SHORT COMMUNICATION Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells Yoko Shoji-Kasai, 1 Makoto Itakura, 1 Masakazu Kataoka 2 , Saori Yamamori 1 and Masami Takahashi 1 1 Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo 194±8511, Japan 2 Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Wakasato 4±17±11, Nagano-shi, Nagano 380±8553, Japan Keywords: GFP, phosphorylation, phorbol ester, VMAT, VAChT Abstract In order to elucidate the molecular mechanism of phorbol ester-induced potentiation of neurotransmitter release, changes in the subcellular distribution of secretory vesicles were studied in PC12 cells. Dopamine (DA) and acetylcholine containing vesicles were selectively labelled by expressing green ¯uorescent protein-conjugated vesicular monoamine transporter and vesicular acetylcholine transporter, respectively. In the resting state, these vesicles were distributed throughout the cytoplasm. Phorbol-12- myristate-13-acetate (PMA), but not the inactive analogue 4a-PMA, induced a redistribution of both types of secretory vesicles near the plasma membrane, and this change was abolished by a protein kinase C (PKC) inhibitor, bisindolylmaleimide I (BIS). PMA also induced a marked enhancement of depolarization-induced DA release and phosphorylation of SNAP-25 at Ser 187 . BIS completely inhibited PMA-induced SNAP-25 phosphorylation but suppressed PMA-induced enhancement of DA release only partially. These results suggest that PMA enhances neurotransmitter release from PC12 cells by both PKC-dependent and PKC- independent mechanisms, and PKC enhances neurotransmitter release by recruiting secretory vesicles to the plasma membrane. Introduction Synaptic transmission is mediated by neurotransmitters released from nerve terminals. Neurotransmitters are stored in synaptic vesicles and released into the synaptic cleft by exocytosis of vesicle contents, which involves docking and fusion of the vesicle membrane with the presynaptic plasma membrane (Sudhof, 1995; Lin & Scheller, 2000). Neurotransmitter release is regulated both positively and negatively by multiple mechanisms and its regulation is believed to be one of the important mechanisms of synaptic plasticity underlying learning and memory (Jessell & Kandel, 1993). In the short term, various protein kinases have been implicated in presynaptic regulation in a wide range of neuronal preparations; however, the precise mechanisms are still poorly understood (Greengard et al., 1993). Clonal rat pheochromocytoma PC12 cells have been used to study the regulation of neurotransmitter release (Greene & Tischler, 1982). These cells synthesize and store dopamine (DA) and acetylcholine (ACh) in large dense-core vesicles (LDCV) and small synaptic vesicles (SSV), respectively, and release these neurotransmitters by Ca 2+ -dependent exocytosis (De Camilli, 1991). Previously, we showed that phorbol ester treatment induced phosphorylation of SNAP-25 at Ser 187 and potentiation of Ca 2+ -induced DA and ACh release from PC12 cells (Shimazaki et al., 1996; Iwasaki et al., 2000). Staurosporine, a nonselective inhibitor of protein kinase, abolished SNAP-25 phosphorylation but suppressed the potentiation of neurotransmitter release only partially, suggesting that phorbol ester potentiated neurotransmitter release by two distinct phosphorylation- dependent and independent mechanisms (Iwasaki et al., 2000). In order to further elucidate the molecular mechanism of phorbol ester- induced potentiation of neurotransmitter release, we studied the changes in the subcellular localization of secretory vesicles before and after treatment with phorbol-12-myristate-13-acetate (PMA) using green ¯uorescent protein (GFP) technology (Cubitt et al., 1995). We found that both LDCV and SSV translocated to plasma membrane after PMA-treatment and this translocation was blocked by a speci®c protein kinase C (PKC) inhibitor. Materials and methods Materials The sources of the following materials are; PMA and BIS, Calbiochem-Novabiochem (La Jolla, CA); 4a-PMA, Alexis Biochemicals (San Diego, CA); Dulbecco's modi®ed Eagle's medium, horse serum, and Lipofectiamine 2000, Life Technologies (Rockville, MD); precolostrum newborn calf serum, Mitsubishi Kagaku (Tokyo, Japan). Cell culture and transfection A DNA fragment corresponding to rat vesicular monoamine trans- porter 2 (VMAT2) and vesicular acetylcholine transporter (VAChT) were isolated by the polymerase chain reaction and subcloned into either pEGFP-N2 vector or pDsRed1-N1 vector (Clontech, Palo Alto, Correspondence: Dr Masami Takahashi, as above. E-mail: masami@libra.ls.m-kagaku.co.jp Received 10 October 2001, revised 12 February 2002, accepted 6 March 2002 European Journal of Neuroscience, Vol. 15, pp. 1390±1394, 2002 ã Federation of European Neuroscience Societies