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Effects of organic and inorganic Ca2+-antagonists on acetylcholine-induced contraction in molluscan (Mytilus edulis) smooth muscle
Gen. Pharmac. Vol. 24, No. 6, pp. 1419-1423, 1993 Printed in Great Britain. All rights reserved
0306-3623/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
EFFECTS OF ORGANIC AND INORGANIC Ca2÷-ANTAGONISTS ON ACETYLCHOLINE-INDUCED CONTRACTION IN MOLLUSCAN (MYTILUS EDULIS) SMOOTH MUSCLE YUKO MIYAHARA, YASUO KIZAWA, MASAKAZU SANO and HAJIME MURAKAMI* Department of Physiology and Anatomy', Nihon University College of Pharmacy, Funabashi, Chiba 274, Japan [Tel. (0474)65-4716; Fax (0474)65-2158] (Received 22 March 1993) Abstract--1. Effect of Ca2+-antagonist on the contractile response to acetylcholine (ACh) in molluscan (Mytilus edulis) smooth muscle was investigated. 2. ACh-induced contraction was remarkably reduced by exposure to Ca2+-deprived medium. 3. The organic Ca2+-blockers, verapamil, diltiazem and nicardipine, reduced the concentration-response curve for ACh in a concentration-dependent manner. 4. The inorganic Ca2+-bloekers, MnClv NiCI2, COC12 and CdC12, also reduced the concentration-response curve for ACh concentration-dependently. 5. ACh significantly increased the amounts of inositol 1,4,5-trisphosphate (IP3) in the ABRM. 6. ACh-induced contraction in the ABRM might therefore be mediated through an influx of extracellular Ca 2+ and CaZ+-release from IP3 sensitive intracellular pools.
INTRODUCTION
Molluscan (Mytilus edulis) smooth muscle, anterior byssus retractor muscle ( A B R M ) is known to cause a catch contraction by acetylcholine (ACh) (Twarog, 1954; Murakami et al., 1985). ACh-induced contraction was reportedly mediated through an action on nicotinic-like A C h receptors (Takayanagi et al., 1983), but the receptors seemed to be slightly different from the vertebrate nicotinic A C h receptors in the A B R M (Zeimal and Vulfius, 1973; Takayanagi et al., 1983; M u r a k a m i et al., 1984). Murakami et aL (1985) reported the effects of Ca2+-antagonists on the ACh-induced contraction in the A B R M , and showed that some of these antagonists inhibited the concentration-response curve for ACh. We recently demonstrated that FMRF-NH2-induced contraction was resistant to organic Ca2+-an tagonists, especially 1,4-dihydropyridine derivatives in the A B R M (Kizawa et al., 1991). In this study, to obtain further evidence of ACh-induced Ca 2÷ mobilization in the A B R M , we investigated the effects of organic and inorganic Ca2+-antagonists on AChinduced contraction. The release of intracellular pools of Ca 2+ is known to be elicited by the generation of IP3 in response to a variety of hormones and neurotransmitters acting on excitable and non-excitable cells alike (Hokin,
*To whom all correspondence should be addressed.
1985; Berridge, 1987; Berridge and Irvine, 1989; Chuang, 1989; Ferris and Snyder, 1992). We measured the amounts of IP3 in the ACh-treated A B R M to determined the effect of A C h on phosphatidylinositol turnover. MATERIALS AND METHODS
Animals and preparation of muscle bundle Sea mussels, Mytilus edulis L., collected from Ise Bay (Japan) were stored in circulating artificial sea water at a temperature of about 10°C and used within a week after collection. The anterior byssus retractor muscle (ABRM) was dissected into a muscle bundle of about I mm in diameter and 1.5 cm in length. The bundle was bathed in a physiological solution of the following composition (mM): NaC1, 475; KC1, 10,0; CaCI2.2H20, 2.0; MgCI2,6H20, 20.0; Tris-HCl, 10.0 (pH 7.4). Each preparation was set up vertically for isometric tension recording in a 5 ml organ bath containing physiological solution, maintained at 20°C and bubbled with air. The resting tension was adjusted to 0.2 g for a period of approximately 1 hr. Isometric tension was recorded with a Nihon Kohden (Japan) TB-651T transducer, connected to a Nihon Kohden EP-601G amplifier and a Rikadenki (Japan) R-2 recorder. Mechanical response Two pieces of ABRM were dissected from the body of a single specimen of M. edulis, one bundle was used as the control preparation and the other as the drug-treated preparation. Cumulative concentration-response curve for ACh was obtained twice, and the drug-treated preparation was incubated in a drug-containing solution during the second concentration-response curve. In some experiments, the concentration-response curve for ACh was obtained in Ca 2+-free solution, which had been prepared by omitting CaC12 and adding 2 mM EGTA.
