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Calcium reversal: Inhibition by Ca ion of sustained contraction in Ca-free medium induced by various agonists in rat uterine smooth muscle
Gen. Pharmac. Vol. 16, No. 2, pp. 133-136, 1985 Printed in Great Britain. All rights reserved
0306-3623/85 $3.00+0.00 Copyright © 1985 Pergamon Press Ltd
C A L C I U M REVERSAL: I N H I B I T I O N BY Ca I O N OF S U S T A I N E D C O N T R A C T I O N IN C a - F R E E M E D I U M I N D U C E D BY V A R I O U S A G O N I S T S IN R A T U T E R I N E SMOOTH MUSCLE KIYOSHI SAKAI, KUNIKO HIGUCHI-NAKAMURA a n d MASAATSU-KouJIRO UCHIDA* Department of Molecular Pharmacology, Meiji College of Pharmacy, 1-35-23 Nozawa Setagayaku Tokyo, 154 Japan (Received 9 July 1984) Abstract 1. Various agonists induced sustained contractions of estrogen-dominated rat uterine smooth muscle in Ca-free salt solution containing 0.2 mM EGTA after incubation of the muscle with 3 mM EGTA for 1 hr. 2. The magnitudes of contraction varied with agonists. (bradykinin>oxytocin>vasopressin>PGF2~ > angiotensin II > acetylcholine > PGE 2> 5-hydroxytryptamine. 3. Addition of 10 4 Ca ion reduced the tension developed: Ca ion inhibited these contractions when they were sufficiently large (marked inhibition on bradykinin-, oxytocin-, and vasopressin-induced contractions; definite one on PGF2~-induced contraction), as observed previously with oxytocin-induced contraction under the same conditions and named "Ca Reversal".
INTRODUCTION
MATERIALS AND METHODS
Previously we reported t h a t low c o n c e n t r a t i o n s of Ca ion inhibited c o n t r a c t i o n o f uterine s m o o t h muscle by oxytocin in Ca-free salt solution (Sakai a n d Uchida, 1980). W e f o u n d conditions in which rat uterine s m o o t h muscle showed sustained c o n t r a c t i o n to oxytocin in the absence o f external Ca ion after t r e a t m e n t with a high c o n c e n t r a t i o n of E G T A (Sakai et al., 1981; Sakai et al., 1982). This c o n t r a c t i o n was reversed to relaxation by a d d i t i o n of a low concentration of Ca ion a n d this p h e n o m e n o n was n a m e d " C a reversal". Ca reversal is manifested in the presence of a C a channel blocker such as D600, which inhibits massive influx o f Ca ion to block its contractile action, just as " A d r e n a l i n e reversal" is manifested in the presence of a n a-blocker, which blocks the rise of b l o o d pressure (a-action) induced by adrenaline a n d manifests the inhibitory action (r-action) of adrenaline (Dale, 1906). W e are elucidating the m e c h a n i s m s of oxytocininduced sustained c o n t r a c t i o n in Ca-free m e d i u m a n d its " C a reversal". C o n t r a c t i o n in Ca-free m e d i u m m a y be evoked by the release of stored Ca ion in the cell. Repeated c o n t r a c t i o n can be evoked by recycling o f Ca ion between myofilaments a n d so-called sarcoplasmic reticulum, which are separated from the cell surface by proliferated cytoplasmic m e m b r a n e s a n d by estrogen d o m i n a t i o n . Thus the sustained contraction is repeated w i t h o u t decay. O n the other h a n d , " C a reversal" is evoked by influx o f m i n u t e a m o u n t s of Ca ion a n d its binding to the intracellular "site o f Ca reversal" or the " i n h i b i t o r y Ca-receptor", which has very high affinity for Ca ion (Sakai et al., 1984, 1985a). Here we report " C a reversal" o f c o n t r a c t i o n o f rat m y o m e t r i u m induced by various agonists other t h a n oxytocin.
