calcimycin and 1-oleoyl-2-acetylglycerol

Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication.

Topics: Adenosine Triphosphatases; Animals; Blood Platelets; Calcimycin; Calcium-Transporting ATPases; Carrier Proteins; Cation Transport Proteins; Cell Membrane; Cyclic AMP; Diglycerides; Electric Conductivity; Enzyme Activation; Fluorescent Antibody Technique; Isoenzymes; Models, Biological; Phosphatidylinositols; Potassium; Protein Kinase C; Purkinje Cells; Rats; Serotonin; Sodium; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Time Factors

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold). Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Humans; Ionophores; Mitogen-Activated Protein Kinases; Neutrophils; Protein Kinase C; Up-Regulation

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

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.

Topics: Animals; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Diglycerides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblast Growth Factor 2; Imidazoles; Interleukin-6; Ionophores; Mice; Mitogen-Activated Protein Kinases; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Pyridines; Time Factors

We have measured the levels of thioredoxin, thioredoxin reductase and glutaredoxin enzyme activity in mouse skin following topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter. The specific activity of thioredoxin and thioredoxin reductase in extracts from normal epidermis increased by 40 and 50%, respectively, after single or multiple application of TPA. Multiple applications (twice per week for 2 weeks) of TPA increased glutaredoxin activity by >300%. Induction of the proteins lasted several days. Other PKC activators, like 12-O-retinoylphorbol 13-acetate, mezerein, 1-oleoyl-2-acetylglycerol and the calcium ionophore A23187, also induced all the enzyme activities. Phorbol and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, weak activators of PKC, selectively induced the thioredoxin system only and did not influence glutaredoxin activity. Multiple applications of TPA to tumor initiated (7,12-dimethyl[a]benzanthracene-treated) skin resulted in elevated levels of both the thioredoxin and glutaredoxin systems when examined 6 days after the last phorbol ester treatment. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activities by TPA and calcium ionophores may play a general role in the epigenetic mechanism of tumor promotion via thiol redox control mechanisms.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcimycin; Calcium; Carcinogens; Cocarcinogenesis; Diglycerides; Diterpenes; Enzyme Activation; Enzyme Induction; Epidermis; Female; Fluocinolone Acetonide; Gene Expression Regulation; Glutaredoxins; Glutathione; Ionophores; Mice; Oxidation-Reduction; Oxidoreductases; Phorbol Esters; Protein Kinase C; Proteins; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Thioredoxin-Disulfide Reductase; Thioredoxins; Tosylphenylalanyl Chloromethyl Ketone; Tretinoin

Protein kinase C (PKC) plays an important role in the differentiation of cells, however, little is known about its relationship to bone metabolism. We have previously demonstrated that staurosporine concentration dependently transformed the cultured rat osteoblasts into stellate cells. In this study, we further investigated the possible mechanisms and significance of the morphological changes of osteoblasts induced by staurosporine. The morphological changes induced by staurosporine were inhibited by microtubule depolymerizers or elevated intracellular calcium, however, actin depolymerizers enhanced the effects of staurosporine. Fluorescence labeling showed that staurosporine caused the dissolution of the actin microfilaments, but left the microtubules and vimentin filaments intact. PKC activators partially antagonized the morphological changes induced by staurosporine. Inhibition of protein kinase A or calmodulin-dependent kinase is less effective in the induction of stellate cell formation. These results suggest that the morphological changes of osteoblasts induced by staurosporine may be partly due to PKC inhibition, but other mechanisms may also be involved.

Topics: Actin Cytoskeleton; Actins; Animals; Animals, Newborn; Calcimycin; Calcium; Carcinogens; Cells, Cultured; Cytochalasins; Diglycerides; Enzyme Inhibitors; Fluorescent Dyes; Ionophores; Microtubules; Osteoblasts; Protein Kinase C; Rats; Rats, Wistar; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate; Vimentin

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 microM or 50 microM OAG for 10 min resulted in meiosis II completion in approximately 80% of instances. By contrast, at a lower concentration (25 microM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 microM OAG, the cytosolic Ca2+ concentration ([Ca2+]i) increased transiently in all the eggs examined, whereas after the addition of 50 microM OAG, [Ca2+]i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca2+ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Female; Ionophores; Maturation-Promoting Factor; Meiosis; Mesothelin; Mice; Microscopy, Fluorescence; Naphthalenes; Oocytes; Parthenogenesis; Protein Kinase C; Spindle Apparatus

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca2+, since exocytosis requires Ca2+ and a potentiation of Ca2+ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca2+ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca2+ accumulation was reduced by inhibitors of phospholipase A2 and voltage-sensitive Ca2+ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A2 was also indicated by the ability of staurosporine to reduce the Ca2+ accumulation induced by arachidonic acid or the phospholipase A2 activator melittin. Similarly, the synergistic facilitation of evoked Ca2+ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca2+ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.

Topics: Acetylcholine; Animals; Arachidonic Acid; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Glutamic Acid; Lipids; Mossy Fibers, Hippocampal; Norepinephrine; Phorbol 12,13-Dibutyrate; Phospholipases A; Phospholipases A2; Protein Kinase C; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Staurosporine

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca(2+)-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.

