ucn-1028-c and mastoparan

Wortmannin is a specific phosphatidylinositol 3-kinase inhibitor, and it inhibits secretion in many cell types. Previously we reported that high K+ and external ATP stimulated a Ca2+ influx and [3H]-noradrenaline ([3H]NA) release from rat pheochromocytoma cell line 12 (PC12) cells in the presence of extracellular CaCl2. Addition of phorbol 12-myristate 13-acetate (PMA) stimulated [3H]NA release by itself and enhanced the maximal responses of high K+ and ATP. In this study, we investigated the effects of wortmannin on NA release from PC12 cells. Wortmannin inhibited the [3H]NA release induced by high K+ and ATP, and the stimulatory effects of PMA, in a dose-dependent manner. Wortmannin caused 50% inhibition of high K+-induced [3H]NA release at a concentration of 2.78+/-0.68 microM (n = 5). The increased cytosolic free Ca2+ concentrations ([Ca2+]i), induced by ATP, were not inhibited by wortmannin. Wortmannin inhibited PMA-induced phosphorylation of a 80-kDa protein in the cytosol fraction of PC12 cells. Calphostin C, a specific protein kinase C inhibitor, also inhibited high K+-, ATP-, and PMA-induced NA release, and the phosphorylation of the 80-kDa protein induced by PMA. Mastoparan, an amphiphilic tetradecapeptide from wasp venom, stimulated NA release in the presence or absence of extracellular CaCl2. Neither wortmannin nor calphostin C inhibited the NA release induced by mastoparan. These findings suggest that wortmannin inhibits the PKC-dependent pathway, not [Ca2+]i mobilization, resulting in the inhibition of NA release from PC12 cells.

Topics: Adenosine Triphosphate; Androstadienes; Animals; Calcium; Enzyme Inhibitors; Intercellular Signaling Peptides and Proteins; Naphthalenes; Norepinephrine; PC12 Cells; Peptides; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Potassium; Protein Kinase C; Rats; Secretory Rate; Tetradecanoylphorbol Acetate; Wasp Venoms; Wortmannin

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

Topics: Amino Acid Isomerases; Amino Acid Sequence; Animals; Biological Transport; Carrier Proteins; Cell Compartmentation; Cell Line; Cross-Linking Reagents; Cyclophilins; Cyclosporine; Endoplasmic Reticulum; Fluorescent Antibody Technique; Golgi Apparatus; Guanosine 5'-O-(3-Thiotriphosphate); Heat-Shock Proteins; HSP47 Heat-Shock Proteins; Intercellular Signaling Peptides and Proteins; Mice; Molecular Sequence Data; Naphthalenes; Peptides; Peptidylprolyl Isomerase; Polyribosomes; Precipitin Tests; Procollagen; Protein Binding; Protein Biosynthesis; Recombinant Proteins; Wasp Venoms

We have reported that erythropoietin induces a dose-dependent increase in cytosolic calcium ([Cai]) in single human peripheral blood BFU-E derived erythroblasts which is specific for stage of differentiation and that this increase is modulated by erythropoietin through an ion channel permeable to Ca2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Cai] stimulated by erythropoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosine kinases, staurosporine (100 nM), blocked the increase in [Cai] over 20 min following erythropoietin stimulation. However, erythropoietin-induced calcium influx was unaffected by preincubation of cells with specific inhibitions of protein kinase C (calphostin C) or the cAMP- or cGMP-dependent kinases (KT 5720, HA 1004), and [Cai] did not increase following stimulation with phorbol 12-myristate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C nor protein kinase A mediate the erythropoietin-induced [Cai] increase. In contrast, preincubation with genistein, a tyrosine kinase inhibitor, blocked the erythropoietin induced increase in [Cai]. To further study calcium entry in erythroblasts, we determined mastoparan, a peptide from wasp venom, induced a dose-dependent rise in [Cai] in erythroblasts which required external calcium. Stimulation of erythroid precursors with 10 microM mastoparan resulted in an increase in [Cai] from 52 +/- 3 nM to 214 +/- 36 nM which peaked at 20 min. The mastoparan-induced [Cai] increase was also dependent on tyrosine phosphorylation since it was blocked by preincubation with genistein. These results demonstrate that both erythropoietin and mastoparan stimulate calcium entry by a mechanism which has a genistein sensitive step and suggest that tyrosine kinase activation is required for the rise in [Cai] to occur.

Topics: Adult; Alkaloids; Analog-Digital Conversion; Biological Transport; Bucladesine; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Erythroid Precursor Cells; Erythropoietin; Genistein; Humans; Image Processing, Computer-Assisted; Indoles; Intercellular Signaling Peptides and Proteins; Intracellular Fluid; Isoflavones; Isoquinolines; Naphthalenes; Peptides; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Pyrroles; Signal Transduction; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Videotape Recording; Wasp Venoms