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Inhibition by calcium antagonists of ionophore-mediated calcium transport in liposomes
European Journal of Pharmacology, 83 (1982) 21-24
21
Elsevier BiomedicalPress
I N H I B I T I O N BY CALCIUM A N T A G O N I S T S T R A N S P O R T IN L I P O S O M E S
OF I O N O P H O R E - M E D I A T E D C A L C I U M
MICHEL DELEERS, MARIANNE MAHY and WILLY J. MALAISSE* Laboratory of Experimental Medicine, Brussels University. Medical School, Brussels, Belgium
Received 28 April 1982, accepted 2 June 1982
M. DELEERS, M. MAHY and W.J. MALAISSE, Inhibition by calcium antagonists of ionophore-mediated calcium transport in liposomes, European J. Pharmacol. 83 (1982) 21-24. Verapamil, gallopamil and suloctidil inhibited the process of calcium exchange-diffusion mediated by the ionophore bromolasalocid in liposomes of variable fluidity. This supports the view that the calcium antagonist capacity of these drugs is primarily attributable to blockade of calcium transport across cell membranes. Organic calcium antagonists
Ionophore Liposomes Calcium
1. Introduction
2. Materials and methods
Organic calcium antagonists, such as verapamil and gallopamil, are thought to selectively block the entry of ionized calcium into cells and, therefore, are often referred to as calcium channel blockers (Braunwald, 1980). The process of calcium transport across membranes can be facilitated by exposure of the tissue to ionophores such as bromolasalocid. Verapamil inhibits the calciumdependent functional response evoked by ionophores (Somerset al., 1976). In a two-phase bulk system or in the Pressman cell, organic calcium antagonists competitively inhibit ionophore-mediated calcium translocation (Malaisse et al., 1977a, 1981; Couturier and Malaisse, 1980). The present study demonstrates that organic calcium antagonists also inhibit calcium transport when the ionophore is inserted in a phospholipid bilayer instead of moving freely in an organic solvent.
Distearoyl-phosphatidylcholine (DSPC), dipalmitoyl-phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and cholesterol were purchased from Sigma Chemicals (St. Louis, Missouri), The ionophore bromolasalocid (Br- X537A) was a gift from Hoffman-La Roche (Nutley, New Jersey). Suloctidil was a gift from Continental Pharma (Mont-Saint Guibert, Belgium), and verapamil and gallopamil from Knoll A G (Ludwigshafen, FRG). Multilamellar liposomes formed of suitable lipids and bromolasalocid were prepared, washed and incubated as described elsewhere (Deleers and Malaisse, 1980a) in a Tris-HCl buffer (20 mM, pH 7.4) containing NaC1 120 mM, CaC12 0.2 mM and, when required, suloctidil, verapamil or gallopamil. The final suspension incubated at 37°C contained 5 mg of lipids per ml of medium. The efflux of 45Ca from liposomes was monitored as previously described (Deleers and Malaisse, 1980a) and expressed either as the amount of 45Ca released by the liposomes (in percent of their initial 45Ca content) or as a fractional outflow rate, which represented the mean value derived from measure-
• To whom all correspondenceshould be addressed: Labora-
tory of Experimental Medicine, 115 boulevard de Waterloo, B-1000 Brussels, Belgium. OOl4-2999/82/oooo-0o0o/$o2.75 © 1982 ElsevierBiomedicalPress
22 ments made after 5, 10 and 15 min incubation. All results are presented as the mean (-+S.E.M.) together with the n u m b e r of individual determinations (in parentheses). The fluorescence polarization of liposomes labelled with 1,6-diphenyl-l,3,5-hexatriene (1 m o l / 8 0 0 mol of lipid) was measured at 37°C with an Elscint MV l a microviscosimeter (Shinitzky and Barenholz, 1978). These measurements yielded viscosities of 6.36, 4.89, 3.82 and 2.12P for DSPC:cholesterol (3:1 molar ratio), D P P C : cholesterol (2:1 molar ratio), D M P C : c h o l e s t e r o l (2 : 1 and 3 : 1 molar ratio) liposomes, respectively.
