Altered protein kinase c activation of na+:ca2+ exchange in mesangial cells from salt sensitive rats. Mashburn, Nicole A., M. Tino Unlap, Jeanette Runquist, Amy Alderman, Gail V. W. Johnson, and P. Darwin Bell. Nephrology Research and Training Center, Departments of Medicine, Physiology, and Psychiatry, University of Alabama at Birmingham, Birmingham, AL 35294
APStracts 5:0214F, 1998.
The purpose of these studies was to determine if there is a defect in protein kinase C (PKC) regulation of the Na+:Ca2+ exchanger in cultured mesangial cells from Dahl/Rapp salt sensitive (S) and salt resistant (R) rats. R and S mesangial cells were cultured, grown on coverslips and loaded with fura 2 for measurement of single cell cytosolic calcium concentration ([Ca2+]i) in a microscope based photometry system. Studies were performed in cells that were exposed to serum (serum fed) and in cells that were serum deprived for 24 hrs. Baseline [Ca2+]i measured in a 150 mM NaCl containing Ringer's solution were similar between R and S mesangial cells in both serum fed and serum deprived groups although baseline [Ca2+]i were uniformly higher in the serum deprived groups. Exchanger activity was assessed by reducing extracellular Na (Nae) from 150 to 2 mM which resulted in movement of Na+ out of and Ca2+ into these cells (reverse mode Na+:Ca2+ exchange). PKC was activated in these cells with 15 min exposure to 100 nM PMA. In the absence of PMA, the change in [Ca2+]i (D[Ca2+]i) with reduction in Nae, was similar between R and S mesangial cells in both serum fed and serum deprived groups although the magnitude of D[Ca2+]i was enhanced by serum deprivation. In both serum fed and serum deprived groups, PMA significantly increased D[Ca2+]i in R but not S mesangial cells. Upregulation of exchanger activity in R mesangial cells could be abolished by prior 24 hr exposure to PMA, a maneuver that downregulates PKC activity. Other studies were performed to evaluate exchanger protein expression using monoclonal and polyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70 and 120 kDa proteins in the crude membrane fraction of R but not S mesangial cells, an increase which was abrogated by prior 24 hr PMA pretreatment and corresponded to reduction in the 70 kDa protein in the crude cytosolic fraction. These data demonstrate that PKC enhances Na+:Ca2+ exchange activity in mesangial cells from R but not from S rats, suggesting that there may be a defect in the PKC-Na+:Ca2+ exchange regulation pathway in mesangial cells of salt sensitive rats. 

Received 18 September 1998; accepted in final form 10 December
1998.
APS Manuscript Number F305-7.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 18 December 1998