Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons

Abstract

The role of phosphorylation events on the activation and modulation of the osmosensitive 3H-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was not decreased when [Ca 2+] i rise evoked by hyposmolarity was prevented by EGTA-AM (50 μM) or depleted by treatment with 1 μM ionomycin in Ca 2+-free medium. Accordingly, OTR was not inhibited by Ca 2+-dependent signaling events. The calmodulin (CAM) blocker W-7 (50 μM) potentiated OTR while the Ca 2+/CAM kinase blocker KN-93 (10 μM) was without effect. Blockade of PKC by H-7, H-8 (50 μM) and Gö6976 (1 μM), as well as activation by phorbol myristate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 μM) and 8-BrcAMP (10 μM) did not change OTR. Protein tyrosine phosphorylation seems to be of crucial importance in the activation and modulation of OTR, as it was markedly inhibited (90%) by tyrphostine A23 (50 μM) and potentiated by the tyrosine phosphatase inhibitor ortho-vanadate (100 μM). The PI3 kinase blocker wortmannin 100 nM essentially abolished OTR but LY294002 (10–100 μM) was without effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of wortmannin may be exerted not in PI3 kinase but instead on phospholipases, which are also sensitive to this blocker. The hyposmotic stimulus induced activation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 μM) only marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomenon, not directly involved in OTR activation.