The effects of a novel vasodilator, LP-805, on cytosolic Ca2+ concentrations and on tension in rabbit isolated femoral arteries.

Abstract

1. LP-805, 8-tert-butyl-6,7-dihydropyrrolo-[3,2-e]-5-methylpyrazolo- [1,5a]-pyrimidine-3-carbonitrile, is a newly synthesized potent vasodilator. To investigate the cellular mechanisms of vasorelaxation induced by LP-805, we simultaneously determined the effects of LP-805 on cytosolic Ca2+ concentrations ([Ca2+]i) and on tension of smooth muscle of rabbit femoral arterial strips, with or without the endothelium, using front-surface fluorometry and fura-2. 2. In the absence of the endothelium, LP-805, in a concentration-dependent manner, decreased [Ca2+]i and tension during the contraction induced by K(+)-depolarization, at relatively low concentrations ([K+]o < or = 30 mM). The decreases in [Ca2+]i and tension were fully antagonized by treatment with 2 x 10(-6) M glibenclamide. The [Ca2+]i-tension relationship in the LP-805-induced relaxation was similar to that of K(+)-depolarization-induced contractions. 3. LP-805, in a concentration-dependent manner (IC50 for inhibition of tension; 1.7 x 10(-6) M), decreased both [Ca2+]i and tension during the steady-state of contractions induced by 1 x 10(-7) M noradrenaline (NA) in the strips without the endothelium. Glibenclamide completely inhibited these reductions of [Ca2+]i and tension. At the steady-state of relaxation induced by LP-805 during NA-induced contraction, [Ca2+]i-tension relation was shifted to the left of that obtained with high K(+)-induced contraction. 4. NA induced transient increases in [Ca2+]i and tension in the absence of extracellular Ca2+. LP-805 (up to 3 x 10(-6) M) had no effect on these intracellular Ca2+ mobilisation and tension development induced by NA. 5. In strips with an intact endothelium, LP-805 decreased both [Ca2+]i and tension during contraction induced by 1 x 10(-7) M NA. The concentration-response curve for inhibition of [Ca2+]i and tension obtained in the presence of the endothelium was shifted to the left from that obtained in the absence of endothelium. IC50 for the inhibition of tension obtained in the strips with the endothelium was 4.0 x 10(-7) M. Treatment with 1 x 10(-4) M NG-nitro-L-arginine (L-NOARG) attenuated reductions of both [Ca2+]i and tension induced by LP-805 and the concentration-response curve shifted to the right and overlapped that obtained in the absence of the endothelium. Treatment with glibenclamide almost fully overcame the reduction of [Ca2+]i induced by LP-805, while the reversion of tension was 50% at most. 6. In the presence of the endothelium with L-NOARG, LP-805 reduced the tension to the extent of that expected from the reduction of [Ca2'ji, as based on the [Ca2+]i-tension relationship obtained with LP-805 in the absence of endothelium. On the contrary, in the presence of the endothelium without L-NOARG, LP-805 induced a greater reduction of tension than expected from the reduction of [Ca2+J1.This effect became more apparent after treatment with glibenclamide.7. These results suggest that: (1) LP-805 relaxes smooth muscle mainly by activating ATP-sensitive K+channels of smooth muscle and by releasing endothelium-derived relaxing factor (EDRF). (2) Activation of ATP-sensitive K+ channels decrease [Ca2+]i and thereby relax smooth muscle with no effect on Ca2"-sensitivity of the contractile apparatus of smooth muscle or on the agonist-induced Ca2"-release process. (3) EDRF induced by LP-805 relaxes smooth muscle not only by decreasing [Ca2+]i but also decreasing Ca2+-sensitivity of the contractile apparatus of smooth muscle. In the presence of an intact endothelium, a decrease in Ca2+-sensitivity of the contractile apparatus may play an important role in LP-805-induced relaxation.