3. Results Fig. 1 illustrates the results of two series of experiments designed to investigate the influence of three distinct calcium antagonists u p o n the
process of Ca exchange-diffusion ion liposomes containing the ionophore bromolasalocid. In liposomes formed of D S P C and cholesterol (3:1 molar ratio) and containing bromolasalocid (5 m o l / 1 0 0 0 mol of lipid), suloctidil, which was added to the aqueous phase at an initial concentration of 0.5 mM, dramatically decreased the rate of 45Ca efflux as judged from either the initial fractional outflow rate or the radioactive content of the medium at the 40th rain of incubation (table 1, first column). N o significant effect of verapamil or gallopamil was seen in these relatively rigid liposomes, although the a m o u n t of 45Ca released in the medium tended to be lower in the presence than in the absence of these agents. In liposomes formed of D P P C and cholesterol (2:1 molar ratio) and containing bromolasalocid ( 5 m o l / 1 0 0 0 mol of lipid), suloctidil again decreased both the initial fractional outflow rate and the radioactive content of the medium. As j u d g e d by the latter criteria, verapamil and gallopamil
lOO~
90 c
2 8O
§ 7o m
6O 5O
i
0
10
|
20 TIME (re|n)
|
30
n
40
n
10
~
2 TIME (rain)
|
j
30
40
Fig. I. Time course for the decrease in 45Ca content (expressed in percent of the initial value) of DPPC :cholesterol (2 : l molar ratio; left panel) and DMPC : cholesterol (2:1 molar ratio; right panel) liposomes containing bromolasalocid (5 tool/1000 tool of lipid) and incubated at 37°C either in the absence of any calcium antagonist (closed circles) or in the presence of 0.5 mM verapamil (open circles), gallopamil (open triangles) or suloctidil (closed triangles). Mean values (+ S.E.M.) refer to 8-16 individual experiments.
23 TABLE 1 Effect of calcium antagonists upon ionophore-mediated 45Ca transport in liposomes. The concentrations of bromolasalocid (5 mol/1000 mol of lipid) and calcium-antagonists (0.5 mM) were the same in all experiments. Mean values for 45Ca fractional outflow rate (FOR) and release at the 40th min of incubation (40 min) are shown together with the number of individual experiments (n) and statistical significance of the difference between control and experimental values (a P<0.05; b P<0.02; c P<0.01; and d P<0.001). * Molar ratio. Liposomes
DSPC : cholesterol, 3 : 1 * n
Control Suloctidil Verapamil Gallopamil Liposomes
Control Suloctidil Verapamil Gallopamil
8 8 8 8
DPPC : cholesterol, 2 : 1 *
FOR
40 min
n
FOR
40 min
2.26 + 0.37 0.35_+0.25 d 2.53 + 0.31 2.33 ± 0.64
31.7 + 2.9 5.5_+ 1.3 d 27.9 + 3.7 24.3 ± 3.8
16 8 8 8
3.25 + 0.23 2.35+0.34 a 3.05 --+0.43 2.80 + 0.29
52.2 + 1.6 27.8_+3.1 d 36.4-+ 3.6 a 30.9+ 2.7 d
DMPC : cholesterol, 2 : 1 *
DMPC : cholesterol, 3 : I *
n
FOR
40 min
16 8 16 8
1.66+0.30 0.49+0.42 a 0.75 -+0.18 b 0.64+0.19 a
40.3+ 1.7 4.5_+2.8 d 13.9+ 1.4 '~ 11.7± 1.7 d
also i n h i b i t e d 45Ca release, b u t less so than suloctidil (table 1, second column). I n liposomes formed of D M P C a n d cholesterol (2:1 or 3 : 1 m o l a r ratio) a n d c o n t a i n i n g b r o m o l a salocid (5 m o l / 1 000 mol of lipid), all three calcium antagonists significantly i n h i b i t e d 45Ca outflow. T h e i n h i b i t o r y action of suloctidil was again more m a r k e d t h a n that of the two other drugs (table 1, third a n d fourth columns). W h e t h e r a calcium a n t a g o n i s t was present or not, the efficiency of i o n o p h o r e - m e d i a t e d 45Ca transport in liposomes formed of D M P C a n d cholesterol was greater at a high ( 3 : 1 ) t h a n at a low (2: 1) D M P C : c h o l e s t e r o l m o l a r ratio. O n the contrary, the relative magnitude of the i n h i b i t o r y action of the calcium antagonists was greater at a low t h a n at a high D M P C : cholesterol m o l a r ratio, I n the liposomes formed of D M P C a n d cholesterol ( 2 : 1 m o l a r ratio) a n d c o n t a i n i n g bromolasalocid ( 5 m o l / 1 0 0 0 mol of lipid), the m a g n i t u d e of the i n h i b i t o r y action of verapamil u p o n i o n o p h o r e - m e d i a t e d 45Ca transport depended on the c o n c e n t r a t i o n of the calcium antagonist, Thus, as the initial c o n c e n t r a t i o n of verapamil was decreased from 0.50 to 0.15 m M , the a m o u n t of
