Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+).
Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), 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 Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)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 Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.
Abrenica
B
B
Department of Oral Biology and Physiology, Division of Stroke and Vascular Disease, University of Manitoba, Winnipeg, Manitoba, Canada.
Gilchrist
J S
JS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006321:Heart / Q000196:embryology; D007211:Indoles / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007211:Indoles; D007476:Ionophores; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D015759:Ionomycin; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid