Effects of 8‐bromo cyclic GMP and verapamil on depolarization‐evoked Ca 2+ signal and contraction in rat aorta

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 BMP on Ca 2+ movements and contraction in rat isolated endothelium‐denuded aorta stimulated by KC1 depolarizing solution using the permeant analogue 8‐bromo cyclic BMP (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 μ m ); 45 Ca 2+ influx in KC1‐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.