Binding sites for 1,4-dihydropyridine Ca2+-channel modulators in rat intestinal smooth muscle

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The contractile response of intestinal smooth muscle to depolarization is characterized by a phasic and a tonic component which are differently sensitive to blockade by 1,4-dihydropyridines. As this difference in sensitivity could be related to different binding sites associated with distinct calcium channels, we analyzed the binding of the calciumantagonist 1,4-dihydropyridine (+)PN 200-110 [isopropyl-4-(2,1,3-benzodiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxycarbonyl-pyridine-3-carboxylate] in longitudinal smooth muscle of the rat ileum. We carried out saturation binding experiments on intact tissue exposed to physiological and depolarizing (100 mmol l-1 K+) solution, and on different membrane fractions: the total microsomal fraction, the light microsomal fraction (enriched with plasma membranes) and the mitochondrial fraction. Binding of 3H(+)PN 200-110 to the intact longitudinal smooth muscle of rat ileum appeared to be voltage-dependent, KD decreased in depolarized tissue whereas Bmax was unchanged (change in membrane potential was assessed by measuring the distribution of 3H-tetraphenylphosphonium bromide). In membrane fractions two binding sites were detected, a high-affinity site associated with plasma membrane and a low-affinity site presumably associated with mitochondria (abundant in the fractions where the cytochrome c oxidase activity was high, and undetectable in the light microsomes poor in cytochrome c oxidase activity). The KD value of the high-affinity binding in isolated membrane fractions was similar to the KD value measured in intact depolarized tissue. The low affinity binding increased at high ionic strength and did not display any stereoselectivity.(ABSTRACT TRUNCATED AT 250 WORDS)