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Effects of calcium antagonists on muscarinic receptor subtypes in the rat brain
168
Brain Research, 422 (1987) 168-171 Elsevier
BRE22511
Effects of calcium antagonists on muscarinic receptor subtypes in the rat brain Sadao Katayama, Shozo Kito, Rie Miyoshi and Hiroaki Matsubayashi Third Departmentof Internal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan) (Accepted 23 June 1987) Key words: Calcium antagonist; Muscarinic receptor; Ml-receptor; Rat cerebral cortex
The effects of several calcium antagonists, including nicardipine, nifedipine, verapamil, and diltiazem, on muscarinic acetylcholine antagonist binding were studied in the P2 fraction of the rat cerebral cortex using either [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]pirenzipine as the radioactive ligand. (1) The potency of [3H]QNB binding inhibition was in the order nicardipine > verapamil > diltiazem > nifedipine. The IC50values of nicardipine, verapamii, and diltiazem were 2.56 x 10-6 M, 1.28 x 10-5 M, and 6.00 × 10-5 M, respectively. (2) The inhibition of [3H]QNB binding by nicardipine was significantly decreased in the presence of Ca ions. (3) In saturation experiments of [3H]QNB binding in the presence of nicardipine, the Kd value appeared to be significantly affected, but the BmaXvalue was unchanged. This indicated that nicardipine probably inhibited [3H]QNB binding allosterically. On the other hand, (4) nicardipine inhibited [3H]pirenzipine binding completely with an IC50value of 7.87 × 10-7 M. It was concluded that nicardipine had an inhibitory effect on Ml-receptor binding. The dihydropyridine ( D H P ) derivatives, verpamil and diltiazem are members of a group of compounds that have been termed 'calcium antagonists', since at low concentrations they inhibit transmembrane calcium 14,15,19'20. D H P s such as nitrendipine, nifedipine, nicardipine and their analogs bind with high affinity to various tissues 2,3,6,9,12,13,16,17. [3H]Nitrendipine binding is interfered with through the presence of Ca ions 5,1°. Effects of the D H P s 18 and verapamil 8,zz on muscarinic acetylcholine receptor ( m - A C h R ) binding have been investigated. A m o n g the DHPs, nicardipine inhibits [3H]QNB binding most intensely in a stereospecific manner, since its high lipid solubility changes membrane fluidity 18. In the rat brain, D600, a methoxy derivative of verapamil, appears to act as a reversible competitive inhibitor of [3H]QNB binding to m - A C h R 8. According to their different affinity for pirenzepine (PZ), muscarinic receptors have recently been divided into two classes, termed M t- and M2-receptors, for high- and low-affinity P Z binding sites, respectively. In the present study, we compare the potency of
the effects of nicardipine, nifedipine, verapamil and diltiazem on [3H]QNB binding to m - A C h R in the rat brain, and the effect of nicardipine on [3H]QNB binding in the presence of Ca and Mg ions. Effects of nicardipine on high affinity [3H]pZ binding sites (M lreceptors) were also investigated. The freshly excised cerebral cortex from male Sprague-Dawley rats each weighing 180-220 g was homogenized in 10 tissue volumes of 0.32 M sucrose using a Teflon-in-glass homogenizer. The resulting homogenate was centrifuged at 900 g for 10 rain. The supernatant was then centrifuged at 11,500 g for 20 min. The pellet was washed twice by successive resuspensions in 50 mM Tris-HCl buffer (pH 7.4) and centrifuged at 11,500 g for 20 min. The membranes were frozen in aliquots at - 2 0 °C until use at which time they were washed once again. The final tissue concentration was 30 mg of the original wet tissue/ml. To study the effects of divalent cations of [3H]QNB binding inhibition by nicardipine, E D T A - t r e a t e d membranes were used to chelate the Ca and Mg ions. They were prepared by incubating the membranes in
Correspondence: S. Katayama, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734 Japan. 0006-8993/87/$03.50 ~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
169 TABLE I Inhibition of [3H]QNB binding to m-AChR in the rat cerebral cortex by various calcium antagonists
Values shown are means _+S.E.M. of 3 replicate experiments, each performed in triplicate. The Hill coefficient and the IC50 values were calculated by computer analysis using the method of Damping Gauss-Newton.