Wistar rats, weighing 17(~220g, were ovariectomized. After a recovery period of 5 days or more, the rats were given estradiol benzoate s.c. (0.1 mg/kg) once a day for 3 days and then sacrificed. The uterine horns were removed and mounted in a 30ml o r g a n b a i h cont/tifiing-LockeRinger solution of the following composition (mM): NaC1 154, KCI 5.63, CAC122.16, MgC12 2.10, NaHCO 3 5.95 and glucose 5.55. Ca-free solution was of the same composition except that CaC12 was omitted. The muscles were equilibrated in normal Locke--Ringer solution for 1 hr with a resting tension of 0.5g and then incubated with 3mM EGTA in Ca-free solution for I hr with a resting tension of 0.2 g. Then the solution was replaced by Ca-free solution containing 0.2 mM EGTA. Ten minutes after change of the medium, contraction was induced by addition of one of the agonists listed below. In control experiments, contraction of one of the pair of uterine horns from a single animal was induced by oxytocin (10 2 U/ml). After 2 ~ 3 0 min, when the contraction reached a plateau, Ca ion was added cumulatively. In some cases, D600 was applied and 10 min later, Ca ions were added cumulatively. Before addition, EGTA was adjusted to pH 7.4 with NaOH and all other drugs were dissolved in water. The agonists tested were vasopressin (3 × l0 2 U/ml, arginine-vasopressin from Sigma Co.), bradykinin (10 ~M from Sigma Co.), PGF2, (10 SM from Upjohn Japan Co.), PGF 2(3 x 10 a M from Upjohn Japan Co.), angiotensin II (3 x 10 8 M, 5isoleucine-angiotensin II from Sigma Co.), 5-HT (3 x 10 4 M from Wako Pure Chemicals Co.) and acetylcholine (3 x 10-3M from Daiichi Pharmaceuticals Co.). Except for vasopressin and bradykinin, these concentrations of the agonists induced their own maximal responses which were tested by adding the agonists cumulatively to the muscle under the same conditions. For other chemicals: oxytocin and estradiol benzoate were from Teikoku Hormone Manufacturing Co., EGTA (glycol ether diaminetetraacetic acid) from Dojin Laboratories Co. and D600 (a-isopropyl-a-[N-methyl-N-homoveratryl)-y-aminopropyl]-3,4,5-trimethoxyphenylacetonitrile) from A. G. Knoll.
*Author to whom all correspondence should be addressed,
RESULTS As s h o w n in Fig. 1, vasopressin a n d b r a d y k i n i n induced large a n d sustained c o n t r a c t i o n s in Ca-free 133
134
KIYOSHI SAKAI et al.
Table 1. Contraction and " C a reversal" in rat m y o m e t r i u m with various agonists in Ca-free solution containing 0.2 m M E G T A . Rat m y o m e t r i u m was incubated in Ca-free solution containing 3 m M E G T A for 1 hr and then in Ca-free solution with 0.2 m M E G T A and contractile responses were recorded isometrically. The magnitudes o f the maximal response were measured by adding the agonist cumulatively. The doses that induced the m a x i m a l responses, and the m a x i m a l responses, were listed as g-value of tension developments. They were compared with the m a x i m a l response to oxytocin in the same conditions which was obtained with the other uterine horn o f the same rat. Contraction for testing " C a reversal" was induced by bradykinin (10 s M, which g a v e 52% o f the m a x i m a l response induced by oxytocin), vasopressin (3 x 10-2 U/ml, which gave 6 4 % o f oxytocin-induced one) or other agonist (in a concentration that gave its maximal response). Ten minutes after adding D600 (3 x 10 6 M, with vasopressin, bradykinin and acetylcholine, or 3 x l0 5 M for PGs, antiotensin It and 5 HT), Ca ions were added cumulatively and " C a reversal" was evaluated as follows: + + , Marked; + , Definite; ± , Slight; , N o t detected. Values are m e a n s +- SE and N = n u m b e r o f experiments.
Agonist Bradykinin Vasopressin PGF 2 Angiotensin II Acetylcholine PGE 2 5-HT
Dose
Tension in g
% of oxytocin-induced m a x i m a l tension
3 x 10 6 M 3 × 10 i U/ml lxl0 5M 3 x 10 8 M I x 10 3 M 1 x 10 a M 1 × 1 0 5M
0.74+-0.08 0.45+-0,01 0.35+-0,04 0.16+-0.01 0.10+-0.02 0.06+-0.02 0.08+-0.01
166.2+ 18.4 99.6+- 1.8 58.2+-6.1 23.4+- 1.2 18.3+-3.6 16.1+-4.1 15.8+-3.0
solution with 0.2 m M E G T A . As shown in Table 1, bradykinin induced much more contraction than oxytocin and the maximal response to bradykinin was the largest among the agonists. Vasopressin induced a similar maximal contraction to oxytocin: PGF2o induced about half the maximal response which was induced by oxytocin and other agonists such as PGE2 and acetylcholine, and 5HT, whose maximal responses were about one-fifth or less of that induced by
