The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.
Tumor necrosis factor alpha (TNFα) and its type 1 receptor (TNFR1) are implicated in several autoimmune diseases, including rheumatoid arthritis, and are associated with complications at the cardiovascular level. Using human cardiomyocytes, vascular smooth muscle, vascular endothelial, and endocardial endothelial cells coupled to indirect immunofluorescence, our results showed the presence of TNFR1 at the levels of the plasma membrane (including the cytosol) and mostly at the level of the nuclear membranes (including the nucleoplasm). The distribution of the receptor is different between cell types; however, the density is significantly higher at the nuclear level in all 4 cell types. The density of the receptor was the highest in contractile cells including the cardiomyocytes and vascular smooth muscle cells, compared with endothelial cells including endocardial endothelial and vascular endothelial cells. Using the Ca2+ probe Fluo-3 coupled to quantitative confocal microscopy, our results showed that the cytokine induced a sustained Ca2+ increase in both the cytosol and nucleoplasm of all 4 cell types. This increase was more significant at the nuclear level, mainly in endothelial cells. Our results demonstrated the presence of TNFR1 at both the cell and nuclear membranes of cardiovascular cells, and that its activation modulated both cytosolic and nuclear Ca2+.
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