Na+/Ca2+ exchanger in epithelial cells of the porcine cortical thick ascending limb

Intracellular Ca2+ concentration ([Ca2+](i)) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+](i) was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+](i)) had basal [Ca2+](i) of 86 +/- 2 nM. Removal of extracellular Na (Na-o(+)) or voltage depolarization with KCl (in the presence of Na-o(+)) resulted in a rapid and reversible maximal elevation of [Ca2+](i) (1,023 +/- 72 nM, n = 28), which was dependent on the presence of external Ca2+ (Ca-o(2+)). The rise in [Ca2+](i) was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+](i) with either removal of Na-o(+) or voltage depolarization were dependent on pretreatment with ouabain and increases in [Na+](i). The presence of a Na+/Ca2+ exchanger was confirmed with hybridization techniques, and the isoform was identified by sequencing the alternative splicing site within the intracellular loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tissue and single CTAL cells by reverse transcription-polymerase chain reaction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D, which is characteristic of one isoform (NACA3) of the renal Na+/Ca2+ exchanger. The results provide both functional and molecular evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.