herbimycin and chelerythrine

Mitogen-activated protein (MAP) and tyrosine kinases play an important role in regulating smooth muscle contraction of the gastrointestinal (GI) tract. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. Thus, the role of MAP and tyrosine kinases on the pacemaker potentials of colonic ICCs was investigated.. Cultured ICCs were prepared from mice colons, and their pacemaker potentials were recorded using whole-cell patch clamping.. In current-clamping mode, colonic ICCs displayed spontaneous pacemaker potentials. SB203580 (a p38 MAP kinase inhibitor), SP600125 (a c-jun NH2-terminal kinase (JNK) inhibitor), genistein and herbimycin A (tyrosine kinase inhibitors) blocked the generation of pacemaker potentials. However, PD98059 (a p42/44 MAP kinase inhibitor) had no effects on pacemaker potentials. LY-294002 (phosphoinositide 3-kinase inhibitor) also reduced the pacemaker potential frequency but calphostin C and chelerythrine (protein kinase C inhibitors) had no effects. However, PD98059, SB203589, SP600125, genistein, herbimycin A, LY-294002, and calphostin C had no effect on normal pacemaker activity in small intestinal ICCs.. Endogenous p38 MAP kinases, JNKs, tyrosine kinases, and PI3-kinases participate in the generation of pacemaker potentials in colonic ICCs but not in ICCs of the small intestine.

Topics: Animals; Anthracenes; Benzophenanthridines; Cells, Cultured; Chromones; Colon; Flavonoids; Genistein; Imidazoles; Interstitial Cells of Cajal; Intestine, Small; Membrane Potentials; Mice; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Protein-Tyrosine Kinases; Pyridines; Rifabutin

The effect of protein tyrosine kinases (PTK) on L-type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10(-6) M) sensitive L-type channels were measured using the patch-clamp technique. The current density of L-type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5 x 10(-6) M) led to an increase of L-type currents. Intracellular application of pp60c-src (30 U/ml) via the patch pipette led to a transient decrease of L-type currents. The protein kinase A (PKA) and PKG blocker H9 (10(-6) M) showed no effect on L-type currents. However, the protein kinase C blocker chelerythrine (10(-5) M) reduced these currents. Up-regulation of PKC by 10(-6) M 4beta-phorbol-12 myristate-13 acetate (PMA) led to a decrease of L-type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60(c-src) in PMA-treated cells led to a transient increase of L-type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L-type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS-RPE cells, this disturbed regulation of L-type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.

Topics: Alkaloids; Animals; Animals, Newborn; Benzophenanthridines; Benzoquinones; Calcium; Calcium Channels; Calcium Channels, L-Type; Cells, Cultured; Enzyme Inhibitors; Fibroblast Growth Factor 2; Genistein; Lactams, Macrocyclic; Membrane Potentials; Nifedipine; Patch-Clamp Techniques; Phenanthridines; Phenols; Pigment Epithelium of Eye; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Reference Values; Retinal Degeneration; Rifabutin; Tetradecanoylphorbol Acetate; Up-Regulation

W/Wv mice are deficient in tissue mast cells, and mast cells cultured from these mice do not proliferate in response to the c-kit ligand, stem cell factor (SCF). In this paper, we report that mouse bone marrow cultured mast cells derived from W/Wv mice do adhere to fibronectin in the presence of SCF and exhibit chemotaxis to SCF, and we explore this model for the understanding of c-kit-mediated signaling pathways. Both in vitro and in vivo (in intact cells) phosphorylation experiments demonstrated a low residual level of W/Wv c-kit protein phosphorylation. SCF-induced responses in W/Wv mast cells were abolished by the tyrosine kinase inhibitor herbimycin A and by the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin but were not affected by protein kinase C inhibitors. These observations are consistent with the conclusions that Wv c-kit initiates a signaling process that is PI 3-kinase dependent and that mutated Wv c-kit retains the ability to initiate mast cell adhesion and migration.

Topics: Alkaloids; Androstadienes; Animals; Benzophenanthridines; Benzoquinones; Bone Marrow Cells; Cell Adhesion; Cell Division; Cells, Cultured; Chemotaxis; Enzyme Inhibitors; Indoles; Kinetics; Lactams, Macrocyclic; Maleimides; Mast Cells; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Phenanthridines; Proto-Oncogene Proteins c-kit; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Signal Transduction; Stem Cell Factor; Wortmannin

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.

Topics: Alkaloids; Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzophenanthridines; Benzoquinones; Brain; Carcinogens; Cells, Cultured; Chemotaxis; Dose-Response Relationship, Drug; Enzyme Inhibitors; Lactams, Macrocyclic; Microglia; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Wistar; Rifabutin; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate

The hemolytically inactive complement component complex C5b67, designated iC5b67, can signal human polymorphonuclear leukocytes (PMN) both as a pertussis toxin-inhibitable agonist for chemotaxis and as an antagonist for C5a- and FMLP-stimulated chemotaxis and superoxide production. The signaling pathways utilized by iC5b67 have been further investigated. In contrast to mastoparan, iC5b67 failed to directly activate G proteins to stimulate inositol phosphate formation in COS cells that had been transfected with G alpha 16. In COS cells co-transfected with both G alpha 16 and the C5a receptor, iC5b67 could neither activate phospholipase C nor inhibit C5a receptor-mediated activation of phospholipase C. iC5b67 stimulated GTPase activity in a membrane-enriched fraction from PMN. These data support the hypothesis that iC5b67 signals through a unique receptor, likely G protein linked, but distinct from the C5a receptor. iC5b67 was able to mobilize intracellular stores to elicit increases in intracellular Ca2+. Based on the effects of herbimycin A, wortmannin, and chelerythrine on iC5b67-induced PMN chemotaxis, iC5b67 signaling involved activation of tyrosine and phosphatidylinositol 3-kinases, but not protein kinase C. Relevant to the capacity of iC5b67 to antagonize PMN superoxide production, iC5b67 induced rapid and sustained increases in intracellular cAMP, which others have shown can inhibit superoxide formation. Although iC5b67 antagonizes C5a and FMLP receptor-mediated superoxide generation, iC5b67 had no effect on PMA-induced superoxide formation. The distinct agonist and antagonist signaling pathways activated by iC5b67 in the PMN diverge soon after initial iC5b67 receptor-mediated transduction steps.

Topics: Alkaloids; Androstadienes; Animals; Antigens, CD; Benzophenanthridines; Benzoquinones; Calcium; Cell Line, Transformed; Chemotaxis, Leukocyte; Chlorocebus aethiops; Complement C5; Complement System Proteins; Cyclic AMP; Enzyme Inhibitors; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Naphthalenes; Neutrophils; Peptides; Pertussis Toxin; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein-Tyrosine Kinases; Quinones; Receptor, Anaphylatoxin C5a; Receptors, Complement; Recombinant Fusion Proteins; Respiratory Burst; Rifabutin; Signal Transduction; Superoxides; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella; Wasp Venoms; Wortmannin