n 8 8 8 8
FOR
40 min
2.79+0.34 0.98+0.17 d 1.66 ± 0.15 c 1.82+0.31
46.3+ 1.7 17.3_+2.1 d 22.3 + 1.5 d 30.3 + 1.7 d
45Ca released a t - t h e 40th m i n increased from 13.9 -+ 1.4 (n = 16) to 28.2 - 1.8 (n = 8) percent, as c o m p a r e d with a control value (no verapamil) of 40.3 -+ 1.7 per cent (n = 16).
4. Discussion The present results d e m o n s t r a t e that drugs currently qualified as organic calcium antagonists or calcium c h a n n e l blockers i n h i b i t dose-relatedly the process of i o n o p h o r e - m e d i a t e d Ca exchange-diffusion across artificial bilayers. We have previously shown that the efficiency of the process of 45Ca transport in the present system d e p e n d s on such factors as the Ca 2+ c o n c e n t r a t i o n gradient, rigidity of the bilayer, c o n c e n t r a t i o n of i o n o p h o r e , stoichiometry of the C a - i o n o p h o r e complex a n d h y d r o p h o b i c interactions in the liposomal matrix (Deleers a n d Malaisse, 1980a,b; 1982; Deleers et al., 1981). The present results agree with these concepts in that the efficiency of 45Ca transport increased with increasing fluidity in liposomes formed of the same c o m b i n a t i o n of lipids, b u t did n o t always parallel the viscosity values in lipo-
24
somes formed of different combinations of lipids. The concentrations of Ca 2÷ and ionophore used in the present series of experiments were selected t o achieve a 45Ca fractional outflow rate close to that found in living cells (Herchuelz et al., 1979). If one ignores the partition of the calcium antagonist between the aqueous medium and liposomal matrix, the molar ratio of ionophore to calcium antagonist (approximately 1 : 10) in the suspension of liposomes was of the same order of magnitude as that required in a two-phase bulk system to detect inhibition of ionophore-mediated 45Ca translocation (Malaisse et al., 1981). In the present system, suloctidil w a s a m o r e potent inhibitor of aSCa transport than either verapamil or gallopamil. This is at variance with the situation found in certain biological systems. For instance, using the process of glucose-stimulated insulin release as the calcium-dependent functional variable, we observed that the EDs0 for inhibition of insulin release was close to 1.25 # M for suloctidil as distinct from 1.00/~M for verapamil and 0.60/~M for gallopamil (Malaisse, 1977; Malaisse et al., 1976, 1977b). In considering this difference, it should be realized that suloctidil is more lipophilic than verapamil or gallopamil and interacts with phospholipids in bitayer membranes (Chatelain and Ruysschaert, 1981). In conclusion, the present data lend support to the view that the inhibitory action of calcium antagonists upon calcium-dependent functional processes may be attributable to primary interference with the process of Ca transport across cell membranes. Indeed, the inhibition by calcium antagonists of ionophore-mediated Ca exchangediffusion across artificial bitayers may serve as a model for the interference of these drugs with the transport of Ca across cell membranes as mediated by either native ionophores of calcium channels (Barritt, 1981). In distinguishing between the latter two modalities of Ca transport, it should be kept in mind that ionophores may themselves be arranged to form ionic channels (Boheim et al., 1976, 1978). Acknowledgements This work was supported in part by a grant from the Belgian Ministry of Scientific Policy. We thank C. Demesmaeker for secretarial help.
