Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs 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 nuclear membrane of bone-forming (NIC3T3-E1) osteoblasts. Intact nuclei fluoresced 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 antireceptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 (similar to 500 kDa) and IP3R-1 (similar to 250 kDa). Band intensity was reduced by just similar to 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes 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 nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+ pathway.