okadaic-acid and 1-oleoyl-2-acetylglycerol

Lipophosphoglycan (LPG), a major surface molecule from Leishmania donovani, stimulated ornithine decarboxylase (ODC) activity in macrophages in a dose- and time-dependent manner. LPG stimulated the rapid increase in ODC activity within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. However, LPG-induced ODC activity was a transient event because 3 hr after exposure to LPG, no stimulation of ODC activity was detectable. ODC activity appeared to be coupled to the activation of protein kinase C (PKC) in macrophages, as activators of PKC caused a rapid increase in the ODC activity. Macrophages pretreated with LPG for 1 hr became unresponsive to subsequent stimulation by the PKC activators 1-oleoyl-2-acetyl-glycerol and the calcium ionophore A23187. In contrast, the ability of macrophages to express ODC activity in response to the cyclic AMP analogue dibutyryl cyclic AMP was not impaired by LPG.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Anti-Bacterial Agents; Bucladesine; Calcimycin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Glycosphingolipids; Leishmania donovani; Leishmaniasis, Visceral; Lipopolysaccharides; Macrophages; Mice; Okadaic Acid; Ornithine Decarboxylase; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Time Factors

The crucial enzyme in diacylglycerol-mediated signaling is protein kinase C (PKC). In this paper we provide evidence for the existence and role of PKC in maize. A protein of an apparent molecular mass of 70 kDa was purified. The protein showed kinase activity that was stimulated by phosphatidylserine and oleyl acetyl glycerol (OAG) in the presence of Ca2+. Phorbol 12-myristate 13-acetate (PMA) replaced the requirement of OAG. [3H]PMA binding to the 70-kDa protein was competed by unlabeled PMA and OAG but not by 4alpha-PMA, an inactive analog. The kinase phosphorylates histone H1 at serine residue(s), and this activity was inhibited by H-7 and staurosporine. These properties suggest that the 70-kDa protein is a conventional serine/threonine protein kinase C (cPKC). Polyclonal antibodies raised against the polypeptide precipitate the enzyme activity and immunostained the protein on Western blots. The antibodies also cross-reacted with a protein of expected size from sorghum, rice, and tobacco. A rapid increase in the protein level was observed in maize following PMA treatments. In order to assign a possible role of PKC in gene regulation, the nitrate reductase transcript level was investigated. The transcript level increased by PMA, not by 4alpha-PMA treatments, and the increase was inhibited by H-7 but not by okadaic acid. The data show the existence and possible function of PKC in higher plants.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Chromatography, Gel; Diglycerides; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Enzymologic; Histones; Hydrogen-Ion Concentration; Isoenzymes; Kinetics; Lipid Metabolism; Nitrate Reductase; Nitrate Reductases; Okadaic Acid; Phosphatidylserines; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Zea mays

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.

Topics: Animals; Antiemetics; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Calmodulin-Binding Proteins; Calponins; Carcinogens; Carotid Arteries; Chelating Agents; Diglycerides; Egtazic Acid; Electrophoresis; Enzyme Inhibitors; Imidazoles; Microfilament Proteins; Okadaic Acid; Organ Culture Techniques; Peptide Fragments; Phorbol 12,13-Dibutyrate; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Staurosporine; Sulfonamides; Swine; Trifluoperazine; Vasodilator Agents

1. Activation of human D2(s) dopamine receptors with quinpirole (10 nM) inhibits omega-conotoxin GVIa-sensitive, high-threshold calcium currents when expressed in differentiated NG108-15 cells (55% inhibition at +10 mV). This inhibition was made irreversible following intracellular dialysis with the non-hydrolysable guanosine triphosphate analogue GTP-gamma-S (100 microM), and was prevented by pretreatment with pertussis toxin (1 microgram ml-1 for 24 h). 2. Stimulation of protein kinase C with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (100 microM), also attenuated the inhibition of the sustained calcium current but did not affect the receptor-mediated decrease in rate of current activation. Similarly, okadaic acid (100 nM), a protein phosphatase 1/2A inhibitor, selectively occluded the inhibition of the sustained current. 3. The depression of calcium currents by quinpirole (10 nM) was enhanced following intracellular dialysis with 100 microM cyclic adenosine monophosphate (cyclic AMP, 72.8 +/- 9.8% depression), but was not mimicked by the membrane permeant cyclic GMP analogue, Sp-8-bromoguanosine-3',5':cyclic monophosphorothioate (100 microM). 4. Inhibition of calcium currents was only partly attenuated by 100 ms depolarizing prepulses to +100 mV immediately preceding the test pulse. However, following occlusion of the sustained depression with okadaic acid (100 nM) the residual kinetic slowing was reversed in a voltage-dependent manner (P < 0.05). 5. Thus pertussis toxin-sensitive G-proteins liberated upon activation of human D2(short) dopamine receptors inhibited high-threshold calcium currents in two distinct ways. The decrease in rate of calcium current activation involved a voltage-dependent pathway, whereas the sustained inhibition of calcium current involved, in part, the voltage-resistant phosphorylation by cyclic AMP-dependent protein kinases and subsequent dephosphorylation by protein phosphatases 1/2A.

