Kip Sertac N, Strehler Emanuel E
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
Am J Physiol Renal Physiol. 2003 Jan;284(1):F122-32. doi: 10.1152/ajprenal.00161.2002. Epub 2002 Aug 21.
Plasma membrane Ca(2+) ATPases (PMCAs) are ubiquitous in Ca(2+)-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na(+)/Ca(2+) exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring (45)Ca(2+) flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca(2+) transport was provided by Na(+)/Ca(2+) exchanger and one-third by PMCAs, as determined in Na(+)-free media and using various PMCA inhibitors (La(3+), vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca(2+) efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca(2+) transport to approximately 140%, whereas antisense treatment reduced Ca(2+) flux approximately 45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca(2+) reabsorption in the distal kidney.
质膜钙ATP酶（PMCAs）在包括肾脏在内的钙转运器官中普遍存在。我们使用逆转录聚合酶链反应（RT-PCR）在犬肾Madin-Darby细胞（MDCK）中检测到了PMCA1b、PMCA2b（罕见）和PMCA4b。在蛋白质水平上，仅易于检测到PMCA1和PMCA4，且它们在基底外侧膜中高度富集。钠/钙交换体NCX1在转录本和蛋白质水平上也被检测到。一项在静息条件下测量跨MDCK细胞单层的（45）钙通量的功能测定表明，在无钠培养基中并使用各种PMCA抑制剂（镧（3+）、钒酸盐、氯米帕明和三氟拉嗪）测定，顶基侧钙转运的三分之二由钠/钙交换体提供，三分之一由PMCAs提供。通过过表达PMCA4b或对内源性PMCA4b进行反义敲低，证明了PMCA4b对基底外侧钙外流的重要性。与对照相比，过表达PMCA4b使顶基侧钙转运增加至约140%，而反义处理使钙通量降低约45%。因此，MDCK系统是研究远端肾脏中PMCA亚型在钙重吸收中的特定作用和调节的功能研究的理想模型。
