Brain Research, 540 (1991) 331-334 Elsevier
331
BRES 24520
Characteristics of [3H]ryanodine binding to the rabbit cerebral microsomes Tomoyuki Kawai, Yasunori Ishii, Yuji Imaizumi and Minoru Watanabe Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467 (Japan) (Accepted 30 October 1990) Key words: Ryanodine; Binding assay; Brain microsome; Adenine nucleotide; Caffeine; Ca-induced Ca release
Specific binding of [3H]ryanodine to the rabbit cerebral microsomes was dependent on free Ca2÷ at micromolar concentrations and significantly increased by AMP-PNP and caffeine. Scatchard analysis showed a high and a low affinity binding site. The results suggest the presence of ryanodine binding sites which are activated by Ca2÷ but with low efficacy, and greatly modified by adenine nucleotide, Mg2÷ and also by caffeine.
Ryanodine, a plant alkaloid, has been extensively used as a potent inhibitor of Ca-induced Ca release from intracellular store sites, because of its specific binding to and action on the Ca release channels of the sarcoplasmic reticulum (SR) in muscular tissues 6. Experiments in SR preparations have demonstrated that the ryanodine binding is activated by Ca 2÷ and modified by drugs affecting the Ca release from the SR. Ryanodine binding has been shown to parallel the activity of Ca release channels in the SR 10'13'14. There are differences, however, in the properties of ryanodine binding between the skeletal muscle and myocardial SR 1°'12. Studies in neuronal tissues including central and peripheral neurons have demonstrated using caffeine the presence of intracellular Ca store sites similar to the SR 7"11A8"2°-22, and suggest possible roles of Ca-induced Ca release in raising intracellular free Ca concentration 5' 7,21. Recently, ryanodine was found to inhibit the Ca release from the caffeine-sensitive Ca store sites in sympathetic ganglia 4'2° and sensory neurons 21. The presence of ryanodine-binding sites and Ca release channels has also been reported in the rat brain microsomes 1. The pharmacological characteristics of ryanodine binding, however, are not known in neuronal tissues. The present study examined the effects of agents known to affect the Ca release, on the [3H]ryanodine binding to the cerebral microsomes, and showed significant modulation by adenine nucleotide and caffeine. Rabbit cerebrum was homogenized with 8 strokes of a glass homogenizer in the physiological buffer solution
containing 140 mM KCI, 10 mM NaCI, 25 mM Tris-HCl (pH = 7.2) and 1 mM dithiothreitol. After centrifugation at 500 g for 15 min, the pellet was rehomogenated and centrifuged again. The collected supernatant was centrifuged at 10,000 g for 20 min (Hitachi, R P R 20-3) and then the supernatant was centrifuged at 110,000 g for 60 min (Beckman, 80Ti). The pellet was resuspended in the buffer solution and was used immediately for ryanodine binding assay. The microsomes of the cerebellum and the superior cervical ganglion of the rabbit were also used. They were obtained by the same procedures. Protein concentrations were determined according to Lowry et al. 8. For [3H]ryanodine binding assay, the microsomes (1 mg of protein) were incubated with 10 nM [3H]ryanodine (60.0 Ci/mmol, New England Nuclear) in the physiological buffer solution (1 ml) containing various concentrations of free Ca 2+ for 60 min at 37 °C. Concentrations of ryanodine more than 10 nM were prepared as admixtures of radioactive and non-radioactive ryanodine. Free Ca 2÷ concentrations were obtained by titrating with E G T A using an apparent association constant (12.3 x 106 M-I). Non-specific binding was determined in the presence of 10/~M non-radioactive ryanodine (purchased from Wako Pure Chemicals). Incubations were terminated by quick dilution with 5 vols of ice-cold medium, vacuum filtration through glass fiber filters (Whatman GF/C), and rapid washing 3 times with 5 vols of cold medium. The receptor-bound radioactivity was analyzed by liquid scintillation counting. The measurements were carried
Correspondence: M. Watanabe, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dohri 3-1, Mizuho-ku, Nagoya 467, Japan. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