1419
YUKO MIYAHARAet al.
1420
100.
100.
B
A
o~
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.o_
so
50 ltO
o
O, -4
Log [acetylcholine (M)]
Log [acetylcholine (M)]
Fig. 1. Effect of verapamil (A) or diltiazem (B) on the concentration-response curve of ACh in the ABRM. O: control, 0 : treated with 10 -5 M verapamil or diltiazem for 15 min, l : treated with 10-4 M verapamil or diltiazem for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
Measurement of inositol 1,4,5-trisphosphate (IP3) formation
Chemicals and reagents
The ABRM tissue was equilibrated in the physiological solution and bubbled with air for I hr at room temperature. Then the tissues were incubated with 10 -5 M ACh for 5, 10, 30 and 60s. The reaction was terminated by immersing the tissues in liquid nitrogen. The tissues were then homogenized in 1.0 ml of ice-cold 3.6% (w/v) perchloric acid and the resulting homogenates were centrifuged at 2000 g for 15 min at 4°C (Lang and Lewis, 1989). The supernatant was neutralized to pH 7.5 with ice-cold KOH solution, and KC104 was precipitated and removed by centrifugation. The final supernatant was purified with minicolumn (Amprep SAX, Amersham, U.K.). One hundred ~ul aliquots of eluate were used for IP 3 assay. The amount of IP 3 in the test sample was measured as described by Seishima et al. (1988).
Acetylcholine chloride (Daiichi Seiyaku Co., Ltd., Tokyo, Japan), verapamil (Eisai Co., Ltd., Tokyo, Japan), diltiazem (Tokyo Tanabe Seiyaku Co., Ltd., Tokyo, Japan), TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride), nifedipine and nicardipine (Sigma Chemical Co., St Louis, MO), CdCI2, NiCI 2 and MnCI 2 (Wako Junyaku Co., Ltd., Tokyo, Japan), EGTA (ethyleneglycol bis(fl-aminoethylether)-N, N'-tetraacetic acid; Dojindo Laboratories, Kumamoto, Japan) and o-myo-inositol 1,4,5trisphosphate (IP3) assay kit (Amersham, U.K.) were used. Other chemicals purchased were of analytical grade. Nifedipine was dissolved in ethanol, and the final ethanol concentration in physiological solution did not exceed 0. 1%
(v/v). RESULTS
Analysis of responses and statistical methods ACh-induced response was expressed as a percentage of the maximum effect reached in the first concentration-response curve in the control and the drug-treated preparations, respectively. Numerical results were expressed as means 4-SEM and statistical analyses were performed using Student's t-test or Duncan's multiple range test. A P value less than 0.05 was considered a significant difference.
A
o
1001
Effect o f organic Ca2+-antagonists on the concentration-response curve for ACh T h e c o n c e n t r a t i o n - r e s p o n s e curves for A C h were reduced by organic Ca2+-antagonists, nifedipine, nicardipine, diltiazem a n d verapamil c o n c e n t r a t i o n dependently (Figs 1 a n d 2). T h e m a x i m u m response
A
0t
100t t
o
0
0
-s
-:4
Log [acetylcholine (M)]
-3
-6
-s
-4
Log [acetylcholine (M)]
Fig. 2. Effect of nifedipine (A) or nicardipine (B) on the concentration-response curve of ACh in the ABRM. C): control, O: treated with 10-6 M nifedipine or nicardipine for 15 min,ll: treated with 10-5 M nifedipine or nicardipine for 15 min, A: treated with 3 x 10 -5 M nifedipine for 15 min, &: treated with 10 -4 M nicardipine. Each value is presented as a mean with SEM (bar) of 6 experiments.
ACh-induced contraction in the ABRM 100.
100.
50
50
0
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,
-
,
-
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.
,
-6 -5 -4 -3 Log [acetylcholine (M)]
-
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.
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.
,
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-
,
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Fig. 3. Effect of CdCI2 (A) or NiC12(B) on the concentration-response curve of ACh in the ABRM. C): control, Q: treated with 1 mM CdCI2 or NiCI2 for 15 min, I : treated with 10mM CdC12 or NiClz for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
to ACh was significantly reduced by these antagonists in a concentration dependent manner. The potency order was diltiazem > verapamil > nicardipine > nifedipine.
Effect of inorganic Ca2+-antagonists on the concentration-response curve for ACh Effect of inorganic Ca2+-antagonists, COCI2, CdCl2, NiCl2 and MnCl2 on the concentrationresponse curve for ACh is shown in Figs 3 and 4. These inorganic Ca2+-antagonists noncompetitively inhibited the contractile response to ACh. The potency order was CdCl2 > NiC12 > MnC12 > CoCl 2.
