okadaic-acid and chelerythrine

Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.

Topics: Animals; Benzophenanthridines; Biological Transport; Enzyme Inhibitors; Erythrocytes; Fluorides; Genistein; K Cl- Cotransporters; Okadaic Acid; Ouabain; Phosphoprotein Phosphatases; Potassium; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quercetin; Rana temporaria; Staurosporine; Symporters; Tyrphostins

Pulmonary inflammation is a characteristic of many lung diseases. Increased levels of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), have been correlated with lung inflammation. In this study, we demonstrated that various inflammatory agents, including lipopolysaccharide, 12-o-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid and ceramide, were able to induce IL-1beta and TNF-alpha productions in human lung epithelial cells (A-549), fibroblasts (HFL1), and lymphoma cells (U-937). Berberine, the protoberberine alkaloid widely distributed in the plant kingdom, was capable of suppressing inflammatory agents-induced cytokine production in lung cells. Inhibition of cytokine production by berberine was dose-dependent and cell type-independent. Moreover, the suppression of berberine on the cytokine production resulted from the inhibition of inhibitory kappaB-alpha phosphorylation and degradation. In conclusion, our findings suggested the potential role of berberine in the treatment of pulmonary inflammation.

Topics: Alkaloids; Anti-Inflammatory Agents; Benzophenanthridines; Berberine; Cell Survival; Ceramides; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblasts; Humans; Hydrogen Peroxide; I-kappa B Proteins; Inflammation; Interleukin-1beta; Isoquinolines; Lipopolysaccharides; Lung; NF-KappaB Inhibitor alpha; Okadaic Acid; Phosphorylation; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; U937 Cells

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g(Cl)) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g(Cl) increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g(Cl) by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g(Cl) in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g(Cl) that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g(Cl) in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCalpha, epsilon, and isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g(Cl). In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.

Topics: Alkaloids; Animals; Benzophenanthridines; Chlorides; Electrophysiology; Gene Expression Regulation, Enzymologic; Hindlimb Suspension; Insulin-Like Growth Factor I; Isoenzymes; Male; Muscle, Skeletal; Okadaic Acid; Phorbol 12,13-Dibutyrate; Phosphoprotein Phosphatases; Protein Kinase C; Rats; Rats, Wistar; Sarcolemma

Hypotonic shock rapidly inhibits Cl(-) secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKC alpha and beta subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKC alpha and PKC beta subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 micromol l(-1)) inhibited Cl(-) secretion that was high, increased Cl(-) secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Western; Chlorides; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Electrophysiology; Epithelium; Focal Adhesion Protein-Tyrosine Kinases; Fundulidae; Imidazoles; Immunohistochemistry; Ion Channels; Isoquinolines; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Nova Scotia; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phenanthridines; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Saline Solution, Hypertonic; Sulfonamides; Tamoxifen; Water-Electrolyte Balance

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

Topics: Adenosine Triphosphate; Alkaloids; Aminoimidazole Carboxamide; Animals; Antimycin A; Benzophenanthridines; Brefeldin A; Carbazoles; Cell Hypoxia; Cells, Cultured; Connexin 43; Cycloheximide; Deoxyglucose; Flavonoids; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Maleimides; Myocardial Contraction; Myocytes, Cardiac; Okadaic Acid; Ouabain; Phenanthridines; Phosphorylation; Potassium Cyanide; Protein Processing, Post-Translational; Pyridines; Pyrroles; Rats; Recombinant Fusion Proteins; Ribonucleotides; Staurosporine; Tacrolimus; Tetradecanoylphorbol Acetate

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Topics: Adult; Agatoxins; Alkaloids; Benzophenanthridines; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Signaling; Cell Size; Diglycerides; Dimethyl Sulfoxide; Enzyme Inhibitors; Erythrocytes; Humans; Ion Transport; Ionophores; Marine Toxins; Okadaic Acid; Osmotic Fragility; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Protein Kinase C; Spider Venoms; Staurosporine; Tetradecanoylphorbol Acetate

