HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Corrigendum All Versions of this Article: 292/5/C1895    most recent 00404.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Awumey, E. M. Articles by Bukoski, R. D. Search for Related Content PubMed PubMed Citation Articles by Awumey, E. M. Articles by Bukoski, R. D.

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

This article has been cited by other articles:

				C. G. Phillips, M. T. Harnett, W. Chen, and S. M. Smith Calcium-Sensing Receptor Activation Depresses Synaptic Transmission J. Neurosci., November 12, 2008; 28(46): 12062 - 12070. [Abstract] [Full Text] [PDF]

				E. M. Awumey, S. K. Hill, D. I. Diz, and R. D. Bukoski Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2363 - H2370. [Abstract] [Full Text] [PDF]