sq-23377 and chelerythrine

Tight control of lung liquid (LL) regulation is vital for pulmonary function. The aim of this work was to determine whether PKC activation is involved in the physiological regulation of LL volume in a whole lung preparation. Rat lungs were perfused with a modified Ringer solution, and the lumen was filled with the same solution without glucose. LL volume was measured during a control period and after modulating drugs were administered, and net LL transepithelial movement (J(v)) was calculated. When the PKC activator PMA (10(-5) M) and the Ca(2+) ionophore ionomycin (10(-6) M) were instilled into the lung together, J(v) was significantly reduced (P = 0.03). This reduction was blocked by the PKC inhibitor chelerythrine chloride (10(-6) M; P = 0.56) and by a second PKC inhibitor GF109203X (10(-5) M; P = 0.98). When PMA and ionomycin were added with the β-adrenergic agonist terbutaline, the terbutaline-induced increase in J(v) was abolished. Addition of PMA and ionomycin with the epithelial Na(+) channel (ENaC) blocker amiloride had no additional inhibitory effect. Together, these results suggest that PKC is likely to be involved in LL absorption, and the ability of PMA/ionomycin to block the terbutaline-induced increase in J(v) suggests that the downstream target of PKC is ENaC.

Topics: Amiloride; Animals; Benzophenanthridines; Biological Transport; Cells, Cultured; Electric Impedance; Enzyme Inhibitors; Epithelial Cells; Epithelial Sodium Channels; Indoles; Ionomycin; Isotonic Solutions; Lung; Male; Maleimides; Protein Kinase C; Rats; Rats, Wistar; Ringer's Solution; Signal Transduction; Sodium Channel Blockers; Terbutaline; Tetradecanoylphorbol Acetate

The effect of hyposmotic shock on exocytosis was examined in isolated hepatocytes of turbot, a marine flatfish, using the molecular probe FM1-43. Sudden exposure to a reduced osmolality caused an increase in cell exocytic activity related to the osmotic gradient between intra- and extracellular fluids. Cytoskeletal microtubules could contribute to this hyposmotic-induced exocytosis since colchicine inhibited the process. Protein kinase C, phosphatidylinositol-3 kinase, phospholipases A2, C and D could constitute key enzymes in the mechanism since their inhibition by specific agents altered the hyposmotic-induced exocytic activity. Moreover, arachidonic acid and derivates from the 5-lipoxygenase pathway as well as calcium could participate in the process. As regulatory volume decrease (RVD) exhibited by turbot hepatocytes following hyposmotic stimulation involves similar features, a potential role of exocytosis in volume regulation is suggested. In particular, exocytosis could serve RVD by contributing to ATP release since this latter process similarly appeared to be phospholipase D-dependent and related to the osmotic gradient. This study provides the first evidence of a volume-sensitive exocytosis that could aim at volume constancy in a marine teleost fish cell type.

Topics: 1-Butanol; Alkaloids; Androstadienes; Animals; Benzophenanthridines; Cell Membrane; Colchicine; Cytochalasin B; Cytoskeleton; Estrenes; Exocytosis; Flatfishes; Fluorescent Dyes; Hepatocytes; Indomethacin; Ionomycin; Isoquinolines; Masoprocol; Osmotic Pressure; Phenanthridines; Pyridinium Compounds; Pyrrolidinones; Quaternary Ammonium Compounds; Quinacrine; Signal Transduction; Sulfonamides; Thapsigargin; Wortmannin

The effects of IgA immune complex (IgA-IC) on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with IgA-IC for 24 hours significantly decreased the contractile responses to angiotensin II (10(-6) M) and phorbol 12-myristate 13-acetate (PMA, 10(-6) M). Pretreatment of mesangial cells with the protein kinase C (PKC) inhibitor, chelerythrine (10(-6) M), eliminated the difference in contractile responses to angiotensin II or PMA between the control and IgA-IC groups indicating IgA-IC may inhibit the activity of PKC. The contractile responses to ionomycin were not significantly different between IgA-IC treated and control mesangial cells, suggesting that the contractile machinery is not impaired by IgA-IC. Intracellular calcium, [Ca2+]i measured by changes in fura-2 level in response to ATP or bradykinin, was significantly inhibited in IgA-IC treated mesangial cells, compared to control cells. In contrast, treatment with thapsigargin did not result in significant differences in [Ca2+]i between IgA-IC and control mesangial cells, suggesting that a negligible role of endoplasmic reticulum in the effects of IgA-IC. Using PKC specific antibodies, IgA-IC significantly increased the particulate fraction of PKC-iota of mesangial cells to 141+/-13% of control, without significantly changing the protein content of PKC-alpha, -delta and -lambda in the cytosolic and particulate fractions. In summary, IgA-IC inhibits the contractile responses of cultured mesangial cells to agonists by inhibiting the activation of PKC and [Ca2+]i.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcium; Cell Size; Cells, Cultured; Enzyme Activators; Enzyme Inhibitors; Glomerular Mesangium; Immunoblotting; Immunoglobulin A; Ionomycin; Ionophores; Isoenzymes; Mice; Muscle Contraction; Phenanthridines; Protein Kinase C; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.

