In smooth muscle, Ca2+ is released from the intracellular stores at frequent discharge sites (FDSs; Gordienko et al. 1998) giving rise to ‘Ca2+ sparks,’ which have been suggested to play a dual role: they can precede and summate into Ca2+ waves causing contraction, but also activate BK channels, hyperpolarise the membrane and thus oppose the contraction (Jaggar et al. 2000). Our aim was to investigate the effect of nitric oxide donors and noradrenaline to find out if they elicit Ca2+ sparks from different locations in the cell, thus activating different effectors.
Guinea-pigs were killed by cervical dislocation followed by exsanguination. Single cells were obtained by enzymatic digestion of segments of small mesenteric arteries (less than 200 µm external diameter) loaded with calcium indicator, fluo-3 AM, and imaged using a laser scanning confocal microscope. Statistical significance was tested with Student’s t test.
NO donors S-nitroso-N-acetyl-penicillamine (SNAP; 50 µM) and sodium nitroprusside (SNP; 100 µM) did not change the position of FDSs and slightly depressed their activity, but did not affect the global [Ca2+]i significantly. Administration of noradrenaline (1-10 µM) increased spark discharge frequency at existing FDS(s) (without changing their position or activating new FDSs) and Ca2+ sparks summated into a generalised Ca2+ wave lasting from ~7 to ~20 s, after which the [Ca2+]i decreased to resting levels and spark discharge ceased. 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 3Ì,5Ì-mono-phosphate (100 µM), a membrane-permeable cGMP analogue. 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.This work was supported by the British Heart Foundation.
- Gordienko, D.V., Bolton, T.B. & Cannell, M.B. (1998). J. Physiol. 507, 707-720. abstract
Jaggar, J.H., Porter, V.A., Lederer, W.J. & Nelson, M.T. (2000). Am. J. Physiol. 278, C235-256.