Novel biochemical and functional insights into nuclear Ca2+ transport through IP(3)Rs and RyRs in osteoblasts

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nu clear membrane of bone-forming (NIC3T3-E1) osteoblasts. Intact nuclei fluor esced intensely with anti-RyR (Ab(34)) and anti-IP3R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific antirecep tor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-Ry R-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respe ctively, to full-length RyR-1 (similar to 500 kDa) and IP3R-1 (similar to 2 50 kDa). Band intensity was reduced by just similar to 20% after brief tryp tic proteolysis of intact nuclei; this confirmed that isolated nuclear memb ranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2 via Ca2+-ATPase activation (1 mM ATP and similar to 100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope land nucle oplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+](np) elevation. Taken together, the results prov ide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts thr ough nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivab ly explain the direct regulation of osteoblastic gene expression by hormone s that use the IP3-Ca2+ pathway.