(..9 ¢.9
0
I 10
,
i 8
i I -"9---"~ *6 4 2 -LOG(DRUG) Fig. 1. Inhibition of specific [3H]QNB binding to rat brain muscarinic receptor by nicardipine (0), nifedipine (*), diltiazem (~') and verapamil (O). The concentration of [3H]QNB is 0.22 riM.
10 m M E D T A for 20 min at r o o m temperature. Then 10 m M Ca or Mg ions were added to the incubation mixture containing the E D T A - t r e a t e d membranes. For [3H]QNB binding, assays of the cerebral cortex contained 4 mg wet tissue, while for [3H]pZ binding assays 6 mg wet cerebral cortex tissue was used. The tissues were incubated in the assay buffer, 0.1 M phosphate buffer p H 7.4, containing the radioligand and drugs. The final assay volumes were 2 ml for both [3H]QNB (30.1 Ci/mmol, New England Nuclear, NEN) and [3H]pZ (76.0 Ci/mmol, N E N ) binding. Incubations were carried out in the dark using the D H P s at 25 °C for 60 min. The m e m b r a n e - b o u n d [3H]QNB or [3H]PZ was trapped at the end of the incubation period by rapid vacuum filtration of the incubation mixture over W h a t m a n GF/B glass fiber filters. The filters were rinsed with 3 aliquots (3 ml) of the same buffer at 0 °C. Specific binding was defined as that binding which was decreased in the presence
~
u-
u
u-
0
--~-~-~.
loo
50
o
1'2
'
1'o
Drug
% Inhibition of [3H]QNB binding
Hill coefficient
IC5o
Nicardipine Nifedipine Verapamil Diltiazem
95.0 + 1.2 7.80 + 2.1 98.3 + 0.3 99.9 _+0.1
0.79 + 0.12 1.04 + 0.04 1.00 + 0.06
2.56 x 10-6 1.28x 10-5 6.00 x 10-5
,
'
8'
'
6'
4
-LOG(DRUG)
Fig. 2. Inhibition of specific [3H]QNB binding by nicardipine. Tissue membranes were treated without EDTA (C)), with EDTA (O), EDTA + Mg ions (~r), and EDTA + Ca ions (~).
of I x 10-5 M atropine in the incubation mixture. All assays were run in triplicate and repeated 3 times. The trapped radioactivity was subsequently measured by a liquid scintillation counter. Protein was determined by the method of Lowry et al. using a bovine serum albumin standard. Table I and Fig. 1 show [3H]QNB binding inhibition by various Ca antagonists. The maximal concentrations of nicardipine, nifedipine, verapamil, and diltiazem were 1 x 10-4 M, 1 x 10 -4 M, 1 × 10-3 M, and 1 x 10 -2 M, respectively, which were the highest final concentrations attainable in the incubation mixture used in the experiments. At these concentrations, nicardipine, verapamil, and diltiazem almost completely inhibited [3H]QNB binding. The Hill coefficient and the ICs0 values were 0.79 and 2.56 x 10-6 M for nicardipine, 1.04 and 1.28 x 10 -5 M for verapamil, and 1.00 and 6.00 x 10 -5 M for diltiazem. Maximal inhibition by nifedipine was only 7.80 + 2.1%. The rank order of potency of [3H]QNB binding inhibition was nicardipine > verapamil > diltiazem > nifedipine. As the next step, the effects of Ca and Mg ions on [3H]QNB binding inhibition by nicardipine were investigated: Fig. 2 and Table II show that the IC50 values of [3H]QNB binding inhibition by nicardipine were not affected in incubation media containing chelated Ca and Mg ions, or in the presence of the Mg ions, but they were significantly decreased in the presence of Ca ions. Through saturation experiments, we investigated the effects of nicardipine on [3H]QNB binding. Scatchard plots of these experiments are shown in Fig. 3. It appeared that [3H]QNB was bound with a K d value of 7.4 × 10-11 M and that nicardipine decreased the
170 TABLE II
Inhibition of [JH]QNB bindingby nicardipine
O ¢ ,or -
The tissue membrane was preincubated with 10 mM EDTA for 20 rain at room temperature. MgCI2 and CaCI2 were added to the incubation mixture at a final concentration of 10 mM. Values shown are means _+ S.E.M. of 3 replicate experiments. Hill coefficient and the IC50value were calculated by computer analysis using the method of Damping Gauss-Newton.
