PHOSPHORYLATION SIGNALS OF THE INSULIN-RECEPTOR IN HUMAN RED-CELLS - ACTIVATION OF NA+ H+ EXCHANGE/

We have a great deal of knowledge about how insulin initiates its phos phorylation cascade. However, which transduction pathways in the human red cell (RBC) mediate activation of Na+/H+ exchange (NHE) by insulin in hypertensive subjects, is not known. In this paper, we have tested the hypothesis that insulin increases the V-max of NHE-1 in vitro, by increasing its phosphorylated state. To this end, we prepared a polyc lonal antibody AB-C1 that recognized in Western blots a monomeric band of the NHE-1 of 110-115 kDa, and a dimeric band of 230 kDa among RBC membrane proteins. RBCs from fasting young subjects were incubated wit h insulin (100 mu U/mL) for different time periods, and membranes were prepared by hypotonic lysis. The time course of tyrosine and serine/t hreonine (S/T) phosphorylation by their corresponding protein kinases were followed by incubation of the membranes in appropriate media. Ins ulin initiated the following phosphorylation events: (i) activation of tyrosine protein kinase in less than 2 min, that decayed at 15 min (i i) stimulation of the S/T protein kinase after 5 min at a rate 2-fold greater than that of tyrosine phosphorylation which persisted up to 45 min (iii) phosphorylation of the NHE-1 immunoprecipitate after 10 min , which persisted with oscillations up to 60 min. The behavior of the V-max of NHE-1 in its state of low affinity for Na+ also paralelled th e time course of the phosphorylation of the immunoprecipitate. These e ffects of insulin are in contrast to the effects of growth factors (EG F, and thrombin), that do not increase the V-max of NHE-1 while the af finity for internal H+ is markedly increased. The results support the hypothesis that the stimulatory action of insulin on the V-max of NHE- 1 human RBC can be due to an enhanced S/T phosphorylation of the cytos olic loop of NHE-1. (C) 1997, Medikal Press.