f
__/U.
d
Fig. 1. Representative contraction of rat myometrium in Cafree solution containing 0.2 mM EGTA and its "Ca reversal". Rat myometrium was incubated in Ca-free solution with 3 mM EGTA for 1 hr and then soaked in Ca-free solution with 0.2 mM EGTA. Vasopressin (3 x 10 2 U/ml, • in a and b) or bradykinin (I0 8M, I in c and d) was applied at the points indicated and contractile response was recorded isometrically. After full development of tension, Ca ions were added cumulatively. Their final concentration was 10~4MatA, 3×10 4MatB, 10 3 M a t C a n d 3 x l 0 3Mat D. In experiments b and d, D600 (3 x 10 6 M) was added at X. Dots (.) above traces show values above scale. Long horizontal lines under the traces show base lines. Short horizontal bars at the end of traces show a time lapse of 10min. Vertical bars at the ends of traces show tension development of 0.5 g.
N
"'Ca reversal"
6 6 7 4 5 7 4
+ + + + + +-
oxytocin, induced a sustained but small contraction in Ca-free solution. Angiotensin II-induced contraction, which was also small, decayed slowly. In Fig. 1, the vasopressin concentration was to produce 64% of contraction height of the maximal response to oxytocin, and bradykinin was to produce 52% of that. Addition of 10 -4 M Ca ion induced transient contraction followed by " C a reversal", i.e. decrease of initial tension development. A n d this Ca reversal was manifested by D600 (3 x 10 -6 M ) a s the transient contraction was abolished. In bradykinininduced contraction Ca ion at 10 -4 M evoked contractions but not " C a reversal". However, the same amount of Ca ion evoked " C a reversal" in the presence of D600. Slight " C a reversal" of the contraction by PGF2o was observed only in the presence of D600 (Table 1). " C a reversal" was difficult to detect in angiotensin II-induced contraction since the contraction decayed. N o " C a reversal" of the smaller contractions induced by other agonists was observed even in the presence of D600.
DISCUSSION
Previously we reported " C a reversal" of oxytocininduced contraction of rat myometrium in Ca-free solution (Sakai and Uchida, 1980). This phenomenon indicates the inhibitory role of Ca ion in the receptorcoupled response. We were interested in whether this phenomenon was restricted to oxytocin-induced contraction or whether it was also observed with other agonists. In ordinary salt solution, K+-depolariza tion-induced contraction is used to test whether the effect of a drug is specific to receptors. But in Ca-free solution, K ion does not of course induce any contraction. Thus we sought agonists that induce contraction like oxytocin in Ca-free solution. Some agonists induced similar contractions to oxytocin but others induced smaller contractions, although almost all of them were sustained. Irrespective of the magnitude of contraction, sustained contraction of smooth muscle in Ca-free solution was first reported by us (Sakai and Uchida, 1980), on uterine smooth muscle, and later similar contraction was reported on vascular smooth muscle (Casteels et al., 1982; Heaslip and
135
Ca reversals Rahman, 1982), and on vas deferens (Ashoori and Tomita, 1983). The mechanism of uterine smooth muscle contraction in Ca-free solution has now been elucidated most clearly, and is to be reported by us elsewhere (Sakai et al., 1985b). Usually, contraction of smooth muscle in Ca-free solution is transient as reported recently by Lalanne et al. (1984), on acetylcholine-, and angiotensin Ill-induced contraction of rat myometrium. We observed "Ca reversal" of the large contraction induced by bradykinin and vasopressin. In PGF2~induced contraction, which was only half that induced by oxytocin, the "Ca reversal" was small, and with other agonists, which induced even smaller contractions, "Ca reversal" was not apparent. Thus there was an intimate correlation between the magnitude of contraction in Ca-free solution and the appearance of Ca reversal. These results showed that Ca reversal is not specific or restricted to the oxytocin receptor mediated response and that Ca reversal, or the inhibitory action of Ca ion, is a general phenomenon and its mechanisms may be the same in various agonists as in oxytocin. The correlation described above would suggest that Ca reversal plays a regulatory role in Ca ion-activated systems in which the inhibitory action of Ca ion is masked by activating action of Ca ion per se. As shown in Fig. 1, D600 abolished contractions and tones by inhibiting massive influx of Ca ion, revealing the inhibitory action of Ca ion. In a preliminary communication (Sakai and Uchida, 1981) and in a foregoing paper (Sakai et al., 1984), we calculated the concentration of{ree Ca ion needed to induce Ca reversal and round that 10-" M (threshold concentration) and 10 -7 M (for maximal inhibition) Ca ions were sufficient to induce Ca reversal. The surprisingly small concentration of Ca ion may control activating responses induced by Ca ion per se. This concentration is the intracellular concentration of free Ca ion in the resting state of the cell. In a previous paper we reported that the site of this inhibition by Ca ion is intracellular (Sakai et al., 1984), and therefore it should be exerted intracellularly before activation by raised internal Ca ions (Fig. 2). We postulate that this inhibition by submicromolar Ca ion is concerned with threshold phenomenon.
