Confocal microscopy to analyze cytosolic and nuclear calcium in cultured vascular cells

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Abstract
With the development of calcium-sensitive fluorescent dyes and videomicroscopic imaging, several investigators have located the changes in intracellular calcium in the cytoplasm, in the perinuclear region, and possibly in the nucleus. However, the presence of calcium in the nucleus is often difficult to ascertain because the fluorescence derived from the perinuclear area interferes with that of the nucleus. We have used confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of fluo 3 (fluo 3-AM) and rhod 2 (rhod 2-AM)] to analyze the cytosolic and nuclear calcium distribution in vascular smooth muscle and endothelial cells studied at rest and after stimulation with receptor-dependent (angiotensin, vasopressin) and receptor-independent (KCl) stimuli. With fluo 3-AM, the baseline fluorescence was located in the cytoplasm but was slightly higher in the nucleus. With all stimuli, the fluorescence intensity increased in both compartments but remained more pronounced within the nucleus. Yet, after calibration, the cytosolic calcium concentration was greater than that of the nucleus at rest and was equally high after stimulation, suggesting different properties of fluo 3 in the cytosol and in the nucleus. With rhod 2-AM, baseline fluorescence was low in the nucleus and high in the cytosol. Cell stimulation caused an initial increase in cytosolic calcium with no change in the nucleus followed by a rise in both compartments. Thus the stimulation of vascular cells is associated with marked increases in cytosolic and nuclear calcium. Fluo 3-AM seems to be a better indicator of nuclear calcium than rhod 2-AM. The increases in nuclear calcium induced by angiotensin II and vasopressin may contribute to their cell proliferative effect.