100
-6--~
&
6--0
LL
,,,
\
U~
o
Treated membrane EDTA (-) EDTA (+) EDTA (+), Mg 10 mM EDTA (+), Ca 10mM
% Inhibition Hill of [3H]QNB coefficient binding
IC5o
90.9 + 0.5 90.7 + 0.3
0.76 + 0.07 0.75 _+0.03
2.65 x 10-6 2.88 x 10-6
89.9 + 0.5
0.75 -+ 0.04
3.37 x 10-6
64.0_+10.0
0.60+0.10
3.98 x 10-5
0
To 15 w 10
o Z
o
o
~ •
o I
2
.(., BOUND
6
8
I
12
10
I
t
8
6
4
-LOG(DRUG)
apparent affinity of [3H]QNB binding, without significantly changing the n u m b e r of binding sites. We investigated the effects of nicardipine on M 1receptors through its inhibition of [3H]pZ binding (Fig. 4). Nicardipine inhibited [3H]pZ binding almost completely at a concentration of 1 x 10 -4 M. The Hill coefficient and the IC50 value were 0.77 and 7.87 x 10-7 M, respectively. The effects of calcium channel antagonists on m - A C h R have been investigated in several laboratories 8'18'22. Nevertheless, no comparative studies on the effects of D H P s , verapamil and diltiazem on mA C h R binding have hitherto been reported. Our studies proved that the rank order of potency of [3H]QNB binding inhibition was nicardipine > verapamil > diltiazem > nifedipine.
UJ or i,
1
lO
(fmolslmg protein) XlO2
Fig. 3. Scatchard plot analysis of the mechanism of action of nicardipineinhibiting specific [3H]QNB binding, measured as described in Methods, in the absence (©) of nicardipine and in the presence of 1 x 10-6 M (Q) nicardipine. Interception with the x-axis is not significantly different. The data present the mean binding values of 3 experiments, each done in triplicate.
Fig. 4. Inhibition of specific [3H]pZ binding to rat brain by nicardipine. [3H]pZ binding was measured in 3 separate experiments. The concentration of [3H]pZ is 0.25 nM. Ehlert et al. have shown that the effects of divalent cations on [3H]nitrendipine binding are inhibitory in the heart 5. From our observations, it appears that the inhibition of [3H]QNB binding by nicardipine was decreased in the presence of Ca ions in rat cerebral cortex. This finding suggests that the inhibitory effects of nicardipine on [3H]QNB binding are not provoked merely by a nicardipine-induced change in membrane fluidity. After Scatchard analysis of the specific [3H]QNB binding, the Bma~ value was not influenced by nicardipine, but the K d value was significantly increased in the presence of nicardipine. This marked effect of nicardipine on the Kd value indicates that nicardipine inhibits [3H]QNB binding in a feasibly allosteric manner. Furthermore, nicardipine had effects not only on [3H]QNB binding, but also on [3H]pZ binding. The ICso value of [3H]QNB binding inhibition by nicardipine was higher than that of [3H]pZ binding. It is, therefore, reasonable to think that nicardipine interacted with Ml-receptors more intensely than with M2-receptors. The association between M2-receptors and potassium channels has been supported by the facts that acetylcholine hyperpolarizes central neurons by acting on M2-receptors 4, and that phencyclidine and 4-aminopyridine as potassium channel blockers inhibit [3H]QNB binding completely and allosterically 1A1'21. Lai et al. have considered that ME-receptors have functional and structural couplings with the potassium channel. As for Mr-receptors, it has been known that Ml-receptors couple with PI turnover 23, and through acting on it, the intracellular Ca ion concentration increases. Several calcium antagonists inhibit the mus-
171 carinic r e c e p t o r - m e d i a t e d cyclic G M P f o r m a t i o n 7. This evidence and our d a t a suggest that the mechanism by which nicardipine c o m p l e t e l y and possibly
allosterically inhibits [3H]QNB binding, is analogous to the one existing b e t w e e n ME-receptors and potas-
