Stimulation-Induced Regional Alteration of Ca2+ Levels in Single Arterial Smooth Muscle Cells

Abstract

Contraction in vascular smooth muscle is normally triggered by an increase in the cytosolic free Ca2+ concentration. We employed the fluorescent Ca indicator, fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in arterial myocytes and the changes elicited by norepinephrine (NE) and by alterations in the Na− electrochemical gradient. In modified Kreb's solution containing 1.8 mM Ca2+, these bovine tail artery myocytes were relaxed and able to maintain normal membrane potentials. The cells contracted rapidly when exposed to NE or high K+ solution. They then relaxed slowly when the activator was washed away. A rise in Ca2+ concentration, [Ca2+], was elicited by NE as well as by Ca2+ entry via Na+/Ca2+ exchange. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed in resting cells prior to activation. During NE-evoked contractions, intracellular [Ca2+] in-creased an average of two- to threefold, and the distribution of [Ca2+] became much more heterogeneous. Upon recovery from activation, the cells relaxed, usually attaining >90% of their original resting length. In contrast to the relatively uniform Ca2+ distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. Similarly, reducing the Na+ electrochemical gradient and, presumably, activating Na+/Ca2+ exchange elicited a rise in [Ca2+]. In cells exposed to increased [Na+]i and reduced [Na+]o, as in cells following recovery from NE stimulation, discrete areas of high [Ca2+] were apparent. The large spatial variation induced by cell activation implies that Ca2+ was sequestered at localized sites throughout the cell.