Contract
SUMMARY
Various agonists induce sustained contractions of rat myometrium in Ca-free medium containing 0.2 mM EGTA after incubation of the muscle with 3 mM EGTA for 1 hr. The magnitude of contraction varied with the agonist. Addition of 10 -4 M Ca ion reduced the tension developed: Ca ion inhibited these contractions w h e n they were sufficiently large, as observed with oxytocin-induced contractions under the same conditions and named "Ca reversal". This inhibition is not restricted to oxytocin-receptormediated responses but is a general phenomenon. Acknowledgements--The authors thank Knoll A.G. for a
kind gift of D600 and Upjohn Japan Co. for kind gifts of PGs. This work was partly supported by a fund from Eiko Wealth Co., Atugi.
REFERENCES
Ashoori F. and Tomita T. (1983) Mechanical responses to n0radrenaline in Ca-free solution in the rat vas deferens. J. Physiol. Lond. 338, 165-172. Casteels R., Raeymaekers L., Suzuki H. and Van Eldere J. (1982) Tension response and Ca-45 release in vascular smooth muscle in Ca-free solution. Pfliigers Arch. 393, 139-143, Dale H. H. (1906) On some physiologicalactions of ergot. J. Physiol. Lond. 43, 163-206. Heaslip R. J. and Rahman R. G. (1982) Evidence of the "existence of two distinct pools of intracellular calcium in the rat aorta accessibleto mobilization by norepinephrine. J. Pharmac. exp. Ther. 221, 7-11. Lalanne C,, Mironneau C., Mironneau J. and Salivineau J. P. (1984) Contraction of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca-free medium. Br. J. Pharmac. 81, 317-322. Sakai K., Higuchi-Nakamura K. and Uchida M. (1982) Oxytocin-induced Ca-free contraction of rat uterine smooth muscle: Effects of preincubation with EGTA and drugs. Gen. Pharmac. 13, 392-399. Sakai K. and Uchida M. (1980) A Calcium reversal phenomenon: Differentiation of excitatory and inhibitory roles of Ca ion in uterine smooth muscle contraction. Jap. J. Pharmac. 30, 394-396. Sakai K. and Uchida M. (1981) Calcium reversal. Inhibition
--
ion
"~"~
(Yin)
II Activation
Resting Ca
t
Ca2+ > 10 -s M Co re1 ease
. . . . . .
Threshold
. . . . . . . .
10 -7
M
<_ C a z +
Inhibition
Co influx Ca reversal -4-
~'~
(Yang)
Fig. 2. Possible scheme for "Yin-Yang" roles of intracellular Ca ions in contraction of rat myometrium. "Yin-Yang" theory was first proposed for cyclic AMP (Yin) and cyclic GMP (Yang). But in our hypothesis a single substance--Ca ion--plays both "Yin" (inhibitory at sub-micromolar concentrations) and "Yang" (activating at supra-mieromolar concentration). The original meaning of "Yang" in Chinese or Japanese is "sunny" or positive, and "Yin" is "shadowy" or negative.
136
KIYOSHI SAKAIel aL
by Ca 2~ of smooth muscle contraction: Inhibition of Cafree contraction of rat myometrium by sub-micromolar concentration of Ca 2~. Abstr. 8th Int. Congr. Pharmacology, p. 678. Sakai K., Yamagishi T., Higuchi-Nakamura K. and Uchida M. K. (1984) Ca reversal: Intracellular localization of site of inhibition by a sub-micromolar concentration of Ca ion in uterine smooth muscle shown by studies with EGTA. Gen. Pharmac. 15, 549 552. Sakai K., Yamagishi T. and Uchida M. K. (1985a) Ca Reversal: Stimulation of "Inhibitory Ca-receptor" by
intracellularly released minute amount of Ca ion from mitochondria to induce "Ca reversal". Gen. Pharmac. Submitted. Sakai K., Yamagishi T. and Uchida M. K. (1985b) Contraction of rat myometrium in Ca-free solution is evoked by release of Ca ion from sarcoplasmic reticulum in contact with myofilaments. Gen. Pharmac. To be submitted. Sakai K., Yamaguchi T. and Uchida M. K. (1981) Oxytocininduced Ca-free contraction of rat uterus smooth muscle: Effects of divalent cations and drugs. Archs int. Pharmacodyn. ThOr. 250, 40-54.
