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The relaxing effect of SKF 38393 on the catch contraction of Mytilus smooth muscle
Gen. Pharrnac. Vol. 17, No. 6, pp. 685-687, 1986 Printed in Great Britain
0306-3623/86 $3.00+0.00 Pergamon Journals Ltd
T H E R E L A X I N G E F F E C T OF SKF 38393 O N T H E C A T C H CONTRACTION OF M Y T I L U S SMOOTH MUSCLE HAJIME MURAKAMI, l TOSHINORI ISHIKAWA2 a n d MASAKAZU SANO3 Departments of ~Physiology and 2 Biology, Nihon University, School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan and 3Department of Oral Physiology, School of Dentistry, Showa University, Shinagawa-Ku, Tokyo 142, Japan (Received 24 February 1986) A b s t r a c t - - 1 . The relaxing effect of SKF 38393 on the catch contraction in the anterior byssus retractor
muscle (ABRM) of Mytilus and the effect of SKF 38393 on the cyclic AMP (cAMP) levels in the ABRM were investigated. 2. The catch contraction was relaxed by SKF 38393 in a dose-dependent manner. 3. The dose-response curves of SKF 38393 were shifted in parallel to the right by butaclamol (3 × 10 -6 and 6 x 10 -6 M ) and haloperidol (3 x 10 -6 and 3 × 10 -5 M). Their pA 2 values were 5.69 + 0.05 and 5.80 __+0.07. 4. The cAMP levels in the ABRM were not altered by SKF 38393 at a concentration (10 -5 M) sufficient to relax. 5. These findings indicate that the dopamine receptors of the ABRM are D-I like receptors but somewhat different from those of the vertebrates.
INTRODUCTION D o p a m i n e relaxes the catch c o n t r a c t i o n o f the a n t e r i o r byssus r e t r a c t o r muscle ( A B R M ) o f Mytilus. T h e existence o f specific d o p a m i n e receptors mediating relaxation in the A B R M has been s h o w n by p h a r m a c o l o g i c a l ( T w a r o g a n d Cole, 1972; M u n e o k a et al., 1978; T a k a y a n a g i et al., 1981; Y o s h i d a et al., 1981; Ishii a n d T a k a y a n a g i , 1982a; M u r a k a m i et al., 1983), r a d i o l i g a n d binding ( I w a y a m a a n d T a k a y a nagi, 1982) a n d photo-affinity labeling techniques (Ishii a n d T a k a y a n a g i , 1982b). K e b a b i a n a n d Calne (1979) have divided the dopa m i n e receptors into two types, namely D-I a n d D-2. Moreover, selective d o p a m i n e receptor agonists a n d a n t a g o n i s t s according to the n o m e n c l a t u r e of them h a v e also been discovered (Stoof a n d K e b a b i a n , 1984). S K F 38393 (1-phenyl-2,3,4,5-tetrahydro-(l-H)-3benzazepine-7,8-diol) has been identified by Pendelton et al. (1978) to be a d o p a m i n e receptor agonist, a n d it acts on the renal D-1 receptors selectively ( S t o o f a n d K e b a b i a n , 1984) a n d in a d d i t i o n is b o u n d to the D-1 receptors in the b r a i n (Scatton a n d Dubois, 1985). A t present S K F 38393 is the m o s t selective D-1 agonist. In the A B R M , since b r o m o c r i p t i n e , a selective D-2 agonist is ineffective in relaxing the catch c o n t r a c t i o n (Ishii a n d T a k a y a n a g i , 1982a), d o p a m i n e receptors in the A B R M do n o t a p p e a r to be D-2 receptors. In the present study, in o r d e r to define which subtype o f d o p a m i n e receptors is involved in the d o p a m i n e - i n d u c e d relaxation, we have investigated the effect of S K F 38393 o n the catch c o n t r a c t i o n in the A B R M , a n d also investigated w h e t h e r the S K F 38393 causes the increase in the cyclic A M P ( c A M P ) level in the A B R M or not, because the response t h a t is m e d i a t e d by D-1 receptors is associated with the 685
adenylate 1979).
