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 mes angial 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 deprive d for 24 h. Baseline [Ca2+](i) values measured in a Ringer solution contain ing 150 mM NaCl were similar between R and S MCs in both serum-fed and seru m-deprived groups, although baseline [Ca2+](i) values were uniformly higher in the serum-deprived groups. Exchanger activity was assessed by reducing extracellular Na (Na-e) from 150 to 2 mM, which resulted in movement of Na out of and Ca2+ into these cells (reverse-mode Na+/ Ca2+ exchange). PKC wa s activated in these cells with 15-min exposure to 100 nM phorbol 12-myrist ate 13-acetate (PMA). In the absence of PMA, the change in [Ca2+](i) (Delta [Ca2+](i)) with reduction in Na-e was similar between R and S MCs in both s erum-fed and serum-deprived groups, although the magnitude of Delta[Ca2+](i ) was enhanced by serum deprivation. In both serum-fed and serum-deprived g roups, PMA significantly increased Delta[Ca2+](i) in R but not S MCs. Upreg ulation of exchanger activity in R MCs could be abolished by prior 24-h exp osure to PMA, a maneuver that downregulates PKC activity. Other studies wer e performed to evaluate exchanger protein expression using monoclonal and p olyclonal antibodies. Immunoblots of PMA-treated cells revealed an increase in the levels of 70- and 120-kDa proteins in the crude membrane fraction o f R but not S MCs, an increase which was abrogated by prior 24-h PMA pretre atment and corresponded to reduction in the 70-kDa protein in the crude cyt osolic fraction. These data demonstrate that PKC enhances Na+/ Ca2+ exchang e 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.