ucn-1028-c and midostaurin

The binding of Ab (IgG)-opsonized particles by FcgammaRs on macrophages results in phagocytosis of the particles and generation of a respiratory burst. Both IgG-stimulated phagocytosis and respiratory burst involve activation of protein kinase C (PKC). However, the specific PKC isoforms required for these responses have yet to be identified. We have studied the involvement of PKC isoforms in IgG-mediated phagocytosis and respiratory burst in the mouse macrophage-like cell line, RAW 264.7. Like primary monocyte/macrophages, their IgG-mediated phagocytosis was calcium independent and diacylglycerol sensitive, consistent with novel PKC activation. Respiratory burst in these cells was Ca2+ dependent and inhibited by staurosporine and calphostin C as well as by the classic PKC-selective inhibitors Gö 6976 and CGP 41251, suggesting that classic PKC is required. In contrast, phagocytosis was blocked by general PKC inhibitors but not by the classic PKC-specific drugs. RAW 264.7 cells expressed PKCs alpha, betaI, delta, epsilon, and zeta. Subcellular fractionation demonstrated that PKCs alpha, delta, and epsilon translocate to membranes during phagocytosis. In Ca2+-depleted cells, only novel PKCs delta and epsilon increased in membranes, and the time course of their translocation was consistent with phagosome formation. Confocal microscopy of cells transfected with green fluorescent protein-conjugated PKC alpha or epsilon confirmed that these isoforms translocated to the forming phagosome in Ca-replete cells, but only PKC epsilon colocalized with phagosomes in Ca2+-depleted cells. Taken together, these results suggest that the classic PKC alpha mediates IgG-stimulated respiratory burst in macrophages, whereas the novel PKCs delta and/or epsilon are necessary for phagocytosis.

Topics: Animals; Biological Transport; Calcium; Cell Line; Cell Membrane; Diglycerides; Enzyme Inhibitors; Green Fluorescent Proteins; Intracellular Membranes; Isoenzymes; Luminescent Proteins; Macrophages; Mice; Naphthalenes; Phagocytosis; Phagosomes; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase C-epsilon; Respiratory Burst; Staurosporine

Process extension by oligodendrocytes (OLs) is a critical early step in myelin formation. We have previously reported that the basal- or phorbol ester-enhanced process outgrowth by adult human OLs is mediated by oligodendroglial protein kinase C (PKC). Recently, we demonstrated that astrocytes facilitated process outgrowth by adult human OLs through the interaction between astrocyte-derived basic fibroblast growth factor (bFGF) and astrocyte extracellular matrix (ECM). If PKC is central to the signal transduction cascade that leads to process formation by OLs, then the effects of bFGF and astrocyte ECM should also involve PKC. In the current study, we have addressed the involvement of PKC in the bFGF- and astrocyte ECM- enhanced process formation by adult human OLs by using a selective inhibitor of PKC, calphostin C. The results show that calphostin C dose-dependently reduced process extension elicited by bFGF and astrocyte ECM, at IC50 concentrations of 24.5 and 26.6 nM, respectively. At the concentrations of calphostin C that inhibited process extension by adult human OLs, necrosis (measured by lactate dehydrogenase release) and apoptosis (determined by using a fluorescent terminal deoxynucleotidyl transferase assay) of OLs did not occur. Finally, we demonstrate that another specific inhibitor of PKC, CGP 41251, also reduced process formation that is elicited by bFGF and astrocyte ECM. Thus, all process-extending agents for adult human OLs identified to date signal through PKC, further implicating PKC of OLs as being central to the production of process extension, an early event in myelinogenesis.

Topics: Astrocytes; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix; Fibroblast Growth Factor 2; Humans; Naphthalenes; Oligodendroglia; Phorbol 12,13-Dibutyrate; Protein Kinase C; Staurosporine

The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.

Topics: Alkaloids; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Cells, Cultured; Cyclic AMP; Cyclooxygenase Inhibitors; Egtazic Acid; Endothelin-1; Enzyme Inhibitors; Epoprostenol; Heart; Heart Ventricles; Kinetics; Myocardium; Naphthalenes; Nifedipine; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Staurosporine; Thionucleotides

The hypothesis was tested that 9 kinase inhibitors with diverse specificities for protein kinase C (PKC), including staurosporine and four of its analogues, interfere differently with PKC derived from either the cytosolic or particulate fractions of MCF-7 breast carcinoma cells. GF 109203X inhibited the enzyme identically in either preparation. CGP 41251 and calphostin C inhibited cytosolic PKC more effectively than membrane-derived PKC with ratios of IC50 (cytosolic PKC) over IC50 (membrane-derived PKC) of 0.07 and 0.04, respectively. The other six agents inhibited membrane-derived PKC more potently than cytosolic enzyme. Staurosporine and RO 31 8220 exhibited IC50 ratios of 12.3 and 21.6, respectively. The results suggest that there are dramatic differences between kinase inhibitors in their divergent effects on cytosolic and membrane-derived PKC which should be borne in mind in the interpretation of their pharmacological properties.

Topics: Alkaloids; Breast Neoplasms; Cell Membrane; Cytosol; Humans; Immunoblotting; Indoles; Naphthalenes; Phorbol 12,13-Dibutyrate; Polycyclic Compounds; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

Pretreatment of LPS-induced RAW 264.7 cells with three PKC inhibitors suggests that induction of TNF-alpha, nitric oxide (NO), gelatinase B (Gel B) and IL-6 involves at least three distinct signalling pathways. We confirmed the PKC dependence of TNF-alpha and NO productions and found that Gel B was inhibited by Calphostin C (CAL), but potentiated by staurosporine (STAR) and CGP 41 251. IL-6 production was stimulated by the three inhibitors. Our results indicate that up-regulation of Gel B, TNF-alpha and NO seems to involve PKC at different levels, whereas up-regulation of IL-6 production appears to be PKC-independent. However, IL-6 production in RAW 264.7 cells seems to be down-regulated by a PKC-dependent feedback mechanism.

Topics: Alkaloids; Animals; Biological Assay; Blotting, Northern; Cell Line, Transformed; Collagenases; Gene Expression; Interleukin-6; Macrophages; Matrix Metalloproteinase 9; Mice; Naphthalenes; Nitric Oxide; Polycyclic Compounds; Protein Kinase C; RNA, Messenger; Staurosporine; Tumor Necrosis Factor-alpha