Effect of nitric oxide donors and noradrenaline on Ca2+ release sites and global intracellular Ca2+ in myocytes from guinea-pig small mesenteric arteries

In smooth muscle the spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) occurs at preferred locations called frequent discharge sites (FDSs) giving rise to localized intracellular Ca2+ transients (Ca2+ sparks). Laser scanning confocal microscopy of fluo-3-loaded single myocytes freshly isolated from small mesenteric arteries of guinea-pig was used to investigate the action of nitric oxide (NO) donors and noradrenaline on the position and activity of FDSs and on global intracellular Ca2+ concentration ([Ca2+](i)). In 8% of cells 'microsparks', Ca2+ release events smaller in duration, spread and amplitude than Ca2+ sparks were observed. The location of the initiation point of Ca2+ sparks observed during line-scan imaging was found to 'jitter' by +/- 0.41 mum. However, the general position of an FDS within the cell did not change; most FDSs were close (within 1.2 +/- 0.1 mum) to the cell membrane and often multiple FDSs occurred in one confocal plane of the cell. In the resting state, NO donors S-nitroso-N-acetylpenicillamine (SNAP; 50 mum) and sodium nitroprusside (SNP; 100 mum) did not change the general position of FDSs and slightly depressed their activity, but did not affect the global [Ca2+](i) significantly. Application of noradrenaline (1-10 mum) increased Ca2+ spark frequency at existing FDS(s) leading to a Ca2+ wave. The increase in FDS activity and in global [Ca2+](i) produced by noradrenaline were inhibited by the presence of SNAP or SNP but not by 8-bromoguanosine cyclic 3',5'-monophosphate (8-Br-cGMP; 100 mum). In the presence of 1H-[1,2,4] oxadiazolo, [4,3-a] quinoxalin-1-one (ODQ), inhibitor of soluble guanylate cyclase, SNAP and SNP still exerted their effects on the noradrenaline response. These results suggest that SNAP and SNP inhibit the noradrenaline-evoked rise in global [Ca2+]i by a cGMP-independent mechanism and that part of this effect is due to inhibition of the activity of FDSs; moreover, only the activity, but not the position, of FDSs is changed by either stimulant or inhibitory substances.