cyclase
system
(Kebabian
and
Calne,
MATERIALS AND METHODS
Sea mussels, Mytilus edulis L., collected from the east coast of Tokyo Bay were used in the present experiment. The animals were stored in circulating artificial seawater (ASW) at about 10°C and used within 8 days after collection. Muscle bundles (about 1 mm dia.) dissected from the ABRM were suspended in a 5 ml organ bath filled with oxygenated ASW (mmol/l: NaC1475, KCI 10, CaCI2-2H20 10, MgC12.6H20 20, Tris-HC1 10, pH 7.4) and kept at 20 + I°C, to be held at about 80% of in situ length. Responses to drugs were recorded on an ink-writing recorder isometrically. To relax the muscle sufficiently, repetitive pulses of stimulation (15V, 2msec, 5Hz) were applied to the muscle for 5 sec. After an equilibration period of 30 min, the muscle was exposed to I 0 - 4 M acetylcholine (ACh) for 2 min to produce the catch contraction and washed with ASW for 5 rain, and then SKF 38393 was applied. The relaxation induced by a 10 rain exposure to SKF 38393 was estimated. The relaxation induced by SKF 38393 was expressed as a % of the maximum relaxation induced by 10 -6 M serotonin. To estimate the antagonism between SKF 38393 and antagonists, butaclamol (BUTA) and haloperidol (HALO), after a 2 min exposure to ACh (10 -4 M) and washing with ASW, one of the antagonists was applied for 5 rain. Subsequently in the presence of the antagonist, SKF 38393 was applied according to the previously described methods (Takayanagi et al., 1981; Murakami et al., 1983). Estimation o f c A M P levels The cAMP formed in the ABRM was estimated by the method of Steiner et aL (1972). Two bundles of the ABRM were equilibrated for 1 hr in test tubes filled with ASW at room temperature and bubbled with air. After a 2min treatment with SKF 38393, the muscle bundles were frozen in liquid nitrogen. One bundle was used for measuring the control level of cAMP and the other for estimating any change of cAMP concentration after treatment with
686
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MURAKAMI
SKF 38393. The muscles were homogenized in 1 ml of cold trichloroacetic acid (6% w/v) which contained 0.5 pmol of [~H]cAMP to estimate the recovery of cAMP. The homogenate was centrifuged at 3000 g at 0°C for 15 rain, supernatant was then acidified with 1 N HC1, and the trichloroacetic acid was extracted with ether. The cAMP samples were lyophilized. The lyophilized samples were dissolved with sodium acetate buffer (pH6.2), and used for the estimation of cAMP, and for calculating the recovery the radioactivity was counted with a liquid scintillation spectrometer (Aloka LSC-9000) in 10 ml toluene scintillator containing 4 g 2,5-diphenyloxazole, 0.1g 1,4-bis-2-(4methyl-5-phenyloxazolyl)-benzene, 300ml methanol and 700 ml toluene.
Control
0-
SKF with 3 X 1 0 - 6 M H A L O
[ ~
SKF with 3 X 1 0 - 5 M H A L O
100 -7
-6
-5
-4
S K F ( l o g M)
Protein assay Protein concentrations were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
Fig. 2. Effect of HALO on the dose-response curve of SKF 38393. Relaxation is presented as a percentage of the maximum relaxation induced by serotonin (10 6 M). Each point is the mean + SE of six experiments. Spontaneous relaxation of catch contraction is shown as a horizontal dotted line and is 23 +_ 3.2% (mean + SE). SKF: SKF 38393.
Estimation of p A 2 values The pA 2 values of the antagonists were calculated from the parallel shift of the dose-response curve of SKF 38393 (Van Rossum, 1963).
The effects of B U T A and H A L O on the S K F 38393induced relaxation
Statistical analysis Statistical significance was evaluated using Student's t-test. Drugs The drugs used and their sources were as follows: acetylcholine chloride (Sigma), haloperidol.HC1 (Sigma), serotonin (5-hydroxytriptamine creatinine sulfate, Wako pure chemical Industries Ltd), butaclamol. HCI (Research Biochemicals Inc.), SKF38393.HCI (1-phenyl-2,3,4,5-tetrahydro-(1-H)-3-benzazepine-7,8-diol hydrochloride, Research Biochemicals Inc.), cAMP (adenosine 3',5'-cyclic monophosphate-Na, Sigma), [3H]cAMP (specific activity: 36.4 Ci/mmol). Other chemicals used were of analytical grade.
