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Calcium Release by Diltiazem from Isolated Sarcoplasmic Reticulum of Rabbit Skeletal Muscle
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(98)00009-3 All rights reserved
Gen. Pharmac. Vol. 31, No. 3, pp. 463–468, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Calcium Release by Diltiazem from Isolated Sarcoplasmic Reticulum of Rabbit Skeletal Muscle Ahmad Reza Dehpour,1* Kazem Mousavizadeh1 and Siavash Gerayesh-Nejad2 1 Department of Pharmacology and 2Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
ABSTRACT. 1. The effect of diltiazem on isolated sarcoplasmic reticulum (SR) from rabbit skeletal muscle was studied. To observe calcium movement into and out of the SR, a fluorescent chelate probe technique with chlortetracycline (CTC) as a reagent was employed. 2. Tris-ATP-induced calcium accumulation by the isolated SR was associated with a rise in the CTC fluorescence. The effect of ATP was dose dependent. 3. Diltiazem (631024M, 231023M) prevented ATP-induced calcium accumulation by the SR. 4. Addition of EGTA to the media chelates external calcium and caused calcium release that can be reversed by further addition of calcium chloride. Similarly diltiazem caused a rapid release of accumulated calcium from the SR, which is not reversed by the addition of calcium chloride. 5. It seems that the effect of diltiazem may be related to SR membrane-bound calcium being available for release. gen pharmac 31;3:463–468, 1998. 1998 Elsevier Science Inc. KEY WORDS. Calcium, diltiazem, sarcoplasmic reticulum, skeletal muscle, rabbit INTRODUCTION Organic calcium antagonists, also known as calcium channel blockers, are a chemically heterogenous group of drugs, which antagonize, with varying degrees of potency and specificity, calcium-dependent events (Schwartz and Triggle, 1984). Many of these compounds appear to exert their major pharmacological action through inhibition of calcium influx through voltage-dependent channels in cell membranes (Fleckenstein, 1977). To examine the role of extracellular calcium in skeletal muscle contraction, numerous studies have been carried out with calcium channel blockers, such as diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle (Curtis, 1994). Voltage-clamp studies have demonstrated the existence of a slow inward calcium current in frog (Sanches and Stefani, 1978; Stanfield 1977) and mammalian (Donaldson and Beam, 1983; Walsh et al. 1986) skeletal muscles. Calcium antagonists such as diltiazem and verapamil, which block this current (Gonzalez-Serratos et al., 1982; Palade and Almers, 1985; Walsh et al., 1986), paradoxically appear to enhance mechanical activity (Dorrschedit-Kafer, 1977; Gonzalez-Serratos et al., 1982). These conflicting and contradictory results appear to result from multiple sites of action of these drugs in skeletal muscle (Su, 1988; Valle-Aguilera, 1984). Verapamil- and diltiazem-binding sites have now been identified in skeletal muscle membranes (Galizzi et al., 1985). Some investigators suggested that diltiazem may affect the function of the sarcoplasmic reticulum (SR) of cardiac papillary and muscle (Abdelmeguid and Feher, 1994; Himori et al., 1975; Nakajima et al., 1976) and rabbit skeletal muscle (Wang et al., 1984). The present study was undertaken to investigate the effects of diltiazem on isolated SR from rabbit skeletal muscles, employing a fluorescent chelate probe as a means of observing calcium movements into and out of the SR.
*To whom all correspondence should be addressed. Received 7 January 1997.
MATERIAL AND METHODS
Microsomes containing sarcoplasmic reticulum Albino rabbits of either sex weighing 1–2 kg were used. The animals were killed by a blow on the head. Microsomes were prepared according to the method described by Martonosi (1968), by homogenizing rabbit thigh muscle with 4 volumes of 5 mM tris–0.1 M KCl buffer, pH. 7.4. The myofibrils were removed by centrifugation at 1,000g for 20 min. This step was repeated once more. The microsomal fraction was obtained by centrifugation of the supernatant at 28,000g for 60 min.
