rottlerin and chelerythrine

Topics: Acetophenones; Adaptation, Physiological; Animals; Benzophenanthridines; Benzopyrans; Cell Hypoxia; Cytoprotection; Gene Expression Regulation; Genistein; Hypoxia; L-Lactate Dehydrogenase; Male; Myocytes, Cardiac; Oxygen; Phosphatidylinositol 3-Kinases; Primary Cell Culture; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Wortmannin

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

Topics: Acetylcholinesterase; Amyloid beta-Peptides; Benzophenanthridines; Binding Sites; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Humans; Isoquinolines; Kinetics; Molecular Docking Simulation; Structure-Activity Relationship

Activation of protein kinase C (PKC) has been implicated in the protection of ischemic preconditioning (IPC), but the exact role of PKC in early and late hepatic IPC is still unclear. The present study was conducted in order to investigate the differential role of PKC during early and late hepatic IPC. Rats were subjected to 90 min of partial hepatic ischemia followed by 3 (early IPC) and 24 h (late IPC) of reperfusion. IPC was induced by 10 min of ischemia following 10 min of reperfusion prior to sustained ischemia, and chelerythrine, a PKC inhibitor, was injected 10 min before IPC (5 mg/kg, i.v.). Chelerythrine abrogated the protection of early IPC, as indicated by increased serum aminotransferase activities and decreased hepatic glutathione content. While the IPC-treated group showed a few apoptotic cell deaths during both phases, chelerythrine attenuated these changes only at late IPC and limited IPC-induced inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) overexpression. Membrane translocation of PKC-δ and -ε during IPC was blocked by chelerythrine. Our results suggest that PKC might play a differential role in early and late IPC; activation of PKC-δ and -ε prevents necrosis in early IPC through preservation of redox state and prevents apoptosis in late IPC with iNOS and HO-1 induction. Therefore, PKC represents a promising target for hepatocyte tolerance to ischemic injury, and understanding the differential role of PKC in early and late IPC is important for clinical application of IPC.

Topics: Acetophenones; Animals; Apoptosis; Benzophenanthridines; Benzopyrans; Cytochromes c; Heme Oxygenase-1; Ischemic Preconditioning; Liver Diseases; Male; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Rats; Reperfusion Injury

Neuromedin S (NMS), a peptide structurally related to NMU, has been identified in the mammalian heart tissues. However to date, any role of NMS in cardiomyocytes and the relevant mechanisms still remain unknown. In this study, we identified a novel functional role of NMS in modulating L-type Ca(2+) channels in adult rat ventricular myocytes, in which NMU type 2 receptors (NMUR2), but not NMUR1, are endogenously expressed. We found that NMS at 0.1 µM reversibly increased I(Ba) by ~29.7%. Intracellular infusion of GDP-β-S or a selective antibody raised against the G(i)-protein blocked the stimulatory effects of NMS. The classical and novel protein kinase C (nPKC) antagonist calphostin C or chelerythrine chloride, as well as the phospholipase C (PLC) inhibitor U73122, abolished NMS responses, whereas a classical PKC antagonist Gö6976 or a PKA antagonist PKI 5-24 had no such effects. Pretreatment of cells with PKC-δ specific inhibitor rottlerin or intracellular application of a PKC-δ-derived inhibitory peptide, δV1-1, abolished NMS responses, while an inactive control peptide had no effects. In summary, NMS acting through NMUR2 increases I(Ba) via a G(i)α-protein-dependent PKC-δ pathway in rat ventricular myocytes.