RESULTS S K F 38393 relaxed the catch c o n t r a c t i o n o f the A B R M in a d o s e - d e p e n d e n t m a n n e r as s h o w n in Figs 1 a n d 2, a l t h o u g h the relaxing response to S K F 38393 was less p o t e n t t h a n t h a t to dopamine. ( ~ 0
To investigate whether the S K F 38393 acted o n the d o p a m i n e receptors in the A B R M or not, the effects of the competitive d o p a m i n e receptor a n t a g o n i s t s in the A B R M , B U T A a n d H A L O on the S K F 38393induced relaxation were investigated. As s h o w n in Fig. 1 the d o s e - r e s p o n s e curves of S K F 38393 were shifted in parallel to the right by B U T A at the c o n c e n t r a t i o n s of 3 x 10 -6 a n d 6 x 10 -6 M, a n d pA2 value of B U T A at a c o n c e n t r a t i o n o f 3 x 10 -6 M was almost equal to t h a t of B U T A at a c o n c e n t r a t i o n of 6 x 10 -6 M. The pA 2 value of B U T A against S K F 38393 was 5.69 + 0.05. Moreover, as s h o w n in Fig. 2 the d o s e - r e s p o n s e curves o f S K F 38393 were also shifted in parallel to the right by H A L O at the c o n c e n t r a t i o n s of 3 x 10 6 a n d 3 x 10 -5 M, a n d the pA 2 value of H A L O at a c o n c e n t r a t i o n of 3 x 1 0 6 M was a l m o s t equal to t h a t of H A L O at a c o n c e n t r a t i o n of 3 x 10 - 5 M . The pA 2 value of H A L O against S K F 38393 was 5.80 + 0.07. These findings indicate t h a t S K F 38393 behaves as a d o p a m i n e r g i c agonist for the d o p a m i n e receptors in the A B R M .
The influence o f S K F 38393 on the c A M P levels in ABRM The c A M P levels in the A B R M was not altered significantly by S K F 38393 at a c o n c e n t r a t i o n of
Control S K F with 3 X 1 0 - 6 M
BUTA
SKF with6XtO-6M
BUTA
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Fig. 1. Effect of BUTA on the dose-response curve of SKF 38393. Relaxation is presented as a percentage of the maximum relaxation induced by serotonin (10 -6 M). Each point is the mean +__SE of six experiments. Spontaneous relaxation of catch contraction is shown as a horizontal dotted line and is 23 + 3.2% (mean + SE). SKF:SKF 38393.
o
0 C
SKF3889a
10-5M Fig. 3. Effect of SKF 38393 on the cAMP levels in the ABRM. Values are presented as the mean with SE of eight experiments. C: untreated ABRM.
SKF 38393 on Mytilus smooth muscle 10 5M which relaxed the catch contraction of the ABRM sufficiently as shown in Fig. 3. The cAMP levels in the untreated ABRM were 6.76 + 0.36 pmol/ mg protein (n = 8) and those in the SKF38393treated muscle were 6.13+0.27pmol/mg protein (n =8). Thus, these findings indicate that the SKF 38393-induced relaxation of catch contraction does not link to the adenylate cyclase system. DISCUSSION A selective D-2 receptor agonist, bromocriptine is ineffective in subserving the relaxation of catch contraction and further a selective D-2 receptor antagonist, sulpride does not possess the ability to prevent the dopamine-induced relaxation of catch contraction (Ishii and Takayanagi, 1982a). Thus dopamine receptors mediating the relaxation of catch contraction do not appear to be D-2 receptors. However, these investigations do not provide the convincing evidence that dopamine receptors in the ABRM are D-1 receptors. Therefore, we tried to solve the problem by using the most selective D-1 receptor agonist, SKF 38393 (Stoof and Kebabian, 1984) as a means of proving the pharmacological property of the dopamine receptors in the ABRM. In result, SKF 38393 relaxed the catch contraction in a dose-dependent manner, as did dopamine. The dopamine-induced relaxation of catch contraction is antagonized by BUTA and HALO in a competitive manner (Yoshida et al., 1981; Murakami et al., 1983). Therefore, the effects of BUTA and HALO on the SKF 38393induced relaxation of catch contraction were investigated. The SKF 38393-induced relaxation was antagonized by BUTA and HALO in a competitive manner and their pA 2 values against SKF 38393 were 5.69_+ 0.05 (BUTA) and 5.80_+ 0.07 (HALO) respectively. These pA: values obtained here are similar to those obtained by Yoshida et al. (1981) and Murakami et al. (1986), i.e. the pA2 value of BUTA against dopamine is 6.13 _+ 0.04 and that of HALO against dopamine is 6.0_+ 0.2. Theoretically two different agonists acting on the same receptor yield a similar pA 2 value against a common antagonist (Arunlakshana and Shild, 1959). Considering