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 (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 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 ([Ca²⁺]_i) with reduction in Na_e was similar between R and S MCs in both serum-fed and serum-deprived groups, although the magnitude of [Ca²⁺]_i was enhanced by serum deprivation. In both serum-fed and serum-deprived groups, PMA significantly increased [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.

sodium/calcium exchanger; Dahl rat; immunoblots; translocation

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