Topics: Acetophenones; Animals; Benzophenanthridines; Benzopyrans; Calcium Channels, L-Type; Carbazoles; Cells, Cultured; Electrophysiological Phenomena; Estrenes; GTP-Binding Protein alpha Subunits, Gi-Go; Heart Ventricles; Male; Muscle Cells; Neuropeptides; Oligopeptides; Peptide Fragments; Protein Kinase C-delta; Pyrrolidinones; Rats; Receptors, Neurotransmitter; Type C Phospholipases

We have previously showed that a phospholipase A₂ isolated from Lachesis muta snake venom and named LM-PLA₂-I displayed particular biological activities, as hemolysis, inhibition on platelet aggregation, edema induction and myotoxicity. In the present work, we evaluated the effect of LM-PLA₂-I on the survival of axotomized rat retinal ganglion cells kept in vitro, as well as its mechanism of action. Our results clearly showed that treatment with LM-PLA₂-I increased the survival of ganglion cells (100% when compared to control cultures) and the treatment of LM-PLA₂-I with p-bromophenacyl bromide abolished this effect. This result indicates that the effect of LM-PLA₂-I on ganglion cell survival is entirely dependent on its enzymatic activity and the generation of lysophosphatidylcholine (LPC) may be a prerequisite to the observed survival. In fact, commercial LPC mimicked the effect of LM-PLA₂-I upon ganglion cell survival. To investigate the mechanism of action of LM-PLA₂-I, cultures were treated with chelerythrine chloride, BAPTA-AM, rottlerin and also with an inhibitor of c-junc kinase (JNKi). Our results showed that rottlerin and JNK inhibitor abolished the LM-PLA₂-I on ganglion cell survival. Taken together, our results showed that LM-PLA₂-I and its enzymatic product, LPC promoted survival of retinal ganglion cells through the protein kinase C pathway and strongly suggest a possible role of the PLA₂ enzyme and LPC in controlling the survival of axotomized neuronal cells.

Topics: Acetonitriles; Acetophenones; Animals; Benzophenanthridines; Benzopyrans; Benzothiazoles; Cell Survival; Cells, Cultured; Crotalid Venoms; Egtazic Acid; Enzyme Inhibitors; Lysophosphatidylcholines; Phospholipases A2; Rats; Retinal Ganglion Cells

Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kgH(2)O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 μM chelerythrine or 50 μM rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined.

Topics: Acetophenones; Animals; Benzophenanthridines; Benzopyrans; Kidney Tubules, Collecting; Male; Membrane Transport Proteins; Osmolar Concentration; Permeability; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Rats, Sprague-Dawley; Urea; Urea Transporters; Vasopressins

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

Topics: Acetophenones; Animals; Benzophenanthridines; Benzopyrans; Cell Line; Chlorocebus aethiops; Diptera; Drug Evaluation, Preclinical; Enzyme Inhibitors; Fluorescent Antibody Technique; HEK293 Cells; Humans; Immunoblotting; Insecta; Protein Kinase C-epsilon; Rift Valley fever virus; RNA Interference; Vero Cells

Previous investigations of retrograde survival signaling by nerve growth factor (NGF) and other neurotrophins have supported diverse mechanisms, but all proposed mechanisms have in common the generation of survival signals retrogradely transmitted to the neuronal cell bodies. We report the finding of a retrograde apoptotic signal in axons that is suppressed by local NGF signaling. NGF withdrawal from distal axons alone was sufficient to activate the pro-apoptotic transcription factor, c-jun, in the cell bodies. Providing NGF directly to cell bodies, thereby restoring a source of NGF-induced survival signals, could not prevent c-jun activation caused by NGF withdrawal from the distal axons. This is evidence that c-jun is not activated due to loss of survival signals at the cell bodies. Moreover, blocking axonal transport with colchicine inhibited c-jun activation caused by NGF deprivation suggesting that a retrogradely transported pro-apoptotic signal, rather than loss of a retrogradely transported survival signal, caused c-jun activation. Additional experiments showed that activation of c-jun, pro-caspase-3 cleavage, and apoptosis were blocked by the protein kinase C inhibitors, rottlerin and chelerythrine, only when applied to distal axons suggesting that they block the axon-specific pro-apoptotic signal. The rottlerin-sensitive mechanism was found to regulate glycogen synthase kinase 3 (GSK3) activity. The effect of siRNA knockdown, and pharmacological inhibition of GSK3 suggests that GSK3 is required for apoptosis caused by NGF deprivation and may function as a retrograde carrier of the axon apoptotic signal. The existence of a retrograde death signaling system in axons that is suppressed by neurotrophins has broad implications for neurodevelopment and for discovering treatments for neurodegenerative diseases and neurotrauma.

