Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca2+]i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca2+ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca2+ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca2+]i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP3Rs) is an important determinant of the [Ca2+]i dynamics and recruits neighboring Ca2+-release sites to generate [Ca2+]i waves; (v) [Ca2+]i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca2+-dependent K+ and Cl- channels sense the local changes in [Ca2+]i during a Ca2+ spark and thereby may couple changes in [Ca2+]i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mIcat) does not “mirror” changes in [Ca2+]i, thus reflecting the complexity of [Ca2+]i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca2+ release, either spontaneous or caffeine-induced, does not augment mIcat; (ix) intracellular flash release of Ca2+ is less effective in augmentation of mIcat than flash release of IP3, suggesting that IP3 may sensitize muscarinic cationic channels to Ca2+; (x) intracellular flash release of IP3 fails to augment mIcat in SMCs, in which [Ca2+]i was strongly buffered, suggesting that IP3 exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca2+]i rather than depletion of the IP3-dependent Ca2+ store; and (xi) predominant expression of IP3R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP3R-mediated Ca2+ release and muscarinic cationic channel opening.