Topics: Alkaloids; Benzophenanthridines; Calcineurin; CD4-Positive T-Lymphocytes; CD40 Antigens; CD40 Ligand; Cyclosporine; Enzyme Inhibitors; Humans; Ionomycin; Jurkat Cells; Lymphoma, T-Cell; Phenanthridines; Phosphoric Monoester Hydrolases; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Up-Regulation

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cells, Cultured; Enzyme Activation; Inositol 1,4,5-Trisphosphate; Ionomycin; Male; Naphthalenes; Phenanthridines; Phorbol Esters; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Submandibular Gland; Thapsigargin

We have previously demonstrated an obligatory requirement for intracellular reactive oxygen species generation during T lymphocyte activation, and have proposed that intracellular reactive oxygen species may act as signalling agents in the regulation of certain cellular processes, for example, during cell cycle entry. To test this hypothesis, we have been interested to determine which, if any, cell cycle entry events are affected by oxidative signalling. In earlier studies, we have identified the transcription factors NF-kappa B and AP-1 as molecular targets for oxidative signalling processes during cell cycle entry, and have shown that oxidative signalling is involved in the regulation of early changes in gene expression during the G0 to G1 phase transition. To extend these initial observations, we have examined the effect of antioxidant treatment on the activity of the mitogen-activated protein kinases erk1 and erk2, as members of a signal transduction pathway known to directly regulate transcription factor function. Using as a probe cysteamine, an aminothiol compound with both antioxidant and antiproliferative activity, we have identified erk2, a key element of the MAP kinase pathway, as being responsive to oxidative signalling during lymphocyte activation. These observations provide further evidence to suggest a role for intracellular oxidant generation as a regulatory mechanism during cell cycle entry, and establish a link between oxidative signalling and other aspects of the intracellular signalling network that is activated in response to mitogenic stimulation.

Topics: Alkaloids; Antioxidants; Benzophenanthridines; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cyclosporine; Cysteamine; DNA-Binding Proteins; Humans; Interphase; Ionomycin; Jurkat Cells; Lymphocyte Activation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Monocytes; Phenanthridines; Reactive Oxygen Species; Signal Transduction; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factor AP-1

The cellular regulation mechanism of Na-K-Cl cotransport was studied in dispersed acinar cells of the guinea pig nasal gland by a microfluorimetric imaging method using the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Addition of 1 micron acetylcholine (ACh) induced an immediate increase in intracellular Na+ concentration ([Na+]i) by 36.7 +/- 9.9 mM, which was almost completely abolished by the addition of atropine. The increased [Na+]i after cholinergic stimulation was due to the external (Cl-)-dependent cotransport system (about 80% of the total Na+ influx) and the dimethyl amiloride-sensitive (Na+)-H+ exchange system (of about 20%). The ACh-induced increase in [Na+]i was dependent on extracellular Ca2+ and was prevented by pretreatment with 8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate or O-O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid tetraacetoxymethylester. Addition of 1 microns ionomycin mimicked the ACh-induced increase in [Na+]i which was dependent on external Cl-. Moreover, both a calmodulin antagonist trifluoperazine and a myosin light chain kinase inhibitor ML-7 reduced the ACh-induced response in [Na+]i. However, the following treatment did not affect the basal [Na+]i nor the ACh-induced increase in [Na+]i: (i) addition of dibutyryl cAMP, 8-Br-cGMP, or phorbol 12-myristate 13-acetate, (ii) pretreatment of protein kinase inhibitors, H-89, H-8, H-7 or chelerythrine, (iii) prevention of cytosolic Cl- efflux by the addition of diphenylamine-2-carboxylic acid or, (iv) prevention of cytosolic K+ efflux by the addition of charybdotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Biological Transport; Bucladesine; Bumetanide; Calcium; Calmodulin; Carrier Proteins; Charybdotoxin; Cholinergic Fibers; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Exocrine Glands; Furosemide; Guinea Pigs; Ionomycin; Ionophores; Isoquinolines; Nasal Cavity; Phenanthridines; Protein Kinase Inhibitors; Protein Kinases; Second Messenger Systems; Signal Transduction; Sodium; Sodium-Potassium-Chloride Symporters; Sulfonamides; Tetradecanoylphorbol Acetate; Trifluoperazine; Vasoactive Intestinal Peptide