Structural and functional analysis of na+/ca2+ exchange in distal
convoluted tubule cells.
White, Kenneth E., Frank A. Gesek, and Peter A. Friedman.
Department of Pharmacology and Toxicology, Dartmouth Medical
School, Hanover, NH 03755-3835
APStracts 3:0086F, 1996.
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 RT-PCR with RNA
from an immortalized mouse DCT cell line. Northern blots probed with
a [32P]-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 a Na+ ionophore inhibited the
rise of [Ca2+]i. The intracellular Na+ concentration, [Na+]i,
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 approximately 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.
Received 26 December 1995; accepted in final form 25 April 1996.
APS Manuscript Number F425-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96