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 (Nao+) or voltage depolarization with KCl (in the presence of Nao+) 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+ (Cao2+). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Nao+ 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.
Dai
L J
LJ
Department of Medicine, University of British Columbia, Vancouver Hospital, Canada.
Ritchie
G
G
Bapty
B
B
Raymond
L
L
Quamme
G A
GA
D000818:Animals; D017920:Antiporters / Q000032:analysis* / Q000378:metabolism; D001483:Base Sequence; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D019831:Sodium-Calcium Exchanger; D013552:Swine
D017920:Antiporters; D019831:Sodium-Calcium Exchanger