References Barritt, G.J., 1981, Calcium transport across cell membranes: progress toward molecular mechanisms, Trends Biochem. Sci. 6, 322. Braunwald, E., 1980, Introduction: calcium channel blockers, A m . J . Cardiol. 46, 1045. Boheim, G., G. Irmscher and G. Jung, 1978, Trichotoxin A-40, a new membranes exciting peptide. Part B. Voltage-dependent pore formation in bilayer lipid membranes and comparison with other alamethicin analogues, Biochim. Biophys. Acta 507, 485. Boheim, G., K. Janko, D. Leibfritz, T. Ooka, W.A. K6nig and G. Jung, 1976, Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in Black lipid films, Biochim. Biophys. Acta 433, 182. Chatelain, P. and J.M. Ruysschaert, 1981, Suloctidil-phospholipid interaction in bilayer membranes: effects on lipid dynamics, Biochem. Pharmacol. 30, 313. Couturier, E. and W.J. Malaisse, 1980, An ionophoretic model for the study of calcium-calcium exchange, J. Inorg. Biochem. 12, 57. Deicers, M., E. Couturier and W.J. Malaisse, 1981, Ionomycinmediated calcium transport in rigid and fluid liposomes, Cell Calcium 2, 159. Deicers,M. and W.J. Malaisse, 1980a, Ionophore-mediated calcium exchange diffusion in liposomes, Biochem. Biophys. Res. Commun. 85, 650. Deleers, M. and W.J. Malaisse, 1980b, Influence of membrane fluidity upon ionophore-mediated calcium transport in liposomes, Biomed. Res. 1,400. Deicers, M. and W.J. Malaisse, 1982, Influence of phorbol esters on ionophore-mediated calcium exchange-diffusion in liposomes, Chem. Phys. Lipids (in press). Herchuelz, A., C. Delcroix and W.J. Malaisse, 1979, Regulation of calcium fluxes in rat pancreatic islets. Quantification of calcium movements, Biochem. Med. 22, 156. Malaisse, W.J., 1977, Calcium-antagonists and islet function. x. Effect of suloctidil, Arch. Int. Pharmacodyn. 238, 339. Malaisse, W.J., G. Devis, G.D. Pipeleers and G. Somers, 1976, Calcium-antagonists and islet function. IV. Effect of D600, Diabetologia 12, 77. Malaisse, W.J., G. Devis and G. Somers, 1977a, Inhibition by verapamil of ionophore-mediated calcium translocation, Experientia 33, 1035. Malaisse, W.J., A. Herchuelz, J. Levy and A. Sener, 1977b, Calcium-antagonists and islet function, lII. The possible site of action of verapamil, Biochem. Pharmacol. 30, 313. Malaisse, W.J., G. Somers, I. Valverde and E. Couturier, 1981, Organic calcium-antagonists and calcium ionophores, Arzneim. Forsch. 31,628. Shinitzky, M. and Y. Barenholz, 1978, Fluidity parameters of lipid regions determined by fluorescence polarization, Biochem. Biophys. Acta 515, 367. Somers, G., G. Devis and W.J. Malaisse, 1976, Analogy between native and exogenous ionophores in the pancreatic B-cell, FEBS Lett. 66, 20.
