Voltage-dependent binding of 1,4-dihydropyridine Ca2+ channel antagonists and activators in cultured neonatal rat ventricular myocytes.
Binding of 1,4-dihydropyridine Ca2+ channel ligands was characterized as a function of membrane potential using saturation, competition, and kinetic measurements in cultured neonatal rat ventricular myocytes. The 1,4-dihydropyridine antagonist [3H]PN 200-110 bound to polarized cells (5.8 mM K+) with a KD value of 3.53 X 10(-9) M and a Bmax value of 50.1 fmol/mg of protein. In depolarized cells (50 mM K+), a KD value of 6.33 X 10(-11) M was found, reflecting a 55-fold increase in affinity; Bmax did not change upon depolarization. Dissociation rates (k-1) of [3H]PN 200-110 binding were faster in polarized cells (0.53 min-1) than in depolarized cells (0.018 min-1), but association rates (k1 of 2.17 X 10(8) and 2.27 X 10(8) min-1M-1 were not different in polarized and depolarized cells. The KD values calculated from the ratio of k-1/k1 accorded well with those determined from equilibrium binding assays. The enantiomers of Bay K 8644 and 202-791 and a series of nifedipine analogs inhibited specific binding of [3H]PN 200-110 in depolarized cells. In polarized cells, the affinities of the S-enantiomers (activators) were close to those in depolarized cells; however, the affinities of R-enantiomers (antagonists) were 50- to 65-fold lower. The effects of both (S)- and (R)-Bay K 8644 on [3H]PN 200-110 binding were mediated through increased apparent KD values, without changes in Bmax and nH. In depolarized cells, l-D600 and d-D600 partially inhibited [3H]PN 200-110 binding to a maximum of 71% and 56%, respectively; in polarized cells, l-D600 (d-D600 not measured) was ineffective on [3H]PN 200-110 binding. d-(cis)-Diltiazem, but not l-(cis)-diltiazem, partially inhibited (maximum 30%) specific binding of [3H]PN 200-110 in depolarized cells, but potentiated (maximum 79%) binding in polarized cells. The potentiating effect of d-(cis)-diltiazem was mediated through an increase in affinity without change in Bmax of [3H]PN 200-110 binding. (S)-Bay K 8644 potentiated 45Ca2+ uptake into the cells, with an EC50 value of 4.26 X 10(-10) M; concentrations higher than 10(-7) M were inhibitory, producing a biphasic concentration-response relationship. (R)-Bay K 8644 inhibited 80 mM K+-stimulated 45Ca2+ uptake with an IC50 value of 2.11 X 10(-9) M. These pharmacologic values correlate well with the binding affinities.(ABSTRACT TRUNCATED AT 400 WORDS)
Wei
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Department of Biochemical Pharmacology, School of Pharmacy, State University of New York, Buffalo 14260.
Rutledge
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Triggle
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D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000378:metabolism*; D004110:Diltiazem / Q000494:pharmacology; D005711:Gallopamil / Q000494:pharmacology; D006352:Heart Ventricles / Q000378:metabolism*; D066298:In Vitro Techniques; D017275:Isradipine; D008564:Membrane Potentials; D010069:Oxadiazoles / Q000378:metabolism; D011188:Potassium / Q000502:physiology; D051381:Rats
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D010069:Oxadiazoles; D005711:Gallopamil; D004110:Diltiazem; D011188:Potassium; D002118:Calcium; D017275:Isradipine