in the similar way, these findings indicate that dopamine and SKF 38393 act on the common dopamine recognition site and the dopamine recognition site in the ABRM is D-1 receptors. Moreover, the increase in cAMP levels produced by dopamine is a biochemical sign characterizing D-I receptors activation deviced by Kebabian and Calne (1979). In the dopamine sensitive adenylate cyclase assay system, SKF 38393 mimics the ability of dopamine to stimulate adenylate cyclase activity, and consequently SKF 38393 activates the adenylate cyclase by the same mechanism as in the case of dopamine (Setler et al., 1978; Waiting and Dowling, 1981). However, SKF 38393 (10 5 M) at a concentration sufficient to relax the catch contraction failed to increase the cAMP levels in the ABRM significantly. The finding is in good agreement with the observations that dopamine fails to increase the cAMP levels in the ABRM significantly (Ishikawa et al., 1981; Ishii and Takayanagi, 1982a). Consequently, although the dopamine receptors of the ABRM are D-I like receptors, the D-1 like
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receptors are somewhat different from the D-1 receptors in the vertebrate tissues in the point that the D-I like receptor activation by SKF 38393 does not link to the increased cAMP levels. REFERENCES
Arunlakshana O. and Schild H. O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmac. Chemother. 14, 48-58. Ishii Y. and Takayanagi I. (1982a) Effects of some ergot alkaloids on dopamine receptors of molluscan smooth muscle. J. pharm. Dyn. Tokyo 5, 751-753. Ishii Y. and Takayanagi I. (1982b) Specific [3H]haloperidol binding to dopamine receptors in the anterior byssus retractor muscle. Jap. J. Pharmac. 32, 1151 1155. Ishikawa T., Murakami H. and Iwayama Y. (1981) Changes in cAMP and cGMP levels induced by relaxing drugs in acetylcholine- and potassium-treated molluscan smooth muscle. Comp. Bioehem. Physiol. 70C, 171-176. Iwayama Y. and Takayanagi I. (1982) Photoaffinity labeling of dopamine receptors in molluscan smooth muscle. J. Pharm. Pharmac. 34, 729-730. Kebabian J. W. and Calne D. B. (1979) Multiple receptors for dopamine. Nature, Lond. 277, 93-96. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Muneoka Y., Shiba Y. and Kanno Y. (1978) Effects of drugs on the relaxing action of various monoamines in molluscan smooth muscle. Hiroshima J. med. Sei. 27, 163-171. Murakami H., Ishikawa T. and Sano M. (1986) The site of action of dopamine and its related compounds in smooth muscle of Mytilus. Comp. Biochem. Physiol. 84C, 51-54. Murakami H., Satoh T. and Ishikawa T. (1983) Effects of bulbocapnine and butaclamol on the relaxation of catch contraction by some dopaminergic agonists in Mytilus smooth muscle. Comp. Biochem. Physiol. 75C, 227-229, Pendelton R. G., Samler L., Kaiser C. and Redley P. T. (1978) Studies on renal dopamine receptors with SK&F 38393, a new agonist. Eur. J. Pharmac. 51, 19 28. Scatton B. and Dubois A. (1985) Autoradiographic localization of D~ dopamine receptors in the rat brain with [3H]SKF38393. Eur. J. Pharmac. 111, 145 146. Setler P. E., Saran H. M., Zirkle C. L. and Saunders H. L. (1978) The central effects of a novel dopamine agonist. Eur. J. Pharmac. 50, 419-430. Steiner A. L., Parker C. E. and Kipnis D. M. (1972) Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. biol. Chem. 247, ll06-1113. Stoof J. C. and Kebabian J. W. (1984) Two dopamine receptors: Biochemistry, physiology and pharmacology. Life Sci. 35, 2281-2296. Takayanagi I., Murakami H., Iwayama Y., Yoshida Y. and Miki S. (1981) Dopamine receptor in anterior byssus retractor muscle of Mytilus. Jap. J. Pharmae. 31,249-252. Twarog B. M. and Cole R. A. (1972) Relaxation of catch in a molluscan smooth muscle--If. Effects of serotonin, dopamine and related compounds. Comp. Biochem. Physiol. 43A, 331-335. Van Rossum J. M. (1963) Cumulative dose-response curve II, Technique for the making of dose-response curve in isolated organ and the evaluation of drug. Archs int. Pharmacodyn. ThOr. 143, 299-330. Waiting K. J. and Dowling J. E. (1981) Dopaminergic mechanism in the toleost retina I. Dopamine-sensitive adenylate cyclase in homogenates of carp retina; effects of agonist, antagonists and ergots. J. Neurochem. 36, 559-568. Yoshida Y., Takayanagi I. and Murakami H. (1981) Dopamine and its antagonists on molluscan smooth muscle. J. pharm. Dyn. Tokyo 5, 751 753.