21
Elsevier BiomedicalPress
I N H I B I T I O N BY CALCIUM A N T A G O N I S T S T R A N S P O R T IN L I P O S O M E S
OF I O N O P H O R E - M E D I A T E D C A L C I U M
MICHEL DELEERS, MARIANNE MAHY and WILLY J. MALAISSE* Laboratory of Experimental Medicine, Brussels University. Medical School, Brussels, Belgium
Received 28 April 1982, accepted 2 June 1982
M. DELEERS, M. MAHY and W.J. MALAISSE, Inhibition by calcium antagonists of ionophore-mediated calcium transport in liposomes, European J. Pharmacol. 83 (1982) 21-24. Verapamil, gallopamil and suloctidil inhibited the process of calcium exchange-diffusion mediated by the ionophore bromolasalocid in liposomes of variable fluidity. This supports the view that the calcium antagonist capacity of these drugs is primarily attributable to blockade of calcium transport across cell membranes. Organic calcium antagonists
Ionophore Liposomes Calcium
1. Introduction
2. Materials and methods
Organic calcium antagonists, such as verapamil and gallopamil, are thought to selectively block the entry of ionized calcium into cells and, therefore, are often referred to as calcium channel blockers (Braunwald, 1980). The process of calcium transport across membranes can be facilitated by exposure of the tissue to ionophores such as bromolasalocid. Verapamil inhibits the calciumdependent functional response evoked by ionophores (Somerset al., 1976). In a two-phase bulk system or in the Pressman cell, organic calcium antagonists competitively inhibit ionophore-mediated calcium translocation (Malaisse et al., 1977a, 1981; Couturier and Malaisse, 1980). The present study demonstrates that organic calcium antagonists also inhibit calcium transport when the ionophore is inserted in a phospholipid bilayer instead of moving freely in an organic solvent.
Distearoyl-phosphatidylcholine (DSPC), dipalmitoyl-phosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and cholesterol were purchased from Sigma Chemicals (St. Louis, Missouri), The ionophore bromolasalocid (Br- X537A) was a gift from Hoffman-La Roche (Nutley, New Jersey). Suloctidil was a gift from Continental Pharma (Mont-Saint Guibert, Belgium), and verapamil and gallopamil from Knoll A G (Ludwigshafen, FRG). Multilamellar liposomes formed of suitable lipids and bromolasalocid were prepared, washed and incubated as described elsewhere (Deleers and Malaisse, 1980a) in a Tris-HCl buffer (20 mM, pH 7.4) containing NaC1 120 mM, CaC12 0.2 mM and, when required, suloctidil, verapamil or gallopamil. The final suspension incubated at 37°C contained 5 mg of lipids per ml of medium. The efflux of 45Ca from liposomes was monitored as previously described (Deleers and Malaisse, 1980a) and expressed either as the amount of 45Ca released by the liposomes (in percent of their initial 45Ca content) or as a fractional outflow rate, which represented the mean value derived from measure-
• To whom all correspondenceshould be addressed: Labora-
tory of Experimental Medicine, 115 boulevard de Waterloo, B-1000 Brussels, Belgium. OOl4-2999/82/oooo-0o0o/$o2.75 © 1982 ElsevierBiomedicalPress
22 ments made after 5, 10 and 15 min incubation. All results are presented as the mean (-+S.E.M.) together with the n u m b e r of individual determinations (in parentheses). The fluorescence polarization of liposomes labelled with 1,6-diphenyl-l,3,5-hexatriene (1 m o l / 8 0 0 mol of lipid) was measured at 37°C with an Elscint MV l a microviscosimeter (Shinitzky and Barenholz, 1978). These measurements yielded viscosities of 6.36, 4.89, 3.82 and 2.12P for DSPC:cholesterol (3:1 molar ratio), D P P C : cholesterol (2:1 molar ratio), D M P C : c h o l e s t e r o l (2 : 1 and 3 : 1 molar ratio) liposomes, respectively.