Topics: Animals; Calcimycin; Calcium; Cell Cycle; Diglycerides; Enzyme Activation; Exocytosis; Female; In Vitro Techniques; Ionophores; Meiosis; Microscopy, Electron; Oocytes; Protein Kinase C; Swine; Tetradecanoylphorbol Acetate

Fertilization involves the production of inositol trisphosphate and diacylglycerol with a subsequent increase in intracellular calcium concentration ([Ca2+]i) and the activation of a calcium-dependent protein kinase, the so-called protein kinase C (PKC). Methods of parthenogenetic activation have focused on this calcium wave which seems to be large enough to generate all the responses associated with fertilization and even finally inducing the activation of PKC activity. The specific stimulation of PKC by phorbol esters in turn elicits [Ca2+]i oscillations although no reports exist claiming that the mere activation of this protein is capable of sustaining embryonic development. In this paper we describe the effect of different calcium ionophores and phorbol esters as parthenogenetic agents on mouse oocytes compared with ethanol as the standard procedure. Phorbol esters (OAG) fail to activate a significant number of oocytes, with very few reaching blastocyst stage. However, when a calcium ionophore (A23187) is added, the percentage of embryos reaching the blastocyst stage increases to such an extent that it is the best chemical method assayed to date. We conclude that incubation with both compounds combined inhibits feed-back processes between the above reactions and so induces a more physiologic parthenogenetic activation.

Topics: Animals; Calcimycin; Diglycerides; Drug Interactions; Embryonic and Fetal Development; Female; Ionophores; Mice; Oocytes; Parthenogenesis

Phorbol 12-myristate 13-acetate, a potential stimulator of protein kinase C (PKC), inhibited taurine uptake in rat astrocytes. This effect was mimicked by 1-oleoyl-2-acetyl-sn-glycerol, an endogenous stimulator of PKC, and by r-59949, an inhibitor of diacylglycerol kinase. Maximal inhibition was obtained at microM phorbol 12-myristate 13-acetate (PMA) after 1 h of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine, an amino acid that shares the same transporter as taurine, was inhibited to a comparable extent. The effect of PMA was potentiated by cotreatment of the cells with thapsigargin or the Ca2+ ionophore A-23187. However, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N1,N1-tetraacetic acid and verapamil did not prevent the PMA effect. Pretreatment of the cells with calmodulin antagonists W-13 or calmidazolium, prevented the PMA-induced inhibition of taurine uptake. This inhibition was not affected by cycloheximide, actinomycin D, colchicine, or cytochalasin D. The Na(+)-to-Cl(-)-to-taurine coupling ratio was unaffected. Dimethyl amiloride, a selective inhibitor of Na+/H+ antiport, was unable to prevent the effects of PMA. These effects were associated with a decrease in the maximal velocity and an increase in the Michaelis-Menten constant.

Topics: Animals; Astrocytes; Binding, Competitive; Biological Transport; Calcimycin; Calcium; Calcium-Transporting ATPases; Calmodulin; Diglycerides; Enzyme Activation; Kinetics; Protein Kinase C; Rats; Taurine; Tetradecanoylphorbol Acetate; Thapsigargin

During the preimplantation period of development, the first cellular polarization and diversification of the mouse embryo occurs. This process starts at the eight-cell stage and is directly driven by the cytoskeleton. Cell polarization finally leads to the first embryonic epithelium, the trophectoderm, characterized by the presence of cytokeratins. It has not been described whether genomic imprinting, an epigenetic modification of certain genes depending on the parent-of-origin, affects preimplantation development. However, implantation is one of the steps in which an exceptionally high mortality rate is observed in mouse parthenogenetic embryos, a phenomenon that may be influenced by a deficiency in trophectoderm differentiation. To assess this possibility we analyzed the expression of various cytoskeletal proteins in late preimplanted embryos. No differences were observed in the expression of microtubules and microfilaments, but surprisingly, the undifferentiated cells of the parthenogenetic inner cell mass showed distinct cytokeratin staining. This anomalous cytoskeleton expression may be considered as one of the earliest manifestation described to date of the effect of genomic imprinting in development.

Topics: Animals; Blastocyst; Calcimycin; Chorionic Gonadotropin; Diglycerides; Female; Fertilization; Gene Expression; Keratins; Mice; Mice, Inbred Strains; Microscopy, Confocal; Oocytes; Parthenogenesis