0306-3623/86 $3.00+0.00 Pergamon Journals Ltd
T H E R E L A X I N G E F F E C T OF SKF 38393 O N T H E C A T C H CONTRACTION OF M Y T I L U S SMOOTH MUSCLE HAJIME MURAKAMI, l TOSHINORI ISHIKAWA2 a n d MASAKAZU SANO3 Departments of ~Physiology and 2 Biology, Nihon University, School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan and 3Department of Oral Physiology, School of Dentistry, Showa University, Shinagawa-Ku, Tokyo 142, Japan (Received 24 February 1986) A b s t r a c t - - 1 . The relaxing effect of SKF 38393 on the catch contraction in the anterior byssus retractor
muscle (ABRM) of Mytilus and the effect of SKF 38393 on the cyclic AMP (cAMP) levels in the ABRM were investigated. 2. The catch contraction was relaxed by SKF 38393 in a dose-dependent manner. 3. The dose-response curves of SKF 38393 were shifted in parallel to the right by butaclamol (3 × 10 -6 and 6 x 10 -6 M ) and haloperidol (3 x 10 -6 and 3 × 10 -5 M). Their pA 2 values were 5.69 + 0.05 and 5.80 __+0.07. 4. The cAMP levels in the ABRM were not altered by SKF 38393 at a concentration (10 -5 M) sufficient to relax. 5. These findings indicate that the dopamine receptors of the ABRM are D-I like receptors but somewhat different from those of the vertebrates.
INTRODUCTION D o p a m i n e relaxes the catch c o n t r a c t i o n o f the a n t e r i o r byssus r e t r a c t o r muscle ( A B R M ) o f Mytilus. T h e existence o f specific d o p a m i n e receptors mediating relaxation in the A B R M has been s h o w n by p h a r m a c o l o g i c a l ( T w a r o g a n d Cole, 1972; M u n e o k a et al., 1978; T a k a y a n a g i et al., 1981; Y o s h i d a et al., 1981; Ishii a n d T a k a y a n a g i , 1982a; M u r a k a m i et al., 1983), r a d i o l i g a n d binding ( I w a y a m a a n d T a k a y a nagi, 1982) a n d photo-affinity labeling techniques (Ishii a n d T a k a y a n a g i , 1982b). K e b a b i a n a n d Calne (1979) have divided the dopa m i n e receptors into two types, namely D-I a n d D-2. Moreover, selective d o p a m i n e receptor agonists a n d a n t a g o n i s t s according to the n o m e n c l a t u r e of them h a v e also been discovered (Stoof a n d K e b a b i a n , 1984). S K F 38393 (1-phenyl-2,3,4,5-tetrahydro-(l-H)-3benzazepine-7,8-diol) has been identified by Pendelton et al. (1978) to be a d o p a m i n e receptor agonist, a n d it acts on the renal D-1 receptors selectively ( S t o o f a n d K e b a b i a n , 1984) a n d in a d d i t i o n is b o u n d to the D-1 receptors in the b r a i n (Scatton a n d Dubois, 1985). A t present S K F 38393 is the m o s t selective D-1 agonist. In the A B R M , since b r o m o c r i p t i n e , a selective D-2 agonist is ineffective in relaxing the catch c o n t r a c t i o n (Ishii a n d T a k a y a n a g i , 1982a), d o p a m i n e receptors in the A B R M do n o t a p p e a r to be D-2 receptors. In the present study, in o r d e r to define which subtype o f d o p a m i n e receptors is involved in the d o p a m i n e - i n d u c e d relaxation, we have investigated the effect of S K F 38393 o n the catch c o n t r a c t i o n in the A B R M , a n d also investigated w h e t h e r the S K F 38393 causes the increase in the cyclic A M P ( c A M P ) level in the A B R M or not, because the response t h a t is m e d i a t e d by D-1 receptors is associated with the 685
adenylate 1979).
