Altered protein kinase C activation of Na+/Ca2+ exchange in mesangial cells from salt-sensitive rats

The purpose of these studies was to determine whether there is a defect in protein kinase C (PKC) regulation of the Na+/Ca2+ exchanger in cultured mesangial cells (MC) from Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. R and S MCs 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 h. Baseline [Ca2+]i values measured in a Ringer solution containing 150 mM NaCl were similar between R and S MCs in both serum-fed and serum-deprived groups, although baseline [Ca2+]i values 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 phorbol 12-myristate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+]i (Delta[Ca2+]i) with reduction in Nae was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+]i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased Delta[Ca2+]i in R but not S MCs. Upregulation of exchanger activity in R MCs could be abolished by prior 24-h 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 MCs, an increase which was abrogated by prior 24-h 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 MCs from R but not from S rats, suggesting that there may be a defect in the PKC-Na+/Ca2+ exchange regulation pathway in MCs of S rats.