Fluorescence probe technique The microsomes were suspended in histidine buffer to obtain 0.5 mg protein/ml, which was determined by the method of Lowry et al. (1951). The medium consisted of 50 mM histidine, 350 mM sucrose, 30 mM chlortetracycline (CTC); pH was 6.8 and temperature 228C. For CTC fluroscence measurement, microsomes containing SR were incubated for 30 min in the buffer. Fluorescence was measured in an Aminco-Bowman spectrophotofluorometer; excitation and emission wavelength were 390 nm and 530 nm, respectively (Caswell, 1976). After preincubation to stablize baseline levels of CTC fluorescence, the reaction was initiated in a fluorescence cuvette by adding tris-ATP, and the fluorescence was immediately recorded as a function of time. The time for mixing tris-ATP and other agents and for initiating fluorescence measurement was less than 5 sec.
Drugs and chemicals Tris-ATP, diltiazem, caffeine and EGTA were obtained from Sigma (St. Louis, MO, USA).
Statistcal Methods Data presented are given as the mean6SEM, and significances have been tested by Student’s t-test. Levels of significances are denoted by *P,0.05 and **P,0.01.
464
A. R. Dehpour et al.
FIGURE 1. Visualization of calcium accumulation by sarcoplasmic reticulum with the use of chlortetracycline as a fluorescence chelate probe. Additions to the medium were: tris-ATP, 500 mM; EGTA, 500 mM; and calcium chloride, 100 mM. Results are typical of six experiments.
FIGURE 2. Effects of different concentrations of tris-ATP on fluorescence of chlortetracycline-loaded sarcoplasmic reticulum. Results are typical of five experiments.
Diltiazem-Induced Calcium Release from SR
FIGURE 3. Log concentration–response curve for the effect of tris-ATP on calcium accumulation by sarcoplasmic reticulum of rabbit skeletal muscle. Shown are mean6SEM (n56).
465 tle calcium is bound to the membrane surface. But, when it has accumulated within the SR, the concentration of calcium within the vesicle rises to a high level, and a substantial part of it is bound to the membrane surface. CTC is able to penetrate within a membrane and will then bind to the calcium on the inner face of the membrane. The calcium accumulation by isolated SR causes an increase in the fluorescence of CTC. The nature of the data that were obtained is illustrated in Figure 1. Sarcoplasmic reticulum is present in the system, as is CTC. If the uptake of calcium is initiated by the addition of ATP, a rise in the fluorescence is observed, associated with the uptake of calcium. That this is indeed a measure of calcium accumulation is determined by the subsequent addition of EGTA, which chelates external calcium. This creates an increased calcium gradient and causes the calcium release that is associated with a diminution in the fluorescence of CTC (Fig. 1). The effect can be subsequently reversed by further addition of calcium chloride (Fig. 1). ATPinduced calcium accumulation is dose dependent, and calculated ED50 was 4.231025M (Fig. 2 and Fig. 3). Diltiazem (631024, 231023M) diminishes ATP-induced calcium accumulation (Figs. 4–6). Diltiazem, like EGTA, causes a rapid decrease in CTC fluorescence. The effect, however, is not reversed by calcium chloride (Fig. 7). Caffeine, a calcium releaser from calcium-loaded isolated SR, has no significant effect on CTC fluorescence (Fig. 8). The observation is consistent with the evidence that the calcium released by caffeine is that which is free in the cytoplasm within the SR; caffeine does not affect the bound calcium within the SR (Bowman and Rand, 1980).