0306-3623/85 $3.00+0.00 Copyright © 1985 Pergamon Press Ltd
C A L C I U M REVERSAL: I N H I B I T I O N BY Ca I O N OF S U S T A I N E D C O N T R A C T I O N IN C a - F R E E M E D I U M I N D U C E D BY V A R I O U S A G O N I S T S IN R A T U T E R I N E SMOOTH MUSCLE KIYOSHI SAKAI, KUNIKO HIGUCHI-NAKAMURA a n d MASAATSU-KouJIRO UCHIDA* Department of Molecular Pharmacology, Meiji College of Pharmacy, 1-35-23 Nozawa Setagayaku Tokyo, 154 Japan (Received 9 July 1984) Abstract 1. Various agonists induced sustained contractions of estrogen-dominated rat uterine smooth muscle in Ca-free salt solution containing 0.2 mM EGTA after incubation of the muscle with 3 mM EGTA for 1 hr. 2. The magnitudes of contraction varied with agonists. (bradykinin>oxytocin>vasopressin>PGF2~ > angiotensin II > acetylcholine > PGE 2> 5-hydroxytryptamine. 3. Addition of 10 4 Ca ion reduced the tension developed: Ca ion inhibited these contractions when they were sufficiently large (marked inhibition on bradykinin-, oxytocin-, and vasopressin-induced contractions; definite one on PGF2~-induced contraction), as observed previously with oxytocin-induced contraction under the same conditions and named "Ca Reversal".
INTRODUCTION
MATERIALS AND METHODS
Previously we reported t h a t low c o n c e n t r a t i o n s of Ca ion inhibited c o n t r a c t i o n o f uterine s m o o t h muscle by oxytocin in Ca-free salt solution (Sakai a n d Uchida, 1980). W e f o u n d conditions in which rat uterine s m o o t h muscle showed sustained c o n t r a c t i o n to oxytocin in the absence o f external Ca ion after t r e a t m e n t with a high c o n c e n t r a t i o n of E G T A (Sakai et al., 1981; Sakai et al., 1982). This c o n t r a c t i o n was reversed to relaxation by a d d i t i o n of a low concentration of Ca ion a n d this p h e n o m e n o n was n a m e d " C a reversal". Ca reversal is manifested in the presence of a C a channel blocker such as D600, which inhibits massive influx o f Ca ion to block its contractile action, just as " A d r e n a l i n e reversal" is manifested in the presence of a n a-blocker, which blocks the rise of b l o o d pressure (a-action) induced by adrenaline a n d manifests the inhibitory action (r-action) of adrenaline (Dale, 1906). W e are elucidating the m e c h a n i s m s of oxytocininduced sustained c o n t r a c t i o n in Ca-free m e d i u m a n d its " C a reversal". C o n t r a c t i o n in Ca-free m e d i u m m a y be evoked by the release of stored Ca ion in the cell. Repeated c o n t r a c t i o n can be evoked by recycling o f Ca ion between myofilaments a n d so-called sarcoplasmic reticulum, which are separated from the cell surface by proliferated cytoplasmic m e m b r a n e s a n d by estrogen d o m i n a t i o n . Thus the sustained contraction is repeated w i t h o u t decay. O n the other h a n d , " C a reversal" is evoked by influx o f m i n u t e a m o u n t s of Ca ion a n d its binding to the intracellular "site o f Ca reversal" or the " i n h i b i t o r y Ca-receptor", which has very high affinity for Ca ion (Sakai et al., 1984, 1985a). Here we report " C a reversal" o f c o n t r a c t i o n o f rat m y o m e t r i u m induced by various agonists other t h a n oxytocin.
