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

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.

Acute Glucose Overload Abolishes Ca 2+ Oscillation in Cultured Endothelial Cells From Bovine Aorta

“…27 Furthermore, it is generally considered that uptake of Ca 2ϩ into the nucleus plays a role in the Ca 2ϩ homeostasis. 35 In the present experiments, however, it was 36 However, hydrogen peroxide is unlikely to play a role in the present experiments, since catalase as well as deferoxamine did not restore glucose overload impairment of Ca 2ϩ mobilization. Recently, it has been shown that high glucose treatment enhances bradykinin-induced Ca 2ϩ transient via the generation of O 2 Ϫ in endothelial cells.…”

New aspects of nuclear calcium signalling

“…In addition to exhibiting differences in dissociation constant and dynamic range, the indicators can also be bound inside the nucleoplasm and sequestered in Ca 2+ stores (Al-Mohanna et al, 1994;Burnier et al, 1994;Ikeda et al, 1996;Ronde and Nichols, 1997). These problems effectively invalidate the majority of the observations of nuclear-cytoplasmic Ca 2+ gradients.…”

The Inhibition of Ca²⁺ Influx Induced by Hypericin in Cultured Human Retinal Pigment Epithelial Cells Analyzed by Confocal Imaging

“…In recent years, the use of the fluorescent dye fluo-3 in combination with LSCM has increased considerably and has afforded some important new insights into the subcellular variations in Ca 2+ in various cell types [20][21][22]. Fluorescent Ca 2+ indicators can be divided into various groups based on whether they are ratiometric or nonratiometric (single wavelength) indicators.…”