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Use-dependent binding of the calcium channel antagonist (-)-[3H]-D888 in vascular smooth muscle
J Mol Cell Cardiol 22 (Supplement IV) (1990) 154USE-DEPENDENT BINDING OF THE CALCIUM CHANNEL ANTAGONIST (-)-(aH]-D888 IN VASCULAR SMOOTH MUSCLE. L. RAKOTOARISOA, C. MI RONNEAU, J. MIRONNEAU. Laboratoire de Physiologie Cellulaire et Pharmacologic MolBculaire, INSERM JF 88-13, Universite de Bordeaux II, 3 place de la Victoire, 33076 Bordeaux. Binding of the phenylalkylamine calcium antagonist (-)-[3H]-D888 was studied in intact strips of rat portal vein. In 5 mMKsolutions, veins showed specific binding of (-)-[IHI-D888 with a dissociation constant (KD) of 0.36 ) 0.05 nM and a maximum receptor density (B,,,) of 16 + 1 fmol/mg wet weight (n = 6). Unlabelled (-)D888, D600 and diltiazem completely inhibited (-)-[3H]-D888 binding in a competitive manner. Unlabelled 1,4-dihydropyridines ((+)-PN 200110 ; nifedipine; (-)-PN 200-110) also inhibited (-)-[3H]-D888 binding but they acted as negative allosteric effecters of phenylalkylamine binding sites. Increase in extracellular potassium concentration (135 mM) or addition of cromakalim (0.1 to 10 PM) produced a gradual decrease of the number of (-)-[3H]-D888 binding sites. These results suggest that the binding of (-)-[XH]-D888 is usedependent, and that opening of calcium channels is required for the binding of (-)-[XH]-D888 at its sites.
155 EFFECTS
OF
PRODUCTIOIN
CHANCES ,3F
Cardiology,
33600
Emsme,
1070
Brussels,
IN
EXTRA-
PROSTACYCLIN. Pewc,
France;
AND
INTRACELLULAR
l.Ramboer” (OO)lnstitut
and of
K+
lnterdisciplinar
11568 Peglion
AND and
THE
ENDOTHELIAL (‘)lNSERM
Research,
U8
ULB
Campus
Belgium.
The earliest event induced by h$emodynamic shear stress (BAEC) is the activation of a K current and the resulting cruciai role in the Process of mechanochemical tmnsduction. !n order to evaluate the role of hyperpclarisation in the hyc investigate the effects of monovalenta cation ionophores K on the endothelial production of PGI In conclusion, +modification of BAEC mem 2d rane potential extmcellular K conccntrution, do not modulate the busul FG12. Valinomycin, nigericin and ouabain enhanced the nigertcin and ouabain, unlike that of valinamycin, induce intmcellular K+.
156 BIOCHEMICAL
ON
J-M.Boeynaems”,
in
bovine aortic hyperpolarisation
control
of and
of
endothelial might
BAEC function, changes in
play
cells a
we extmceilular
resulting from changes in the or ATP-stimulated production of release of PG! but the action of a time-depen 2 ent depletion of
AND ELECTROPHY5lOLOGlCAL PROPERTIES OF A NOVEL DlHYDROPYRlDlNE ITS ENANTIOMERS 5 12967 AND 5 12968 J.C.R. Randle, A. Lombet, N. Nagel, J.F. Renaud. Fondax-Groupe de Recherche Servier. 7, rue Ampere Puteaux 92800