cyclase
system
(Kebabian
and
Calne,
MATERIALS AND METHODS
Sea mussels, Mytilus edulis L., collected from the east coast of Tokyo Bay were used in the present experiment. The animals were stored in circulating artificial seawater (ASW) at about 10°C and used within 8 days after collection. Muscle bundles (about 1 mm dia.) dissected from the ABRM were suspended in a 5 ml organ bath filled with oxygenated ASW (mmol/l: NaC1475, KCI 10, CaCI2-2H20 10, MgC12.6H20 20, Tris-HC1 10, pH 7.4) and kept at 20 + I°C, to be held at about 80% of in situ length. Responses to drugs were recorded on an ink-writing recorder isometrically. To relax the muscle sufficiently, repetitive pulses of stimulation (15V, 2msec, 5Hz) were applied to the muscle for 5 sec. After an equilibration period of 30 min, the muscle was exposed to I 0 - 4 M acetylcholine (ACh) for 2 min to produce the catch contraction and washed with ASW for 5 rain, and then SKF 38393 was applied. The relaxation induced by a 10 rain exposure to SKF 38393 was estimated. The relaxation induced by SKF 38393 was expressed as a % of the maximum relaxation induced by 10 -6 M serotonin. To estimate the antagonism between SKF 38393 and antagonists, butaclamol (BUTA) and haloperidol (HALO), after a 2 min exposure to ACh (10 -4 M) and washing with ASW, one of the antagonists was applied for 5 rain. Subsequently in the presence of the antagonist, SKF 38393 was applied according to the previously described methods (Takayanagi et al., 1981; Murakami et al., 1983). Estimation o f c A M P levels The cAMP formed in the ABRM was estimated by the method of Steiner et aL (1972). Two bundles of the ABRM were equilibrated for 1 hr in test tubes filled with ASW at room temperature and bubbled with air. After a 2min treatment with SKF 38393, the muscle bundles were frozen in liquid nitrogen. One bundle was used for measuring the control level of cAMP and the other for estimating any change of cAMP concentration after treatment with
686
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SKF 38393. The muscles were homogenized in 1 ml of cold trichloroacetic acid (6% w/v) which contained 0.5 pmol of [~H]cAMP to estimate the recovery of cAMP. The homogenate was centrifuged at 3000 g at 0°C for 15 rain, supernatant was then acidified with 1 N HC1, and the trichloroacetic acid was extracted with ether. The cAMP samples were lyophilized. The lyophilized samples were dissolved with sodium acetate buffer (pH6.2), and used for the estimation of cAMP, and for calculating the recovery the radioactivity was counted with a liquid scintillation spectrometer (Aloka LSC-9000) in 10 ml toluene scintillator containing 4 g 2,5-diphenyloxazole, 0.1g 1,4-bis-2-(4methyl-5-phenyloxazolyl)-benzene, 300ml methanol and 700 ml toluene.
Control
0-
SKF with 3 X 1 0 - 6 M H A L O
[ ~
SKF with 3 X 1 0 - 5 M H A L O
100 -7
-6
-5
-4
S K F ( l o g M)
Protein assay Protein concentrations were measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
Fig. 2. Effect of HALO on the dose-response curve of SKF 38393. Relaxation is presented as a percentage of the maximum relaxation induced by serotonin (10 6 M). Each point is the mean + SE of six experiments. Spontaneous relaxation of catch contraction is shown as a horizontal dotted line and is 23 +_ 3.2% (mean + SE). SKF: SKF 38393.
Estimation of p A 2 values The pA 2 values of the antagonists were calculated from the parallel shift of the dose-response curve of SKF 38393 (Van Rossum, 1963).
The effects of B U T A and H A L O on the S K F 38393induced relaxation
Statistical analysis Statistical significance was evaluated using Student's t-test. Drugs The drugs used and their sources were as follows: acetylcholine chloride (Sigma), haloperidol.HC1 (Sigma), serotonin (5-hydroxytriptamine creatinine sulfate, Wako pure chemical Industries Ltd), butaclamol. HCI (Research Biochemicals Inc.), SKF38393.HCI (1-phenyl-2,3,4,5-tetrahydro-(1-H)-3-benzazepine-7,8-diol hydrochloride, Research Biochemicals Inc.), cAMP (adenosine 3',5'-cyclic monophosphate-Na, Sigma), [3H]cAMP (specific activity: 36.4 Ci/mmol). Other chemicals used were of analytical grade.
