CALCIUM-INDUCED ACTIVATION OF THE RAT VASCULAR MYOCYTE NA+ H+ EXCHANGER ISOFORM-1/

An established intermediate phenotype of human hypertension and diabet ic nephropathy is an elevation of Na+/H+ exchanger (NHE) activity, but the mechanism for this is unclear. This phenotype is maintained in va scular myocytes from the spontaneously hypertensive rat (SHR) compared with the normotensive Wistar Kyoto rat (WKY). Since intracellular cal cium levels ([Ca2+](i)) following agonist stimulation were elevated in cells from both hypertensive humans and SHR, we have examined the rol e of calcium-calmodulin (CaM) in the mechanism of increased NHE activi ty in vascular myocytes of SHR by determining the activity and phospho rylation 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 act ivity was measured using fluorometry and NHE-1 phosphorylation by immu noprecipitating the exchanger from P-32-orthophosphate-labeled cells w ith a polyclonal NHE-1-specific antibody. The ionophore A23187 increas ed [Ca2+](i) in both cell types to approximately 700 to 800 nmol . L(- 1), and led to stimulation of NHE-1 activity only in WKY myocytes, wit h 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 ef fect on unstimulated NHE-1 activity in both cell types. lonomycin also elevated [Ca2+](i) in both cell types to approximately 1,000 nmol . 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 incre ase 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 e xtracted from SHR cells to a lesser extent than that from WKY cells. W e conclude that calcium-induced NHE-1 activation in WKY cells was not mediated by CaM kinase II. The elevated NHE-1 activity and phosphoryla tion of SHR cells was not further modulated by increased [Ca2+](i), an d was also independent of CaM kinase II. Non-phosphorylation-dependent mechanisms of activation of NHE-1 may therefore be responsible for al terations 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. Copyright (C) 1997 by W.B. Saunders Co mpany.