u-0126 and chelerythrine

Angiotensin II (AngII) stimulation of water and NaCl intake is a classic model of the behavioural effects of hormones. In vitro studies indicate that the AngII type 1 (AT(1)) receptor stimulates intracellular pathways that include protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation. Previous studies support the hypotheses that PKC is involved in AngII-induced water, but not NaCl intake and that MAP kinase plays a role in NaCl consumption, but not water intake, after injection of AngII. The present experiments test these hypotheses in rats using central injections of AngII in the presence or absence of a PKC inhibitor or a MAP kinase inhibitor. Pretreatment with the PKC inhibitor chelerythrine attenuated AngII-induced water intake, but NaCl intake was unaffected. In contrast, pretreatment with U0126, a MAP kinase inhibitor, had no effect on AngII-induced water intake, but attenuated NaCl intake. These data support the working hypotheses and significantly extend our earlier findings and those of others. Perhaps more importantly, these experiments demonstrate the remarkable diversity of peptide receptor systems and add support for the surprising finding that intracellular signalling pathways can have divergent behavioural relevance.

Topics: Angiotensin II; Animals; Benzophenanthridines; Butadienes; Dose-Response Relationship, Drug; Drinking Behavior; Male; Mitogen-Activated Protein Kinase Kinases; Nitriles; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Angiotensin; Signal Transduction; Sodium Chloride; Time Factors; Vasoconstrictor Agents; Water

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Topics: Alkaloids; Amino Acid Sequence; Benzophenanthridines; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth Mucosa; Mouth Neoplasms; Nitriles; Phenanthridines; Protein Kinase C; Protein Kinase Inhibitors

Lead (Pb) increases lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), lipid peroxidation (LPO), and liver damage. In this study, we investigated the role of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) and the causal relationships between TNF-alpha, NO, and LPO in Pb-increased LPS-induced liver damage in rats. Treatment with PKC and p42/44 MAPK inhibitors significantly reduced Pb + LPS-induced NO, TNF-alpha, LPO, and liver damage, which was revealed by elevated serum levels of aspartate aminotransferase and alanine aminotransferase. Pb + LPS coexposure significantly increased phosphorylation of p42/44 MAPK and TNF-alpha expression in peripheral blood cells; however, exposure to Pb + LPS did not induce TNF-alpha, NO, or LPO production and p42/44 MAPK activation in the liver. Pentoxifylline, a TNF-alpha inhibitor, also reduced liver damage but did not alter NO or LPO in Pb + LPS-treated rats. Thus, Pb increased LPS-induced liver damage through PKC and p42/44 MAPK modulation of TNF-alpha and oxidative stress, but modulation of TNF-alpha did not affect NO or LPO in rats.

Topics: Alanine Transaminase; Alkaloids; Animals; Aspartate Aminotransferases; Benzophenanthridines; Butadienes; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Lead; Lipid Peroxidation; Lipopolysaccharides; Liver; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide; Nitriles; Oxidative Stress; Phenanthridines; Phosphorylation; Protein Kinase C; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Cell Movement; Chromones; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, Dominant; Genetic Vectors; Immunoblotting; Isometric Contraction; Mice; Morpholines; Myosin Light Chains; Nitriles; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Plasmids; Precipitin Tests; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; RNA Interference; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection

Na+ channels in the dendrites of rat CA1 pyramidal neurons display a profound activity-dependent inactivation, termed slow inactivation, that limits excitability in the dendrites even at low physiological rates of firing. The magnitude of this slow inactivation is powerfully modulated by a protein kinase C-dependent process. Because activation of kinases is a rapid and common feature of a number of seizure models, we hypothesized that a loss of slow inactivation of Na+ channels might exacerbate other changes in excitability. Thus, we observed the effects of a brief (5 min) chemical convulsant treatment on Na+ currents and action potentials in hippocampal slices. We found that slow inactivation decreased significantly and remained decreased for at least 30 min after return to control conditions. Pretreatment with either chelerythrine, a protein kinase C inhibitor, or U0126, a mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitor, blocked this reduction of slow inactivation. These results demonstrate that a brief period of hyperexcitability leads to a rapid, protein kinase-dependent loss of slow inactivation of Na+ channels that would contribute to and perhaps prolong the hyperexcitable state.

Topics: Action Potentials; Alkaloids; Animals; Benzophenanthridines; Butadienes; Convulsants; Dendrites; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Male; Nitriles; Patch-Clamp Techniques; Phenanthridines; Phosphotransferases; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Sodium Channels; Time Factors

A number of different agents, such as growth factors, cytokines and phorbol esters have been shown to modulate trabecular meshwork cell function. These studies were designed to evaluate the role extracellular signal-regulated kinase (ERK) pathway plays in mediating the responses to platelet-derived growth factor-BB (PDGF-BB) and phorbol 12-myristate 13-acetate (PMA) in trabecular meshwork cells. The human trabecular meshwork cell line, HTM-3, and the bovine trabecular meshwork (BTM) cells were treated with either PDGF-BB or PMA and the activation of ERK 1/2 evaluated. The effects of the MAP kinase kinase (MEK) inhibitor U0126, and the PKC inhibitor chelerythrine on ERK 1/2 were also determined. In a separate group of experiments, cells were treated with PDGF-BB or PMA and the secretion of matrix metalloproteinase-2 (MMP-2) evaluated. The addition of PDGF-BB or PMA produced time- and dose-dependent activation of ERK 1/2. Pretreatment with U0126 or chelerythrine significantly reduced ERK 1/2 activation induced by PDGF-BB or PMA. The addition of PDGF-BB or PMA stimulated the secretion of MMP-2. This secretory response was inhibited by pretreatment with the MEK inhibitor U0126. In trabecular meshwork cells, PDGF-BB and PMA activate ERK 1/2 by a PKC-dependent mechanism. Activation of ERK 1/2 by these agents in trabecular meshwork cells leads to the secretion of MMP-2. These studies provide evidence that ERK pathway is an important mechanism for integrating various signals that regulate trabecular function.

Topics: Alkaloids; Animals; Benzophenanthridines; Butadienes; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Humans; Immunoblotting; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phenanthridines; Precipitin Tests; Proto-Oncogene Proteins c-sis; Tetradecanoylphorbol Acetate; Trabecular Meshwork

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Amides; Animals; Benzamides; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Indoles; Inhibitory Concentration 50; Isoquinolines; Lithium; Magnesium; Nitriles; Phenanthridines; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Pyridines; Sirolimus; Substrate Specificity; Sulfonamides