Siczkowski M, Quinn P A, Ng L L
Department of Medicine and Therapeutics, Leicester Royal Infirmary, UK.
Metabolism. 1997 Mar;46(3):250-6. doi: 10.1016/s0026-0495(97)90249-3.
An established intermediate phenotype of human hypertension and diabetic nephropathy is an elevation of Na+/H+ exchanger (NHE) activity, but the mechanism for this is unclear. This phenotype is maintained in vascular myocytes from the spontaneously hypertensive rat (SHR) compared with the normotensive Wistar Kyoto rat (WKY). Since intracellular calcium levels ([Ca2+]i) following agonist stimulation were elevated in cells from both hypertensive humans and SHR, we have examined the role of calcium-calmodulin (CaM) in the mechanism of increased NHE activity in vascular myocytes of SHR by determining the activity and phosphorylation state of NHE isoform-1 (NHE-1) in cells from SHR and WKY when [Ca2+]i was elevated by the ionophores A23187 or ionomycin. NHE activity was measured using fluorometry and NHE-1 phosphorylation by immunoprecipitating the exchanger from 32P-orthophosphate-labeled cells with a polyclonal NHE-1-specific antibody. The ionophore A23187 increased [Ca2+]i in both cell types to approximately 700 to 800 nmol x L(-1), and led to stimulation of NHE-1 activity only in WKY myocytes, with no effect on SHR cells. An inhibitor of CaM kinase II (KN-62) failed to abolish stimulation of NHE-1 by A23187 in WKY cells, and had no effect on unstimulated NHE-1 activity in both cell types. Ionomycin also elevated [Ca2+]i in both cell types to approximately 1,000 nmol x L(-1) and activated NHE-1 activity in only WKY cells. Activation of NHE-1 in WKY cells by an increased [Ca2+]i was not mediated by an increase in NHE-1 phosphorylation, whether in the presence or absence of KN-62. The elevated NHE-1 phosphorylation in SHR cells was not affected by elevated [Ca2+]i or KN-62. Calmodulin-agarose beads bound NHE-1 extracted from SHR cells to a lesser extent than that from WKY cells. We conclude that calcium-induced NHE-1 activation in WKY cells was not mediated by CaM kinase II. The elevated NHE-1 activity and phosphorylation of SHR cells was not further modulated by increased [Ca2+]i, and was also independent of CaM kinase II. Non-phosphorylation-dependent mechanisms of activation of NHE-1 may therefore be responsible for alterations of NHE-1 activity in these cells, such as the direct binding of CaM to NHE-1. This direct binding of CaM to NHE-1 may be impaired in SHR compared with WKY cells.
人类高血压和糖尿病肾病一种既定的中间表型是钠氢交换体（NHE）活性升高，但其机制尚不清楚。与正常血压的Wistar Kyoto大鼠（WKY）相比，自发性高血压大鼠（SHR）血管平滑肌细胞中维持了这种表型。由于高血压患者和SHR细胞在激动剂刺激后细胞内钙水平（[Ca2+]i）均升高，我们通过测定离子载体A23187或离子霉素使[Ca2+]i升高时SHR和WKY细胞中NHE亚型1（NHE-1）的活性和磷酸化状态，研究了钙调蛋白（CaM）在SHR血管平滑肌细胞NHE活性增加机制中的作用。使用荧光法测量NHE活性，并用多克隆NHE-1特异性抗体从32P-正磷酸盐标记的细胞中免疫沉淀交换体来检测NHE-1磷酸化。离子载体A23187使两种细胞类型中的[Ca2+]i均升高至约700至800 nmol·L-1，且仅在WKY平滑肌细胞中刺激NHE-1活性，对SHR细胞无影响。CaM激酶II抑制剂（KN-62）未能消除A23187对WKY细胞中NHE-1的刺激，且对两种细胞类型中未刺激的NHE-1活性均无影响。离子霉素也使两种细胞类型中的[Ca2+]i升高至约1000 nmol·L-1，且仅在WKY细胞中激活NHE-1活性。无论有无KN-62，[Ca2+]i升高对WKY细胞中NHE-1的激活均不是由NHE-1磷酸化增加介导的。SHR细胞中升高的NHE-1磷酸化不受[Ca2+]i升高或KN-62的影响。钙调蛋白琼脂糖珠与从SHR细胞中提取的NHE-1的结合程度低于从WKY细胞中提取的NHE-1。我们得出结论，WKY细胞中钙诱导的NHE-1激活不是由CaM激酶II介导的。SHR细胞中升高的NHE-1活性和磷酸化不受[Ca2+]i升高的进一步调节，且也独立于CaM激酶II。因此，NHE-1激活的非磷酸化依赖性机制可能是这些细胞中NHE-1活性改变的原因，例如CaM与NHE-1的直接结合。与WKY细胞相比，SHR细胞中CaM与NHE-1的这种直接结合可能受损。