We have recently demonstrated that in quiescent fibroblasts protein kinase C (PKC) epsilon(95) is phosphorylated at Ser(729), Ser(703), and Thr(566) and that upon passage of quiescent cells phosphorylation at Ser(729) is lost, giving rise to PKCepsilon(87). Ser(729) may be rephosphorylated later, suggesting cycling between PKCepsilon(87) and PKCepsilon(95). Here we show that the dephosphorylation at Ser(729) is insensitive to okadaic acid, calyculin, ascomycin C, and cyclosporin A, suggesting that dephosphorylation at this site is not mediated through protein phosphatases 1, 2A or 2B. We demonstrate that this dephosphorylation at Ser(729) requires serum and cell readhesion and is sensitive to rapamycin, PD98059, chelerythrine, and Ro-31-8220. These results suggest that the phosphorylation status of Ser(729) in the hydrophobic domain at Ser(729) is regulated independently of the phosphorylation status of other sites in PKCepsilon, by a mTOR-sensitive phosphatase. The mitogen-activated protein kinase pathway and PKC are also implicated in regulating the dephosphorylation at Ser(729).

Topics: 3T3 Cells; Alkaloids; Animals; Anti-Bacterial Agents; Benzophenanthridines; Blotting, Western; Calcineurin; Cell Adhesion; Cell Line; Culture Media; Cyclosporine; Down-Regulation; Enzyme Inhibitors; Flavonoids; Indoles; Isoenzymes; MAP Kinase Signaling System; Marine Toxins; Methionine; Mice; Models, Biological; Okadaic Acid; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Phosphorylation; Plasmids; Precipitin Tests; Protein Binding; Protein Kinase C; Protein Kinase C-epsilon; Protein Structure, Tertiary; Serine; Signal Transduction; Sirolimus; Tacrolimus; Transfection

By recording the calcium transients evoked by voltage-clamp depolarizing pulse with arsenazo III as a calcium indicator, it has been shown that 1 micromol/l phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) agonist, causes a transient potentiation and then a depression of the calcium transients of twitch muscle fibers in frogs. PDBu also produces an initial translocation and activation of PKC, which is followed by a down-regulation. To find out whether the effect of PDBu on the calcium transients depends on PKC, a correlated study of the effect of phorbol esters on calcium transients and PKC activity was performed. The calcium transients and PKC activity were similarly affected by PDBu in ordinary and cold-accommodated frogs, but the effects occurred more quickly in the latter. However, they still changed in parallel as in ordinary frogs. 1 or 10 micromol/l, 4-alpha-phorbol, a PKC-inactive analogue of phorbol ester, caused a partial depression of the calcium transients in cold-accommodated frogs, while PKC activity was not affected. Moreover, the transient potentiation of the calcium transients induced by 1 micromol/l PDBu could be antagonized by the PKC inhibitors 10 micromol/l chelerythrine chloride or 10 micromol/l polymyxin B (PMB). All these results suggest that: (1) the transient potentiation of calcium transients induced by PDBu is caused by activation of PKC; (2) phorbol ester can depress the calcium transients by a mechanism that is independent of PKC.

Topics: Alkaloids; Animals; Anti-Bacterial Agents; Benzophenanthridines; Calcium Signaling; Cold Temperature; Down-Regulation; Enzyme Inhibitors; In Vitro Techniques; Muscle Contraction; Muscle Fibers, Skeletal; Okadaic Acid; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Polymyxin B; Protein Kinase C; Ranidae

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

Topics: Alkaloids; Animals; Aorta; Benzophenanthridines; Calcium Channels; Calcium Channels, L-Type; Cell Line; Enzyme Activation; Enzyme Inhibitors; Ion Channel Gating; Kinetics; Membrane Potentials; Muscle, Smooth, Vascular; Okadaic Acid; Patch-Clamp Techniques; Phenanthridines; Phorbol 12,13-Dibutyrate; Probability; Protein Kinase C; Rats; Staurosporine