332 out in duplicate. Averaged data were shown as mean + S.E.M. Statistical significance was determined using Student's t-test. Fig. 1 shows a saturation isotherm and Scatchard plot of the specific binding of [3H]ryanodine at a free Ca 2+ concentration of 50/~M to the microsomes of the rabbit cerebrum. The Scatchard plot revealed a high and a low affinity binding site; averaged values (3 experiments) of the dissociation constant, /(ol, and maximum number of binding sites, Bmax~, for high affinity site were 29.3 + 3.1 nM and 35.0 _+ 4.4 fmol/mg of protein, respectively. The Kd2 and Bmax2 for low affinity site were 401 + 26 nM and 320 + 29 fmol/mg of protein, respectively. The value of K m in cerebral microsomes is in a range observed in the sarcoplasmic reticulum (SR) from skeletal and cardiac muscle (2-200 nM) 6. The low affinity binding site (Kd; 28 n M - 3 a M ) has also been reported in the cardiac SR preparations 9"1°'12 and the skeletal muscle SR 9. The specific binding of ryanodine was greatly affected by Ca 2+ concentration, and the relationship between the amount of binding and -log [Ca 2+] was bell-shaped (Fig. 2). The concentrations of Ca 2+ for half activation and inhibition of ryanodine binding were approximately 7 a M and 0.4 mM, respectively. The optimal [Ca 2+] for the binding was 50-100 aM. A similar Ca-dependence on ryanodine binding is reported in the skeletal muscle SR 2'12. It is known that the ryanodine binding in the skeletal muscle SR is modulated by caffeine 14 or adenine nucleotides 2'1°'14. Fig. 3 shows the effects of 10 mM caffeine and/or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP), a non-hydrolysable derivative of ATP, on the ryanodine binding to the cerebral microsomes at Ca 2+ concentrations corresponding to that in the resting state (0.2 a M ) or during the excitation (2 aM) of the cell and at the optimal Ca 2+ concentration (50 aM) for the binding. Caffeine increased the specific binding of ryanodine at all Ca 2+ concentrations tested, showing that caffeine lowered the threshold concentration of Ca 2+ for activation of the binding. In contrast to caffeine, AMPPNP had no detectable effect at 0.2 /~M Ca 2+, and dramatically enhanced the specific binding of ryanodine at 2 a M and 50 a M Ca 2+. In experiments performed at 50 /~M Ca 2+ (not shown), AMP-PNP exhibited its maximal effect at 1 mM (ECso; < 0.5 mM). Caffeine at 10 and 30 mM increased the binding to 267% and 448% of the control, respectively. As shown in Fig. 3C, maximal effect caused by t mM AMP-PNP was further increased by 10 mM caffeine by 2-fold at 50 ,uM Ca 2+, thereby the increment was greater than the increase caused by caffeine alone. Quite similar results were obtained at lower Ca 2+ concentrations. These results suggest different rather than the same regulatory sites of action in ryanodine-binding molecule for caffeine and
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Fig. 1. Specific binding of [3H]ryanodine to cerebral microsomes shown as saturation isotherm and Scatchard plot (inset). The specific binding was determined at a free Ca2+ concentration of 50 /~M as described in the text. The dissociation constant Kdi and maximum number of binding sites Bmaxl for high-affinity site were 25.4 nM and 28 fmol/mg of protein, respectively. KalE and Bm,x2for low affinity sites were 443 nM and 262 fmol/mg of protein, respectively. adenine nucleotide, in line with the previous observation in the skeletal muscle SR 14. Fig. 3C denotes inhibitory effects of 1 mM Mg 2+ examined at 50 a M Ca 2+. In the absence of caffeine or AMP-PNP, Mg 2+ nearly abolished the specific binding of ryanodine. This inhibitory effect was recovered in part by caffeine and/or AMP-PNP. In the presence of caffeine and AMP-PNP, Mg 2+ inhibited only 30% of specific ryanodine binding, Ruthenium red, a potent inhibitor of Ca release and Ca release channels in the muscle SR 17, also inhibited at 10/aM the specific ryanodine binding to 14.3% and to 5.1% of the control in the absence and presence of 10 mM caffeine and 1 mM AMP-PNP, respectively, at the optimal [Ca 2+] (not shown). Inositol 1,4,5-trisphosphate (IP3) induces the Ca release from the brain microsomes 15 and also stimulates the activation of the ryanodine-sensitive Ca release channels l'lg. The specific ryanodine binding in cerebral microsomes, however, was not affected by 10 a M IP3 in
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333 the absence or presence of 1 m M A M P - P N P at 50/~M Ca 2+ (not shown) in agreement with previous report 14. GTP-),-S at 0.1 m M also had essentially no effects (not shown). The specific binding of ryanodine was found in the microsomal fraction of the cerebellum to a lesser extent than that of the cerebrum (Fig. 4), suggesting that the ryanodine binding sites are distributed in the region of brain with different densities in contrast to the IP 3