Topics: Acetophenones; Animals; Apoptosis; Axonal Transport; Axons; Benzophenanthridines; Benzopyrans; Caspase 3; Cell Survival; Cells, Cultured; Colchicine; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Nerve Growth Factor; Nerve Growth Factors; Neurons; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Receptor, trkA; RNA, Small Interfering; Signal Transduction; Sympathetic Nervous System

Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.

Topics: Acetophenones; Aldosterone; Animals; Benzophenanthridines; Benzopyrans; Biological Transport; Blotting, Western; Cell Line; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epithelial Sodium Channels; Immunoprecipitation; Kidney Cortex; Kidney Tubules, Collecting; Mice; Microscopy, Confocal; Protein Kinase C; RNA Interference; Spironolactone

It has previously been shown that inhibitors of protein kinase C (PKC) attenuate retinal glutamate uptake in situ. The aim of the current study was to determine whether PKCdelta-mediated inhibition differentially reduces the transport of glutamate into retinal Müller cells when compared with retinal neurons. The influence of two different types of PKC inhibitors on the uptake of [3H]D-aspartate was therefore compared in the intact retina, mixed retinal cultures, and Müller cell-enriched retinal cultures. It was found that 25 microM of the pan-isoform PKC inhibitor, chelerythrine, reduced [3H]D-aspartate uptake by 78%, 71%, and 68% in isolated retinas, mixed neuronal/glial cultures, and Müller cell-enriched cultures, respectively. Importantly, 20 microM of the PKCdelta-selective inhibitor rottlerin also reduced the uptake of D-aspartate to similar extents in all three systems, and the reductions were statistically similar to those found for the pan-specific PKC inhibitor. Neither pan-isoform nor PKCdelta-selective activators stimulated glutamate uptake in either culture system or the intact retina. The current results suggest that specific PKC inhibitors are quantitatively similar in reducing the uptake of glutamate into retinal neurons and Müller cells.

Topics: Acetophenones; Alkaloids; Animals; Aspartic Acid; Benzophenanthridines; Benzopyrans; Cell Culture Techniques; Enzyme Inhibitors; Excitatory Amino Acid Transporter 1; Glutamic Acid; Immunoenzyme Techniques; Neuroglia; Protein Kinase C-delta; Rats; Retina

Using non-selective and selective protein kinase C (PKC) epsilon and delta isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters pPKCepsilon and/or pPKCdelta in cytosolic and mitochondrial fractions.. Male Sprague-Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg)+/-postcon; Rottlerin (PKCdelta inhibitor, 0.3 mg/kg)+/-postcon; KIE1-1 (PKCepsilon inhibitor, 3.8 mg/kg)+/-postcon. A subset of rats was employed to assess pPKCepsilon and/or pPKCdelta in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts.. Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39+/-2% vs. 53+/-1% in control, P<0.001). Treatment with chelerythrine alone or the PKCepsilon antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKCepsilon antagonism (50+/-2% and 50+/-1%, respectively, P<0.002). Inhibition of PKCdelta reduced infarct size to values comparable to that in postcon group (36+/-3% vs. 39+/-2%). However, postcon in the presence of PKCdelta inhibitor did not enhance the infarct-sparing effects (38+/-2%). In addition, pPKCepsilon in postcon hearts was significantly higher in the total cell homogenate (10338+/-1627 vs. 4165+/-608 in Isch/RP, arbitrary units), and pPKCdelta translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP.. These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Blotting, Western; Cytosol; Male; Mitochondria, Heart; Models, Animal; Myocardial Infarction; Myocardial Reperfusion; Myocardium; Necrosis; Peptides; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Random Allocation; Rats; Rats, Sprague-Dawley; Signal Transduction

