The membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. insulin secretion using rat islets. When islets had been incubated for 10 min with high potassium, G?-6976, an inhibitor of conventional PKC, and PKC- pseudosubstrate fused to antennapedia peptide (Antp-PKC19C31) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by G?-6976 and Antp-PKC19C31. However, when islets were stimulated for 60 min with high glucose, both G?-6976 and Antp-PKC19C31 reduced glucose-induced insulin secretion. Similar results were obtained by transfection of dominant-negative PKC- using adenovirus vector. Taken together, PKC- is usually activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is usually inhibitory on depolarization-induced insulin secretion 1996; Yedovitzky 1997), with the dominant classical Ca2+CDAG-sensitive isoform being PKC-. Although the role of PKC in Ca2+-mobilizing, agonist-induced insulin release has been established, the possible role of PKC in nutrient-stimulated insulin secretion is still a matter of argument (Hii 1987; Jones 1991; Deeney 1996; Harris 1996; Yedovitzky 1997; Zawalich & Zawalich, 2001; Carpenter 2004). It is accepted that this function of PKC well correlates with its subcellular localization. In many tissues, activation of PKC is usually associated with translocation from your cytosol to the membrane-associated state. It was exhibited that a stimulatory concentration of glucose promoted translocation of PKC- from your cytosol to the plasma membrane in rat pancreatic islets (Deeney 1996; Ganesan 1990). These results suggest that PKC- is usually activated by glucose. Nevertheless, the role of activated PKC is still controversial. For example, studies conducted on cells with down-regulated PKC activity suggested that PKC is not directly involved in insulin secretion promoted by glucose (Hii 1987). However, it has been pointed out that down-regulation of PKC is dependent on the type of isoform (Yaney 2002), and down-regulation also affects various cellular functions including the insulin content of the cells (Yaney 2002). Therefore, the role of PKC, especially its isoform-specific role, should be considered cautiously. In this regard, involvement of PKC in glucose-induced insulin secretion has been shown by a study using isoform-specific inhibitors of PKC translocation. Yedovitzky (1997) reported that an inhibitor of PKC- inhibited glucose-induced insulin secretion. Similarly, an inhibitor of PKC-? inhibited glucose-induced Metyrosine insulin secretion from pancreatic islets (Yedovitzky 1997). Their results clearly showed the isoform-specific role of PKC in glucose-induced insulin secretion but do not thoroughly explain the controversial data around the role of PKC in nutrient-stimulated insulin secretion (Hii 1987; Jones 1991; Persaud & Jones, 1995; Harris 1996; Harris 1999; Carpenter 2004). Recent advances in the use of green fluorescent protein (GFP) have allowed us to investigate the regulation of PKC activity in intact living cells by monitoring translocation of the GFP-tagged PKC (Oancea & Meyer, 1998; Almholt 1999). Using this approach, we obtained a slightly different aspect of regulation of PKC in cells: standard PKC and novel PKC are activated by depolarization-evoked Ca2+ influx through voltage-dependent calcium channels (VDCCs) in insulinoma cells, INS-1 (Mogami 2003). This raises an intriguing possibility that PKC in pancreatic cells is usually Mouse monoclonal to KLHL13 activated by brokers that depolarize the cell membrane. Since cells are excitable, many brokers induce calcium influx in these cells. It is therefore crucial to determine whether or not these brokers activate PKC as calcium-mobilizing agonists do, and if so, to determine the role of PKC in insulin secretion induced by these brokers. The present study was conducted to investigate the role of calcium access in the activation mechanism of PKC-, a dominant standard PKC isoform expressed in pancreatic cells (Oancea & Meyer, 1998). The role of PKC- in Ca2+-evoked insulin secretion was also evaluated. Methods Chemicals Fura-2-AM was Metyrosine purchased from Sigma (St Louis, MO, USA). A PKC- inhibitor peptide, antennapedia-PKC19C31 (Antp-PKC19C31) (RRMKW KKRFA RKGAL RQKNV), and a PKC-? inhibitor peptide, antennapedia-PKC149C164 (Antp-PKC149C164) (RRMKW KKERM RPRKR QGAVR RRV), were synthesized by Takara Shuzo Co., Ltd (Tokyo, Japan). Both inhibitors are tandemly synthesized peptides comprising peptides derived from the third -helix of the homeodomain of antennapedia Metyrosine (residues 52C58, known as penetratin) (Derossi 1998; Fischer 2000), and the PKC pseudosubstrate peptides (PKC- residues 19C31 and PKC-? residues 149C164) (Harris 1999; Zoukhri 1997). Antennapedia peptide (Antp), used as a control, was also synthesized by Takara Shuzo. PKC inhibitor G?-6976 (12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-52000) and PKC-?CGFP (Shirai 1998) were kindly provided by Dr Naoaki Saito of Kobe University or college (Kobe, Japan). To obtain brighter fluorescence, the GFP in MARCKSCGFP was replaced with pEGFP-N2 as previously explained (Mogami 2003). A GFP-tagged C1 region of PKC (C1-GFP) was produced from a DNA clone of CKR1, which was subcloned into an expression plasmid for mammalian cells, pTB701 (Ono 1988), as previously explained (Mogami 2003). To obtain higher transfection efficiency and brighter fluorescence, the C1 was inserted into pEGFP-N1 vector. A cDNA fragment of PKC for the C1 region with an 2002) were kindly provided by Dr Y..