ACh-induced contraction in Ca2+-lowering medium The concentration-response curve for ACh was depressed in the Ca2+-lowering medium (Ca2+-free solution containing 2 mM EGTA) for 30 min in the ABRM (Fig. 5). Figure 6 shows the time course of
contractile response to ACh (10 -5 M) after exposure to the Ca2+-deprived medium in the ABRM. The ACh-induced contraction was reduced to 66.3 + 2.7, 56.5 + 4.0, 34.0 + 3.7, 18.5 + 1.8 and 12.0 + 2.5% (mean with SEM of 6 experiments) at, respectively, 5, 10, 15, 20 or 30 min after exposure to the Ca~+-de prived medium, suggesting that influx of extracellular Ca 2+ is necessary for the contractile response to ACh. After incubation of ABRM in Ca 2+-deprived medium for 5 min, the remaining ACh-induced contraction was reduced to 9.5 + 2.0% (mean with SEM of 9 experiments) by 10min pretreatment with 0.1 mM TMB-8 (data not shown). The ACh-induced response may thus be due to release of intracellular pooled Ca 2+
Estimation of the amounts of IP~ in the A B R M The resting level of IP 3 was 100.8 + 4.7 pmol/mg protein (mean with SEM of 5 experiments). The
100.
lOO.
g g
g 50
~
o
so
o
Log [acetylcholine M)]
Log [acetylchoUne (M)]
Fig. 4. Effect of MnCI2 (A) or CoCIz (B) on the concentration-response curve of ACh in the ABRM. C): control, @: treated with 1 mM MnCI2or COC12for 15 min, I1: treated with 10 mM MnCI2 or COC12for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
1422
YUKOMIYANARAet al. 500
100.
400
o~ "~. 300.
c-
.o 50.
200,
C 0
o 100. 0 ,
-7
-6
"4
"5
"3
Log [acetylcholine (M)]
amounts of IP 3 were increased to 210.8 +20.0, 402.2 ___39.5, 360.2 + 65.3 and 165.9 + 31.2 pmol/mg protein (mean with SEM of 5 experiments) by incubation of the ABRM with ACh (10 -5 M) for, respectively, 5, 10, 30 or 60s (Fig. 7). DISCUSSION We compared the effects of organic and inorganic Ca2+-antagonists on ACh-induced contraction in the ABRM. Under our experimental conditions both organic and inorganic Ca2+-blockers reduced the contraction in a concentration-dependent manner. These observations corroborate previous reports (Muneoka, 1973, 1974; Murakami et al., 1985), and confirm that ACh-evoked Ca2÷-mobilization for the contraction is differerent from that evoked by FMRF-NH2(Kizawa et al., 1991). Moreover, ACh-
1 0 0 . = =
50
o lo
2o
-
,
I0
-
,
•
20
,
30
•
,
40
-
,
50
-
,
60
Time (sec)
Fig. 5. Effect of lowering external C a 2+ o n the ACh-induced contraction in the ABRM. Q): control, O: treated with Ca2+-free medium (contained 2mM EGTA) for 30min. Each value is presented as a mean with SEM (bar) of 6 experiments.
0
0
3'0
Time (rain) Fig. 6. The time course of contractile response to ACh (10-5 M) after exposure to the Ca2+-free medium (containing 2 mM EGTA) in the ABRM. Each value is presented as a mean with SEM (bar) of 6 experiments.
Fig. 7. Effect of ACh (10-SM) on IP3 formation in the ABRM. The amounts of IP3 were determined as described in Methods. The resting level of IP3 was 100.8 __+4.7 pmol/mg protein (mean with SEM of 5 experiments. • Significantlydifferent from the value for the control (0s) at P < 0.01.