RESULTS Microsomes containing SR from rabbit skeletal muscle exhibit a high ATPase activity, indicating a high content of SR membrane (Martonosi, 1968). Isolated SR from rabbit skeletal muscle was used, and a fluorescent chelate probe as a means of observing calcium movements into and out of the SR was employed. With the probe technique, we used CTC, which fluoresces with great intensity when it binds with calcium on a membrane surface (Caswell, 1976). The initial external calcium concentration is very low, and very lit-
DISCUSSION Diltiazem, like EGTA, exerts multiple actions both on the contractile system and on the SR of skinned skeletal muscle fibers (Ishizuka and Endo, 1983). Diltiazem has been reported to potentiate twitch tension in bullfrog skeletal muscle (Walsh et al., 1988) as well as frog and mouse skeletal muscle fibers (Walsh et al., 1984). The mechanical potentiation produced by diltiazem in skeletal muscle contrasts with the inhibitory action of this drug in smooth and cardiac
FIGURE 4. Chlortetracycline fluorescence decreases after pretreatment of the medium with 631024 M and 231023 M diltiazem. Calcium accumulation is initiated by addition of 500 mM tris-ATP. Results are typical of six experiments.
466
A. R. Dehpour et al.
FIGURE 5. Log concentration–response curves of tris-ATP alone (A) and in the presence of 631024M (B) and 231023M (C) diltiazem. *P,0.05 and **P,0.01. Shown are mean6SEM (n56).
muscle. The twitch potentiative action of the drug has been attributed to lowering of mechanical threshold, increased sensitivity of the contractile proteins for calcium and induction of calcium release from the SR (Su, 1988). There is some evidence that calcium channel blockers can penetrate the cell membrane and accumulate in the cytosol and may interact with intracellular calcium binding proteins (Wang et al., 1984). This evidence provides a rational for studying the possible effects of these drugs on intracellular organelle systems pertinent to calcium-transport activities. In this regard, the SR is
the primary system involved in the control of intacellular calcium through the action of a calcium pump, which is the SR membranebound calcium-ATPase (Simonides and VanHardeweld, 1990; Wang et al., 1984). In the present study, we used CTC to monitor alteration in the SR calcium level. CTC is considered a specific probe for monitoring mobilization of membrane-associated calcium (Schneider et al., 1983). Diltiazem causes a rapid decrease in CTC fluorescence of the calcium-loaded SR. Thus membrane-bound calcium in the SR may be targeted by diltiazem. This finding is con-
FIGURE 6. Release of calcium from sarcoplasmic reticulum by diltiazem. Similar results were obtained in six other experiments.
Diltiazem-Induced Calcium Release from SR
467
FIGURE 7. Diltiazem-induced calcium release from sarcoplasmic reticulum not reversed by addition of calcium chloride. Similar results were obtained in six other experiments. firmed by the observation that caffeine, a calcium releaser from calcium-loaded isolated SR, has no effect on the CTC fluorescence. However, we did not actually measure calcium release directly. The concentrations of diltiazem used here are very high, but this is consistent with the results of Walsh et al. (1986), who reported that all tonic effects of diltiazem on skeletal muscle occurred over a concentration range of 50–500 mM. Wang et al. (1984) reported that diltiazem at a concentration of 400 mM activates skeletal muscle SR calcium-ATPase and causes the early release of calcium after binding. The membrane effects of diltiazem described in this study may not be directly involved in its pharmacological and clinical actions. However, the data suggest some interesting and different ef-
FIGURE 8. Lack of effect of caffeine (20 mM) on chlortetracycline fluorescence. Results are typical of five experiments.