3. Results Fig. 1 illustrates the results of two series of experiments designed to investigate the influence of three distinct calcium antagonists u p o n the
process of Ca exchange-diffusion ion liposomes containing the ionophore bromolasalocid. In liposomes formed of D S P C and cholesterol (3:1 molar ratio) and containing bromolasalocid (5 m o l / 1 0 0 0 mol of lipid), suloctidil, which was added to the aqueous phase at an initial concentration of 0.5 mM, dramatically decreased the rate of 45Ca efflux as judged from either the initial fractional outflow rate or the radioactive content of the medium at the 40th rain of incubation (table 1, first column). N o significant effect of verapamil or gallopamil was seen in these relatively rigid liposomes, although the a m o u n t of 45Ca released in the medium tended to be lower in the presence than in the absence of these agents. In liposomes formed of D P P C and cholesterol (2:1 molar ratio) and containing bromolasalocid ( 5 m o l / 1 0 0 0 mol of lipid), suloctidil again decreased both the initial fractional outflow rate and the radioactive content of the medium. As j u d g e d by the latter criteria, verapamil and gallopamil
lOO~
90 c
2 8O
§ 7o m
6O 5O
i
0
10
|
20 TIME (re|n)
|
30
n
40
n
10
~
2 TIME (rain)
|
j
30
40
Fig. I. Time course for the decrease in 45Ca content (expressed in percent of the initial value) of DPPC :cholesterol (2 : l molar ratio; left panel) and DMPC : cholesterol (2:1 molar ratio; right panel) liposomes containing bromolasalocid (5 tool/1000 tool of lipid) and incubated at 37°C either in the absence of any calcium antagonist (closed circles) or in the presence of 0.5 mM verapamil (open circles), gallopamil (open triangles) or suloctidil (closed triangles). Mean values (+ S.E.M.) refer to 8-16 individual experiments.
23 TABLE 1 Effect of calcium antagonists upon ionophore-mediated 45Ca transport in liposomes. The concentrations of bromolasalocid (5 mol/1000 mol of lipid) and calcium-antagonists (0.5 mM) were the same in all experiments. Mean values for 45Ca fractional outflow rate (FOR) and release at the 40th min of incubation (40 min) are shown together with the number of individual experiments (n) and statistical significance of the difference between control and experimental values (a P<0.05; b P<0.02; c P<0.01; and d P<0.001). * Molar ratio. Liposomes
DSPC : cholesterol, 3 : 1 * n
Control Suloctidil Verapamil Gallopamil Liposomes
Control Suloctidil Verapamil Gallopamil
8 8 8 8
DPPC : cholesterol, 2 : 1 *
FOR
40 min
n
FOR
40 min
2.26 + 0.37 0.35_+0.25 d 2.53 + 0.31 2.33 ± 0.64
31.7 + 2.9 5.5_+ 1.3 d 27.9 + 3.7 24.3 ± 3.8
16 8 8 8
3.25 + 0.23 2.35+0.34 a 3.05 --+0.43 2.80 + 0.29
52.2 + 1.6 27.8_+3.1 d 36.4-+ 3.6 a 30.9+ 2.7 d
DMPC : cholesterol, 2 : 1 *
DMPC : cholesterol, 3 : I *
n
FOR
40 min
16 8 16 8
1.66+0.30 0.49+0.42 a 0.75 -+0.18 b 0.64+0.19 a
40.3+ 1.7 4.5_+2.8 d 13.9+ 1.4 '~ 11.7± 1.7 d
also i n h i b i t e d 45Ca release, b u t less so than suloctidil (table 1, second column). I n liposomes formed of D M P C a n d cholesterol (2:1 or 3 : 1 m o l a r ratio) a n d c o n t a i n i n g b r o m o l a salocid (5 m o l / 1 000 mol of lipid), all three calcium antagonists significantly i n h i b i t e d 45Ca outflow. T h e i n h i b i t o r y action of suloctidil was again more m a r k e d t h a n that of the two other drugs (table 1, third a n d fourth columns). W h e t h e r a calcium a n t a g o n i s t was present or not, the efficiency of i o n o p h o r e - m e d i a t e d 45Ca transport in liposomes formed of D M P C a n d cholesterol was greater at a high ( 3 : 1 ) t h a n at a low (2: 1) D M P C : c h o l e s t e r o l m o l a r ratio. O n the contrary, the relative magnitude of the i n h i b i t o r y action of the calcium antagonists was greater at a low t h a n at a high D M P C : cholesterol m o l a r ratio, I n the liposomes formed of D M P C a n d cholesterol ( 2 : 1 m o l a r ratio) a n d c o n t a i n i n g bromolasalocid ( 5 m o l / 1 0 0 0 mol of lipid), the m a g n i t u d e of the i n h i b i t o r y action of verapamil u p o n i o n o p h o r e - m e d i a t e d 45Ca transport depended on the c o n c e n t r a t i o n of the calcium antagonist, Thus, as the initial c o n c e n t r a t i o n of verapamil was decreased from 0.50 to 0.15 m M , the a m o u n t of
n 8 8 8 8
FOR
40 min
2.79+0.34 0.98+0.17 d 1.66 ± 0.15 c 1.82+0.31
46.3+ 1.7 17.3_+2.1 d 22.3 + 1.5 d 30.3 + 1.7 d
45Ca released a t - t h e 40th m i n increased from 13.9 -+ 1.4 (n = 16) to 28.2 - 1.8 (n = 8) percent, as c o m p a r e d with a control value (no verapamil) of 40.3 -+ 1.7 per cent (n = 16).