induced contraction was significantly reduced by exposure to a Ca2+-lowering solution in the ABRM. These findings suggest that the ACh-induced response results from the influx of extracellular Ca 2+ possibly through the L-type Ca2+-channels. The inhibitory effects of dihydropyridine Ca2+-antagonists on ACh-induced contraction were weaker than those of other organic antagonists. This observation was similar to their effect on K+-induced contraction in the ABRM (Kizawa et aL, 1991). We therefore believe that there are some differences in Ca2+-channels gated by ACh between the ABRM and the mammalian smooth muscle. After exposure to the Ca2÷-deprived medium for 30min, ACh-induced contraction still remained, however, the ACh-induced response was almost abolished by TMB-8. This response might be caused either by Ca2+-release from intracellular pools or by another yet unknown mechanism. There is substantial evidence that IP 3 releases Ca 2+ from intracellular pools followed by activation of membrane surface receptors in various cells (Hokin, 1985; Berridge, 1987; Berridge and Irvine, 1989; Chuang, 1989; Ferris and Snyder, 1992). As reported by Hellwig and Achazi (1991), ACh accelerated the accumulation of inositol 1,4,5-trisphosphate (IP3) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the ABRM with [3H]-inositol. The present study also demonstrated that ACh increased the accumulation of IP3 in the ABRM without exogenous application of inositol. Thus, ACh-induced contraction in the ABRM may be partly mediated by Ca2+-release from IP 3 sensitive intraceilular pools. We conclude that both influx of extracellular Ca 2÷ via L-like Ca 2+ channels and Ca2÷-release from IP 3
ACh-induced contraction in the ABRM
1423
Hokin L. E. (1985) Receptors and phosphoinositide-generated second messengers. Ann. Rev. Biochem. 54, 205-235. Kizawa Y., Inudoh S., Arai Y., Uemura Y., Ohura M., Matsuura R., Tsukimura T. and Murakami H. (1991) Organic Ca2+-antagonist-resistant response to FMRFNH a on the molluscan smooth muscle. Gen. Pharmac. 22, 959-964. Lang B. D. and Lewis M. J. (1989) Endothelium-derived relaxing factor inhibits the formation of inositol trisphosphate by rabbit aorta. J. Physiol. 411, 45-52. Muneoka Y. (1973) Calcium-dependent acetylcholine contracture of molluscan catch muscle in sodium-free solution. Comp. Gen. Pharmac. 4, 277-284. Muneoka Y. (1974) Mechanical responses in potassium-depolarized smooth muscle Mytilus Edulis. Comp. Biochem. Physiol. 47A, 61-70. Murakami H., Ishikawa T. and Watanabe H. (1984) Acetylcholine release elicited by electrical stimulation and its Acknowledgement--Tiffs research was supported in part by sites of action in smooth muscle of Mytilus. Comp. a Research Grant from Nihon University (A91-066). Biochem. Physiol. 77C, 273-277. Murakami H., Ishikawa T. and Watanabe H. (1985) Effects of Ca-antagonists and antispasmodic drugs on contraction by ACh in molluscan smooth muscle. Comp. BioREFERENCES chem. Physiol. 80C, 167-173. Berridge M. J. (1987) Inositol trisphosphate and diacyl- Seishima M., Yada Y., Nagao S., Mori S. and Nozawa Y. (1988) Defective formation of inositol 1,4,5-trisphosphate glycerol: two interacting second messengers. Ann. Rev. in bradykinin-stimulatedfibroblasts from progressive sysBiochem. 56, 159-193. temic sclerotic patients. Biochem. Biophys. Res. Commun. Berridge M. J. and Irvine R. F. (1989) Inositol phosphates 156, 1077-1082. and cell signaling. Nature 341, 197-204. Chuang D. M. (1989) Neurotransmitter receptors and phos- Takayanagl I., Suzuhigashi K., Ishii Y. and Koike K. (1983) Pharmacological properties of acetylcholine receptor in phoinositide turnover. Ann. Rev. Pharmac. Toxicol. 29, molluscan smooth muscle. Jap. J. Pharmac. 33, 698-701. 71-110. Ferris C. D. and Snyder S. H. (1992) Inositol 1,4,5-trispho- Twarog B. M. (1954) Response of a molluscan smooth muscle to acetylcholine and 5-hydroxytryptamine. J. Cell. sphate-activated calcium channels. Ann. Rev. Physiol. 54, Comp. Physiol. 44, 141-164. 469-488. Hellwig G. and Achazi R. K. (1991) ACh and 5-HT induced Zeimal E. V. and Vulfius E. A. (1973) Pharmacology of cholinergic systems in molluscs. In International Encyclochanges in the concentration of cytosolic inositol trisphopedia of Pharmacology and Therapeutics. Section 85, sphate (InsP3) and inositol bisphosphate (InsP2) in the Vol. 1. Comparative Pharmacology (Edited by Michelson ABRM of Mytilus edulis L. Comp. Biochem. Physiol. M. J.), pp. 111-168. Pergamon Press, Oxford. 100C, 343-348.
sensitive intraceilular stores might be involved in ACh-induced contraction in the ABRM. In the Ca .'+ uptake pathway from extracellular space, the results obtained here are different from those obtained in the FMRF-NH2-induced contraction (Kizawa et al., 1991). Moreover, in our preliminary experiment, the Ca 2+ mobilization from intracellular stores for both contractions appears to be induced by a similar mechanism, possibly by one which is IP3 dependent. Thus, the discrepancy between Ca2+-mobilization mechanisms required for both contractions remains to be elucidated.
