Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells

被引:30
作者
White, KE
Gesek, FA
Friedman, PA
机构
来源
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL FLUID AND ELECTROLYTE PHYSIOLOGY | 1996年 / 271卷 / 03期
关键词
intracellular calcium; calcium transport; reverse transcription polymerase chain reaction; sodium/calcium exchanger; kidney;
D O I
10.1152/ajprenal.1996.271.3.F560
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca2+-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7:12:1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a P-32-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb + DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+](i)), in single cells. In the presence of an outward Na+ gradient, [Ca2+](i) increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+](i). Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+](i). The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+](i). Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at similar to 125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na+-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.
引用
收藏
页码:F560 / F570
页数:11
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