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Topics: Acetamides; Acetophenones; Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Benzopyrans; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Histones; Humans; Indomethacin; Kinetics; Phenanthridines; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Pyrans; Spiro Compounds; Tetradecanoylphorbol Acetate; 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

Inhibition of protein kinase C (PKC) antagonizes ischemic preconditioning of myocardium. Opening of mitochondrial adenosine triphosphate (ATP)-dependent potassium (mitoK(ATP)) channels and subsequent oxidation of mitochondria are known to contribute to ischemic preconditioning. We therefore tested the effects of PKC inhibitors on flavoprotein oxidation, measured by flavoprotein fluorescence, as an index of mitoK(ATP) activity in ventricular myocytes from guinea pigs. The PKC inhibitors chelerythrine (1 and 5 microM) and bisindolylmaleimide (100 and 400 nM) strongly increased flavoprotein oxidation in a dose-dependent manner. Specific inhibition of PKC-delta by rottlerin produced persistent flavoprotein oxidation. Inhibition of the production of inositol (1,4,5)-triphosphate by neomycin (0.5 mM) abolished chelerythrine- but not rottlerin-induced flavoprotein oxidation. Inhibition of PKC promotes flavoprotein oxidation via production of inositol (1,4,5)-triphosphate, possibly through the PKC-delta isoform. We speculate that although a certain degree of mitochondrial flavoprotein oxidation causes cardioprotective effects, excessive and/or persistent oxidation abolishes any beneficial actions. Instead of a simple mediator, PKC may act as a regulator of the mitoK(ATP) channel to prevent excessive mitochondrial oxidation.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Calibration; Cell Separation; Female; Flavoproteins; Guinea Pigs; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate; Male; Maleimides; Mitochondria, Heart; Myocytes, Cardiac; Neomycin; Oxidation-Reduction; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Protein Synthesis Inhibitors; Spectrometry, Fluorescence

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Bicarbonates; Bile; Bile Ducts, Intrahepatic; Chelating Agents; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dopamine; Dopamine Agonists; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Epithelium; Liver; Male; Phenanthridines; Protein Kinase C; Quinolines; Rats; Rats, Inbred F344; Receptors, Dopamine D2; Secretin

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2). Herein, we determined whether some kinases of this pathway connect BDNF to the cyclic AMP response element -binding protein (CREB), a transcription factor also involved in plasticity and survival. Crude cytosolic and nuclear fractions were prepared from hippocampal slices of adult rat, and then kinase involvement in CREB phosphorylation was studied with a combination of pharmacologic inhibition and antibody depletion. In addition, the regional localization of this signaling pathway was immunohistochemically investigated. We show that: (i). the BDNF-stimulated CaMKII cascade phosphorylates the key positive regulatory site of CREB via its end MAPKAPK-2 component; (ii). this process appears to be highly localized in the outermost cell layer of the dentate gyrus. The present findings suggest that CaMKII is involved in neurotrophic-dependent activation of CREB in the dentate gyrus. Such a signaling process could be important for controlling synaptic plasticity in this major area for the afferent inputs to the hippocampal formation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Alkaloids; Androstadienes; Animals; Antibodies; Benzophenanthridines; Benzopyrans; Blotting, Western; Brain-Derived Neurotrophic Factor; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Carbazoles; Cell Nucleus; Chromones; Cyclic AMP Response Element-Binding Protein; Cytosol; Enzyme Inhibitors; Flavonoids; Hippocampus; Imidazoles; Immunohistochemistry; In Vitro Techniques; Indoles; Intracellular Signaling Peptides and Proteins; Male; Morpholines; Naphthalenes; Phenanthridines; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Pyridines; Pyrroles; Rats; Signal Transduction; Time Factors; Tyrphostins; Wortmannin