1 Aronstam, R.S., Elderfrawi, M.E., Elderfrawi, A.T., Albuquerque, E.X., Jim, K.F. and Triggle, D.J., Sites of action of phencyclidine III. Interactions with muscarinic receptors, Mol. Pharmacol., 18 (1980) 179-184. 2 Bolger, G.T., Gengo, E.M., Luchowski, E.M., Siegel, H., Triggle, D.J. and Janis, R.A., High affinity binding of a calcium channel antagonist to smooth and cardiac muscle, Biochem. Biophys. Res. Commun., 104 (1982) 1604-1609. 3 Chatelain, P., Demol, D. and Roba, J., Inhibition by suloctidil of [3H]nitrendipine binding to cerebral cortex membranes, Biochem. Pharmacol., 33 (1984) 1099-1103. 4 Egan, T.M. and North, R.A., Acetylcholine hyperpolarizes central neurones by acting on an M2 muscarinic receptor, Nature (London), 319 (1986) 405-407. 5 Ehlert, F.J., Itoga, E., Roeske, W.R. and Yamamura, H.I., The interaction of [3H]nitrendipine with receptors for calcium antagonists in the cerebral cortex and heart of rats, Biochem. Biophys. Res. Commun., 104 (1982) 937-943. 6 Ehlert, F.J., Roeske, W.R., Itoga, E. and Yamamura, H.I., The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart cerebral cortex, and ileum of rats, Life Sci., 30 (1982) 2191-2202. 7 EI-Fakahany, E. and Richelson, E., Effect of some calcium antagonists on muscarinic receptor-mediated cyclic GMP formation, J. Neurochem., 40 (1983) 705-710. 8 Fairhurst, A.S., Whittaker, M.L. and Ehlert, F.J., Interactions of D600 (metoxyverapamil) and local anesthetics with rat brain a-adrenergic and muscarinic receptors, Biochem. Pharmacol., 29 (1980) 155-162. 9 Gould, R.J., Murphy, K.M.M. and Snyder, S.H., Tissue heterogeneity of calcium channel antagonist binding sites labeled by [3H]nitrendipine, Mol. Pharmacol., 25 (1984) 235-241. 10 Gould, R.J., Murphy, K.M.M. and Snyder, S.H., [3H]nitrendipine labeled calcium channels discriminate inorganic calcium agonists and antagonists, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3656-3660. 11 Lai, W.S., Ramkumar, V. and E1-Fakahany, E.E., Possible allosteric interaction of 4-aminopyridine with rat brain muscarinic acetylcholine receptors, J. Neurochem., 44 (1985) 1936-1942. 12 Marangos, P.J., Patel, J., Miller, C. and Martino, M., Specific calcium antagonist binding sites in brain, Life Sci., 31 (1982) 1575-1585.
13 Murphy, K.M.M. and Snyder, S.H., Calcium antagonist receptor binding sites labeled with [3H]nitrendipine, Eur. J. Pharmacol., 77 (1982) 201-202. 14 Nachshen, D.A. and Blaustein, M.P., The effects of some organic 'calcium antagonists' on calcium influx in presynaptic nerve terminals, Mol. Pharmacol., 16 (1979) 579-586. 15 Norris, D.K. and Bradford, H.F., On the specificity of verapamil as a calcium channel-blocker, Biochem. Pharmacol., 34 (1985) 1953-1956. 16 Sarmintto, J.G., Janis, R.A., Katz, A.M. and Triggle, D.J., Comparison of high affinity binding of calcium channel blocking drugs to vascular smooth muscle and cardiac sarcolemmal membranes, Biochem. Pharmacol., 33 (1984) 3119-3123. 17 Schoemaker, H.S., Itoga, E., Boles, R.G., Roeske, W.R., Ehlert, F.J., Kito, S. and Yamamura, H.I., Temperature dependence and allosteric modulation by verapamil and diltiazem of [3H]nitrendipine binding in the rat brain, Proc. Workshop Nitrendipine, New York, 1982, pp. 1-40. 18 Thayer, S.A., Welcome, M., Chhaba, A. and Fairhurst, A.S., Effects of dihydropyridine calcium channel blocking drugs on rat brain muscarinic and a-adrenergic receptors, Biochem. Pharmacol., 34 (1985) 175-180. 19 Triggle, C.R., Swamy, V.C. and Triggle, D.J., Calcium antagonists and contractile responses in rat vas deferens and guinea pig ileal smooth muscle, Can. J. Physiol. Pharmacol., 57 (1979) 804-818. 20 Turner, T.J. and Goldin, S.M., Calcium channels in rat brain synaptosomes: identification and pharmacological characterization, J. Neurosci., 5 (1985) 841-849. 21 Vincent, J.P., Cavey, D., Kamenka, J.M., Geneste, P. and Lazduski, M., Interaction of phencylclidines with the muscarinic and opiate receptors in the central nervous system, Brain Research, 152 (1978) 176-182. 22 Waelbroeck, M., Robberecht, P., Neef, P. and Christophe, J., Effects of verapamil on the binding properties of rat heart muscarinic receptors: evidence for an allosteric site, Biochem. Biophys. Res. Commun., 121 (1984) 340-345. 23 Watson, M., Roeske, W.R., Vickroy, T.W., Smith, T.L., Akiyama, K., Gulya, K., Duckies, S.P., Serra, M., Adem, A., Nordberg, A., Gehlert, D.R., Wamsley, J.K. and Yamamura, H.I., Biochemical and functional basis of putative muscarinic receptor subtypes and its implications, TIPS, Suppl. (1986) 46-55.
