The renal Na+:Ca2+ exchanger (NCX) plays an important role in regulation of cytosolic calcium concentration [Ca 2+]i in contractile cells such as vascular smooth muscle and mesangial cells (MCs). NCX regulation of [Ca2+]i is accomplished by exchanging one cytosolic Ca2+ ion for three extracellular Na+ ions. Previously, we cloned, sequenced, and expressed MC exchangers from Dahl/Rapp salt resistant (R; RNCX1) and salt sensitive (S; SNCX1) rats. RNCX1 (alternative splice site encoded by B, D; isoleucine at amino acid 218), but not SNCX1 (B, D, and F; phenylalanine at 218), exhibited up-regulation following agonist-induced protein kinase C (PKC) activation. Using RT-PCR, we initiated studies to identify NCX1 isoform(s) from cultured MCs of the parental Sprague Dawley (SD) rat strain. We identified a novel isoform, denoted SDNCX1.10, that contains isoleucine at 218 and exons B, D, E, and F at the alternative splice site. SDNCX1.10 was then stably expressed in opossum proximal tubule kidney (OK-PTH) cells where we were able to evaluate functional activity by assessing, in forward and reverse mode, exchange activity. OK-PTH cells expressing SDNCX1.10 reduced elevations in [Ca2+] i, induced by 1 mM ATP via purinergic receptors, back to baseline levels 5-fold faster than cells expressing vector alone. Down-regulation of PKC, following prolonged exposure (24 h) to PMA resulted in a 66% reduction in ATP-induced increases in [Ca2+]i in SDNCX1.10 transfected cells compared to cells transfected with vector alone. This attenuation suggests that SDNCX1.10 is a PKC-sensitive isoform. KB-R7943, an exchanger-specific inhibitor, attenuated the reverse activity of SDNCX1.10 (by 50% versus vector-only cells) and stimulated the forward mode. Further studies were performed to investigate some of the regulatory properties of SDNCX1.10. We found that this isoform was regulated by [Ca2+]i and was inhibited by decreases in intracellular pH (pHi) and that the activity of this exchanger was not altered by protein kinases A (PKA) and G (PKG). We also compared the regulation of [Ca2+]i between the RNCX1 and SNCX1 isoforms. SNCX1 was much less effective in lowering agonist induced increases in [Ca2+]i back to baseline, suggesting that cells expressing SNCX1 may have an impaired ability to regulate [Ca2+]i. Thus, this finding may help explain the hypertension and renal failure that are a hallmark of salt-sensitive hypertension.
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机译：肾Na + ：Ca 2 + 交换子（NCX）在调节胞质钙浓度[Ca 2 + ] i 在诸如血管平滑肌和肾小球膜细胞（MCs）的收缩细胞中。 [Ca 2 + ] i 的NCX调节是通过将一个胞质Ca 2 + 离子交换为三个胞外Na + 2 + ] i 的升高，这是由嘌呤能受体通过1 mM ATP诱导的，比基线速度快5倍细胞单独表达载体。长时间暴露于PMA（24 h）后，PKC的下调导致SDNCX1中ATP诱导的[Ca 2 + ] i 的增加减少了66％。与仅用载体转染的细胞相比，有10个转染的细胞。这种衰减表明SDNCX1.10是PKC敏感的同工型。交换子特异性抑制剂KB-R7943减弱SDNCX1.10的逆向活性（相对于仅含载体的细胞，降低了50％）并刺激了正向模式。进行了进一步的研究以研究SDNCX1.10的某些调节特性。我们发现这种同工型受[Ca 2 + ] i 的调节，并受到细胞内pH值（pH i ）的降低的抑制，并且交换蛋白的活性没有被蛋白激酶A（PKA）和G（PKG）改变。我们还比较了RNCX1和SNCX1亚型之间[Ca 2 + ] i 的调控。 SNCX1在降低激动剂诱导的[Ca 2 + ] i 增加到基线时的效果要差得多，这表明表达SNCX1的细胞调节[Ca 的能力可能受损。 super> 2 + ] i 。因此，这一发现可能有助于解释高血压和肾衰竭，这是盐敏感性高血压的标志。
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