Wistar rats, weighing 17(~220g, were ovariectomized. After a recovery period of 5 days or more, the rats were given estradiol benzoate s.c. (0.1 mg/kg) once a day for 3 days and then sacrificed. The uterine horns were removed and mounted in a 30ml o r g a n b a i h cont/tifiing-LockeRinger solution of the following composition (mM): NaC1 154, KCI 5.63, CAC122.16, MgC12 2.10, NaHCO 3 5.95 and glucose 5.55. Ca-free solution was of the same composition except that CaC12 was omitted. The muscles were equilibrated in normal Locke--Ringer solution for 1 hr with a resting tension of 0.5g and then incubated with 3mM EGTA in Ca-free solution for I hr with a resting tension of 0.2 g. Then the solution was replaced by Ca-free solution containing 0.2 mM EGTA. Ten minutes after change of the medium, contraction was induced by addition of one of the agonists listed below. In control experiments, contraction of one of the pair of uterine horns from a single animal was induced by oxytocin (10 2 U/ml). After 2 ~ 3 0 min, when the contraction reached a plateau, Ca ion was added cumulatively. In some cases, D600 was applied and 10 min later, Ca ions were added cumulatively. Before addition, EGTA was adjusted to pH 7.4 with NaOH and all other drugs were dissolved in water. The agonists tested were vasopressin (3 × l0 2 U/ml, arginine-vasopressin from Sigma Co.), bradykinin (10 ~M from Sigma Co.), PGF2, (10 SM from Upjohn Japan Co.), PGF 2(3 x 10 a M from Upjohn Japan Co.), angiotensin II (3 x 10 8 M, 5isoleucine-angiotensin II from Sigma Co.), 5-HT (3 x 10 4 M from Wako Pure Chemicals Co.) and acetylcholine (3 x 10-3M from Daiichi Pharmaceuticals Co.). Except for vasopressin and bradykinin, these concentrations of the agonists induced their own maximal responses which were tested by adding the agonists cumulatively to the muscle under the same conditions. For other chemicals: oxytocin and estradiol benzoate were from Teikoku Hormone Manufacturing Co., EGTA (glycol ether diaminetetraacetic acid) from Dojin Laboratories Co. and D600 (a-isopropyl-a-[N-methyl-N-homoveratryl)-y-aminopropyl]-3,4,5-trimethoxyphenylacetonitrile) from A. G. Knoll.
*Author to whom all correspondence should be addressed,
RESULTS As s h o w n in Fig. 1, vasopressin a n d b r a d y k i n i n induced large a n d sustained c o n t r a c t i o n s in Ca-free 133
134
KIYOSHI SAKAI et al.
Table 1. Contraction and " C a reversal" in rat m y o m e t r i u m with various agonists in Ca-free solution containing 0.2 m M E G T A . Rat m y o m e t r i u m was incubated in Ca-free solution containing 3 m M E G T A for 1 hr and then in Ca-free solution with 0.2 m M E G T A and contractile responses were recorded isometrically. The magnitudes o f the maximal response were measured by adding the agonist cumulatively. The doses that induced the m a x i m a l responses, and the m a x i m a l responses, were listed as g-value of tension developments. They were compared with the m a x i m a l response to oxytocin in the same conditions which was obtained with the other uterine horn o f the same rat. Contraction for testing " C a reversal" was induced by bradykinin (10 s M, which g a v e 52% o f the m a x i m a l response induced by oxytocin), vasopressin (3 x 10-2 U/ml, which gave 6 4 % o f oxytocin-induced one) or other agonist (in a concentration that gave its maximal response). Ten minutes after adding D600 (3 x 10 6 M, with vasopressin, bradykinin and acetylcholine, or 3 x l0 5 M for PGs, antiotensin It and 5 HT), Ca ions were added cumulatively and " C a reversal" was evaluated as follows: + + , Marked; + , Definite; ± , Slight; , N o t detected. Values are m e a n s +- SE and N = n u m b e r o f experiments.
Agonist Bradykinin Vasopressin PGF 2 Angiotensin II Acetylcholine PGE 2 5-HT
Dose
Tension in g
% of oxytocin-induced m a x i m a l tension
3 x 10 6 M 3 × 10 i U/ml lxl0 5M 3 x 10 8 M I x 10 3 M 1 x 10 a M 1 × 1 0 5M
0.74+-0.08 0.45+-0,01 0.35+-0,04 0.16+-0.01 0.10+-0.02 0.06+-0.02 0.08+-0.01
166.2+ 18.4 99.6+- 1.8 58.2+-6.1 23.4+- 1.2 18.3+-3.6 16.1+-4.1 15.8+-3.0
solution with 0.2 m M E G T A . As shown in Table 1, bradykinin induced much more contraction than oxytocin and the maximal response to bradykinin was the largest among the agonists. Vasopressin induced a similar maximal contraction to oxytocin: PGF2o induced about half the maximal response which was induced by oxytocin and other agonists such as PGE2 and acetylcholine, and 5HT, whose maximal responses were about one-fifth or less of that induced by
f
__/U.
d
Fig. 1. Representative contraction of rat myometrium in Cafree solution containing 0.2 mM EGTA and its "Ca reversal". Rat myometrium was incubated in Ca-free solution with 3 mM EGTA for 1 hr and then soaked in Ca-free solution with 0.2 mM EGTA. Vasopressin (3 x 10 2 U/ml, • in a and b) or bradykinin (I0 8M, I in c and d) was applied at the points indicated and contractile response was recorded isometrically. After full development of tension, Ca ions were added cumulatively. Their final concentration was 10~4MatA, 3×10 4MatB, 10 3 M a t C a n d 3 x l 0 3Mat D. In experiments b and d, D600 (3 x 10 6 M) was added at X. Dots (.) above traces show values above scale. Long horizontal lines under the traces show base lines. Short horizontal bars at the end of traces show a time lapse of 10min. Vertical bars at the ends of traces show tension development of 0.5 g.
