 5.5 mM glucose, 23.8 mM NaHCO3, 1.5 mM CaC12, 1.0 MM MgC12and 0.01 mM ethylenediamine tetraacetic acid (EDTA). The concentration of CaC12 was changed to 0.3 mM or 7.5 mM in some experiments. Isosmotic 65.4 mM K+ solu tion was made by substituting 60 mM NaCI in the nor mal solution with equimolar KCI. Hyperosmotic 45.4 mM K+ solution was made by increasing the concentra tion of KCI to 45.4 mM. These solutions were saturated with a mixture of 95%02 and 5% CO2 at 37°C (pH 7.4). Muscle tension was recorded isometrically with a force-displacement transducer connected to a polygraph (Nihon Kohden). A passive tension of 1 g for the aortas and 0.2 g for taenia and ileum was initially applied, and the tissues were allowed to equilibrate in a 20-ml bath for 60 min before beginning the experiments. [Ca2+]i was measured simultaneously with isometric muscle contraction as reported previously (6, 7) with a fluorescent Ca 2+ indicator, fura-2 (8). The fura-2 loaded muscle strip was illuminated alternately (48 Hz)

at the excitation wavelengths of 340 nm and 380 nm, and the intensity of 500-nm fluorescence induced by the 340-nm excitation (F340) and that induced by the 380 nm excitation (F380) was measured by a fluorimeter (CAF-100, Japan Spectroscopic, Tokyo). In a prelimi nary experiment, it was confirmed that reserpine is a fluorescent substance with its excitation peak at approx imately 390 nm and emission peak at approximately 500 nm. Since F340 was not affected by reserpine, we used

F340 as an indicator of [Ca"];. Results of the experiments were expressed as the mean ± S.E.M. Values were considered to be signifi cantly different when the P value was less than 0.05 by Student's

t-test.

The following drugs and chemicals were used: reser pine (Sigma Chemicals, St. Louis, MO, USA), Bay k8644 (donated by Bayer AG, Darmstadt, FRG) and acetoxymethyl ester of fura-2 (Dojindo Laboratories, Kumamoto, Japan). In rabbit and rat aortas, 65.4 mM K+ induced sus

tained contractions. Cumulative addition of reserpine inhibited the high K+ induced contraction. The inhibi tory effect of reserpine was antagonized by the increase in external Ca2+ concentration and also by the Ca2+ channel activator, Bay k8644 at 0.1 uM (9) (Table 1). In guinea pig taenia and ileum, 45.4 mM K+ induced sustained contraction. Cumulative addition of reserpine inhibited the high K+ induced contraction. The inhibi tory effect was augmented by a decrease in the external Ca2+ concentration in both taenia and ileum and attenuated by an increase in external Ca2+ concentra tion in the ileum (Table 1). The inhibitory effect in the ileum was antagonized by 0.1 ,uM Bay k8644 (Table 1). Since these results are quite similar to those with verapamil (10) and other Ca2+ channel blockers (11, 12), we determined if the inhibitory effect of reserpine is due to a decrease in [Ca 2+];. As shown in Fig. 1, the contraction induced by 65.4 mM K+ was accompanied by an increase in [Ca2+],. The addition of 0.1,uM reser pine during the contraction inhibited the contraction

Table 1. Concentration of reserpine to induce a half maximum inhibition of high K-'induced contraction (in -log M) in rabbit and rat aortas and guinea pig taenia and ileum

Fig. 1. Effects of 0.1 uM reserpine on contraction (lower trace) and relative [Ca 21]i (upper trace; indicated by F340) induced by 65.4 mM KCl in fura-2-loaded rat aorta. Traced from a typical result out of 4 experiments.

REFERENCES 1 Godfraind, T., Miller, R. and Wibo, M.: Calcium antagonism and calcium entry blockade. Pharmacol. Rev. 38, 321-416 2 3

4

5

6 Fig. 2. [CaZ+];-tension relationship in fura-2-loaded rat aorta. KCI was cumulatively applied in the absence (closed circle) or presence of 1 uM reserpine (open circle). Each point represents mean of 4 to 15 experiments, and S.E.M. is shown by vertical and horizontal bars when it is larger than the symbol. Concentration of KCI is shown near the experimental points. 100% represents the 65.4 mM K+-stimulated muscle tension and [Ca2+]; measured before the cumulative addition of KCI.

and [Ca2+];. As shown in Fig. 2, cumulative application of KCl induced a stepwise increase in muscle tension and [Ca2+]i. In the muscles pretreaed with 1 uM reser pine for 30 min, the increments in muscle tension and [Ca2+]i were inhibited without changing the [Ca2+]; tension relationship. These results suggest that the in hibitory effect of reserpine is due to a decrease in [Ca2+]i, but not to a decrease in Ca2+ sensitivity of contractile elements. In conclusion, it is suggested that reserpine has a Ca2+ channel blockers-like action with no effect on the Ca2+ sensitivity of contractile elements. Acknowledgment Supported Ministry

by

a Grant-in-Aid

of Education,

Science

for and

Scientific Culture,

Research Japan.

from

the

7

8

9

10

11 12

(1986) Karaki, H.: Ca2+ localization and sensitivity in vascular smooth muscle. Trends Pharmacol. Sci. 10, 320-325 (1989) Karaki, H., Sato, K. and Ozaki, H.: Differential effects of verapamil on cytosolic Ca2+ and contraction in norepinephrine stimulated vascular smooth muscle. Japan. J. Pharmacol. 55, 35-42 (1991) Casteels, R. and Login, I.S.: Reserpine has a direct action as a calcium antagonist on mammalian smooth muscle cells. J. Physiol. (Lond.) 340, 403-414 (1983) Karaki, H. and Urakawa, N.: Possible role of endogenous catecholamine in the contraction induced in rabbit aorta by ouabain, sodium depletion and potassium depletion. Eur. J. Pharmacol. 43, 65-72 (1977) Ozaki, H., Sato, K., Satoh, T. and Karaki, H.: Simultaneous recordings of calcium signals and mechanical activity using fluorescent dye fura 2 in isolated strips of vascular smooth muscle. Japan. J. Pharmacol. 45, 429-433 (1987) Sato, K., Ozaki, H. and Karaki, H.: Changes in cytosolic cal cium level in vascular smooth muscle strip measured simul taneously with contraction using fluorescent calcium indicator fura 2. J. Pharmacol. Exp. Ther. 246, 294-300 (1988) Grynkiewicz, G., Poenie, M. and Tsien, R.Y.: A new gen eration of Ca 2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450 (1985) Schramm, M., Towart, G.T.R. and Franchowiak, G.: Novel dihydropyridines with positive inotropic action through activa tion of Ca2+ channels. Nature 303, 535-537 (1983) Karaki, H., Nakagawa, H. and Urakawa, N.: Comparative effects of verapamil and sodium nitroprusside on contraction and 45Ca uptake in the smooth muscle of rabbit aorta, rat aorta and guinea pig taenia coli. Br. J. Pharmacol. 81, 393 400(1984) Karaki, H. and Weiss, G.B.: Calcium channels in smooth muscle. Gastroenterology 87, 960-970 (1984) Flaim, S.F.: Comparative pharmacology of calcium blockers based on studies of vascular smooth muscle. In Calcium Blockers, Edited by Flaim, S.F. and Zelis, R., p. 155-178, Urban & Schwarzenberg, New York (1982)

Calcium channel blocker-like action of reserpine in smooth muscle.

The effect of reserpine on vascular and intestinal smooth muscles was examined. In these muscles, reserpine inhibited the high K(+)-induced contractio...
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