fects of calcium channel blockers, which may undergo complex chemical–membrane interactions. References Abdelmeguid A. E. and Feher J. J. (1994) Effect of low perfusate calcium and diltiazem on cardiac sarcoplasmic reticulum in myocardial stunning. Am. J. Physiol. 266, H406–H414. Bowman W. C. and Rand M. J. (1980) Textbook of Pharmacology. p. 750. Blackwell Scientific, Oxford. Caswell A. H. (1976) Calcium and the sarcoplasmic reticulum. In Horizons in Clinical Pharmacology. (Edited by Ronger A. and Palmer F.) pp. 151– 159. Academic Press, New York. Curtis B. A. (1994) Effect of diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle. J. Muscle Res. Cell Motil. 15, 49–58. Donaldson P. L. and Beam K. G. (1983) Calcium currents in fast-twitch skeletal muscle of the rat. J. Gen. Physiol. 82, 449–468. Dorrschedit-Kafer M. (1977) The action of D600 on frog skeletal muscle: facilitation of excitation–contraction coupling. Pfluegers Arch. 369, 259–267. Fleckenstein A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Annu. Rev. Pharmac. Toxicol. 17, 149–166. Galizzi J. P., Fosset M. and Lazdunski M. (1985) Characterization of the calcium coordination site regulating binding of calcium channel inhibitors d-cis-diltiazem, (1)bepridil and (2)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes. Biochem. Biophys. Res. Commun. 132, 49–55. Gonzalez-Serratos H., Valle-Aguilera R., Lathrop A.D. and Delcarmen Garcia M. (1982) Slow inward calcium currents have no obvious role in muscle excitation–contraction coupling. Nature (Lond.) 298, 292–294. Himori N., Ono H. and Thiara N. (1975) Dual effects of a new coronary vasodilator diltiazem on the contractile force of the blood-perfused papillary muscle of the dog. Jpn. J. Pharmac. 25, 350–352. Ishizuka T. and Endo M. (1983) Effects of diltiazem on skinned skeletal muscle fibers of the African clawed toad. Circ. Res. 52 (Suppl. 1), 110–114. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall F. J. (1951) Protein measurement with the folin phenol reagent. J. Biochem. 193, 265–275. Martonosi A. (1968) Sarcoplasmic reticulum IV: solubilization of microsomal adenosine triphosphate. J. Biol. Chem. 243, 71–81. Nakajima H., Hoshiyama M., Yamashita K. and Kiomoto A. (1976) Electrical and mechanical responses to diltiazem in potassium depolarized myocardium of guinea-pig. Jpn. J. Pharmac. 26, 571–580. Palade P. T. and Almers W. (1985) Slow calcium and potassium currents in
468 frog skeletal muscle: their relationship and pharmacological properties. Pfluegers Arch. 405, 91–101. Sanchez J. A. and Stefani E. (1978) Inward calcium current in twitch muscle fibers of the frog. J. Physiol. (Lond.) 283, 197–209. Schneider A. S., Herz R. and Sonenberg M. (1983) Chlortetracycline as a probe of membrane associated calcium and magnesium: interaction with red cell membranes, phospholipids, and proteins monitored by fluorescence and circular dichroism. Biochemistry 22, 1680–1686. Schwartz A. and Triggle D. J. (1984) Cellular action of calcium channel blocking drugs. Annu. Rev. Med. 35, 325–356. Simonides W. S. and VanHardeweld C. (1990) An assay for sarcoplasmic reticulum Ca-ATPase activity in muscle homogenates. Anal. Biochem. 191, 321–331. Stanfield P. R. (1977) A calcium dependent inward current in frog skeletal muscle fibers. Pfluegers Arch. 368, 267–270.
A. R. Dehpour et al. Su J. Y. (1988) Intracellular mechanisms of verapamil and diltiazem action on striated muscle of the rabbit. Naunyn-Schmiedebergs-Arch-Pharmacol. 338, 297–302. Walsh K. B., Bryant S. H. and Schwartz A. (1984) Diltiazem potentiates mechanical activity in mammalian skeletal muscle. Biochem. Biophys. Res. Commun. 122, 1091–1096. Walsh K. B., Bryant S. H. and Schwartz A. (1986) Effect of calcium antagonist drugs on calcium currents in mammalian skeletal muscle fibers. J. Pharmac. Exp. Ther. 236, 403–407. Walsh K. B., Bryant S. H. and Schwartz A. (1988) Action of diltiazem on excitation–contaction coupling in bullfrog skeletal muscle fibers. J. Pharmac. Exp. Ther. 245, 531–536. Wang T., Tasi L. and Schwartz A. (1984) Effect of verapamil, and diltiazem, nislodipine on sarcoplasmic reticulum. Eur. J. Pharmac. 100, 253–261.