sium channels.
Brain Research, 422 (1987) 168-171 Elsevier
BRE22511
Effects of calcium antagonists on muscarinic receptor subtypes in the rat brain Sadao Katayama, Shozo Kito, Rie Miyoshi and Hiroaki Matsubayashi Third Departmentof Internal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan) (Accepted 23 June 1987) Key words: Calcium antagonist; Muscarinic receptor; Ml-receptor; Rat cerebral cortex
The effects of several calcium antagonists, including nicardipine, nifedipine, verapamil, and diltiazem, on muscarinic acetylcholine antagonist binding were studied in the P2 fraction of the rat cerebral cortex using either [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]pirenzipine as the radioactive ligand. (1) The potency of [3H]QNB binding inhibition was in the order nicardipine > verapamil > diltiazem > nifedipine. The IC50values of nicardipine, verapamii, and diltiazem were 2.56 x 10-6 M, 1.28 x 10-5 M, and 6.00 × 10-5 M, respectively. (2) The inhibition of [3H]QNB binding by nicardipine was significantly decreased in the presence of Ca ions. (3) In saturation experiments of [3H]QNB binding in the presence of nicardipine, the Kd value appeared to be significantly affected, but the BmaXvalue was unchanged. This indicated that nicardipine probably inhibited [3H]QNB binding allosterically. On the other hand, (4) nicardipine inhibited [3H]pirenzipine binding completely with an IC50value of 7.87 × 10-7 M. It was concluded that nicardipine had an inhibitory effect on Ml-receptor binding. The dihydropyridine ( D H P ) derivatives, verpamil and diltiazem are members of a group of compounds that have been termed 'calcium antagonists', since at low concentrations they inhibit transmembrane calcium 14,15,19'20. D H P s such as nitrendipine, nifedipine, nicardipine and their analogs bind with high affinity to various tissues 2,3,6,9,12,13,16,17. [3H]Nitrendipine binding is interfered with through the presence of Ca ions 5,1°. Effects of the D H P s 18 and verapamil 8,zz on muscarinic acetylcholine receptor ( m - A C h R ) binding have been investigated. A m o n g the DHPs, nicardipine inhibits [3H]QNB binding most intensely in a stereospecific manner, since its high lipid solubility changes membrane fluidity 18. In the rat brain, D600, a methoxy derivative of verapamil, appears to act as a reversible competitive inhibitor of [3H]QNB binding to m - A C h R 8. According to their different affinity for pirenzepine (PZ), muscarinic receptors have recently been divided into two classes, termed M t- and M2-receptors, for high- and low-affinity P Z binding sites, respectively. In the present study, we compare the potency of
the effects of nicardipine, nifedipine, verapamil and diltiazem on [3H]QNB binding to m - A C h R in the rat brain, and the effect of nicardipine on [3H]QNB binding in the presence of Ca and Mg ions. Effects of nicardipine on high affinity [3H]pZ binding sites (M lreceptors) were also investigated. The freshly excised cerebral cortex from male Sprague-Dawley rats each weighing 180-220 g was homogenized in 10 tissue volumes of 0.32 M sucrose using a Teflon-in-glass homogenizer. The resulting homogenate was centrifuged at 900 g for 10 rain. The supernatant was then centrifuged at 11,500 g for 20 min. The pellet was washed twice by successive resuspensions in 50 mM Tris-HCl buffer (pH 7.4) and centrifuged at 11,500 g for 20 min. The membranes were frozen in aliquots at - 2 0 °C until use at which time they were washed once again. The final tissue concentration was 30 mg of the original wet tissue/ml. To study the effects of divalent cations of [3H]QNB binding inhibition by nicardipine, E D T A - t r e a t e d membranes were used to chelate the Ca and Mg ions. They were prepared by incubating the membranes in
Correspondence: S. Katayama, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734 Japan. 0006-8993/87/$03.50 ~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
169 TABLE I Inhibition of [3H]QNB binding to m-AChR in the rat cerebral cortex by various calcium antagonists
Values shown are means _+S.E.M. of 3 replicate experiments, each performed in triplicate. The Hill coefficient and the IC50 values were calculated by computer analysis using the method of Damping Gauss-Newton.