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binding sites 3. Fig. 4 also shows the presence of ryanodine binding sites in the microsomes of the superior cervical ganglion. The present experiments were carried out using the medium having physiological ionic composition. The affinity and density of ryanodine receptors obtained here in cerebral microsomes are lower than those recently reported in rat brain microsomes (Ko, 2.5 nM; Bmax, 690 fmol/mg of protein)l; it may be, at least in part, due to difference in assay conditions, high ionic strength 1°'16 and presence of ATP 2'1°'14'16. The value of Bmax may be rather smaller than that reported in heavy SR preparations from skeletal (4-25 pmole/mg protein) and cardiac muscle (0.5-14 pmole/mg protein) 6, even after taking account of the difference in the purity of membrane preparations and the assay conditions. The Ca-dependent ryanodine binding was inhibited by Mg 2÷ and Ruthenium red, and enhanced by adenine nucleotide. These characteristics considerably resemble those in skeletal rather than cardiac SR preparations. In the latter, Mg 2÷ and adenine nucleotide have no or little effect on ryanodine binding ~°'12. The remarkable feature observed in cerebral microsomes is that caffeine potentiates the binding even under the optimal Ca 2+ concentration and in the presence of adenine nucleotide. In the skeletal SR, the modulation by caffeine is observed only when the binding is reduced by addition of Mg 2+, and furthermore, even under these conditions synergism with adenine nucleotide is not observed at optimal [Ca2+] 12'14. This effect of caffeine and Ca-dependency of the ryanodine binding suggest that the development of inhibitory effects of ryanodine on intracellular Ca release is dependent on the activity of the cell or requires the presence of caffeine as observed in peripheral neurons 21. It is established that the ryanodine-receptor molecule
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Fig. 3. Modulatory effects of caffeine (10 mM), 5'-adenylylimidodiphosphate (AMP-PNP, 1 mM) and Mgz+ (1 mM) on specific binding of 10 nM [3H]ryanodine to cerebral microsomes. Free Ca2÷ concentrations in assay medium were 0.2 (A), 2 (B) and 50/~M (C). In C, left and right columns in each pair represent the absence and presence of Mg2÷, respectively. Vertical bars show S.E.M. of 3 experiments. Statistically significant differences from the control are indicated by * (P < 0.05), ** (P < 0.01) and *** (P < 0.001). Significant differences from the value obtained in the absence of Mg2÷ are indicated by #; in C.
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Fig. 4. Specific binding of ryanodine to microsomes from cerebrum, cerebellum and superior cervical ganglion (SCG) of the rabbit. Free Ca2÷ concentration was 50/~M. Number of experiments is indicated in parentheses. The specific binding in cerebellum is significantly different from that in cerebrum (P < 0.001).
334 is the Ca release channel itself in the SR of skeletal and cardiac muscles 6. Since the ryanodine binding parallels
implicated as has been shown in peripheral neurons ~ 7.2o,21. The efficacy of Ca 2+ as an activator, however, may
the activation of Ca release channels, it is considered that ryanodine binds preferably to site(s) which are accessible
be rather low in spite of its indispensability. Activity of Ca release channels is estimated to be less than 6% in the
only when the Ca release channels are in the open state ~°'~3. This feature is very likely to apply the ryanodine binding sites in the cerebrum since the recent
presence of Ca 2+ alone at the optimal concentration and less than 13% in the presence of physiological concentrations of adenine nucleotide and Mg 2+ , if it is assumed
electrophysiological study ~ has shown the modulatory effect of ryanodine, adenine nucleotide and caffeine on
that full activation is induced by an addition of caffeine, Therefore, significant modulatory effects of endogenous
the activity of Ca channels located in the rat brain microsomes incorporated to planner lipid bilayer,
adenine nucleotide and Mg" , and exogenous caffeine on ryanodine binding/Ca release channels are important to
whereas C a - d e p e n d e n t activation is not shown clearly. O n the basis of these considerations, the present results
consider the roles of Ca-induced Ca release from intracellular store sites in the neuronal tissues.
suggest that the Ca release channels in the cerebrum are activated by micromolar concentrations of free Ca- , and that mechanisms of Ca-induced Ca release may be
This work was supported by a grant from the Japanese Ministry of Education, Science and Culture.
