Parathyroid Hormone (PTH)-stimulated NA/CA exchange is blocked by inhibitors of protein kinase C and not by inhibitors of protein kinase a in a novel transfected cell line

The Na/Ca exchanger is expressed exclusively in the distal nephron in kidney, and plays a major role in the basolateral exit of calcium in this segment. Studies in isolated membrane vesicles have shown that the exchanger is regulated by PTH. NCX-1, the first member of the Na/Ca exchanger gene family cloned, is widely expressed in many tissues including kidney, and a variety of tissue-specific alternatively spliced isoforms have been described. In order to evaluate the molecular mechanisms responsible for the regulation of the renal Na/Ca exchanger we stably transfected OK-P cells with a kidney-specific isoform (NACA2). Subclones of the parent cell line were isolated based on transport activity, and immunofluorescence with an exchanger-specific monoclonal antibody. PTH acutely stimulated sodium-dependent calcium uptake in a dose-dependent fashion, with a 1/2 maximal concentration of 0.7 nM. The PTH-induced increase in Na/Ca exchanger activity was completely blocked by the protein kinase C inhibitors chelerythrine and calphostin C. The protein kinase A inhibitor H-89 had no effect. In addition, the PTH stimulation of cAMP could be dissociated from that of the Na/Ca exchanger. PTH stimulated cAMP at a 1/2 maximal concentration that is approximately 40 times higher (30 nM) than that required for 1/2 maximal stimulation of the exchanger. These data suggest that protein kinase C and not protein kinase A participates in the signaling pathways that regulate the renal Na/Ca exchanger.