RESULTS S K F 38393 relaxed the catch c o n t r a c t i o n o f the A B R M in a d o s e - d e p e n d e n t m a n n e r as s h o w n in Figs 1 a n d 2, a l t h o u g h the relaxing response to S K F 38393 was less p o t e n t t h a n t h a t to dopamine. ( ~ 0
To investigate whether the S K F 38393 acted o n the d o p a m i n e receptors in the A B R M or not, the effects of the competitive d o p a m i n e receptor a n t a g o n i s t s in the A B R M , B U T A a n d H A L O on the S K F 38393induced relaxation were investigated. As s h o w n in Fig. 1 the d o s e - r e s p o n s e curves of S K F 38393 were shifted in parallel to the right by B U T A at the c o n c e n t r a t i o n s of 3 x 10 -6 a n d 6 x 10 -6 M, a n d pA2 value of B U T A at a c o n c e n t r a t i o n o f 3 x 10 -6 M was almost equal to t h a t of B U T A at a c o n c e n t r a t i o n of 6 x 10 -6 M. The pA 2 value of B U T A against S K F 38393 was 5.69 + 0.05. Moreover, as s h o w n in Fig. 2 the d o s e - r e s p o n s e curves o f S K F 38393 were also shifted in parallel to the right by H A L O at the c o n c e n t r a t i o n s of 3 x 10 6 a n d 3 x 10 -5 M, a n d the pA 2 value of H A L O at a c o n c e n t r a t i o n of 3 x 1 0 6 M was a l m o s t equal to t h a t of H A L O at a c o n c e n t r a t i o n of 3 x 10 - 5 M . The pA 2 value of H A L O against S K F 38393 was 5.80 + 0.07. These findings indicate t h a t S K F 38393 behaves as a d o p a m i n e r g i c agonist for the d o p a m i n e receptors in the A B R M .
The influence o f S K F 38393 on the c A M P levels in ABRM The c A M P levels in the A B R M was not altered significantly by S K F 38393 at a c o n c e n t r a t i o n of
Control S K F with 3 X 1 0 - 6 M
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-6
-5
-4
SKF (log
M)
Fig. 1. Effect of BUTA on the dose-response curve of SKF 38393. Relaxation is presented as a percentage of the maximum relaxation induced by serotonin (10 -6 M). Each point is the mean +__SE of six experiments. Spontaneous relaxation of catch contraction is shown as a horizontal dotted line and is 23 + 3.2% (mean + SE). SKF:SKF 38393.
o
0 C
SKF3889a
10-5M Fig. 3. Effect of SKF 38393 on the cAMP levels in the ABRM. Values are presented as the mean with SE of eight experiments. C: untreated ABRM.
SKF 38393 on Mytilus smooth muscle 10 5M which relaxed the catch contraction of the ABRM sufficiently as shown in Fig. 3. The cAMP levels in the untreated ABRM were 6.76 + 0.36 pmol/ mg protein (n = 8) and those in the SKF38393treated muscle were 6.13+0.27pmol/mg protein (n =8). Thus, these findings indicate that the SKF 38393-induced relaxation of catch contraction does not link to the adenylate cyclase system. DISCUSSION A selective D-2 receptor agonist, bromocriptine is ineffective in subserving the relaxation of catch contraction and further a selective D-2 receptor antagonist, sulpride does not possess the ability to prevent the dopamine-induced relaxation of catch contraction (Ishii and Takayanagi, 1982a). Thus dopamine receptors mediating the relaxation of catch contraction do not appear to be D-2 receptors. However, these investigations do not provide the convincing evidence that dopamine receptors in the ABRM are D-1 receptors. Therefore, we tried to solve the problem by using the most selective D-1 receptor agonist, SKF 38393 (Stoof and Kebabian, 1984) as a means of proving the pharmacological property of the dopamine receptors in the ABRM. In result, SKF 38393 relaxed the catch contraction in a dose-dependent manner, as did dopamine. The dopamine-induced relaxation of catch contraction is antagonized by BUTA and HALO in a competitive manner (Yoshida et al., 1981; Murakami et al., 1983). Therefore, the effects of BUTA and HALO on the SKF 38393induced relaxation of catch contraction were investigated. The SKF 38393-induced relaxation was antagonized by BUTA and HALO in a competitive manner and their pA 2 values against SKF 38393 were 5.69_+ 0.05 (BUTA) and 5.80_+ 0.07 (HALO) respectively. These pA: values obtained here are similar to those obtained by Yoshida et al. (1981) and Murakami et al. (1986), i.e. the pA2 value of BUTA against dopamine is 6.13 _+ 0.04 and that of HALO against dopamine is 6.0_+ 0.2. Theoretically two different agonists acting on the same receptor yield a similar pA 2 value against a common antagonist (Arunlakshana and Shild, 1959). Considering