In the present study, we investigated the effects of different diacylglycerols in comparison with phorbol 12-myristate 13-acetate (PMA) on eicosanoid-independent phospholipase A2 (PLA2) activation in human platelets and neutrophils. Eicosanoid-independent PLA2 activation was measured under conditions where both cyclooxygenase and lipoxygenases were blocked by BW755C. In the presence of PMA (50 nM), the amount of mass arachidonic acid (AA) released represented 400 and 257% of control (without PMA) in A23187-stimulated platelets and neutrophils, respectively, while 1,2-dioctanoylglycerol (1,2-DiC8) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) had increased the eicosanoid-independent AA release by 150 and 117-134% of control, in platelets and neutrophils, respectively. Our results further demonstrate that 1,3-dioctanoylglycerol (1,3-DiC8), a poor activator of protein kinase C (PKC), is nearly as effective as diacylglycerols, such as OAG and 1,2-DiC8 (activators of PKC) in priming PLA2 activation, but is less effective than PMA as a priming agent. However, all three diacylglycerols were less effective than PMA as priming agents. Furthermore, diacylglycerols including 1,3-DiC8 exerted a much greater effect on PLA2 activation in platelets than in neutrophils. Neither 1,3-DiC8 nor 1,2-DiC8 and OAG had any significant priming effect on the accumulation of palmitic and stearic acids, while PMA caused a substantial accumulation of these fatty acids in platelets, but not in neutrophils. We also found that exogenously added OAG underwent significant hydrolysis even in unstimulated platelets, but not in neutrophils, suggesting that exogenously added OAG may be readily accessible for diacylglycerol (DAG) lipase/PLA1 in platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Arachidonic Acid; Blood Platelets; Calcimycin; Diglycerides; Enzyme Activation; Humans; Neutrophils; Phospholipases A; Phospholipases A1; Phospholipases A2; Protein Kinase C; Tetradecanoylphorbol Acetate

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Arachidonic Acid; Biological Transport; Calcimycin; Diglycerides; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukotriene Antagonists; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Tetradecanoylphorbol Acetate

The human hepatoma cell line, Hep 3B, produces biologically active erythropoietin (Epo) in response to normal physiologic stimuli and thus provides a model system for the study of Epo regulation. The addition of phorbol 12-myristate 13-acetate (PMA) to Hep 3B cells subsequently grown under hypoxic conditions resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 95 +/- 1% with half-maximal inhibition at 8 ng/mL. By Northern blot analysis, Epo mRNA levels were correspondingly decreased after treatment with PMA. Direct measurement of both membrane and cytosolic protein kinase C activity in Hep 3B cells following treatment with PMA demonstrated a biphasic response as a function of time. Membrane-associated protein kinase C activity initially increased but subsequently decreased to baseline levels by 12 hours. The PMA-induced inhibition of hypoxia-induced Epo production was shown to occur as early as 3 hours after PMA addition, suggesting that the initial activation, rather than the subsequent decrease in protein kinase C activity, is of primary importance. The relative specificity of the PMA-induced inhibition of Epo production was demonstrated by 1) the finding that overall protein and RNA synthesis were not similarly decreased as measured by 3H-leucine and 3H-uridine pulse labeling studies and 2) the observation that the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, failed to have any effect on hypoxia-induced Epo production. In addition, the synthetic analog of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore, A23187, inhibited hypoxia-induced Epo production up to 85 +/- 3% and 82 +/- 4%, respectively, in a dose-dependent manner. Taken together, these findings suggest that hypoxia-induced Epo production may be negatively regulated by activators of a protein kinase C-mediated pathway.

Topics: Blotting, Northern; Calcimycin; Carcinoma, Hepatocellular; Diglycerides; DNA; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Leucine; Liver Neoplasms; Protein Kinase C; Radioimmunoassay; RNA; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured; Uridine

Because of the high intracellular Cl- concentration ([Cl-]i) in gastrointestinal smooth muscle, receptor-mediated opening of Cl- channels at the cell resting potential could represent a plausible mechanism for initial receptor-mediated cell depolarization. To test this hypothesis, we characterized activation of large-conductance Cl- channels by the neurokinin-1 (NK-1) receptor agonist [Sar9,Met(O2)11]-substance P, by specific second messengers, and by direct G protein activation in myocytes isolated from the rabbit colon longitudinal muscle layer. In excised inside-out patches, large-conductance ion channels selective for Cl- over Na+ could be induced by holding the patch at pipette potentials values > 60 mV. The channel showed multiple smaller conductance states (< or = 20) but could open and close via a main gate. When the channel was fully open, its slope conductance was 300 pS, with substates as small as 15 pS, comparable to the predominant conductance observed in cell-attached patches. The voltage-activation profile for full conductance was bell-shaped with maximal open probability (Po) for channel opening of approximately 0 mV. In cell-attached patches, addition of the NK-1 agonist to pipette solution activated a channel that corresponded to a subconductance state of the maxi Cl- channel. The voltage-activation profile for this subconductance state showed a maximal Po value for membrane potentials of approximately 0 mV, with rapid inactivation at more positive and partial inactivation at more negative membrane potentials. In excised inside-out patches, both the full and smaller conductance states of the Cl- channel were activated by the nonhydrolyzable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) and inhibited by pertussis toxin (PTX), whereas [Ca2+]i increased channel activity only in concentrations > 1 mM. In cell-attached patches, addition of different Ca2+ ionophores resulted in channel activation in 10% of cells, and activators of protein kinase A or protein kinase C had no effect. These findings are consistent with the hypothesis of a possible role of G protein-coupled Cl- channels in receptor-mediated initial cell depolarization in longitudinal colonic smooth muscle.