0306-3623/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
EFFECTS OF ORGANIC AND INORGANIC Ca2÷-ANTAGONISTS ON ACETYLCHOLINE-INDUCED CONTRACTION IN MOLLUSCAN (MYTILUS EDULIS) SMOOTH MUSCLE YUKO MIYAHARA, YASUO KIZAWA, MASAKAZU SANO and HAJIME MURAKAMI* Department of Physiology and Anatomy', Nihon University College of Pharmacy, Funabashi, Chiba 274, Japan [Tel. (0474)65-4716; Fax (0474)65-2158] (Received 22 March 1993) Abstract--1. Effect of Ca2+-antagonist on the contractile response to acetylcholine (ACh) in molluscan (Mytilus edulis) smooth muscle was investigated. 2. ACh-induced contraction was remarkably reduced by exposure to Ca2+-deprived medium. 3. The organic Ca2+-blockers, verapamil, diltiazem and nicardipine, reduced the concentration-response curve for ACh in a concentration-dependent manner. 4. The inorganic Ca2+-bloekers, MnClv NiCI2, COC12 and CdC12, also reduced the concentration-response curve for ACh concentration-dependently. 5. ACh significantly increased the amounts of inositol 1,4,5-trisphosphate (IP3) in the ABRM. 6. ACh-induced contraction in the ABRM might therefore be mediated through an influx of extracellular Ca 2+ and CaZ+-release from IP3 sensitive intracellular pools.
INTRODUCTION
Molluscan (Mytilus edulis) smooth muscle, anterior byssus retractor muscle ( A B R M ) is known to cause a catch contraction by acetylcholine (ACh) (Twarog, 1954; Murakami et al., 1985). ACh-induced contraction was reportedly mediated through an action on nicotinic-like A C h receptors (Takayanagi et al., 1983), but the receptors seemed to be slightly different from the vertebrate nicotinic A C h receptors in the A B R M (Zeimal and Vulfius, 1973; Takayanagi et al., 1983; M u r a k a m i et al., 1984). Murakami et aL (1985) reported the effects of Ca2+-antagonists on the ACh-induced contraction in the A B R M , and showed that some of these antagonists inhibited the concentration-response curve for ACh. We recently demonstrated that FMRF-NH2-induced contraction was resistant to organic Ca2+-an tagonists, especially 1,4-dihydropyridine derivatives in the A B R M (Kizawa et al., 1991). In this study, to obtain further evidence of ACh-induced Ca 2÷ mobilization in the A B R M , we investigated the effects of organic and inorganic Ca2+-antagonists on AChinduced contraction. The release of intracellular pools of Ca 2+ is known to be elicited by the generation of IP3 in response to a variety of hormones and neurotransmitters acting on excitable and non-excitable cells alike (Hokin,
*To whom all correspondence should be addressed.
1985; Berridge, 1987; Berridge and Irvine, 1989; Chuang, 1989; Ferris and Snyder, 1992). We measured the amounts of IP3 in the ACh-treated A B R M to determined the effect of A C h on phosphatidylinositol turnover. MATERIALS AND METHODS
Animals and preparation of muscle bundle Sea mussels, Mytilus edulis L., collected from Ise Bay (Japan) were stored in circulating artificial sea water at a temperature of about 10°C and used within a week after collection. The anterior byssus retractor muscle (ABRM) was dissected into a muscle bundle of about I mm in diameter and 1.5 cm in length. The bundle was bathed in a physiological solution of the following composition (mM): NaC1, 475; KC1, 10,0; CaCI2.2H20, 2.0; MgCI2,6H20, 20.0; Tris-HCl, 10.0 (pH 7.4). Each preparation was set up vertically for isometric tension recording in a 5 ml organ bath containing physiological solution, maintained at 20°C and bubbled with air. The resting tension was adjusted to 0.2 g for a period of approximately 1 hr. Isometric tension was recorded with a Nihon Kohden (Japan) TB-651T transducer, connected to a Nihon Kohden EP-601G amplifier and a Rikadenki (Japan) R-2 recorder. Mechanical response Two pieces of ABRM were dissected from the body of a single specimen of M. edulis, one bundle was used as the control preparation and the other as the drug-treated preparation. Cumulative concentration-response curve for ACh was obtained twice, and the drug-treated preparation was incubated in a drug-containing solution during the second concentration-response curve. In some experiments, the concentration-response curve for ACh was obtained in Ca 2+-free solution, which had been prepared by omitting CaC12 and adding 2 mM EGTA.
1419
YUKO MIYAHARAet al.
1420
100.
100.
