Effects of 8-bromo cyclic GMP and verapamil on depolarization-evoked Ca~(2+) signal and contraction in rat aorta

摘要

1. The pharmacological action of NO donors is usually attributed to a cellular rise in guanosine 3′:5′-cyclic monophosphate (cyclic GMP), but this hypothesis is based only on indirect evidence. Therefore, we have studied the effects of cyclic GMP on Ca~(2+) movements and contraction in rat isolated endothelium-denuded aorta stimulated by KCl depolarizing solution using the permeant analogue 8-bromo cyclic GMP (BrcGMP). Isometric contraction and fura-2 Ca~(2+) signals were measured simultaneously in preparations treated with BrcGMP and with verapamil. The activation of calcium channels was estimated by measuring the quenching rate of the intracellular fura-2 signal by Mn~(2+) and by the depolarization-dependent influx of ~(45)Ca~(2+). 2. Stimulation with 67 mM KCl-solution evoked an increase in cytosolic Ca~(2+) concentration ([Ca~(2+)]_(cyt)) and a contractile response which were inhibited by pretreatment with verapamil (0.1 μm) or BrcGMP (0.1-1 mM). However, the inhibition of the fura-2 Ca~(2+) signal was significantly higher with verapamil than with BrcGMP, whereas the contraction was inhibited to a similar extent. 3. When preparations were exposed to K~+-depolarizing solution in which the calcium concentration was cumulatively increased, the related increase in fura-2 Ca~(2+) signal was barely affected by BrcGMP, whereas the contractile tension was strongly and significantly inhibited. 4. Cellular Ca~(2+) changes were also estimated with ~(45)Ca~(2+). ~(45)Ca~(2+) influx in resting preparations was significantly reduced by BrcGMP (0.1 mM) but not by verapamil (0.1 μM); ~(45)Ca~(2+) influx in KCl-depolarized preparations was reduced by verapamil but was unaffected by BrcGMP. 5. Measurements of Mn~(2+)-induced quenching of the intracellular fura-2 signal showed that BrcGMP did not affect divalent cation entry in K~+-stimulated preparations, whereas verapamil concentration-dependently inhibited Mn~(2+) entry stimulated by K~+-depolarization. 6. The present results indicate that BrcGMP did not affect voltage-dependent Ca~(2+) channel gating in the rat aorta. For a given fura-2 Ca~(2+) signal, the contraction was lower in preparations exposed to BrcGMP than in the untreated ones, suggesting that the activation of cyclic GMP-dependent kinases reduced the contractile efficacy of calcium. Furthermore, the reduction of depolarization-dependent ~(45)Ca~(2+) uptake reported with sodium nitroprusside, a NO donor, was not observed with biologically active concentrations of BrcGMP, suggesting that this drug could have additional mechanisms of action, unrelated to activation of protein G-kinase.