We recently demonstrated that oral administration of geranylgeranylacetone (GGA), an antiulcer agent, induces heat-shock protein 72 (HSP72) in the rat heart and renders cardioprotection against ischemia/reperfusion injury. However, the signaling pathways remain to be elucidated. The present study tested the hypothesis that oral GGA would activate protein kinase C (PKC), leading to the phosphorylation and translocation of heat-shock factor 1 (HSF1), and thus, promote the expression of HSP72 protein. Rats were classified into four groups: a control (CNT) group (vehicle administration), a GGA group (GGA 200 mg/kg administration), a chelerythrine (CHE)-CNT group (pretreated with intravenous (i.v.) injection of 5 mg/kg CHE before vehicle administration), and a CHE-GGA group (pretreated with CHE before GGA administration). After 24 h administration, oral GGA-induced overexpression of HSP72, increased amount of the phosphorylated form of HSF1 in the nucleus, produced heat-shock element-specific DNA-HSF1 complex, and caused translocation of protein kinase C (PKC)delta, all of which were prevented by pretreatment with CHE. GGA also increased the PKC activity in a particulate fraction, which was prevented by pretreatment with rottlerin, a specific inhibitor of PKCdelta. Isolated-perfused heart experiments revealed that the better functional recovery observed in the GGA group during the reperfusion period following the 20 min of no-flow global ischemia, compared with the CNT group, was abolished by pretreatment with CHE. These results suggest that activation of PKC (translocation of PKCdelta), which primes the phosphorylation of HSF1, plays an essential role in the cardiac overexpression of HSP72 by GGA that leads to cardioprotection.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Diterpenes; DNA-Binding Proteins; Heart; Heat Shock Transcription Factors; Heat-Shock Proteins; Humans; Isoenzymes; Myocardial Reperfusion; Myocardium; Phenanthridines; Phosphorylation; Protein Kinase C; Rats; Transcription Factors

The aim of the present study was to compare the potency of a series of widely used PKC inhibitors acting either at the regulatory (NPC 15437, tamoxifen and D-sphingosine) or at the catalytic domain (Ro 32-0432, chelerythrine and rottlerin) on individual mammalian PKC isoforms of the classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC families, using the yeast phenotypic assay, in order to determine their isoform-selectivity. The PKC inhibitors studied presented differences in their ability to reduce the effect of the appropriate PKC activator (estimated as EC50 ratios) which was interpreted as an index of PKC inhibitory potency. In general, the more marked inhibition was observed on novel PKC isoforms, particularly on PKC-eta. This study indicates promising isoform-selectivity of some PKC inhibitors, namely NPC 15437 for PKC-eta or rottlerin for both novel PKC isoforms. It also suggests that the PKC domain involved in the inhibition does not seem to be relevant for the potency and isoform-selectivity of PKC inhibitors.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Catalysis; Enzyme Inhibitors; Escherichia coli; Indoles; Isoenzymes; Phenanthridines; Piperidines; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Kinase C-delta; Pyrroles; Saccharomyces cerevisiae; Sphingosine; Tamoxifen; Yeasts

We recently observed that treatment of CYP11B2-expressing COS-1 cells with the broad range kinase inhibitor, staurosporine (STS), strongly inhibited aldosterone biosynthesis, indicating that the activity of a kinase might be a prerequisite for steroid hydroxylase activity. In an attempt to identify such kinases, we measured conversion of 11-deoxycortisol (RSS) and 11-deoxycorticosterone (DOC) by V79MZh11B1 and V79MZh11B2 cells, respectively, in the presence of STS and also after treatment with the kinase inhibitors chelerythrine, rottlerin and Gö 6976. The conversion of both substrates by both cell lines was affected in a selective manner by the kinase inhibitors, suggesting that the activity of the novel PKC-delta and either of conventional PKCs or of PKD alter steroid hydroxylation activity, with their influence depending on both the cytochrome P450 tested and on its steroid substrate.

