DEPLETION AND FILLING OF INTRACELLULAR CALCIUM STORES IN VASCULAR SMOOTH-MUSCLE

In vascular smooth muscle, binding of vasoactive substances to surface membrane receptors leads to a rise of intracellular cytoplasmic Ca2+ and to contraction. Cytoplasmic free Ca2+ concentration ([Ca2+](i)) increases through release of Ca2+ from intracellular stores and Ca2+ entry through surface membrane ion channels. Membrane-permeant and membrane-impermeant forms of fura 2 were used to distinguish changes in intracellularly stored Ca2+ ([Ca2+](s)) from changes in [Ca2+](i). The spatiotemporal patterns of the movement of Ca2+ between these two cellular compartments in cultured vascular smooth muscle cells (A7r5 cell line) were visualized with digital imaging fluorescence microscopy. The Ca2+ stores were localized by double staining with a fluorescent organelle-specific dye and the Ca2+ indicator. [Ca2+](s) was measured after accumulation of the membrane-permeant form of fura 2 inside the stores and quenching of the fura 2 fluorescence in the cytoplasmic compartment with manganese. Stimulation with vasopressin led to a transient increase of [Ca2+](i) and a concomitant decrease of [Ca2+](s). After stimulation with vasopressin, [Ca2+](i) returned rapidly to normal resting levels, whereas the recovery of [Ca2+](s) occurred on a much slower time scale. The refilling pathway of depleted stores involved Ca2+ entry into the bulk cytoplasmic compartment before uptake into the stores.