Ca2+ efflux from distal convoluted tubule (DCT) cells may be mediated by Na+/Ca2+ exchange and a Ca2+-ATPase. We previously cloned a NaVCa?+ exchanger from a mouse DCT cell line (JASN, 5:303. 1994). The present studies determined if DCT cells also express a plasma membrane Ca2+-ATPase (PMCA). Four PMCA isoforms, which are the products of separate genes, have been cloned. RNA isolated from DCT cells and mouse brain was reverse-transcribed. The cDNA was amplified by PCR with primers specific for PMCA1 or PMCA2. The primers to PMCA1 yielded an appropriately-sized product of 550 bp in DCT and brain. The primers for PMCA2 revealed the predicted product from brain; DCT was negative. The DCT PMCA1 product was subcloned and sequenced and found to be identical to rat PMCA1 in this region. Northern analysis was performed on poly (A)+ RNA from DCT cells and mouse kidney using a [32P|dCTPlabeled DCT PMCA I PCR product. Transcripts of 7 and 5 kb, consistent with reported PMCA1 mRNA sizes, hybridized with the probe in kidney and DCT cells. Western analysis performed with a monoclonal antibody to PMCA on membrane preparations from mouse kidney cortex, primary cultures of mouse distal tubule cells, and the mouse DCT cell line exhibited a protein of 140 kDa, in agreement with the reported PMCA molecular mass. We conclude that DCT cells express PMCA1 mRNA and PMCA protein. The presence of PMCA3 and PMCA4 cannot be ruled out. Thus, DCT cells contain both PMCA1 and the Na+/Ca2+ exchanger, indicating that Ca2+ efflux from these cells may be mediated by two mechanisms.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology