Posttranslational effects of protein kinase C and insulin on red cell membrane phosphorylation and cation heteroexchange in hypertension.

In the red blood cell membrane, sodium-proton exchange (NHE-1) exchanges intracellular H+, Li+, and Na+ with extracellular Na+. In hypertensives (HT), the maximal velocity of translocation (V(max)) of Na+/H+ and of Na+/Li+ exchange modes are higher, while apparent affinity for external Na+ of Na+/Li+ exchange and Hill's coefficient for H+ activation of Na+/H+ exchange are lower than in normotensive subjects (NT). We have therefore examined the effects of protein kinase C (PKC) and insulin on red blood cell membrane phosphorylation and on the kinetic properties of cation heteroexchange. In red cell from NT, PMA-induced activation of PKC reduced K(m) for H+ of NHE but it did not affect V(max) and K(m) for Na+. In red cell from HT, PMA induced a greater PKC stimulation and membrane phosphorylation of band 3, 4.1, 4.9 than in NT and it did not significantly reduce K(m) for H(i). On the contrary, in HT PKC activation significantly increased Hill's coefficient of NHE. The larger activation of PKC in HT could be due to downregulation secondary to higher membrane calpain activity. Incubation of red cells with insulin decreases K(m) for external Na+ and increases V(max) of Na+/Li+ exchange. Therefore, we have examined the relationships between Na+-activation kinetics of Na+/Li+ exchange and fasting insulin levels. Na+-stimulated Li+ efflux was studied by raising Na+ up to 300 mM isoosmotically to measure K(m) for Na+ and V(max). Li+ efflux saturated at 150 mM external Na+ in NT but not in HT because in HT it exhibited a two fold higher Na+ K(m). V(max) was higher in HT than in NT. In hyperinsulinemic (fasting insulin > 10 μU/ml) HT, V(max) and Na+ K(m) were higher than in normoinsulinemic HT. In NT, hyperinsulinemia was not associated to abnormal kinetic properties of Na+/Li+ exchange. Stepwise multiple regression analysis confirmed that the main determinants of a high K(m) were blood pressure and insulin. Our results show that posttranslational effects of PKC and insulin affect the kinetic properties of NHE-1 in red blood cells and suggest that the differences observed between hypertensives and normotensive subjects can be accounted for by PKC activation and insulin exposure.