(..9 ¢.9
0
I 10
,
i 8
i I -"9---"~ *6 4 2 -LOG(DRUG) Fig. 1. Inhibition of specific [3H]QNB binding to rat brain muscarinic receptor by nicardipine (0), nifedipine (*), diltiazem (~') and verapamil (O). The concentration of [3H]QNB is 0.22 riM.
10 m M E D T A for 20 min at r o o m temperature. Then 10 m M Ca or Mg ions were added to the incubation mixture containing the E D T A - t r e a t e d membranes. For [3H]QNB binding, assays of the cerebral cortex contained 4 mg wet tissue, while for [3H]pZ binding assays 6 mg wet cerebral cortex tissue was used. The tissues were incubated in the assay buffer, 0.1 M phosphate buffer p H 7.4, containing the radioligand and drugs. The final assay volumes were 2 ml for both [3H]QNB (30.1 Ci/mmol, New England Nuclear, NEN) and [3H]pZ (76.0 Ci/mmol, N E N ) binding. Incubations were carried out in the dark using the D H P s at 25 °C for 60 min. The m e m b r a n e - b o u n d [3H]QNB or [3H]PZ was trapped at the end of the incubation period by rapid vacuum filtration of the incubation mixture over W h a t m a n GF/B glass fiber filters. The filters were rinsed with 3 aliquots (3 ml) of the same buffer at 0 °C. Specific binding was defined as that binding which was decreased in the presence
~
u-
u
u-
0
--~-~-~.
loo
50
o
1'2
'
1'o
Drug
% Inhibition of [3H]QNB binding
Hill coefficient
IC5o
Nicardipine Nifedipine Verapamil Diltiazem
95.0 + 1.2 7.80 + 2.1 98.3 + 0.3 99.9 _+0.1
0.79 + 0.12 1.04 + 0.04 1.00 + 0.06
2.56 x 10-6 1.28x 10-5 6.00 x 10-5
,
'
8'
'
6'
4
-LOG(DRUG)
Fig. 2. Inhibition of specific [3H]QNB binding by nicardipine. Tissue membranes were treated without EDTA (C)), with EDTA (O), EDTA + Mg ions (~r), and EDTA + Ca ions (~).
of I x 10-5 M atropine in the incubation mixture. All assays were run in triplicate and repeated 3 times. The trapped radioactivity was subsequently measured by a liquid scintillation counter. Protein was determined by the method of Lowry et al. using a bovine serum albumin standard. Table I and Fig. 1 show [3H]QNB binding inhibition by various Ca antagonists. The maximal concentrations of nicardipine, nifedipine, verapamil, and diltiazem were 1 x 10-4 M, 1 x 10 -4 M, 1 × 10-3 M, and 1 x 10 -2 M, respectively, which were the highest final concentrations attainable in the incubation mixture used in the experiments. At these concentrations, nicardipine, verapamil, and diltiazem almost completely inhibited [3H]QNB binding. The Hill coefficient and the ICs0 values were 0.79 and 2.56 x 10-6 M for nicardipine, 1.04 and 1.28 x 10 -5 M for verapamil, and 1.00 and 6.00 x 10 -5 M for diltiazem. Maximal inhibition by nifedipine was only 7.80 + 2.1%. The rank order of potency of [3H]QNB binding inhibition was nicardipine > verapamil > diltiazem > nifedipine. As the next step, the effects of Ca and Mg ions on [3H]QNB binding inhibition by nicardipine were investigated: Fig. 2 and Table II show that the IC50 values of [3H]QNB binding inhibition by nicardipine were not affected in incubation media containing chelated Ca and Mg ions, or in the presence of the Mg ions, but they were significantly decreased in the presence of Ca ions. Through saturation experiments, we investigated the effects of nicardipine on [3H]QNB binding. Scatchard plots of these experiments are shown in Fig. 3. It appeared that [3H]QNB was bound with a K d value of 7.4 × 10-11 M and that nicardipine decreased the
170 TABLE II
Inhibition of [JH]QNB bindingby nicardipine
O ¢ ,or -
The tissue membrane was preincubated with 10 mM EDTA for 20 rain at room temperature. MgCI2 and CaCI2 were added to the incubation mixture at a final concentration of 10 mM. Values shown are means _+ S.E.M. of 3 replicate experiments. Hill coefficient and the IC50value were calculated by computer analysis using the method of Damping Gauss-Newton.