Topics: Animals; Bucladesine; Calcimycin; Cell Adhesion; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Colon; Cytosol; Diglycerides; Electric Conductivity; Electrophysiology; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Ion Channels; Ionomycin; Male; Meglumine; Membrane Potentials; Membrane Proteins; Muscle, Smooth; NAD; Pertussis Toxin; Phorbol 12,13-Dibutyrate; Rabbits; Receptors, Neurokinin-2; Receptors, Neurotransmitter; Substance P; Tetraethylammonium; Tetraethylammonium Compounds; Virulence Factors, Bordetella

Histamine-containing cells isolated from rabbit fundic mucosa were found in a small cell elutriation fraction (cells with diameter about 9-12 microns) enriched in mucus and endocrine cells and containing less than 1% mast cells (F1 cells). Gastrin (HG-17), pentagastrin and CCK-8 (C-terminal octapeptide of cholecystokinin) dose-dependently stimulated histamine release (EC50, respectively, 0.126 +/- 0.03, 0.92 +/- 0.15 and 0.211 +/- 0.025 nM) and somatostatin inhibited this release. PGE1, PGE2 and PGD2 alone were unable to enhance histamine release even at high concentrations but, when used in combination with gastrin of CCK-8, the release of histamine caused by these peptides was potentiated (about 1.5- to 2-fold). Carbachol also enhanced the liberation of histamine but with a weaker potency and efficacy than the gastrointestinal peptides (EC50: 1.50 +/- 0.06 microM). The use of specific muscarinic antagonists for M1-, M2- and M3-type receptors led us to conclude that an M1 receptor might be involved in the muscarinic-induced stimulation of histamine release. Activators of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2-acetyl-glycerol (OAG) as well as the calcium ionophore, A23187, induced histamine release, whereas agents which increased intracellular cAMP content were devoid of effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Calcimycin; Carbachol; Colforsin; Diglycerides; Dose-Response Relationship, Drug; Gastric Fundus; Gastric Mucosa; Gastrins; Histamine Release; Male; Muscarinic Antagonists; Prostaglandins; Rabbits; Sincalide; Somatostatin; Tetradecanoylphorbol Acetate

The diacylglycerol DiC8 (1,2-dioctanoyl-sn-glycerol; 100 mumol l-1) was found to induce acrosomal loss in 70% of wallaby spermatozoa and 40% of possum spermatozoa after incubation for 120 min. If 10 mumol calcium ionophore l-1 was present, the time required to reach these end points was reduced to 30 and 60 min, respectively. The diacylglycerol OAG (1-oleoyl-2-acetyl-sn-glycerol; 100 mumol l-1) produced some acrosomal loss, particularly when 10 mumol calcium ionophore l-1 was present, but when the ionophore was absent, results were equivocal. The other diacylglycerol tested DOG (1,2-dioleoyl-sn-glycerol; 100 mumol l-1) did not induce significant acrosomal loss in possum or wallaby spermatozoa. The concentration of DiC8 required to induce acrosomal loss in marsupial spermatozoa (50-100 mumol l-1) was relatively high compared with that for placental mammals, suggesting a less specific mode of action than enzyme activation. Of the diacylglycerols tested alone, only DiC8 led to significant loss of motility. In wallabies, this was detectable after incubation for 60 min and in possums after incubation for 120 min. The ultrastructure of the DiC8-induced acrosomal loss was essentially identical to the acrosome reaction described for a broad range of placental mammals. A membrane vesicle shroud covered the acrosomal surface of the sperm head. The vesicles appeared to be formed by multiple point fusions between the plasma membrane and the outer acrosomal membrane. The mechanism of DiC8-induced acrosomal loss remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acrosome; Animals; Calcimycin; Cells, Cultured; Diglycerides; Exocytosis; Macropodidae; Male; Marsupialia; Microscopy, Electron; Sperm Motility

Dispersed rat gastric mucosal cells (F0) were separated into five fractions (F1-F5) by counterflow elutriation, with F1 representing the smallest and F5 the largest cell diameters. In F0-F5, leukotriene B4 (LTB4) release in response to 10(-5) M calcium ionophore A-23187 was 7.68 +/- 1.26, 51.6 +/- 10.8, 72.4 +/- 10.4, 7.1 +/- 0.7, 5.7 +/- 0.6, and 11.6 +/- 3.4 pg.10(6) cells-1 x 30 min-1. In the identical fractions, sulfidopeptide release in response to A-23187 was 200.6 +/- 20.5, 1,116.0 +/- 166.6, 1,309.4 +/- 163.2, 189.8 +/- 25.8, 108.0 +/- 18.0, and 158.4 +/- 54.0 pg.10(6) cells-1 x 30 min-1. High-pressure liquid chromatography verified the radioimmunologically determined LTB4 and identified LTC4 as the only sulfidopeptide LT released. LT release from F2 cells in response to A-23187 was time and dose dependent, reaching maximal stimulation at 10(-5) M A-23187. This response was blocked by the dual inhibitor of cyclooxygenase and lipoxygenases, BW755C (2 x 10(-5)-2 x 10(-4) M), by the selective 5-lipoxygenase inhibitor L-651,392 (10(-7)-10(-5) M), and by MK-886 (10(-9)-10(-7) M), which blocks translocation of 5-lipoxygenase. The postreceptor stimuli dibutyryl adenosine 3',5'-cyclic monophosphate, forskolin, 12-O-tetradecanoyl-phorbol-13-acetate, and oleyl-acetyl-glycerol failed to induce LT release. However, 10(-4) M arachidonic acid increased basal LT release up to eightfold and increased A-23187-stimulated LT release by an additional 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Arachidonic Acid; Bucladesine; Calcimycin; Cell Separation; Colforsin; Diglycerides; Female; Gastric Mucosa; Histamine; In Vitro Techniques; Interleukin-1; Interleukin-2; Kinetics; Leukotriene B4; Leukotriene C4; Lipoxygenase Inhibitors; N-Formylmethionine Leucyl-Phenylalanine; Pentagastrin; Phenothiazines; Platelet Activating Factor; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.