B
A
o~
¢-
.o_
so
50 ltO
o
O, -4
Log [acetylcholine (M)]
Log [acetylcholine (M)]
Fig. 1. Effect of verapamil (A) or diltiazem (B) on the concentration-response curve of ACh in the ABRM. O: control, 0 : treated with 10 -5 M verapamil or diltiazem for 15 min, l : treated with 10-4 M verapamil or diltiazem for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
Measurement of inositol 1,4,5-trisphosphate (IP3) formation
Chemicals and reagents
The ABRM tissue was equilibrated in the physiological solution and bubbled with air for I hr at room temperature. Then the tissues were incubated with 10 -5 M ACh for 5, 10, 30 and 60s. The reaction was terminated by immersing the tissues in liquid nitrogen. The tissues were then homogenized in 1.0 ml of ice-cold 3.6% (w/v) perchloric acid and the resulting homogenates were centrifuged at 2000 g for 15 min at 4°C (Lang and Lewis, 1989). The supernatant was neutralized to pH 7.5 with ice-cold KOH solution, and KC104 was precipitated and removed by centrifugation. The final supernatant was purified with minicolumn (Amprep SAX, Amersham, U.K.). One hundred ~ul aliquots of eluate were used for IP 3 assay. The amount of IP 3 in the test sample was measured as described by Seishima et al. (1988).
Acetylcholine chloride (Daiichi Seiyaku Co., Ltd., Tokyo, Japan), verapamil (Eisai Co., Ltd., Tokyo, Japan), diltiazem (Tokyo Tanabe Seiyaku Co., Ltd., Tokyo, Japan), TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride), nifedipine and nicardipine (Sigma Chemical Co., St Louis, MO), CdCI2, NiCI 2 and MnCI 2 (Wako Junyaku Co., Ltd., Tokyo, Japan), EGTA (ethyleneglycol bis(fl-aminoethylether)-N, N'-tetraacetic acid; Dojindo Laboratories, Kumamoto, Japan) and o-myo-inositol 1,4,5trisphosphate (IP3) assay kit (Amersham, U.K.) were used. Other chemicals purchased were of analytical grade. Nifedipine was dissolved in ethanol, and the final ethanol concentration in physiological solution did not exceed 0. 1%
(v/v). RESULTS
Analysis of responses and statistical methods ACh-induced response was expressed as a percentage of the maximum effect reached in the first concentration-response curve in the control and the drug-treated preparations, respectively. Numerical results were expressed as means 4-SEM and statistical analyses were performed using Student's t-test or Duncan's multiple range test. A P value less than 0.05 was considered a significant difference.
A
o
1001
Effect o f organic Ca2+-antagonists on the concentration-response curve for ACh T h e c o n c e n t r a t i o n - r e s p o n s e curves for A C h were reduced by organic Ca2+-antagonists, nifedipine, nicardipine, diltiazem a n d verapamil c o n c e n t r a t i o n dependently (Figs 1 a n d 2). T h e m a x i m u m response
A
0t
100t t
o
0
0
-s
-:4
Log [acetylcholine (M)]
-3
-6
-s
-4
Log [acetylcholine (M)]
Fig. 2. Effect of nifedipine (A) or nicardipine (B) on the concentration-response curve of ACh in the ABRM. C): control, O: treated with 10-6 M nifedipine or nicardipine for 15 min,ll: treated with 10-5 M nifedipine or nicardipine for 15 min, A: treated with 3 x 10 -5 M nifedipine for 15 min, &: treated with 10 -4 M nicardipine. Each value is presented as a mean with SEM (bar) of 6 experiments.
ACh-induced contraction in the ABRM 100.
100.
50
50
0
1421
0 ,
-7
-
,
-
,
-
,
.
,
-6 -5 -4 -3 Log [acetylcholine (M)]
-
,
-2
-7
.
,
•
,
.
,
.
,
-6 -5 -4 -3 Log [acetylcholine (M)]
-
,
-2
Fig. 3. Effect of CdCI2 (A) or NiC12(B) on the concentration-response curve of ACh in the ABRM. C): control, Q: treated with 1 mM CdCI2 or NiCI2 for 15 min, I : treated with 10mM CdC12 or NiClz for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
to ACh was significantly reduced by these antagonists in a concentration dependent manner. The potency order was diltiazem > verapamil > nicardipine > nifedipine.
Effect of inorganic Ca2+-antagonists on the concentration-response curve for ACh Effect of inorganic Ca2+-antagonists, COCI2, CdCl2, NiCl2 and MnCl2 on the concentrationresponse curve for ACh is shown in Figs 3 and 4. These inorganic Ca2+-antagonists noncompetitively inhibited the contractile response to ACh. The potency order was CdCl2 > NiC12 > MnC12 > CoCl 2.
