d-609 and chelerythrine

ACh-induced contraction of esophageal circular muscle (ESO) depends on Ca2+ influx and activation of protein kinase Cepsilon (PKCepsilon). PKCepsilon, however, is known to be Ca2+ independent. To determine where Ca2+ is needed in this PKCepsilon-mediated contractile pathway, we examined successive steps in Ca2+-induced contraction of ESO muscle cells permeabilized by saponin. Ca2+ (0.2-1.0 microM) produced a concentration-dependent contraction that was antagonized by antibodies against PKCepsilon (but not by PKCbetaII or PKCgamma antibodies), by a calmodulin inhibitor, by MLCK inhibitors, or by GDPbetas. Addition of 1 microM Ca2+ to permeable cells caused myosin light chain (MLC) phosphorylation, which was inhibited by the PKC inhibitor chelerythrine, by D609 [phosphatidylcholine-specific phospholipase C inhibitor], and by propranolol (phosphatidic acid phosphohydrolase inhibitor). Ca2+-induced contraction and diacylglycerol (DAG) production were reduced by D609 and by propranolol, alone or in combination. In addition, contraction was reduced by AACOCF(3) (cytosolic phospholipase A(2) inhibitor). These data suggest that Ca2+ may directly activate phospholipases, producing DAG and arachidonic acid (AA), and PKCepsilon, which may indirectly cause phosphorylation of MLC. In addition, direct G protein activation by GTPgammaS augmented Ca2+-induced contraction and caused dose-dependent production of DAG, which was antagonized by D609 and propranolol. We conclude that agonist (ACh)-induced contraction may be mediated by activation of phospholipase through two distinct mechanisms (increased intracellular Ca2+ and G protein activation), producing DAG and AA, and activating PKCepsilon-dependent mechanisms to cause contraction.

Topics: Adrenergic beta-Antagonists; Alkaloids; Animals; Antibodies; Azepines; Benzophenanthridines; Bridged-Ring Compounds; Calcium; Calcium Signaling; Cats; Dose-Response Relationship, Drug; Enzyme Inhibitors; Esophagus; Estrenes; Female; Guanosine 5'-O-(3-Thiotriphosphate); Isoenzymes; Male; Muscle Contraction; Muscle, Smooth; Myosin Light Chains; Naphthalenes; Norbornanes; Phenanthridines; Phosphodiesterase Inhibitors; Phospholipase D; Phosphorylation; Propranolol; Protein Kinase C; Protein Kinase C-epsilon; Pyrrolidinones; Quercetin; Second Messenger Systems; Thiocarbamates; Thiones; Type C Phospholipases

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Topics: Alkaloids; Benzophenanthridines; Bridged-Ring Compounds; Cell Adhesion; Enzyme Inhibitors; Fibronectins; Humans; Integrins; Leukemia, T-Cell; Lymphocyte Activation; Neomycin; Norbornanes; Phenanthridines; Phosphodiesterase Inhibitors; Protein Kinase C; Protein Synthesis Inhibitors; Sensitivity and Specificity; Signal Transduction; Substrate Specificity; T-Lymphocytes; Thiocarbamates; Thiones; Tumor Cells, Cultured; Type C Phospholipases

The cystogenesis event of Toxoplasma gondii is poorly understood. In order to throw light on it, the effect of tumor necrosis factor-alpha (TNF-alpha) was studied in the Prugniaud strain of the organism. This showed that TNF-alpha increased the number of cysts formed in vitro in human MRC5 fibroblasts. The sphingomyelinase pathway may be involved in mediating the TNF effect, since ceramide (natural form in permeabilized cells or cell-permeable analogue) could mimic the action of TNF. More precisely, our results strongly suggest the involvement of an acidic sphingomyelinase in mediating the effect of TNF; indeed, D609 inhibited both the TNF effect and cyst formation, while arachidonic acid had no effect. Moreover, protein kinase (PKC) seems also to play a role in the process, since phorbol-12-myristate-13-acetate (PMA) enhanced the cyst formation. However, chelerythrine chloride did not prevent the TNF effect, suggesting that several host-cell pathways can affect the cystogenesis event. Taken together, these results suggest the active participation of host-cell components in the cystogenesis of Toxoplasma gondii.

Topics: Alkaloids; Animals; Benzophenanthridines; Bridged-Ring Compounds; Cell Line; Ceramides; Enzyme Inhibitors; Fibroblasts; Humans; Norbornanes; Phenanthridines; Protein Kinase C; Sphingomyelin Phosphodiesterase; Tetradecanoylphorbol Acetate; Thiocarbamates; Thiones; Toxoplasma; Tumor Necrosis Factor-alpha