Topics: Adenosine Triphosphate; Animals; Brain; Calcimycin; Calcium; Cells, Cultured; Coronary Vessels; Diglycerides; Ion Channel Gating; Membrane Potentials; Muscle, Smooth, Vascular; Potassium Channels; Probability; Protein Kinase C; Rats; Swine; Tetradecanoylphorbol Acetate

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.

Topics: Animals; Bucladesine; Calcimycin; Chloroquine; Colforsin; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Female; Goldfish; Melitten; Ovarian Follicle; Ovary; Phospholipases A; Phospholipases A2; Prostaglandins; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.

Topics: Arachidonic Acid; Aristolochic Acids; Calcimycin; Diglycerides; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phenanthrenes; Phosphatidate Phosphatase; Phospholipase D; Phospholipases A; Phospholipases A2; Polymers; Prostaglandins B; Sphingosine; Superoxides; Tetradecanoylphorbol Acetate

The cell surface lipophosphoglycan (LPG) from Leishmania donovani promastigotes is a potent inhibitor of purified protein kinase C (PKC) activity in vitro. In this study, we have investigated the effect of LPG on the activation process of PKC in murine bone marrow-derived macrophages. The extent and kinetics of calcium ionophore A23187-induced [3H] phorbol dibutyrate binding to macrophages were not affected by LPG pretreatment or infection with either wild-type or LPG-deficient promastigotes, indicating no effect on the association of calcium-dependent PKC with the plasma membrane. In contrast, LPG inhibited the phosphorylation of both the PKC-specific VRKRTRLLR substrate peptide and MARCKS, and endogenous PKC substrate, in 1-oleoyl-2-acetyl-glycerol-stimulated macrophages. These observations provide direct evidence that LPG effectively inhibits PKC activity in intact macrophages. Finally, depletion of PKC rendered macrophages more permissive for the proliferation of L. donovani, suggesting that inhibition of PKC-dependent events contributes to the survival of this parasite within its host cell.

Topics: Amino Acid Sequence; Animals; Antigens, Protozoan; Blotting, Northern; Calcimycin; Cell Membrane; Diglycerides; Dose-Response Relationship, Drug; Female; Glycosphingolipids; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Leishmania donovani; Macrophage Activation; Macrophages; Membrane Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Myristoylated Alanine-Rich C Kinase Substrate; Oligopeptides; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Proteins; Proto-Oncogene Proteins c-fos; RNA; Time Factors

The stimulatory effect of FMLP 5 nmol.L-1, OAG 25 nmol.L-1 and calcimycin 10 nmol.L-1 on luminol-dependent chemiluminescence (CL) was observed in human neutrophils (Neu). The rest level of Neu intracellular free calcium ([Ca]i) measured by Ca(2+)-sensitive probe Quin 2/AM was calculated to be 200 +/- s 19 nmol.L-1. By the addition of FMLP 0.1 nmol.L-1 or calcimycin 0.5 nmol.L-1, the peak [Ca]i increased to 769 +/- 104 nmol.L-1 and 953 +/- 53 nmol.L-1, respectively. Ketotifen (50-300 mumol.L-1) inhibited Neu CL in a dose-dependent manner with activator FMLP. Inhibition was also seen when Neu CL was activated by calcimycin and OAG, respectively. However, ketotifen did not inhibit Neu [Ca]i increment activated by FMLP and calcimycin.

Topics: Calcimycin; Calcium; Diglycerides; Humans; Ketotifen; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Respiratory Burst

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaloids; Animals; Blotting, Northern; Blotting, Southern; Calcimycin; Cell Division; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Down-Regulation; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Calcitriol; Receptors, Steroid; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, on high-affinity Na(+)-dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with TPA led to concentration- and time-dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022. The effects of TPA were potentiated by the Ca2+ ionophore A23187. Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons. We conclude that PKC activates the sodium-dependent high-affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Binding, Competitive; Calcimycin; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Glutamates; Glutamic Acid; Isoquinolines; Kinetics; Neuroglia; Neurons; Piperazines; Tetradecanoylphorbol Acetate

The effect of purified lipophosphoglycan (LPG) of Leishmania donovani on signal transduction and gene expression in murine bone marrow-derived macrophages was investigated. LPG stimulated the rapid expression of both c-fos and TNF genes within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. Macrophages pretreated with LPG for 3 h became unresponsive to subsequent stimulation with LPS and the activators of protein kinase C, 1-oleoyl-2-acetyl-glycerol, and calcium ionophore A23187. Moreover, LPG induced a rapid down-modulation of TNF receptors. In contrast, the ability of macrophages to express the c-fos gene in response to the cAMP analogue, dibutyryl cAMP, was not impaired by LPG. Fragmentation of LPG revealed that the inhibitory activity of LPG required both the repeating phosphorylated disaccharides and the phosphosaccharide core. Collectively, these data demonstrate that LPG selectively impaired signal transduction in macrophages and suggest a role for this molecule in the survival of the parasite within the macrophage.