N
"'Ca reversal"
6 6 7 4 5 7 4
+ + + + + +-
oxytocin, induced a sustained but small contraction in Ca-free solution. Angiotensin II-induced contraction, which was also small, decayed slowly. In Fig. 1, the vasopressin concentration was to produce 64% of contraction height of the maximal response to oxytocin, and bradykinin was to produce 52% of that. Addition of 10 -4 M Ca ion induced transient contraction followed by " C a reversal", i.e. decrease of initial tension development. A n d this Ca reversal was manifested by D600 (3 x 10 -6 M ) a s the transient contraction was abolished. In bradykinininduced contraction Ca ion at 10 -4 M evoked contractions but not " C a reversal". However, the same amount of Ca ion evoked " C a reversal" in the presence of D600. Slight " C a reversal" of the contraction by PGF2o was observed only in the presence of D600 (Table 1). " C a reversal" was difficult to detect in angiotensin II-induced contraction since the contraction decayed. N o " C a reversal" of the smaller contractions induced by other agonists was observed even in the presence of D600.
DISCUSSION
Previously we reported " C a reversal" of oxytocininduced contraction of rat myometrium in Ca-free solution (Sakai and Uchida, 1980). This phenomenon indicates the inhibitory role of Ca ion in the receptorcoupled response. We were interested in whether this phenomenon was restricted to oxytocin-induced contraction or whether it was also observed with other agonists. In ordinary salt solution, K+-depolariza tion-induced contraction is used to test whether the effect of a drug is specific to receptors. But in Ca-free solution, K ion does not of course induce any contraction. Thus we sought agonists that induce contraction like oxytocin in Ca-free solution. Some agonists induced similar contractions to oxytocin but others induced smaller contractions, although almost all of them were sustained. Irrespective of the magnitude of contraction, sustained contraction of smooth muscle in Ca-free solution was first reported by us (Sakai and Uchida, 1980), on uterine smooth muscle, and later similar contraction was reported on vascular smooth muscle (Casteels et al., 1982; Heaslip and
135
Ca reversals Rahman, 1982), and on vas deferens (Ashoori and Tomita, 1983). The mechanism of uterine smooth muscle contraction in Ca-free solution has now been elucidated most clearly, and is to be reported by us elsewhere (Sakai et al., 1985b). Usually, contraction of smooth muscle in Ca-free solution is transient as reported recently by Lalanne et al. (1984), on acetylcholine-, and angiotensin Ill-induced contraction of rat myometrium. We observed "Ca reversal" of the large contraction induced by bradykinin and vasopressin. In PGF2~induced contraction, which was only half that induced by oxytocin, the "Ca reversal" was small, and with other agonists, which induced even smaller contractions, "Ca reversal" was not apparent. Thus there was an intimate correlation between the magnitude of contraction in Ca-free solution and the appearance of Ca reversal. These results showed that Ca reversal is not specific or restricted to the oxytocin receptor mediated response and that Ca reversal, or the inhibitory action of Ca ion, is a general phenomenon and its mechanisms may be the same in various agonists as in oxytocin. The correlation described above would suggest that Ca reversal plays a regulatory role in Ca ion-activated systems in which the inhibitory action of Ca ion is masked by activating action of Ca ion per se. As shown in Fig. 1, D600 abolished contractions and tones by inhibiting massive influx of Ca ion, revealing the inhibitory action of Ca ion. In a preliminary communication (Sakai and Uchida, 1981) and in a foregoing paper (Sakai et al., 1984), we calculated the concentration of{ree Ca ion needed to induce Ca reversal and round that 10-" M (threshold concentration) and 10 -7 M (for maximal inhibition) Ca ions were sufficient to induce Ca reversal. The surprisingly small concentration of Ca ion may control activating responses induced by Ca ion per se. This concentration is the intracellular concentration of free Ca ion in the resting state of the cell. In a previous paper we reported that the site of this inhibition by Ca ion is intracellular (Sakai et al., 1984), and therefore it should be exerted intracellularly before activation by raised internal Ca ions (Fig. 2). We postulate that this inhibition by submicromolar Ca ion is concerned with threshold phenomenon.