An immobilized hepatocyte preparation was used to show that both vasopressin and glucagon could desensitize the ability of glucagon to increase intracellular cyclic AMP concentrations. This process was not dependent on any influx of extracellular Ca2+ and was not mediated by any rise in the intracellular level of Ca2+. The protein kinase C-selective inhibitors chelerythrine, staurosporine and calphostin C acted as potent inhibitors of the desensitization process but with various degrees of selectivity regarding their ability to inhibit the desensitizing actions of glucagon and vasopressin. The protein phosphatase inhibitor okadaic acid was just as potent as vasopressin and glucagon in causing desensitization. Treatment of hepatocyte membranes with alkaline phosphatase restored to near control levels the ability of glucagon to stimulate adenylate cyclase activity in membranes from both glucagon- and vasopressin-treated (desensitized) hepatocytes. It is suggested that the desensitization of glucagon-stimulated adenylate cyclase activity involves a reversible phosphorylation reaction with the likely target being the glucagon receptor itself.

Topics: Adenylyl Cyclases; Alkaline Phosphatase; Alkaloids; Animals; Arginine Vasopressin; Benzophenanthridines; Cell Adhesion; Cells, Cultured; Collagen; Cyclic AMP; Drug Tolerance; Ethers, Cyclic; Glucagon; Liver; Male; Naphthalenes; Okadaic Acid; Phenanthridines; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Signal Transduction; Staurosporine

In order to characterize the phosphorylation of a approximately 20k M(r) protein present in the mitochondrial fraction of the rat retina, chelerythrine chloride, a well-known protein kinase C inhibitor, was tested for activity. Instead of the expected inhibition of the kinase reaction by chelerythrine the phosphorylation of the approximately 20k M(r) protein was stimulated by a factor of 3 at 150 microM. This unique stimulatory action of chelerythrine could be eliminated by the addition of 10 mM dithiothreitol. A suggested mechanism of action for dithiothreitol in the elimination of the increased phosphorylation of the approximately 20k M(r) protein by chelerythrine is the addition of the thiol group of dithiothreitol to the iminium bond of chelerythrine. Taurine, a known inhibitor of the phosphorylation of retinal proteins was also tested in combination with chelerythrine for its effects on the phosphorylation of the approximately 20k M(r) protein. A non-competitive relationship was observed when chelerytrhine was used as the variable activator and taurine as the fixed inhibitor (30 mM). The stimulatory effect of chelerythrine on the phosphorylation of proteins was not limited to retinal tissue but was also observed in the P2 fraction of brain cortex. Chelerythrine demonstrated only inhibitory effects on the phosphorylation of proteins in a heart mitochondrial fraction.

Topics: Alkaloids; Animals; Autoradiography; Benzophenanthridines; Brain; Dithiothreitol; Dose-Response Relationship, Drug; Electrophoresis; Ethers, Cyclic; Heart; Mitochondria; Okadaic Acid; Phenanthridines; Phosphorylation; Protein Kinase C; Rats; Rats, Wistar; Retina

Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.

Topics: Alkaloids; Animals; Benzophenanthridines; Ethers, Cyclic; Hypoxia; In Vitro Techniques; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Marine Toxins; Okadaic Acid; Oxazoles; Phenanthridines; Phosphates; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase C; Proteins; Rabbits; Reference Values

Aromatic L-amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12-myristate 13-acetate administered intracerebroventricularly transiently increased AAAD activity by 30-50% over control values within approximately 30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of Vmax with little change of Km for either L-3,4-dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester-induced increase of AAAD. Moreover, okadaic acid, a serine/threonine-selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C-mediated pathways modulate AAAD activity in vivo.

Topics: Alkaloids; Animals; Aromatic-L-Amino-Acid Decarboxylases; Benzophenanthridines; Cerebral Ventricles; Corpus Striatum; Ethers, Cyclic; Injections, Intraventricular; Kinetics; Levodopa; Male; Mesencephalon; Mice; Okadaic Acid; Phenanthridines; Phosphoprotein Phosphatases; Protein Kinase C; Protein Phosphatase 1; Pyridoxal Phosphate; Reference Values; Regression Analysis; Tetradecanoylphorbol Acetate