PKC-dependent inhibition of CA2+-dependent exocytosis from astrocytes.
Astrocytes release various bioactive substances via Ca(2+) - 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 Ser(95), Ser(120), and Ser(160) in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser(110) 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 Ca(2+) - 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 Ca(2+) - and SNARE-dependent exocytosis in astroglial cells.
Department of Biochemistry, Kitasato University School of Medicine, Sagamihara-shi, Kanagawa, Japan.
Keiichi
K
Yasuda
Makoto
M
Itakura
Kyota
K
Aoyagi
Tsukiko
T
Sugaya
Etsuko
E
Nagata
Hideshi
H
Ihara
Masami
M
Takahashi
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D013058:Mass Spectrometry; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D033921:Vesicular Transport Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; C498858:Snap23 protein, rat; D033921:Vesicular Transport Proteins; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D002118:Calcium