Nucleoplasmic Ca2+ loading is regulated by mobilization of perinuclear Ca2+

Regulation of nucleoplasmic calcium (Ca2+) concentration may occur by the mobilization of perinuclear luminal Ca2+ pools involving specific Ca2+ pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca2+, we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca2+ fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca2+ channels (RyR2), sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca2+ channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca2+ from 100 to 1600 nM Ca2+ was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca2+ loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca2+ loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca2+ loading at low concentrations of extranuclear added Ca2+. Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca2+ fluxes. (C) 2000 Harcourt Publishers Ltd.