calyculin-a and chelerythrine

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

Topics: Alkaloids; Animals; Apoptosis; Benzophenanthridines; Cardiomegaly; Cardiomyopathy, Dilated; Caspase 3; Caspases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Genes, Dominant; Heart Ventricles; Marine Toxins; Mice; Mice, Transgenic; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Organ Specificity; Oxazoles; Phenanthridines; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Transduction, Genetic

The eel intestinal epithelium responds to an acute hypertonic challenge by a biphasic increase of the rate of Cl(-) absorption (measured as short circuit current, Isc, and creating a negative transepithelial potential, V(te), at the basolateral side of the epithelium). While the first, transient phase is bumetanide-insensitive, the second, sustained phase is bumetanide-sensitive, reflecting activation of the apically located Na(+)-K(+)-2Cl(-) (NKCC) cotransporter, which correlates with the cellular RVI response. Here, we investigated the involvement of the cytoskeleton and of serine/threonine phosphorylation events in the osmotic stress-induced ion transport in the eel intestinal epithelium, focusing on the sustained RVI phase, as well as on the previously uncharacterized response to hypotonic stress. The study was carried out using confocal laser scanning microscopy, a quantitative F-actin assay, and transepithelial electrophysiological measurements (V(te) and Isc) in Ussing chambers. Hypertonic stress did not detectably alter either net F-actin content or F-actin organization. In contrast, a brief exposure to hypotonic stress decreased the total cellular F-actin content in eel intestinal epithelium by about 15%, detectable morphologically mainly as a decrease in the intensity of the apical brush border F-actin labeling.The bumetanide-sensitive response of V(te) and Isc to hypertonicity was potently inhibited by treatment with either cytochalasin, latrunculin A, colchicine, the protein kinase C (PKC) inhibitor chelerythrine, the myosin light chain kinase (MLCK) inhibitor ML-7, or the serine/threonine protein phosphatase inhibitor Calyculin A, but was unaffected by the PKA inhibitor H-89. The electrophysiological response of the epithelium to hypotonic stress was characterized by a sustained decrease of V(te) and Isc, which was smaller and recovered faster in the presence of either cytochalasin, latrunculin A, or colchicine. It is concluded that in eel intestinal epithelium, the changes in ion transport in response to both hyper- and hypotonic stress require the integrity of both F-actin and microtubules. In addition, the shrinkage-induced activation of NKCC appears to require the activity of both PKC and MLCK. It is suggested that NKCC regulation by hypertonic stress involves an interaction between the cytoskeleton and protein phosphorylation events.

Topics: Actins; Alkaloids; Anguilla; Animals; Azepines; Benzophenanthridines; Bridged Bicyclo Compounds, Heterocyclic; Bumetanide; Cell Size; Colchicine; Cytochalasins; Cytoskeleton; Enzyme Inhibitors; Hypertonic Solutions; Hypotonic Solutions; Intestinal Mucosa; Ion Transport; Isoquinolines; Marine Toxins; Microtubules; Naphthalenes; Osmotic Pressure; Oxazoles; Phenanthridines; Phosphorylation; Protein Serine-Threonine Kinases; Sulfonamides; Thiazoles; Thiazolidines

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

Because extensive kinase inhibition during anoxia has previously been reported, we investigated the role of kinase inhibition in anoxic cell injury by studying the effects of kinase inhibitors on a membrane-microvillar cytoskeleton linker protein, ezrin, in rabbit renal proximal tubules. Like anoxia, kinase inhibitors caused ezrin dephosphorylation in a dose-dependent manner under normoxia. The kinase inhibitor chelerythrine also induced ezrin dissociation from the cytoskeleton, i.e., causing it to lose its membrane-cytoskeleton linker function. Blockage of kinase inhibitor-induced ezrin dephosphorylation by a phosphatase inhibitor, calyculin A, ameliorated ezrin dissociation. Stimulation of the kinase during anoxia did not improve ezrin phosphorylation, suggesting that anoxia-induced kinase inhibition might be due to the lack of the substrate ATP. Finally, in vitro study of ezrin phosphatase revealed no increase in its activity during anoxia, suggesting the principal role of kinase inhibition in the loss of the linker function of ezrin during anoxia. Our results provide, for the first time at the molecular level, a mechanistic insight into anoxic cell injury caused by unopposed phosphatase action.

Topics: Alkaloids; Animals; Benzophenanthridines; Cytoskeletal Proteins; Enzyme Inhibitors; Female; Hypoxia; Kidney Tubules, Proximal; Marine Toxins; Oxazoles; Phenanthridines; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Protein Kinase C; Rabbits; Reference Values

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