Topics: Animals; Blotting, Northern; Calcimycin; Diglycerides; DNA-Binding Proteins; Drug Antagonism; Female; Glycosphingolipids; Leishmania donovani; Macrophages; Mice; Mice, Inbred BALB C; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA; Signal Transduction; Tumor Necrosis Factor-alpha

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Animals; Arachidonic Acids; Calcimycin; Calcium; Carcinogens; Chickens; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Male; Pituitary Gland; Protein Kinase C; Second Messenger Systems; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Transforming Growth Factor alpha

One aspect of human neutrophil (PMN) function during inflammation is formation of platelet-activating factor (PAF), leukotriene B4 (LTB4), and 5-hydroxyeicosatetraenoic acid (5-HETE), but production of these lipid mediators is limited if PMN are directly stimulated with soluble, physiologic agonists. In vitro, PMN activities can be enhanced by the process of primed-stimulation where cells are sequentially treated with non-stimulatory concentrations of different agonists. Many agents that prime PMN also induce production of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols. Therefore, we investigated whether diglycerides were involved in priming PMN for production of lipid mediators. We previously described the ability of the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and its alkylacylglycerol analog, 1-O-octadecenyl-2-acetylglycerol (EAG), to prime phospholipase A2 (PLA2) for subsequent activation by a second stimulus. However, while OAG also primed 5-lipoxygenase activity (LTB4 and 5-HETE production), EAG priming inhibited LTB4 and 5-HETE formation. We now report the effects of diglyceride priming on acetyltransferase activation (PAF formation). PMN, prelabeled with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine, were primed with OAG or EAG before stimulation. Neither OAG nor EAG induced formation of labeled PAF. Treatment of PMN with the chemotactic peptide, N-formyl-met-leu-phe (FMLP), induced low but significant production of PAF; PAF formation doubled in PMN primed with 20 microM OAG before FMLP stimulation while priming with 20 microM EAG more than tripled the level of PAF. Calcium ionophore strongly induced PAF formation; OAG priming before ionophore challenge had no effect but EAG priming further enhanced PAF formation. These results suggests a role for alkylacylglycerols in modulating the production of lipid mediators of inflammation.

Topics: Acetyltransferases; Adult; Arachidonate 5-Lipoxygenase; Calcimycin; Chromatography, Thin Layer; Diglycerides; Enzyme Activation; Humans; In Vitro Techniques; Kinetics; Lipids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases A; Phospholipases A2; Phospholipids; Platelet Activating Factor; Structure-Activity Relationship

In experimental membranous nephropathy, C5b-9 induces noncytolytic glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by metabolites of arachidonic acid. In cultured GEC, sublytic C5b-9 increases cytosolic Ca2+ concentration ([Ca2+]i), activates phospholipase C (PLC), and releases arachidonic acid and eicosanoids. This study examined mechanisms of arachidonic acid production by C5b-9. In GEC labeled with [3H]arachidonate C5b-9 increased free [3H]arachidonic acid and 1,2-[3H]-arachidonoyl-diacylglycerol (DAG), an endogenous activator of protein kinase C (PKC). Elevated [Ca2+]i was not sufficient to account for increased free arachidonic acid. Moreover, in GEC that had been depleted of PKC by preincubation for 18 h with 2 microM phorbol myristate acetate, the C5b-9-induced arachidonate release was inhibited by greater than 75%. Reacylation of phospholipids was not decreased by C5b-9. Homogenates of GEC that had been stimulated with C5b-9 released more [14C]arachidonate from exogenously added 2-[14C]arachidonoyl-phosphatidyl-ethanolamine or 2-[14C]arachidonoyl-phosphatidylcholine than homogenates of unstimulated cells (assayed at a Ca2+ concentration of 2 mM). These experiments demonstrate directly that C5b-9 increased phospholipase A2 (PLA2) activity. PLA2 appeared to be stimulated as a result of PKC activation (probably secondary to increased DAG) in association with elevated [Ca2+]i. The C5b-9-induced activation of PLA2 may lead to release of eicosanoids, which may contribute toward impaired glomerular capillary wall permselectivity in experimental membranous nephropathy.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cells, Cultured; Complement Membrane Attack Complex; Cyclohexanones; Diglycerides; Edetic Acid; Egtazic Acid; Epithelium; Fatty Acids, Nonesterified; Kidney Glomerulus; Kinetics; Lipase; Lipoprotein Lipase; Phospholipases A; Phospholipases A2; Phospholipids; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate

The involvement of protein kinase C in the initiation of free oxygen radical generation by rat leukocytes in response to the nematode Nippostrongylus brasiliensis was investigated. Inhibitors of protein kinase C, trifluoperazine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), inhibited free radical generation in response to N. brasiliensis in vitro. Neither inhibitor affected free radical generation by the cell-free xanthine/xanthine oxidase system, indicating that the agents did not scavenge free radicals; they also failed to affect leukocyte viability. Furthermore, activators of protein kinase C, the calcium ionophore A23187 and the diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol (OAG), enhanced free radical generation by leukocytes in response to N. brasiliensis in vitro. Thus, protein kinase C apparently plays an important role in the initiation of free radical generation in response to N. brasiliensis; since free radicals may play a critical role in worm expulsion, this implies that protein kinase C may also be important in the rejection of N. brasiliensis from the small intestine of the rat.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcimycin; Diglycerides; Enzyme Activation; Female; Free Radicals; Isoquinolines; Leukocytes; Nematode Infections; Nippostrongylus; Oxygen; Piperazines; Protein Kinase C; Rats; Rats, Inbred Strains; Trifluoperazine

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.

Topics: Animals; Calcimycin; Diglycerides; Drug Synergism; Exocytosis; Histamine Release; Kinetics; Male; Mast Cells; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Receptor activation on the cell surface is coupled through a guanine nucleotide regulatory protein to polyphosphoinositide phosphodiesterase. The activation of this enzyme catalyses the hydrolysis of phosphatidylinositol biphosphate. One of the products of this hydrolysis is diacylglycerol, which activates protein kinase C. It can also be activated by tumour-promoting phorbol esters. The synthetic diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) have been used to stimulate protein kinase C in a pure population of rat peritoneal mast cells. Both of them caused histamine release, but the rate of release with TPA or OAG alone was slow. The release was inhibited by blocking the oxidative energy metabolism with antimycin A, and was associated with progressive exocytosis, showing that it is a secretory process. Studies on the interaction between the stimulation of protein kinase C by OAG/TPA and the secretagogues showed a dual effect, both potentiation and inhibition. Antigen (in sensitized cells) and compound 48/80 showed this pattern of response. With the calcium ionophore, A23187, potentiation was the dominant effect, although some inhibition could be shown with TPA. This is possibly related to the large calcium influx which causes translocation of protein kinase C to the membranes and enhances its activity. The potentiation suggests that protein kinase C is involved in the secretion process by the secretagogues, while the inhibition reflects a regulatory function, which is apparently exerted through an inhibition of phosphatidylinositol breakdown. Calcium uptake was enhanced by both TPA and OAG. Protein kinase C may thus contribute to the replenishment of the intracellular calcium stores after the secretory response.

Topics: Animals; Biological Transport; Calcimycin; Calcium; Diglycerides; Histamine Release; Male; Mast Cells; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Diabetes mellitus is associated with high levels of adenosine 3',5'-cyclic monophosphate in tissue and plasma. Diabetes inhibits and insulin stimulates and restores low Km adenosine 3',5'-cyclic monophosphate phosphodiesterase activity. We recently reported that phorbol ester, a tumor promoting agent known to act through protein kinase C also stimulates phosphodiesterase. Here, we address the issue of whether or not the activation of phosphodiesterase by insulin and phorbol ester is different in streptozotocin diabetic adipose tissue. Rat adipose tissue was incubated with insulin, phorbol ester or other known components or effectors of the protein kinase C pathway, i.e. 1,2 dioleoyl-glycerol, 1- oleoyl, 2- acetylglycerol, Ca(++)-Ionophore A 23187, and nifedipine. After incubation, preparation and assay of adenosine 3',5'-cyclic monophosphate phosphodiesterase was made. As in previous data streptozotocin-diabetes inhibits basal phosphodiesterase by about 50% (P less than .02); insulin and phorbol ester each stimulate phosphodiesterase, in streptozotocin-diabetes less than normal (P less than .025); nifedipine inhibits phorbol stimulated phosphodiesterase in streptozotocin-diabetes but not normal (P less than .001); and nifedipine inhibits insulin stimulated phosphodiesterase in normal (84%) and diabetic (97%) (P less than .005). In normal and diabetic tissue, diacyl glycerol and oleoyl-acyl glycerol stimulate phosphodiesterase, are augmented by ionophore and inhibited by nifedipine. In addition 32P incorporation studies and measurements of tyrosine kinase activity are presented which support these differences between normal and diabetic. In summary then, these data suggest common pathways of activation for low Km adenosine 3',5'-cyclic monophosphate phosphodiesterase by insulin and phorbol ester; imply a relationship between two second messenger systems, phosphoinositides and adenosine 3',5'-cyclic monophosphate; and demonstrate a difference in activation of phosphodiesterase between normal and diabetic adipose tissue.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adipose Tissue; Animals; Calcimycin; Diabetes Mellitus, Experimental; Diglycerides; Enzyme Activation; Insulin; Male; Nifedipine; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Tetradecanoylphorbol Acetate

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.