Gen. Pharmac. Vol. 31, No. 3, pp. 463–468, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Calcium Release by Diltiazem from Isolated Sarcoplasmic Reticulum of Rabbit Skeletal Muscle Ahmad Reza Dehpour,1* Kazem Mousavizadeh1 and Siavash Gerayesh-Nejad2 1 Department of Pharmacology and 2Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran
ABSTRACT. 1. The effect of diltiazem on isolated sarcoplasmic reticulum (SR) from rabbit skeletal muscle was studied. To observe calcium movement into and out of the SR, a fluorescent chelate probe technique with chlortetracycline (CTC) as a reagent was employed. 2. Tris-ATP-induced calcium accumulation by the isolated SR was associated with a rise in the CTC fluorescence. The effect of ATP was dose dependent. 3. Diltiazem (631024M, 231023M) prevented ATP-induced calcium accumulation by the SR. 4. Addition of EGTA to the media chelates external calcium and caused calcium release that can be reversed by further addition of calcium chloride. Similarly diltiazem caused a rapid release of accumulated calcium from the SR, which is not reversed by the addition of calcium chloride. 5. It seems that the effect of diltiazem may be related to SR membrane-bound calcium being available for release. gen pharmac 31;3:463–468, 1998. 1998 Elsevier Science Inc. KEY WORDS. Calcium, diltiazem, sarcoplasmic reticulum, skeletal muscle, rabbit INTRODUCTION Organic calcium antagonists, also known as calcium channel blockers, are a chemically heterogenous group of drugs, which antagonize, with varying degrees of potency and specificity, calcium-dependent events (Schwartz and Triggle, 1984). Many of these compounds appear to exert their major pharmacological action through inhibition of calcium influx through voltage-dependent channels in cell membranes (Fleckenstein, 1977). To examine the role of extracellular calcium in skeletal muscle contraction, numerous studies have been carried out with calcium channel blockers, such as diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle (Curtis, 1994). Voltage-clamp studies have demonstrated the existence of a slow inward calcium current in frog (Sanches and Stefani, 1978; Stanfield 1977) and mammalian (Donaldson and Beam, 1983; Walsh et al. 1986) skeletal muscles. Calcium antagonists such as diltiazem and verapamil, which block this current (Gonzalez-Serratos et al., 1982; Palade and Almers, 1985; Walsh et al., 1986), paradoxically appear to enhance mechanical activity (Dorrschedit-Kafer, 1977; Gonzalez-Serratos et al., 1982). These conflicting and contradictory results appear to result from multiple sites of action of these drugs in skeletal muscle (Su, 1988; Valle-Aguilera, 1984). Verapamil- and diltiazem-binding sites have now been identified in skeletal muscle membranes (Galizzi et al., 1985). Some investigators suggested that diltiazem may affect the function of the sarcoplasmic reticulum (SR) of cardiac papillary and muscle (Abdelmeguid and Feher, 1994; Himori et al., 1975; Nakajima et al., 1976) and rabbit skeletal muscle (Wang et al., 1984). The present study was undertaken to investigate the effects of diltiazem on isolated SR from rabbit skeletal muscles, employing a fluorescent chelate probe as a means of observing calcium movements into and out of the SR.
*To whom all correspondence should be addressed. Received 7 January 1997.
MATERIAL AND METHODS
Microsomes containing sarcoplasmic reticulum Albino rabbits of either sex weighing 1–2 kg were used. The animals were killed by a blow on the head. Microsomes were prepared according to the method described by Martonosi (1968), by homogenizing rabbit thigh muscle with 4 volumes of 5 mM tris–0.1 M KCl buffer, pH. 7.4. The myofibrils were removed by centrifugation at 1,000g for 20 min. This step was repeated once more. The microsomal fraction was obtained by centrifugation of the supernatant at 28,000g for 60 min.
