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RECEPTORS AND SIGNAL TRANSDUCTION

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

Emmanuel M. Awumey,¹ Allyn C. Howlett,² James W. Putney, Jr.,³ Debra I. Diz,⁴ and Richard D. Bukoski^1,

¹Cardiovascular Disease Research Program, ²Neuroscience of Drug Abuse Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham; and ³Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park; and ⁴Hypertension, 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) Ca²⁺-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 Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation.

desensitization; protein kinase C

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