Topics: Acetophenones; Alkaloids; Benzophenanthridines; Benzopyrans; Carbazoles; Cell Line; Cortodoxone; Desoxycorticosterone; Enzyme Inhibitors; Hydroxylation; Indoles; Phenanthridines; Protein Kinase C; Staurosporine; Steroid Hydroxylases; Transfection

We have previously demonstrated that cardioprotection induced by the infusion of a selective delta1-opioid agonist is mediated by the specific translocation of PKC-delta to the mitochondria in in vivo rat hearts and via opening of the mitochondrial KATP channel. Ischemic preconditioning (IPC) is also thought to involve the translocation of specific isoforms of PKC and KATP channel activation. Therefore, we utilized the PKC-delta selective antagonist, rottlerin, to assess the effect of inhibition of this isozyme on cardioprotection induced by one-cycle of IPC prior to 30 minutes of ischemia and 2 hours of reperfusion. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Non-preconditioned control animals had an IS/AAR of 59.7 +/- 1.6. IPC significantly reduced the extent of myocardial infarction (6.3 +/- 1.4). Rottlerin, 0.3 mg/kg, did not alter IS/AAR in control animals (55.0 +/- 5.6), and had no significant effect on IS/AAR in preconditioned animals (14.4 +/- 3.8). Additionally, we demonstrated, using a luciferase-based assay to determine the rate of ATP synthesis and state of mitochondrial bioenergetics, that IPC preserves ATP synthesis in the ischemic myocardium and that this preservation is attenuated by the isoform non-selective PKC inhibitor, chelerythrine, but not by the delta-selective antagonist, rottlerin. These data suggest that PKC-delta does not play an important role in IPC and that differences in isoform importance are evident during pharmacological versus ischemia-induced preconditioning.

Topics: Acetophenones; Adenosine Triphosphate; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Enzyme Inhibitors; Hemodynamics; Immunohistochemistry; Ischemic Preconditioning, Myocardial; Isoenzymes; Male; Mitochondria, Heart; Myocardial Infarction; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Rats; Rats, Wistar

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, d-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Topics: Acetophenones; Alkaloids; Amino Acid Transport System X-AG; Animals; Aspartic Acid; Benzophenanthridines; Benzopyrans; Biological Transport; Blotting, Western; Carbazoles; Enzyme Inhibitors; Glutamic Acid; Immunohistochemistry; In Vitro Techniques; Indoles; Isoenzymes; Phenanthridines; Protein Kinase C; Rats; Rats, Inbred Strains; Retina

Stimulation of the delta(1)-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after delta-opioid receptor stimulation with TAN-67 or D-Ala(2)-D-Leu(5)-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 +/- 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 +/- 3.9 and 36.7 +/- 4.7, respectively). The delta(1)-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 +/- 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 +/- 3.2) and TAN-67-induced cardioprotection (55.4 +/- 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 +/- 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-alpha to the sarcolemma, PKC-beta(1) to the nucleus, PKC-delta to the mitochondria, and PKC-epsilon to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-delta selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 +/- 4.8) and PKC-delta translocation without affecting the translocation of PKC-alpha, -beta(1), or -epsilon. These results suggest that PKC-delta is a key second messenger in the cardioprotective effects of delta(1)-opioid receptor stimulation in rats.

Topics: Acetophenones; Alkaloids; Analgesics; Animals; Benzophenanthridines; Benzopyrans; Benzylidene Compounds; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Enzyme Inhibitors; Heart Rate; Indoles; Ischemic Preconditioning, Myocardial; Isoenzymes; Male; Maleimides; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Naltrexone; Narcotic Antagonists; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Quinolines; Rats; Rats, Wistar; Receptors, Opioid, delta

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

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Topics: Acetophenones; Alkaloids; Benzophenanthridines; Benzopyrans; Bryostatins; Carbazoles; Enzyme Activation; Enzyme Inhibitors; Indoles; Isoenzymes; Lactones; Macrolides; Maleimides; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; Recombinant Proteins; Schizosaccharomyces; Sphingosine; Tetradecanoylphorbol Acetate; Transformation, Genetic