Fluorescence probe technique The microsomes were suspended in histidine buffer to obtain 0.5 mg protein/ml, which was determined by the method of Lowry et al. (1951). The medium consisted of 50 mM histidine, 350 mM sucrose, 30 mM chlortetracycline (CTC); pH was 6.8 and temperature 228C. For CTC fluroscence measurement, microsomes containing SR were incubated for 30 min in the buffer. Fluorescence was measured in an Aminco-Bowman spectrophotofluorometer; excitation and emission wavelength were 390 nm and 530 nm, respectively (Caswell, 1976). After preincubation to stablize baseline levels of CTC fluorescence, the reaction was initiated in a fluorescence cuvette by adding tris-ATP, and the fluorescence was immediately recorded as a function of time. The time for mixing tris-ATP and other agents and for initiating fluorescence measurement was less than 5 sec.
Drugs and chemicals Tris-ATP, diltiazem, caffeine and EGTA were obtained from Sigma (St. Louis, MO, USA).
Statistcal Methods Data presented are given as the mean6SEM, and significances have been tested by Student’s t-test. Levels of significances are denoted by *P,0.05 and **P,0.01.
464
A. R. Dehpour et al.
FIGURE 1. Visualization of calcium accumulation by sarcoplasmic reticulum with the use of chlortetracycline as a fluorescence chelate probe. Additions to the medium were: tris-ATP, 500 mM; EGTA, 500 mM; and calcium chloride, 100 mM. Results are typical of six experiments.
FIGURE 2. Effects of different concentrations of tris-ATP on fluorescence of chlortetracycline-loaded sarcoplasmic reticulum. Results are typical of five experiments.
Diltiazem-Induced Calcium Release from SR
FIGURE 3. Log concentration–response curve for the effect of tris-ATP on calcium accumulation by sarcoplasmic reticulum of rabbit skeletal muscle. Shown are mean6SEM (n56).
465 tle calcium is bound to the membrane surface. But, when it has accumulated within the SR, the concentration of calcium within the vesicle rises to a high level, and a substantial part of it is bound to the membrane surface. CTC is able to penetrate within a membrane and will then bind to the calcium on the inner face of the membrane. The calcium accumulation by isolated SR causes an increase in the fluorescence of CTC. The nature of the data that were obtained is illustrated in Figure 1. Sarcoplasmic reticulum is present in the system, as is CTC. If the uptake of calcium is initiated by the addition of ATP, a rise in the fluorescence is observed, associated with the uptake of calcium. That this is indeed a measure of calcium accumulation is determined by the subsequent addition of EGTA, which chelates external calcium. This creates an increased calcium gradient and causes the calcium release that is associated with a diminution in the fluorescence of CTC (Fig. 1). The effect can be subsequently reversed by further addition of calcium chloride (Fig. 1). ATPinduced calcium accumulation is dose dependent, and calculated ED50 was 4.231025M (Fig. 2 and Fig. 3). Diltiazem (631024, 231023M) diminishes ATP-induced calcium accumulation (Figs. 4–6). Diltiazem, like EGTA, causes a rapid decrease in CTC fluorescence. The effect, however, is not reversed by calcium chloride (Fig. 7). Caffeine, a calcium releaser from calcium-loaded isolated SR, has no significant effect on CTC fluorescence (Fig. 8). The observation is consistent with the evidence that the calcium released by caffeine is that which is free in the cytoplasm within the SR; caffeine does not affect the bound calcium within the SR (Bowman and Rand, 1980).