100
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6--0
LL
,,,
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o
Treated membrane EDTA (-) EDTA (+) EDTA (+), Mg 10 mM EDTA (+), Ca 10mM
% Inhibition Hill of [3H]QNB coefficient binding
IC5o
90.9 + 0.5 90.7 + 0.3
0.76 + 0.07 0.75 _+0.03
2.65 x 10-6 2.88 x 10-6
89.9 + 0.5
0.75 -+ 0.04
3.37 x 10-6
64.0_+10.0
0.60+0.10
3.98 x 10-5
0
To 15 w 10
o Z
o
o
~ •
o I
2
.(., BOUND
6
8
I
12
10
I
t
8
6
4
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apparent affinity of [3H]QNB binding, without significantly changing the n u m b e r of binding sites. We investigated the effects of nicardipine on M 1receptors through its inhibition of [3H]pZ binding (Fig. 4). Nicardipine inhibited [3H]pZ binding almost completely at a concentration of 1 x 10 -4 M. The Hill coefficient and the IC50 value were 0.77 and 7.87 x 10-7 M, respectively. The effects of calcium channel antagonists on m - A C h R have been investigated in several laboratories 8'18'22. Nevertheless, no comparative studies on the effects of D H P s , verapamil and diltiazem on mA C h R binding have hitherto been reported. Our studies proved that the rank order of potency of [3H]QNB binding inhibition was nicardipine > verapamil > diltiazem > nifedipine.
UJ or i,
1
lO
(fmolslmg protein) XlO2
Fig. 3. Scatchard plot analysis of the mechanism of action of nicardipineinhibiting specific [3H]QNB binding, measured as described in Methods, in the absence (©) of nicardipine and in the presence of 1 x 10-6 M (Q) nicardipine. Interception with the x-axis is not significantly different. The data present the mean binding values of 3 experiments, each done in triplicate.
Fig. 4. Inhibition of specific [3H]pZ binding to rat brain by nicardipine. [3H]pZ binding was measured in 3 separate experiments. The concentration of [3H]pZ is 0.25 nM. Ehlert et al. have shown that the effects of divalent cations on [3H]nitrendipine binding are inhibitory in the heart 5. From our observations, it appears that the inhibition of [3H]QNB binding by nicardipine was decreased in the presence of Ca ions in rat cerebral cortex. This finding suggests that the inhibitory effects of nicardipine on [3H]QNB binding are not provoked merely by a nicardipine-induced change in membrane fluidity. After Scatchard analysis of the specific [3H]QNB binding, the Bma~ value was not influenced by nicardipine, but the K d value was significantly increased in the presence of nicardipine. This marked effect of nicardipine on the Kd value indicates that nicardipine inhibits [3H]QNB binding in a feasibly allosteric manner. Furthermore, nicardipine had effects not only on [3H]QNB binding, but also on [3H]pZ binding. The ICso value of [3H]QNB binding inhibition by nicardipine was higher than that of [3H]pZ binding. It is, therefore, reasonable to think that nicardipine interacted with Ml-receptors more intensely than with M2-receptors. The association between M2-receptors and potassium channels has been supported by the facts that acetylcholine hyperpolarizes central neurons by acting on M2-receptors 4, and that phencyclidine and 4-aminopyridine as potassium channel blockers inhibit [3H]QNB binding completely and allosterically 1A1'21. Lai et al. have considered that ME-receptors have functional and structural couplings with the potassium channel. As for Mr-receptors, it has been known that Ml-receptors couple with PI turnover 23, and through acting on it, the intracellular Ca ion concentration increases. Several calcium antagonists inhibit the mus-
171 carinic r e c e p t o r - m e d i a t e d cyclic G M P f o r m a t i o n 7. This evidence and our d a t a suggest that the mechanism by which nicardipine c o m p l e t e l y and possibly
allosterically inhibits [3H]QNB binding, is analogous to the one existing b e t w e e n ME-receptors and potas-
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sium channels.