in the similar way, these findings indicate that dopamine and SKF 38393 act on the common dopamine recognition site and the dopamine recognition site in the ABRM is D-1 receptors. Moreover, the increase in cAMP levels produced by dopamine is a biochemical sign characterizing D-I receptors activation deviced by Kebabian and Calne (1979). In the dopamine sensitive adenylate cyclase assay system, SKF 38393 mimics the ability of dopamine to stimulate adenylate cyclase activity, and consequently SKF 38393 activates the adenylate cyclase by the same mechanism as in the case of dopamine (Setler et al., 1978; Waiting and Dowling, 1981). However, SKF 38393 (10 5 M) at a concentration sufficient to relax the catch contraction failed to increase the cAMP levels in the ABRM significantly. The finding is in good agreement with the observations that dopamine fails to increase the cAMP levels in the ABRM significantly (Ishikawa et al., 1981; Ishii and Takayanagi, 1982a). Consequently, although the dopamine receptors of the ABRM are D-I like receptors, the D-1 like
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receptors are somewhat different from the D-1 receptors in the vertebrate tissues in the point that the D-I like receptor activation by SKF 38393 does not link to the increased cAMP levels. REFERENCES
Arunlakshana O. and Schild H. O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmac. Chemother. 14, 48-58. Ishii Y. and Takayanagi I. (1982a) Effects of some ergot alkaloids on dopamine receptors of molluscan smooth muscle. J. pharm. Dyn. Tokyo 5, 751-753. Ishii Y. and Takayanagi I. (1982b) Specific [3H]haloperidol binding to dopamine receptors in the anterior byssus retractor muscle. Jap. J. Pharmac. 32, 1151 1155. Ishikawa T., Murakami H. and Iwayama Y. (1981) Changes in cAMP and cGMP levels induced by relaxing drugs in acetylcholine- and potassium-treated molluscan smooth muscle. Comp. Bioehem. Physiol. 70C, 171-176. Iwayama Y. and Takayanagi I. (1982) Photoaffinity labeling of dopamine receptors in molluscan smooth muscle. J. Pharm. Pharmac. 34, 729-730. Kebabian J. W. and Calne D. B. (1979) Multiple receptors for dopamine. Nature, Lond. 277, 93-96. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Muneoka Y., Shiba Y. and Kanno Y. (1978) Effects of drugs on the relaxing action of various monoamines in molluscan smooth muscle. Hiroshima J. med. Sei. 27, 163-171. Murakami H., Ishikawa T. and Sano M. (1986) The site of action of dopamine and its related compounds in smooth muscle of Mytilus. Comp. Biochem. Physiol. 84C, 51-54. Murakami H., Satoh T. and Ishikawa T. (1983) Effects of bulbocapnine and butaclamol on the relaxation of catch contraction by some dopaminergic agonists in Mytilus smooth muscle. Comp. Biochem. Physiol. 75C, 227-229, Pendelton R. G., Samler L., Kaiser C. and Redley P. T. (1978) Studies on renal dopamine receptors with SK&F 38393, a new agonist. Eur. J. Pharmac. 51, 19 28. Scatton B. and Dubois A. (1985) Autoradiographic localization of D~ dopamine receptors in the rat brain with [3H]SKF38393. Eur. J. Pharmac. 111, 145 146. Setler P. E., Saran H. M., Zirkle C. L. and Saunders H. L. (1978) The central effects of a novel dopamine agonist. Eur. J. Pharmac. 50, 419-430. Steiner A. L., Parker C. E. and Kipnis D. M. (1972) Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. biol. Chem. 247, ll06-1113. Stoof J. C. and Kebabian J. W. (1984) Two dopamine receptors: Biochemistry, physiology and pharmacology. Life Sci. 35, 2281-2296. Takayanagi I., Murakami H., Iwayama Y., Yoshida Y. and Miki S. (1981) Dopamine receptor in anterior byssus retractor muscle of Mytilus. Jap. J. Pharmae. 31,249-252. Twarog B. M. and Cole R. A. (1972) Relaxation of catch in a molluscan smooth muscle--If. Effects of serotonin, dopamine and related compounds. Comp. Biochem. Physiol. 43A, 331-335. Van Rossum J. M. (1963) Cumulative dose-response curve II, Technique for the making of dose-response curve in isolated organ and the evaluation of drug. Archs int. Pharmacodyn. ThOr. 143, 299-330. Waiting K. J. and Dowling J. E. (1981) Dopaminergic mechanism in the toleost retina I. Dopamine-sensitive adenylate cyclase in homogenates of carp retina; effects of agonist, antagonists and ergots. J. Neurochem. 36, 559-568. Yoshida Y., Takayanagi I. and Murakami H. (1981) Dopamine and its antagonists on molluscan smooth muscle. J. pharm. Dyn. Tokyo 5, 751 753.