1 Ashley, R.H., Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planer lipid bilayers, J. Membrane Biol., 111 (1989) 179-189. 2 Imagawa, T., Smith, J.S., Coronado, R. and Campbell, K.P., Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel, J. Biol. Chem., 262 (1987) 16636-16643. 3 Joseph, S.K. and Rice, H.L., The relationship between inositol trisphosphate receptor density and calcium release in brain microsomes, Mol. PharmacoL, 35 (1989) 355-359. 4 Kawai, T. and Watanabe, M., Effects of ryanodine on the spike after-hyperpolarization in sympathetic neurones of the rat superior cervical ganglion, Pflager's Arch.. 413 (1989) 470-475. 5 Kuba, K., Release of calcium ions linked to the activation o1 potassium conductance in a caffeine-treated sympathetic neurone, J. Physiol., 298 (1980) 251-269. 6 Lai, EA. and Meissner, G., The muscle ryanodine receptor and its intrinsic Ca2+ channel activity, J. Bioenerg. Biomembr., 21 (1989) 227-246. 7 Lipscombe, D., Madison, D.V., Poenie, M., Reuter, H., Tsien, R.W. and Tsien, R.Y., Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons, Neuron, 1 (1988) 355-365. 8 Lowry, O.H., Rosebrough, N.J., Farr, D.L. and Randall, R.J., Protein measurements with the Folin-phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 9 McGrew, S.G., Wolleben, C., Siegl, P., Inui, M. and Fleischer, S., Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle, Biochemistry, 28 (1989) 1686-1691. 10 Michalak, M., Dupraz, P. and Shospan-Barmatz, V., Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle, Biochim. Biophys. Acta, 939 (1988) 587-594. 11 Neering, I.R. and McBurney, R.N., Role for microsomal Ca storage in mammalian neurones?, Nature, 309 (1984) 158-160. 12 Pessah, I.N., Waterhouse, A.L. and Casida, J.E., The calcium-
ryanodine receptor complex of skeletal and cardiac muscle, Biochem. Biophys. Res. Commun., 128 (1985) 449-456. 13 Pessah, I.N., Franeini, A.O., Scales, D.J., Waterhouse, AIL. and Casida, J.E., Calcium-ryanodine receptor complex. Solubilization and partial characterization from skeletal muscle junctional sarcoplasmic reticulum vesicles, J. Biol. Chem., 261 (1986) 8643-8648. 14 Pessah, I.N., Stambuk, R.A. and Casida, J.E., CaZ~-activated ryanodine binding: mechanisms of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nucleotides, Mol. Pharmacol., 31 (1987) 232-238, 15 Shah, J., Cohen R.S. and Pant, H.C., Inositol trisphosphateinduced calcium release in brain microsomes, Brain Research, 419 (1987) 1-6. 16 Shoshan-Barmatz, V. and Zarka, A., Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum, ). Biol. Chem., 263 (1988) 16772-16779. 17 Smith, J.S., Coronado, R. and Meissner, G., Single-channel calcium and barium currents of large and small conductance from sarcoplasmic reticulum, Biophys. J., 50 (1986) 921-928. 18 Smith, S.J., MacDermott, A.B. and Weight, EE, Detection of intracellular Ca2+ transients in sympathetic neurones using arsenazo III, Nature, 304 (1983) 350-352. 19 Suarez-Isla B.A., Irribarra, V., Oberhauser, A., Larralde, L., Bull, R., Hidalgo, C. and Jaimovich, E., Inositol (1,4,5)trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes, Biophys. J., 54 (1988) 737-741. 20 Thayer, S.A., Hirning, L.D. and Miller, R.J., The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro, Mol. Pharmacol., 34 (1988) 664-673. 21 Thayer, S.A., Perney, T.M. and Miller, R.J., Regulation of calcium homeostasis in sensory neurons by bradykinin, J. Neurosci., 8 (1988) 4089-4097. 22 Thayer, S.A., Sturek, M. and Miller, R.J., Measurement of neuronal Ca2+ transients using simultaneous microfluorimetry and electrophysiology, Pflager's Arch. Eur. Z Physiol., 412 (1988) 216-223.