Topics: Calcium; Calcium Channel Blockers; Calcium Channels; Cyclic AMP; Diglycerides; Dopamine Agonists; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Ergolines; Ethers, Cyclic; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Neuroblastoma; Neurons; Okadaic Acid; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Pertussis Toxin; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Phosphatase 1; Quinpirole; Receptors, Dopamine D2; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

Norepinephrine (NE) and gamma-aminobutyric acid (GABA) inhibit N-type calcium channels in embryonic chick sensory neurons. We demonstrate here that the modulatory actions of the two transmitters are mediated through distinct biochemical pathways. Intracellular application of the pseudosubstrate inhibitor for protein kinase C blocks the inhibition produced by NE (and the protein kinase C activator oleoylacetylglycerol), but not that produced by GABA. Calcium current inhibition produced by oleoylacetylglycerol occludes inhibition by subsequent application of NE; GABA-mediated inhibition, however, is not eliminated by prior activation of protein kinase C. These results demonstrate that multiple biochemical pathways converge to control N-type calcium channel function.

Topics: Animals; Calcium; Chick Embryo; Diglycerides; Electric Conductivity; Ethers, Cyclic; Nerve Tissue Proteins; Neurons; Neurotransmitter Agents; Okadaic Acid; Phosphoric Monoester Hydrolases; Protein Kinase C; Second Messenger Systems

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although th

Topics: Adenosine Diphosphate; Alkaloids; Blood Platelets; Diacylglycerol Kinase; Diglycerides; Enzyme Activation; Ethers, Cyclic; Humans; Okadaic Acid; Phosphotransferases; Platelet Aggregation; Protein Kinase C; Pyrimidinones; Serotonin; Staurosporine; Thiazoles; Thrombin; Type C Phospholipases

12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Carcinogens; Dexamethasone; Diglycerides; Drug Synergism; Enzyme Activation; Ethers, Cyclic; Histidine Decarboxylase; Isoquinolines; Mast-Cell Sarcoma; Mice; Okadaic Acid; Phorbol Esters; Phosphoprotein Phosphatases; Piperazines; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In Helix aspersa, activation of the cerebral giant serotonin neurones (GSNs) evokes a biphasic, excitatory synaptic response in the M neurones of the buccal ganglia. Local application of serotonin to the current-clamped M neurones also evokes fast and slow depolarizing responses. The slow response is thought to be dependent on calcium ions, whereas sodium ions have been implicated in the fast response. Here we provide further evidence that the slow response results from an increase in conductance to calcium ions, and show that okadaic acid, an antagonist of protein phosphatases 1 and 2A, potentiates the effect of serotonin, suggesting that the response is phosphorylation dependent. Further, agents known to activate protein kinase C, such as 1-oleoyl-2-acetyl-rac-glycerol and active phorbol esters (but not an inactive one) were found to increase the calcium current (actually carried by barium ions) of the M neurones. Such data suggest that the slow synaptic response mediated by serotonin can occur by activation of protein kinase C and phosphorylation of the affected voltage-sensitive calcium channels, or some closely associated protein(s).

Topics: Animals; Calcium; Cheek; Diglycerides; Electrophysiology; Enzyme Activation; Ethers, Cyclic; Helix, Snails; Neurons; Okadaic Acid; Phorbol Esters; Phosphoric Monoester Hydrolases; Protein Kinase C; Second Messenger Systems; Serotonin