The signaling cascade linking insulin receptor stimulation to the activation of Na/H exchanger (NHE) was investigated in human erythrocytes, a simple cell model expressing the NHE1 isoform and protein kinase C (PKC) a and z isoforms only. Our results demonstrate the presence of phosphatidylinositol (PtdIns) 3-kinase in these cells and its activation by insulin. With a similar time-course, insulin also promoted both the translocation and activation of PKC z, but had no effect on PKC a. Inhibition of PtdIns 3-kinase with wortmannin prevented the activation of PKC z by insulin. Stimulation of NHE1 was observed after 10 min of insulin treatment and persisted for at least 60 min. This effect was totally abolished by wortmannin or GF 109203X, an inhibitor of all PKC isoforms, but not by Go È 6976, a specific inhibitor of conventional and novel PKCs (e.g. PKC a). These data indicate that PKC z activation is mediated by a PtdIns 3-kinase-dependent mechanism and that NHE1 stimulation involves the sequential activation of PtdIns 3-kinase and PKC z. In addition, insulin stimulation of NHE1 occurred without altering the phosphorylation state of the exchanger, suggesting that the phosphorylation of an ancillary protein by PKC z would be responsible for activation of the transporter.

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