Ca2+ mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

1 Cardiovascular Disease Research Program, 2 Neuroscience of Drug Abuse Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham; and 3 Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park; and 4 Hypertension, Vascular Diseases Center and Department of Physiology, and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina Submitted 25 July 2006 ; accepted in final form 22 January 2007 The rat dorsal root ganglion (DRG) Ca 2+ -sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca 2+ ] ([Ca 2+ ] e ) from 0.5 to 1 mM resulted in increases in [Ca 2+ ] i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca 2+ ] e -response studies indicate a Ca 2+ sensitivity with an EC 50 of 1.75 ± 0.10 mM. NPS R-467 and Gd 3+ activated the CaR. When [Ca 2+ ] e was successively raised from 0.25 to 4 mM, peak [Ca 2+ ] i , attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca 2+ , 1 and 10 µM NPS R-467, or 50 and 100 µM Gd 3+ , indicating desensitization of the response. Furthermore, Ca 2+ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca 2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca e 2+ . Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca 2+ ] i transients by 49.9 ± 5.2% (at 1 mM Ca 2+ ) and 40.5 ± 6.5% (at 2 mM Ca 2+ ), compared with controls. The findings suggest involvement of PKC in the pathway for Ca 2+ mobilization following CaR activation. desensitization; protein kinase C Address for reprint requests and other correspondence: E. M. Awumey, Cardiovascular Disease Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central Univ., 700 George St., Durha