Spatial and Temporal Resolution of Serotonin-Induced Changes in Intracellular Calcium in a Cultured Arterial Smooth Muscle Cell Line

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Abstract
Ca2+ transients (1–2 µM) evoked by serotonin (5-HT) in cultured A7r5 cells were studied using fura-2 and digital imaging microscopy. Fura-2 was introduced into cells either by incubation with its acetoxymethyl ester analogue fura-2/AM or by transient ATP-induced permeabilization of the sarcolemma such that the free fura-2 entered the cell directly. The distribution of cytoplasmic Ca2+ in unstimulated cells loaded by the former method was heterogeneous, reflecting, in part, separate pools of Ca2+ in the cytosol and sarcoplasmic reticulum (SR). In contrast, the distribution of Ca2+ was uniform in cells loaded with fura-2 by transient permeabilization; this reflected the restriction of fura-2 to the cytosol. Average Ca2+ in these cells was substantially lower than that in fura-2/AM-loaded cells, because SR Ca2+ influences the fura-2 signal from fura-2/AM-loaded cells, but not from cells loaded with free fura-2. The differences in the Ca2+ distribution measured by the two loading methods were also evident during the course of 5-HT-evoked Ca2+ transients. Spatial and temporal resolution of the rising phase of 5-HT-evoked Ca2+ transients in fura-2/AM-loaded cells revealed that the onset of the Ca2+ transients was first manifested as small regions of elevated Ca2+ that subsequently expanded until peak apparent intracellular Ca2+ levels were present in virtually all of the nonnuclear regions of the cells. The rate of rise of Ca2+ varied in different cell regions with the nucleus responding the slowest.

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