Effects of PKA and PKC modulators on high affinity glutamate uptake in primary neuronal cell cultures from rat cerebral cortex.

摘要

In this study, the effects of various agents known to alter protein phosphorylation, via protein kinase C or A, on high affinity glutamate uptake were investigated in primary neuronal cell cultures of rat cerebral cortex. Incubating the culture dishes with chelerythrine or H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide), which inhibit PKC and PKA, respectively, dramatically decreased the glutamate uptake in a dose-dependent manner. Saturation kinetic analysis showed that chelerythrine and H89 decreased the Vmax (chelerythrine: -61%, P < 0.06; -59%, P < 0.05) without affecting the Km of the transport process as compared to the control values. These inhibitory effects were counteracted by the corresponding protein kinase activators, i.e. PMA (phorbol-12-myristate 13-acetate) in the case of PKC and forskolin in the case of PKA, although these protein kinase activators alone did not significantly affect the glutamate uptake. These results provide evidence that, in primary cultures of neuronal cells, the high affinity glutamate uptake may be regulated by both PKA and PKC-mediated phosphorylation processes.