RESULTS Microsomes containing SR from rabbit skeletal muscle exhibit a high ATPase activity, indicating a high content of SR membrane (Martonosi, 1968). Isolated SR from rabbit skeletal muscle was used, and a fluorescent chelate probe as a means of observing calcium movements into and out of the SR was employed. With the probe technique, we used CTC, which fluoresces with great intensity when it binds with calcium on a membrane surface (Caswell, 1976). The initial external calcium concentration is very low, and very lit-
DISCUSSION Diltiazem, like EGTA, exerts multiple actions both on the contractile system and on the SR of skinned skeletal muscle fibers (Ishizuka and Endo, 1983). Diltiazem has been reported to potentiate twitch tension in bullfrog skeletal muscle (Walsh et al., 1988) as well as frog and mouse skeletal muscle fibers (Walsh et al., 1984). The mechanical potentiation produced by diltiazem in skeletal muscle contrasts with the inhibitory action of this drug in smooth and cardiac
FIGURE 4. Chlortetracycline fluorescence decreases after pretreatment of the medium with 631024 M and 231023 M diltiazem. Calcium accumulation is initiated by addition of 500 mM tris-ATP. Results are typical of six experiments.
466
A. R. Dehpour et al.
FIGURE 5. Log concentration–response curves of tris-ATP alone (A) and in the presence of 631024M (B) and 231023M (C) diltiazem. *P,0.05 and **P,0.01. Shown are mean6SEM (n56).
muscle. The twitch potentiative action of the drug has been attributed to lowering of mechanical threshold, increased sensitivity of the contractile proteins for calcium and induction of calcium release from the SR (Su, 1988). There is some evidence that calcium channel blockers can penetrate the cell membrane and accumulate in the cytosol and may interact with intracellular calcium binding proteins (Wang et al., 1984). This evidence provides a rational for studying the possible effects of these drugs on intracellular organelle systems pertinent to calcium-transport activities. In this regard, the SR is
the primary system involved in the control of intacellular calcium through the action of a calcium pump, which is the SR membranebound calcium-ATPase (Simonides and VanHardeweld, 1990; Wang et al., 1984). In the present study, we used CTC to monitor alteration in the SR calcium level. CTC is considered a specific probe for monitoring mobilization of membrane-associated calcium (Schneider et al., 1983). Diltiazem causes a rapid decrease in CTC fluorescence of the calcium-loaded SR. Thus membrane-bound calcium in the SR may be targeted by diltiazem. This finding is con-
FIGURE 6. Release of calcium from sarcoplasmic reticulum by diltiazem. Similar results were obtained in six other experiments.
Diltiazem-Induced Calcium Release from SR
467
FIGURE 7. Diltiazem-induced calcium release from sarcoplasmic reticulum not reversed by addition of calcium chloride. Similar results were obtained in six other experiments. firmed by the observation that caffeine, a calcium releaser from calcium-loaded isolated SR, has no effect on the CTC fluorescence. However, we did not actually measure calcium release directly. The concentrations of diltiazem used here are very high, but this is consistent with the results of Walsh et al. (1986), who reported that all tonic effects of diltiazem on skeletal muscle occurred over a concentration range of 50–500 mM. Wang et al. (1984) reported that diltiazem at a concentration of 400 mM activates skeletal muscle SR calcium-ATPase and causes the early release of calcium after binding. The membrane effects of diltiazem described in this study may not be directly involved in its pharmacological and clinical actions. However, the data suggest some interesting and different ef-
FIGURE 8. Lack of effect of caffeine (20 mM) on chlortetracycline fluorescence. Results are typical of five experiments.