DERIVATIVE,
C. Abraham,
5 J.L.
FRANCE
Dihydropyridines are well known for their ability to bind specifically to membrane proteins associated with voltage dependent Ca2 l channels to reduce the contractility of cardiac and vascular smooth muscles and to control arterial pressure in animal models and humans. The atim of this study was to describe the biochemical and electrophysiological properties of a new Ca * * channel blocker of the dihydropyridine family the 5 11568 ( f ) 2-{2-[2-(Aminoethoxy) ethoxy] methyl)-4-(2.3-dichlorophenyl)-3-ethoxycarbonyl-5methoxycarbonyl-6-methyl-1,4-dihydropyridine and its enantiomers 5 12967 [( +)-5 115681 and 5 12968 [(-)-5 115681. Binding studies have shown that 5 11568 and 5 12968 displaced specifically bound ]3H] PN 200-l 10 from cardiac and vascular smooth muscle preparation with potencies ranging from 5 6 to 51 nM. 5 12967 was found to be 6 to 18 fold less potent than 5 12968 A good correlation was found between ECsa for the inhibition of 4sCa2 + uptake by A7rs aortic smooth muscle cells and binding data Whole-cell patch-clamp studies in both guinea-pig ventricular myocytes and Airs cells at two holding potentials (VH) -50 mV and -100 and 2) tltat 5 tZ~bl( mV have shown 1) that these compounds are eHec1tve blockers of L-lype Cd * + channel (Et& = 70 nM) was 2 to 3-fold more potent than 5 11568 and 30 fold more potent than 5 12967 when VH was adjusted to -5OmV. When VH was adjusted to -1OOmV all compounds were less polent, with EC50 values rangingfrom 500 nM to 3 pti. These results demonstrate conclusively that 5 12968 is the more active enantiomer. Furthermore, the voltage-dependence of its actions indicates that in viva il will exhibit good selectivity for vascular smooth muscle over cardiac muscle. S.52
155 EFFECTS
OF
PRODUCTIOIN
CHANCES ,3F
Cardiology,
33600
Emsme,
1070
Brussels,
IN
EXTRA-
PROSTACYCLIN. Pewc,
France;
AND
INTRACELLULAR
l.Ramboer” (OO)lnstitut
and of
K+
lnterdisciplinar
11568 Peglion
AND and
THE
ENDOTHELIAL (‘)lNSERM
Research,
U8
ULB
Campus
Belgium.
The earliest event induced by h$emodynamic shear stress (BAEC) is the activation of a K current and the resulting cruciai role in the Process of mechanochemical tmnsduction. !n order to evaluate the role of hyperpclarisation in the hyc investigate the effects of monovalenta cation ionophores K on the endothelial production of PGI In conclusion, +modification of BAEC mem 2d rane potential extmcellular K conccntrution, do not modulate the busul FG12. Valinomycin, nigericin and ouabain enhanced the nigertcin and ouabain, unlike that of valinamycin, induce intmcellular K+.
156 BIOCHEMICAL
ON
J-M.Boeynaems”,
in
bovine aortic hyperpolarisation
control
of and
of
endothelial might
BAEC function, changes in
play
cells a
we extmceilular
resulting from changes in the or ATP-stimulated production of release of PG! but the action of a time-depen 2 ent depletion of
AND ELECTROPHY5lOLOGlCAL PROPERTIES OF A NOVEL DlHYDROPYRlDlNE ITS ENANTIOMERS 5 12967 AND 5 12968 J.C.R. Randle, A. Lombet, N. Nagel, J.F. Renaud. Fondax-Groupe de Recherche Servier. 7, rue Ampere Puteaux 92800
DERIVATIVE,
C. Abraham,
5 J.L.
FRANCE
Dihydropyridines are well known for their ability to bind specifically to membrane proteins associated with voltage dependent Ca2 l channels to reduce the contractility of cardiac and vascular smooth muscles and to control arterial pressure in animal models and humans. The atim of this study was to describe the biochemical and electrophysiological properties of a new Ca * * channel blocker of the dihydropyridine family the 5 11568 ( f ) 2-{2-[2-(Aminoethoxy) ethoxy] methyl)-4-(2.3-dichlorophenyl)-3-ethoxycarbonyl-5methoxycarbonyl-6-methyl-1,4-dihydropyridine and its enantiomers 5 12967 [( +)-5 115681 and 5 12968 [(-)-5 115681. Binding studies have shown that 5 11568 and 5 12968 displaced specifically bound ]3H] PN 200-l 10 from cardiac and vascular smooth muscle preparation with potencies ranging from 5 6 to 51 nM. 5 12967 was found to be 6 to 18 fold less potent than 5 12968 A good correlation was found between ECsa for the inhibition of 4sCa2 + uptake by A7rs aortic smooth muscle cells and binding data Whole-cell patch-clamp studies in both guinea-pig ventricular myocytes and Airs cells at two holding potentials (VH) -50 mV and -100 and 2) tltat 5 tZ~bl( mV have shown 1) that these compounds are eHec1tve blockers of L-lype Cd * + channel (Et& = 70 nM) was 2 to 3-fold more potent than 5 11568 and 30 fold more potent than 5 12967 when VH was adjusted to -5OmV. When VH was adjusted to -1OOmV all compounds were less polent, with EC50 values rangingfrom 500 nM to 3 pti. These results demonstrate conclusively that 5 12968 is the more active enantiomer. Furthermore, the voltage-dependence of its actions indicates that in viva il will exhibit good selectivity for vascular smooth muscle over cardiac muscle. S.52