Pharmacological properties of voltage-dependent calcium channels in functional microvessels isolated from rat brain.

Voltage-operated calcium channels were studied in rat intracerebral microvessels. The contractile reactivity to KCl-depolarization was assessed by the measurement of internal diameter of superfused microvessels. Dihydropyridine receptor sites associated with calcium channels were identified and characterized using 3H(+)PN 200-110 [isopropyl-4-(2,1,3-benzodiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- -methoxycarbonyl-pyridine-3-carboxylate]. Depolarization induced by high-KCl solution produced a marked reduction of the internal diameter of cerebral microvessels which was associated with the appearance of rhythmic activity. The vessel contraction was reversible and abolished by nimodipine. Binding studies with 3H(+)PN 200-110 revealed the existence of a single class of specific, stereoselective and voltage-dependent binding sites which bound (+)PN 200-110 with a KD of 88 +/- 6.6 pmol l-1 at 37 degrees C in microvessels incubated in NaCl medium. When microvessels were incubated in KCl-medium, the apparent KD value was reduced to 35 +/- 2 pmol l-1. Bmax was not significantly changed. The effect of KCl was not related to concomitant changes in the Na concentration. The potency of various dihydropyridine derivatives in inhibiting 3H(+)PN 200-110 binding was in agreement with their pharmacological potency in smooth muscle preparations. The effect of PN 200-110 and of nimodipine was stereoselective. Ki values of PN 200-110 and of nimodipine were increased in depolarized preparations, while nifedipine's potency was unchanged. Verapamil was only a partial inhibitor of 3H(+)PN 200-110 binding. The effect of diltiazem was stereoselective: the (+)-cis isomer enhanced the binding and the (-)-cis isomer of diltiazem poorly inhibited the binding of PN 200-110. Results showed that isolated cerebral microvessels possess functional voltage-operated calcium channels, which contain potential-modulated receptors for dihydropyridine calcium entry blockers with characteristics similar to those described in other tissues.