NA+ CA2+ EXCHANGER IN EPITHELIAL-CELLS OF THE PORCINE CORTICAL THICK ASCENDING LIMB/

Intracellular Ca2+ concentration ([Ca2+](i)) plays an important role i n the signal transduction processes within cortical thick ascending li mb (CTAL) cells. Control of [Ca2+](i) was investigated in isolated CTA L cells with microfluorescent techniques. CTAL cells pretreated with o uabain to elevate intracellular Na+ concentration ([Na+](i)) had basal [Ca2+](i) of 86 +/- 2 nM. Removal of extracellular Na (Na-o(+)) or vo ltage depolarization with KCl (in the presence of Na-o(+)) resulted in a rapid and reversible maximal elevation of [Ca2+](i) (1,023 +/- 72 n M, 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 ouab ain and increases in [Na+](i). The presence of a Na+/Ca2+ exchanger wa s confirmed with hybridization techniques, and the isoform was identif ied by sequencing the alternative splicing site within the intracellul ar loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tiss ue and single CTAL cells by reverse transcription-polymerase chain rea ction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained ex ons B and D, which is characteristic of one isoform (NACA3) of the ren al Na+/Ca2+ exchanger. The results provide both functional and molecul ar evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.