PKC-dependent inhibition of CA2+-dependent exocytosis from astrocytes

Astrocytes release various bioactive substances via Ca2+‐ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE)‐dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cells using primary cultured astrocytes and clonal rat glioma C6 cells. Mass spectrometry and Western blot analysis using phospho‐specific antibodies revealed that phorbol 12‐myristate 13‐acetate (PMA) treatment induced the phosphorylation of synaptosomal‐associated protein of 23 kDa (SNAP‐23) on Ser95, Ser120, and Ser160 in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser110 of SNAP‐23 was constitutively phosphorylated in these cells and was dephosphorylated in a PKC‐dependent manner. Exogenously expressed human growth hormone (hGH) accumulated in cytoplasmic granular structures in cultured astrocytes, and its release after ATP‐treatment was Ca2+‐ and SNARE‐dependent. PMA treatment suppressed the ATP‐induced hGH release from astrocytes and this inhibition was reversed by BIS. We also observed PMA‐dependent suppression and an attenuation of that suppression by BIS in ionomycin‐induced hGH release from C6 cells. These results suggest that intracellular activation of PKC suppresses Ca2+‐ and SNARE‐dependent exocytosis in astroglial cells. © 2010 Wiley‐Liss, Inc.