ACh-induced contraction in Ca2+-lowering medium The concentration-response curve for ACh was depressed in the Ca2+-lowering medium (Ca2+-free solution containing 2 mM EGTA) for 30 min in the ABRM (Fig. 5). Figure 6 shows the time course of
contractile response to ACh (10 -5 M) after exposure to the Ca2+-deprived medium in the ABRM. The ACh-induced contraction was reduced to 66.3 + 2.7, 56.5 + 4.0, 34.0 + 3.7, 18.5 + 1.8 and 12.0 + 2.5% (mean with SEM of 6 experiments) at, respectively, 5, 10, 15, 20 or 30 min after exposure to the Ca~+-de prived medium, suggesting that influx of extracellular Ca 2+ is necessary for the contractile response to ACh. After incubation of ABRM in Ca 2+-deprived medium for 5 min, the remaining ACh-induced contraction was reduced to 9.5 + 2.0% (mean with SEM of 9 experiments) by 10min pretreatment with 0.1 mM TMB-8 (data not shown). The ACh-induced response may thus be due to release of intracellular pooled Ca 2+
Estimation of the amounts of IP~ in the A B R M The resting level of IP 3 was 100.8 + 4.7 pmol/mg protein (mean with SEM of 5 experiments). The
100.
lOO.
g g
g 50
~
o
so
o
Log [acetylcholine M)]
Log [acetylchoUne (M)]
Fig. 4. Effect of MnCI2 (A) or CoCIz (B) on the concentration-response curve of ACh in the ABRM. C): control, @: treated with 1 mM MnCI2or COC12for 15 min, I1: treated with 10 mM MnCI2 or COC12for 15 min. Each value is presented as a mean with SEM (bar) of 6 experiments.
1422
YUKOMIYANARAet al. 500
100.
400
o~ "~. 300.
c-
.o 50.
200,
C 0
o 100. 0 ,
-7
-6
"4
"5
"3
Log [acetylcholine (M)]
amounts of IP 3 were increased to 210.8 +20.0, 402.2 ___39.5, 360.2 + 65.3 and 165.9 + 31.2 pmol/mg protein (mean with SEM of 5 experiments) by incubation of the ABRM with ACh (10 -5 M) for, respectively, 5, 10, 30 or 60s (Fig. 7). DISCUSSION We compared the effects of organic and inorganic Ca2+-antagonists on ACh-induced contraction in the ABRM. Under our experimental conditions both organic and inorganic Ca2+-blockers reduced the contraction in a concentration-dependent manner. These observations corroborate previous reports (Muneoka, 1973, 1974; Murakami et al., 1985), and confirm that ACh-evoked Ca2÷-mobilization for the contraction is differerent from that evoked by FMRF-NH2(Kizawa et al., 1991). Moreover, ACh-
1 0 0 . = =
50
o lo
2o
-
,
I0
-
,
•
20
,
30
•
,
40
-
,
50
-
,
60
Time (sec)
Fig. 5. Effect of lowering external C a 2+ o n the ACh-induced contraction in the ABRM. Q): control, O: treated with Ca2+-free medium (contained 2mM EGTA) for 30min. Each value is presented as a mean with SEM (bar) of 6 experiments.
0
0
3'0
Time (rain) Fig. 6. The time course of contractile response to ACh (10-5 M) after exposure to the Ca2+-free medium (containing 2 mM EGTA) in the ABRM. Each value is presented as a mean with SEM (bar) of 6 experiments.
Fig. 7. Effect of ACh (10-SM) on IP3 formation in the ABRM. The amounts of IP3 were determined as described in Methods. The resting level of IP3 was 100.8 __+4.7 pmol/mg protein (mean with SEM of 5 experiments. • Significantlydifferent from the value for the control (0s) at P < 0.01.