fects of calcium channel blockers, which may undergo complex chemical–membrane interactions. References Abdelmeguid A. E. and Feher J. J. (1994) Effect of low perfusate calcium and diltiazem on cardiac sarcoplasmic reticulum in myocardial stunning. Am. J. Physiol. 266, H406–H414. Bowman W. C. and Rand M. J. (1980) Textbook of Pharmacology. p. 750. Blackwell Scientific, Oxford. Caswell A. H. (1976) Calcium and the sarcoplasmic reticulum. In Horizons in Clinical Pharmacology. (Edited by Ronger A. and Palmer F.) pp. 151– 159. Academic Press, New York. Curtis B. A. (1994) Effect of diltiazem upon a rapidly exchanging calcium compartment related to repriming in frog skeletal muscle. J. Muscle Res. Cell Motil. 15, 49–58. Donaldson P. L. and Beam K. G. (1983) Calcium currents in fast-twitch skeletal muscle of the rat. J. Gen. Physiol. 82, 449–468. Dorrschedit-Kafer M. (1977) The action of D600 on frog skeletal muscle: facilitation of excitation–contraction coupling. Pfluegers Arch. 369, 259–267. Fleckenstein A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers and vascular smooth muscle. Annu. Rev. Pharmac. Toxicol. 17, 149–166. Galizzi J. P., Fosset M. and Lazdunski M. (1985) Characterization of the calcium coordination site regulating binding of calcium channel inhibitors d-cis-diltiazem, (1)bepridil and (2)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes. Biochem. Biophys. Res. Commun. 132, 49–55. Gonzalez-Serratos H., Valle-Aguilera R., Lathrop A.D. and Delcarmen Garcia M. (1982) Slow inward calcium currents have no obvious role in muscle excitation–contraction coupling. Nature (Lond.) 298, 292–294. Himori N., Ono H. and Thiara N. (1975) Dual effects of a new coronary vasodilator diltiazem on the contractile force of the blood-perfused papillary muscle of the dog. Jpn. J. Pharmac. 25, 350–352. Ishizuka T. and Endo M. (1983) Effects of diltiazem on skinned skeletal muscle fibers of the African clawed toad. Circ. Res. 52 (Suppl. 1), 110–114. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall F. J. (1951) Protein measurement with the folin phenol reagent. J. Biochem. 193, 265–275. Martonosi A. (1968) Sarcoplasmic reticulum IV: solubilization of microsomal adenosine triphosphate. J. Biol. Chem. 243, 71–81. Nakajima H., Hoshiyama M., Yamashita K. and Kiomoto A. (1976) Electrical and mechanical responses to diltiazem in potassium depolarized myocardium of guinea-pig. Jpn. J. Pharmac. 26, 571–580. Palade P. T. and Almers W. (1985) Slow calcium and potassium currents in
468 frog skeletal muscle: their relationship and pharmacological properties. Pfluegers Arch. 405, 91–101. Sanchez J. A. and Stefani E. (1978) Inward calcium current in twitch muscle fibers of the frog. J. Physiol. (Lond.) 283, 197–209. Schneider A. S., Herz R. and Sonenberg M. (1983) Chlortetracycline as a probe of membrane associated calcium and magnesium: interaction with red cell membranes, phospholipids, and proteins monitored by fluorescence and circular dichroism. Biochemistry 22, 1680–1686. Schwartz A. and Triggle D. J. (1984) Cellular action of calcium channel blocking drugs. Annu. Rev. Med. 35, 325–356. Simonides W. S. and VanHardeweld C. (1990) An assay for sarcoplasmic reticulum Ca-ATPase activity in muscle homogenates. Anal. Biochem. 191, 321–331. Stanfield P. R. (1977) A calcium dependent inward current in frog skeletal muscle fibers. Pfluegers Arch. 368, 267–270.
A. R. Dehpour et al. Su J. Y. (1988) Intracellular mechanisms of verapamil and diltiazem action on striated muscle of the rabbit. Naunyn-Schmiedebergs-Arch-Pharmacol. 338, 297–302. Walsh K. B., Bryant S. H. and Schwartz A. (1984) Diltiazem potentiates mechanical activity in mammalian skeletal muscle. Biochem. Biophys. Res. Commun. 122, 1091–1096. Walsh K. B., Bryant S. H. and Schwartz A. (1986) Effect of calcium antagonist drugs on calcium currents in mammalian skeletal muscle fibers. J. Pharmac. Exp. Ther. 236, 403–407. Walsh K. B., Bryant S. H. and Schwartz A. (1988) Action of diltiazem on excitation–contaction coupling in bullfrog skeletal muscle fibers. J. Pharmac. Exp. Ther. 245, 531–536. Wang T., Tasi L. and Schwartz A. (1984) Effect of verapamil, and diltiazem, nislodipine on sarcoplasmic reticulum. Eur. J. Pharmac. 100, 253–261.