Contract
SUMMARY
Various agonists induce sustained contractions of rat myometrium in Ca-free medium containing 0.2 mM EGTA after incubation of the muscle with 3 mM EGTA for 1 hr. The magnitude of contraction varied with the agonist. Addition of 10 -4 M Ca ion reduced the tension developed: Ca ion inhibited these contractions w h e n they were sufficiently large, as observed with oxytocin-induced contractions under the same conditions and named "Ca reversal". This inhibition is not restricted to oxytocin-receptormediated responses but is a general phenomenon. Acknowledgements--The authors thank Knoll A.G. for a
kind gift of D600 and Upjohn Japan Co. for kind gifts of PGs. This work was partly supported by a fund from Eiko Wealth Co., Atugi.
REFERENCES
Ashoori F. and Tomita T. (1983) Mechanical responses to n0radrenaline in Ca-free solution in the rat vas deferens. J. Physiol. Lond. 338, 165-172. Casteels R., Raeymaekers L., Suzuki H. and Van Eldere J. (1982) Tension response and Ca-45 release in vascular smooth muscle in Ca-free solution. Pfliigers Arch. 393, 139-143, Dale H. H. (1906) On some physiologicalactions of ergot. J. Physiol. Lond. 43, 163-206. Heaslip R. J. and Rahman R. G. (1982) Evidence of the "existence of two distinct pools of intracellular calcium in the rat aorta accessibleto mobilization by norepinephrine. J. Pharmac. exp. Ther. 221, 7-11. Lalanne C,, Mironneau C., Mironneau J. and Salivineau J. P. (1984) Contraction of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca-free medium. Br. J. Pharmac. 81, 317-322. Sakai K., Higuchi-Nakamura K. and Uchida M. (1982) Oxytocin-induced Ca-free contraction of rat uterine smooth muscle: Effects of preincubation with EGTA and drugs. Gen. Pharmac. 13, 392-399. Sakai K. and Uchida M. (1980) A Calcium reversal phenomenon: Differentiation of excitatory and inhibitory roles of Ca ion in uterine smooth muscle contraction. Jap. J. Pharmac. 30, 394-396. Sakai K. and Uchida M. (1981) Calcium reversal. Inhibition
--
ion
"~"~
(Yin)
II Activation
Resting Ca
t
Ca2+ > 10 -s M Co re1 ease
. . . . . .
Threshold
. . . . . . . .
10 -7
M
<_ C a z +
Inhibition
Co influx Ca reversal -4-
~'~
(Yang)
Fig. 2. Possible scheme for "Yin-Yang" roles of intracellular Ca ions in contraction of rat myometrium. "Yin-Yang" theory was first proposed for cyclic AMP (Yin) and cyclic GMP (Yang). But in our hypothesis a single substance--Ca ion--plays both "Yin" (inhibitory at sub-micromolar concentrations) and "Yang" (activating at supra-mieromolar concentration). The original meaning of "Yang" in Chinese or Japanese is "sunny" or positive, and "Yin" is "shadowy" or negative.
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KIYOSHI SAKAIel aL
by Ca 2~ of smooth muscle contraction: Inhibition of Cafree contraction of rat myometrium by sub-micromolar concentration of Ca 2~. Abstr. 8th Int. Congr. Pharmacology, p. 678. Sakai K., Yamagishi T., Higuchi-Nakamura K. and Uchida M. K. (1984) Ca reversal: Intracellular localization of site of inhibition by a sub-micromolar concentration of Ca ion in uterine smooth muscle shown by studies with EGTA. Gen. Pharmac. 15, 549 552. Sakai K., Yamagishi T. and Uchida M. K. (1985a) Ca Reversal: Stimulation of "Inhibitory Ca-receptor" by
intracellularly released minute amount of Ca ion from mitochondria to induce "Ca reversal". Gen. Pharmac. Submitted. Sakai K., Yamagishi T. and Uchida M. K. (1985b) Contraction of rat myometrium in Ca-free solution is evoked by release of Ca ion from sarcoplasmic reticulum in contact with myofilaments. Gen. Pharmac. To be submitted. Sakai K., Yamaguchi T. and Uchida M. K. (1981) Oxytocininduced Ca-free contraction of rat uterus smooth muscle: Effects of divalent cations and drugs. Archs int. Pharmacodyn. ThOr. 250, 40-54.