331
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Characteristics of [3H]ryanodine binding to the rabbit cerebral microsomes Tomoyuki Kawai, Yasunori Ishii, Yuji Imaizumi and Minoru Watanabe Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467 (Japan) (Accepted 30 October 1990) Key words: Ryanodine; Binding assay; Brain microsome; Adenine nucleotide; Caffeine; Ca-induced Ca release
Specific binding of [3H]ryanodine to the rabbit cerebral microsomes was dependent on free Ca2÷ at micromolar concentrations and significantly increased by AMP-PNP and caffeine. Scatchard analysis showed a high and a low affinity binding site. The results suggest the presence of ryanodine binding sites which are activated by Ca2÷ but with low efficacy, and greatly modified by adenine nucleotide, Mg2÷ and also by caffeine.
Ryanodine, a plant alkaloid, has been extensively used as a potent inhibitor of Ca-induced Ca release from intracellular store sites, because of its specific binding to and action on the Ca release channels of the sarcoplasmic reticulum (SR) in muscular tissues 6. Experiments in SR preparations have demonstrated that the ryanodine binding is activated by Ca 2÷ and modified by drugs affecting the Ca release from the SR. Ryanodine binding has been shown to parallel the activity of Ca release channels in the SR 10'13'14. There are differences, however, in the properties of ryanodine binding between the skeletal muscle and myocardial SR 1°'12. Studies in neuronal tissues including central and peripheral neurons have demonstrated using caffeine the presence of intracellular Ca store sites similar to the SR 7"11A8"2°-22, and suggest possible roles of Ca-induced Ca release in raising intracellular free Ca concentration 5' 7,21. Recently, ryanodine was found to inhibit the Ca release from the caffeine-sensitive Ca store sites in sympathetic ganglia 4'2° and sensory neurons 21. The presence of ryanodine-binding sites and Ca release channels has also been reported in the rat brain microsomes 1. The pharmacological characteristics of ryanodine binding, however, are not known in neuronal tissues. The present study examined the effects of agents known to affect the Ca release, on the [3H]ryanodine binding to the cerebral microsomes, and showed significant modulation by adenine nucleotide and caffeine. Rabbit cerebrum was homogenized with 8 strokes of a glass homogenizer in the physiological buffer solution
containing 140 mM KCI, 10 mM NaCI, 25 mM Tris-HCl (pH = 7.2) and 1 mM dithiothreitol. After centrifugation at 500 g for 15 min, the pellet was rehomogenated and centrifuged again. The collected supernatant was centrifuged at 10,000 g for 20 min (Hitachi, R P R 20-3) and then the supernatant was centrifuged at 110,000 g for 60 min (Beckman, 80Ti). The pellet was resuspended in the buffer solution and was used immediately for ryanodine binding assay. The microsomes of the cerebellum and the superior cervical ganglion of the rabbit were also used. They were obtained by the same procedures. Protein concentrations were determined according to Lowry et al. 8. For [3H]ryanodine binding assay, the microsomes (1 mg of protein) were incubated with 10 nM [3H]ryanodine (60.0 Ci/mmol, New England Nuclear) in the physiological buffer solution (1 ml) containing various concentrations of free Ca 2+ for 60 min at 37 °C. Concentrations of ryanodine more than 10 nM were prepared as admixtures of radioactive and non-radioactive ryanodine. Free Ca 2÷ concentrations were obtained by titrating with E G T A using an apparent association constant (12.3 x 106 M-I). Non-specific binding was determined in the presence of 10/~M non-radioactive ryanodine (purchased from Wako Pure Chemicals). Incubations were terminated by quick dilution with 5 vols of ice-cold medium, vacuum filtration through glass fiber filters (Whatman GF/C), and rapid washing 3 times with 5 vols of cold medium. The receptor-bound radioactivity was analyzed by liquid scintillation counting. The measurements were carried
Correspondence: M. Watanabe, Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dohri 3-1, Mizuho-ku, Nagoya 467, Japan. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
