GENISTEIN INHIBITS NA+/CA2+ EXCHANGE ACTIVITY IN PRIMARY RAT CORTICAL NEURON CULTURE

We have examined the possible regulatory effect of tyrosine kinase activity on Ca2+transport observed in the cultured rat cortical neurons. Na+/Ca2+exchange was studied using cells cultured for various time periods. A nearly two fold increase in Ca2+uptake was seen when comparing 3 day and 9 day cultures. Western blot analysis also showed a two fold increase in Na+/Ca2+exchanger (NCX1) protein levels as cells matured in culture. To study the effect of genistein (a specific tyrosine kinase inhibitor) cells were incubated with 100μM genistein (in 1% DMSO) for 1 hour before the assay of Na+/Ca2+exchange activity. There was a significant decrease of Ca2+uptake in genistein treated neurons (control: 4.596±0.205 nmol/mg protein/15min, n=12; genistein: 1.420±0.131 nmol/mg protein/15min, n=12, mean±S.E. P<0.001). Daidzein, an inactive analog of genistein and phorbol myristate acetate (PMA), a PKC activator were without effect. The results suggest that as cells mature in culture, Na+/Ca2+exchange capacity increases, as a result of greater protein expression. Exposure to genistein inhibited Ca2+uptake suggesting that the exchanger may be modulated by tyrosine phosphorylation.