Modulation of Na+/Ca2+ exchange in epithelial cells of porcine thick ascending limb

We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ([Ca2+](i)) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+](i)) from 10 to 20 mM. These cells had normal basal [Ca2+](i) (79 +/- 3 nM). Substitution of extracellular NaCl (50 mM) with KCl resulted in the rapid elevation of [Ca2+](i) to maximal levels of 795 +/- 60 nM (n = 17). The increments of [Ca2+](i) were associated with [Na+](i). We next determined the modulation of Na+/Ca2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca2+-calmodulin inhibitor, resulted in a shift of the [Na+](i) dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in half-maximal [Ca2+](i) increase near normal [Na+](i) of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+](i) was not required to elicit increments in [Ca2+](i). These data indicate that Na+/Ca2+ exchange is present in CTAL, cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.