332 out in duplicate. Averaged data were shown as mean + S.E.M. Statistical significance was determined using Student's t-test. Fig. 1 shows a saturation isotherm and Scatchard plot of the specific binding of [3H]ryanodine at a free Ca 2+ concentration of 50/~M to the microsomes of the rabbit cerebrum. The Scatchard plot revealed a high and a low affinity binding site; averaged values (3 experiments) of the dissociation constant, /(ol, and maximum number of binding sites, Bmax~, for high affinity site were 29.3 + 3.1 nM and 35.0 _+ 4.4 fmol/mg of protein, respectively. The Kd2 and Bmax2 for low affinity site were 401 + 26 nM and 320 + 29 fmol/mg of protein, respectively. The value of K m in cerebral microsomes is in a range observed in the sarcoplasmic reticulum (SR) from skeletal and cardiac muscle (2-200 nM) 6. The low affinity binding site (Kd; 28 n M - 3 a M ) has also been reported in the cardiac SR preparations 9"1°'12 and the skeletal muscle SR 9. The specific binding of ryanodine was greatly affected by Ca 2+ concentration, and the relationship between the amount of binding and -log [Ca 2+] was bell-shaped (Fig. 2). The concentrations of Ca 2+ for half activation and inhibition of ryanodine binding were approximately 7 a M and 0.4 mM, respectively. The optimal [Ca 2+] for the binding was 50-100 aM. A similar Ca-dependence on ryanodine binding is reported in the skeletal muscle SR 2'12. It is known that the ryanodine binding in the skeletal muscle SR is modulated by caffeine 14 or adenine nucleotides 2'1°'14. Fig. 3 shows the effects of 10 mM caffeine and/or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP), a non-hydrolysable derivative of ATP, on the ryanodine binding to the cerebral microsomes at Ca 2+ concentrations corresponding to that in the resting state (0.2 a M ) or during the excitation (2 aM) of the cell and at the optimal Ca 2+ concentration (50 aM) for the binding. Caffeine increased the specific binding of ryanodine at all Ca 2+ concentrations tested, showing that caffeine lowered the threshold concentration of Ca 2+ for activation of the binding. In contrast to caffeine, AMPPNP had no detectable effect at 0.2 /~M Ca 2+, and dramatically enhanced the specific binding of ryanodine at 2 a M and 50 a M Ca 2+. In experiments performed at 50 /~M Ca 2+ (not shown), AMP-PNP exhibited its maximal effect at 1 mM (ECso; < 0.5 mM). Caffeine at 10 and 30 mM increased the binding to 267% and 448% of the control, respectively. As shown in Fig. 3C, maximal effect caused by t mM AMP-PNP was further increased by 10 mM caffeine by 2-fold at 50 ,uM Ca 2+, thereby the increment was greater than the increase caused by caffeine alone. Quite similar results were obtained at lower Ca 2+ concentrations. These results suggest different rather than the same regulatory sites of action in ryanodine-binding molecule for caffeine and
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Fig. 1. Specific binding of [3H]ryanodine to cerebral microsomes shown as saturation isotherm and Scatchard plot (inset). The specific binding was determined at a free Ca2+ concentration of 50 /~M as described in the text. The dissociation constant Kdi and maximum number of binding sites Bmaxl for high-affinity site were 25.4 nM and 28 fmol/mg of protein, respectively. KalE and Bm,x2for low affinity sites were 443 nM and 262 fmol/mg of protein, respectively. adenine nucleotide, in line with the previous observation in the skeletal muscle SR 14. Fig. 3C denotes inhibitory effects of 1 mM Mg 2+ examined at 50 a M Ca 2+. In the absence of caffeine or AMP-PNP, Mg 2+ nearly abolished the specific binding of ryanodine. This inhibitory effect was recovered in part by caffeine and/or AMP-PNP. In the presence of caffeine and AMP-PNP, Mg 2+ inhibited only 30% of specific ryanodine binding, Ruthenium red, a potent inhibitor of Ca release and Ca release channels in the muscle SR 17, also inhibited at 10/aM the specific ryanodine binding to 14.3% and to 5.1% of the control in the absence and presence of 10 mM caffeine and 1 mM AMP-PNP, respectively, at the optimal [Ca 2+] (not shown). Inositol 1,4,5-trisphosphate (IP3) induces the Ca release from the brain microsomes 15 and also stimulates the activation of the ryanodine-sensitive Ca release channels l'lg. The specific ryanodine binding in cerebral microsomes, however, was not affected by 10 a M IP3 in
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333 the absence or presence of 1 m M A M P - P N P at 50/~M Ca 2+ (not shown) in agreement with previous report 14. GTP-),-S at 0.1 m M also had essentially no effects (not shown). The specific binding of ryanodine was found in the microsomal fraction of the cerebellum to a lesser extent than that of the cerebrum (Fig. 4), suggesting that the ryanodine binding sites are distributed in the region of brain with different densities in contrast to the IP 3
