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

The role of phosphorylation events on the activation and modulation of the osmosensitive H-3-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was no t decreased when [Ca2+](i) rise evoked by hyposmolarity was prevented by EG TA-AM (50 muM) or depleted by treatment with 1 muM ionomycin in Ca2+-free m edium. Accordingly, OTR was not inhibited by Ca2+-dependent signaling event s. The calmodulin (CAM blocker W-7 (50 muM) potentiated OTR while the Ca2+/ CAM kinase blocker KN-93 (10 muM) was without effect. Blockade of PKC by H- 7, H-8 (50 muM) and Go6976 (1 muM), as well as activation by phorbol myrist ate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 muM) and 8-BrcAMP (10 muM) did not change OTR. Protein tyrosine phosphorylation seems to be of cr ucial importance in the activation and modulation of OTR, as it was markedl y inhibited (90%) by tyrphostine A23 (50 muM) and potentiated by the tyrosi ne phosphatase inhibitor ortho-vanadate (100 muM). The PI3 kinase blocker w ortmannin 100 nM essentially abolished OTR but LY294002 (10-100 muM) was wi thout effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of w ortmannin may be exerted not in PI3 kinase but instead on phospholipases, w hich are also sensitive to this blocker. The hyposmotic stimulus induced ac tivation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 muM) onl y marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomeno n, not directly involved in OTR activation. (C) 2001 Elsevier Science Ltd. All rights reserved.