induced contraction was significantly reduced by exposure to a Ca2+-lowering solution in the ABRM. These findings suggest that the ACh-induced response results from the influx of extracellular Ca 2+ possibly through the L-type Ca2+-channels. The inhibitory effects of dihydropyridine Ca2+-antagonists on ACh-induced contraction were weaker than those of other organic antagonists. This observation was similar to their effect on K+-induced contraction in the ABRM (Kizawa et aL, 1991). We therefore believe that there are some differences in Ca2+-channels gated by ACh between the ABRM and the mammalian smooth muscle. After exposure to the Ca2÷-deprived medium for 30min, ACh-induced contraction still remained, however, the ACh-induced response was almost abolished by TMB-8. This response might be caused either by Ca2+-release from intracellular pools or by another yet unknown mechanism. There is substantial evidence that IP 3 releases Ca 2+ from intracellular pools followed by activation of membrane surface receptors in various cells (Hokin, 1985; Berridge, 1987; Berridge and Irvine, 1989; Chuang, 1989; Ferris and Snyder, 1992). As reported by Hellwig and Achazi (1991), ACh accelerated the accumulation of inositol 1,4,5-trisphosphate (IP3) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the ABRM with [3H]-inositol. The present study also demonstrated that ACh increased the accumulation of IP3 in the ABRM without exogenous application of inositol. Thus, ACh-induced contraction in the ABRM may be partly mediated by Ca2+-release from IP 3 sensitive intraceilular pools. We conclude that both influx of extracellular Ca 2÷ via L-like Ca 2+ channels and Ca2÷-release from IP 3
ACh-induced contraction in the ABRM
1423
Hokin L. E. (1985) Receptors and phosphoinositide-generated second messengers. Ann. Rev. Biochem. 54, 205-235. Kizawa Y., Inudoh S., Arai Y., Uemura Y., Ohura M., Matsuura R., Tsukimura T. and Murakami H. (1991) Organic Ca2+-antagonist-resistant response to FMRFNH a on the molluscan smooth muscle. Gen. Pharmac. 22, 959-964. Lang B. D. and Lewis M. J. (1989) Endothelium-derived relaxing factor inhibits the formation of inositol trisphosphate by rabbit aorta. J. Physiol. 411, 45-52. Muneoka Y. (1973) Calcium-dependent acetylcholine contracture of molluscan catch muscle in sodium-free solution. Comp. Gen. Pharmac. 4, 277-284. Muneoka Y. (1974) Mechanical responses in potassium-depolarized smooth muscle Mytilus Edulis. Comp. Biochem. Physiol. 47A, 61-70. Murakami H., Ishikawa T. and Watanabe H. (1984) Acetylcholine release elicited by electrical stimulation and its Acknowledgement--Tiffs research was supported in part by sites of action in smooth muscle of Mytilus. Comp. a Research Grant from Nihon University (A91-066). Biochem. Physiol. 77C, 273-277. Murakami H., Ishikawa T. and Watanabe H. (1985) Effects of Ca-antagonists and antispasmodic drugs on contraction by ACh in molluscan smooth muscle. Comp. BioREFERENCES chem. Physiol. 80C, 167-173. Berridge M. J. (1987) Inositol trisphosphate and diacyl- Seishima M., Yada Y., Nagao S., Mori S. and Nozawa Y. (1988) Defective formation of inositol 1,4,5-trisphosphate glycerol: two interacting second messengers. Ann. Rev. in bradykinin-stimulatedfibroblasts from progressive sysBiochem. 56, 159-193. temic sclerotic patients. Biochem. Biophys. Res. Commun. Berridge M. J. and Irvine R. F. (1989) Inositol phosphates 156, 1077-1082. and cell signaling. Nature 341, 197-204. Chuang D. M. (1989) Neurotransmitter receptors and phos- Takayanagl I., Suzuhigashi K., Ishii Y. and Koike K. (1983) Pharmacological properties of acetylcholine receptor in phoinositide turnover. Ann. Rev. Pharmac. Toxicol. 29, molluscan smooth muscle. Jap. J. Pharmac. 33, 698-701. 71-110. Ferris C. D. and Snyder S. H. (1992) Inositol 1,4,5-trispho- Twarog B. M. (1954) Response of a molluscan smooth muscle to acetylcholine and 5-hydroxytryptamine. J. Cell. sphate-activated calcium channels. Ann. Rev. Physiol. 54, Comp. Physiol. 44, 141-164. 469-488. Hellwig G. and Achazi R. K. (1991) ACh and 5-HT induced Zeimal E. V. and Vulfius E. A. (1973) Pharmacology of cholinergic systems in molluscs. In International Encyclochanges in the concentration of cytosolic inositol trisphopedia of Pharmacology and Therapeutics. Section 85, sphate (InsP3) and inositol bisphosphate (InsP2) in the Vol. 1. Comparative Pharmacology (Edited by Michelson ABRM of Mytilus edulis L. Comp. Biochem. Physiol. M. J.), pp. 111-168. Pergamon Press, Oxford. 100C, 343-348.
sensitive intraceilular stores might be involved in ACh-induced contraction in the ABRM. In the Ca .'+ uptake pathway from extracellular space, the results obtained here are different from those obtained in the FMRF-NH2-induced contraction (Kizawa et al., 1991). Moreover, in our preliminary experiment, the Ca 2+ mobilization from intracellular stores for both contractions appears to be induced by a similar mechanism, possibly by one which is IP3 dependent. Thus, the discrepancy between Ca2+-mobilization mechanisms required for both contractions remains to be elucidated.