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binding sites 3. Fig. 4 also shows the presence of ryanodine binding sites in the microsomes of the superior cervical ganglion. The present experiments were carried out using the medium having physiological ionic composition. The affinity and density of ryanodine receptors obtained here in cerebral microsomes are lower than those recently reported in rat brain microsomes (Ko, 2.5 nM; Bmax, 690 fmol/mg of protein)l; it may be, at least in part, due to difference in assay conditions, high ionic strength 1°'16 and presence of ATP 2'1°'14'16. The value of Bmax may be rather smaller than that reported in heavy SR preparations from skeletal (4-25 pmole/mg protein) and cardiac muscle (0.5-14 pmole/mg protein) 6, even after taking account of the difference in the purity of membrane preparations and the assay conditions. The Ca-dependent ryanodine binding was inhibited by Mg 2÷ and Ruthenium red, and enhanced by adenine nucleotide. These characteristics considerably resemble those in skeletal rather than cardiac SR preparations. In the latter, Mg 2÷ and adenine nucleotide have no or little effect on ryanodine binding ~°'12. The remarkable feature observed in cerebral microsomes is that caffeine potentiates the binding even under the optimal Ca 2+ concentration and in the presence of adenine nucleotide. In the skeletal SR, the modulation by caffeine is observed only when the binding is reduced by addition of Mg 2+, and furthermore, even under these conditions synergism with adenine nucleotide is not observed at optimal [Ca2+] 12'14. This effect of caffeine and Ca-dependency of the ryanodine binding suggest that the development of inhibitory effects of ryanodine on intracellular Ca release is dependent on the activity of the cell or requires the presence of caffeine as observed in peripheral neurons 21. It is established that the ryanodine-receptor molecule
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Calf.
+ AMP-PNP
Fig. 3. Modulatory effects of caffeine (10 mM), 5'-adenylylimidodiphosphate (AMP-PNP, 1 mM) and Mgz+ (1 mM) on specific binding of 10 nM [3H]ryanodine to cerebral microsomes. Free Ca2÷ concentrations in assay medium were 0.2 (A), 2 (B) and 50/~M (C). In C, left and right columns in each pair represent the absence and presence of Mg2÷, respectively. Vertical bars show S.E.M. of 3 experiments. Statistically significant differences from the control are indicated by * (P < 0.05), ** (P < 0.01) and *** (P < 0.001). Significant differences from the value obtained in the absence of Mg2÷ are indicated by #; in C.
E
4.
N-V
"10 t-
2
O
m
0 Cerebrum
(15)
Cerebellum
SCC
(5)
(3)
Fig. 4. Specific binding of ryanodine to microsomes from cerebrum, cerebellum and superior cervical ganglion (SCG) of the rabbit. Free Ca2÷ concentration was 50/~M. Number of experiments is indicated in parentheses. The specific binding in cerebellum is significantly different from that in cerebrum (P < 0.001).
334 is the Ca release channel itself in the SR of skeletal and cardiac muscles 6. Since the ryanodine binding parallels
implicated as has been shown in peripheral neurons ~ 7.2o,21. The efficacy of Ca 2+ as an activator, however, may
the activation of Ca release channels, it is considered that ryanodine binds preferably to site(s) which are accessible
be rather low in spite of its indispensability. Activity of Ca release channels is estimated to be less than 6% in the
only when the Ca release channels are in the open state ~°'~3. This feature is very likely to apply the ryanodine binding sites in the cerebrum since the recent
presence of Ca 2+ alone at the optimal concentration and less than 13% in the presence of physiological concentrations of adenine nucleotide and Mg 2+ , if it is assumed
electrophysiological study ~ has shown the modulatory effect of ryanodine, adenine nucleotide and caffeine on
that full activation is induced by an addition of caffeine, Therefore, significant modulatory effects of endogenous
the activity of Ca channels located in the rat brain microsomes incorporated to planner lipid bilayer,
adenine nucleotide and Mg" , and exogenous caffeine on ryanodine binding/Ca release channels are important to
whereas C a - d e p e n d e n t activation is not shown clearly. O n the basis of these considerations, the present results
consider the roles of Ca-induced Ca release from intracellular store sites in the neuronal tissues.
suggest that the Ca release channels in the cerebrum are activated by micromolar concentrations of free Ca- , and that mechanisms of Ca-induced Ca release may be
This work was supported by a grant from the Japanese Ministry of Education, Science and Culture.
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