4. Discussion The present results d e m o n s t r a t e that drugs currently qualified as organic calcium antagonists or calcium c h a n n e l blockers i n h i b i t dose-relatedly the process of i o n o p h o r e - m e d i a t e d Ca exchange-diffusion across artificial bilayers. We have previously shown that the efficiency of the process of 45Ca transport in the present system d e p e n d s on such factors as the Ca 2+ c o n c e n t r a t i o n gradient, rigidity of the bilayer, c o n c e n t r a t i o n of i o n o p h o r e , stoichiometry of the C a - i o n o p h o r e complex a n d h y d r o p h o b i c interactions in the liposomal matrix (Deleers a n d Malaisse, 1980a,b; 1982; Deleers et al., 1981). The present results agree with these concepts in that the efficiency of 45Ca transport increased with increasing fluidity in liposomes formed of the same c o m b i n a t i o n of lipids, b u t did n o t always parallel the viscosity values in lipo-
24
somes formed of different combinations of lipids. The concentrations of Ca 2÷ and ionophore used in the present series of experiments were selected t o achieve a 45Ca fractional outflow rate close to that found in living cells (Herchuelz et al., 1979). If one ignores the partition of the calcium antagonist between the aqueous medium and liposomal matrix, the molar ratio of ionophore to calcium antagonist (approximately 1 : 10) in the suspension of liposomes was of the same order of magnitude as that required in a two-phase bulk system to detect inhibition of ionophore-mediated 45Ca translocation (Malaisse et al., 1981). In the present system, suloctidil w a s a m o r e potent inhibitor of aSCa transport than either verapamil or gallopamil. This is at variance with the situation found in certain biological systems. For instance, using the process of glucose-stimulated insulin release as the calcium-dependent functional variable, we observed that the EDs0 for inhibition of insulin release was close to 1.25 # M for suloctidil as distinct from 1.00/~M for verapamil and 0.60/~M for gallopamil (Malaisse, 1977; Malaisse et al., 1976, 1977b). In considering this difference, it should be realized that suloctidil is more lipophilic than verapamil or gallopamil and interacts with phospholipids in bitayer membranes (Chatelain and Ruysschaert, 1981). In conclusion, the present data lend support to the view that the inhibitory action of calcium antagonists upon calcium-dependent functional processes may be attributable to primary interference with the process of Ca transport across cell membranes. Indeed, the inhibition by calcium antagonists of ionophore-mediated Ca exchangediffusion across artificial bitayers may serve as a model for the interference of these drugs with the transport of Ca across cell membranes as mediated by either native ionophores of calcium channels (Barritt, 1981). In distinguishing between the latter two modalities of Ca transport, it should be kept in mind that ionophores may themselves be arranged to form ionic channels (Boheim et al., 1976, 1978). Acknowledgements This work was supported in part by a grant from the Belgian Ministry of Scientific Policy. We thank C. Demesmaeker for secretarial help.
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