Topics: Acyltransferases; Blood Platelets; Calcimycin; Calcium; Coenzyme A Ligases; Diglycerides; Eicosanoids; Glutathione; Humans; Hydrogen Peroxide; Kinetics; Lysophospholipase; Methylmercury Compounds; Microsomes; Multienzyme Complexes; Phospholipases; Tetradecanoylphorbol Acetate; Thrombin

We have previously characterized a hormonally regulated soluble form of phospholipase A2 (PLA2) in the cultured renal mesangial cell which is similar and possibly identical to the major form in rat kidney. In an attempt to further characterize the mechanisms of regulation of this enzyme we have used epidermal growth factor (EGF), which does not activate polyphosphoinositide-specific phospholipase C in these cells. EGF-enhanced PLA2 activity as assayed by the ability of the soluble extracts of cells to cleave arachidonic acid from the sn-2 position of phosphatidylcholine and phosphatidylethanolamine. This represents a direct demonstration of EGF-induced PLA2 activation which is preserved in a cell-free extract. Phorbol myristate acetate (PMA), as well as 1-oleoyl-2-acetylglycerol, also enhanced PLA2 activity. By contrast, the calcium ionophore A23187 had no effect on extract PLA2 activity. The EGF- and PMA-induced enhanced activity was recovered following fractionation by Mono-Q anion exchange chromatography. The peak of activity comigrated for both agonists, suggesting that both EGF and PMA stimulated the same form of the enzyme. Down-regulation of protein kinase C by pretreatment with PMA resulted in loss of the PMA-induced, but not the EGF-induced, enhancement in PLA2 activity. 8-Bromo-cAMP had no effect upon the PLA2 activity, and did not modulate the EGF effect. Pertussis toxin induced G protein ADP-ribosylation but had no effect upon PLA2 activity, and did not alter the EGF effect. In summary, EGF results in a stable modification of PLA2 activity in glomerular mesangial cells. This enhanced activity is independent of polyphosphoinositide hydrolysis, insensitive to protein kinase C down-regulation, and is not affected by cAMP or pertussis toxin pretreatment of the cells.

Topics: Animals; Arginine Vasopressin; Calcimycin; Cells, Cultured; Chromatography, Ion Exchange; Cytosol; Diglycerides; Enzyme Activation; Epidermal Growth Factor; Glomerular Mesangium; Inositol; Inositol Phosphates; Kinetics; NAD; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

The breakdown of inositol phospholipids is an important transmembrane signalling system that is composed of two kinds of signals: the diacylglycerol-protein kinase C signal, and the inositol trisphosphate-Ca2+ signal. Using membrane-permeable diacylglycerol, I-oleoyl-2-acetylglycerol (OAG), and calcium ionophore, A-23187, the effects of these chemicals on the epidermal adenylate cyclase system were investigated. OAG increased forskolin- and cholera toxin-induced cyclic AMP accumulations, but receptor adenylate cyclase responses were markedly decreased by treatment with OAG. The effects of OAG were inhibited by the protein kinase C inhibitor, H-7. Calcium ionophore, A-23187, had no effect on the epidermal adenylate cyclase responses. Combinations of OAG and A-23187 (as well as the calcium chelator, EGTA), showed that the action of OAG was mostly unaffected by the modulation of intracellular and extracellular Ca2+ concentrations. The results suggest that among the signals triggered by the breakdown of inositol phospholipids, only diacylglycerol-protein kinase C signal is involved in the regulation of the epidermal adenylate cyclase system.

Topics: Adenylyl Cyclases; Animals; Calcimycin; Calcium; Culture Media; Diglycerides; Egtazic Acid; Epidermis; Protein Kinase C; Signal Transduction

Steroidogenesis in teleost fish, as in other vertebrate groups, is mediated by the activation of adenylate cyclase. For the present studies, calcium ionophore A23187 and either phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were used to investigate the possible roles that changes in intracellular calcium content and protein kinase C activation play in steroid production by goldfish preovulatory ovarian follicles incubated in vitro. While ineffective alone, PMA (1.6-400 nM) and OAG (25-100 micrograms/ml) exhibited classical synergism with A23187 (1.0-10 microM), leading to increased testosterone production. The magnitude of these responses was at least tenfold lower than that obtained with human chorionic gonadotropin (hCG), forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Testosterone production stimulated by hCG and forskolin was blocked by addition of PMA but not OAG. Unlike PMA, the inactive phorbol ester 4 alpha-phorbol 12,13-dideconate did not influence basal or stimulated testosterone production. A23187 had a biphasic effect on stimulated testosterone production: a dosage of 0.25 or 1.0 microM potentiated the action of submaximally effective dosages of hCG or forskolin on testosterone production; a higher dosage of 4 microM inhibited stimulated testosterone production by up to 50%. In conclusion, these studies suggest that, in addition to the adenylate cyclase second messenger system, changes in intracellular calcium and activation of protein kinase C may modulate steroidogenesis in goldfish ovarian follicles.

Topics: Animals; Calcimycin; Calcium; Cyprinidae; Diglycerides; Female; Glycerides; Goldfish; Ovarian Follicle; Ovary; Phorbol Esters; Protein Kinase C; Testosterone; Tetradecanoylphorbol Acetate

The acute incubation of mouse bone marrow-derived mast cells with low concentrations of agents known to activate protein kinase C [phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (diC8), and 1-oleoyl-2-acetyl-glycerol (OAG)] caused an enhancement of beta-hexosaminidase release stimulated by the calcium ionophore A23187. Higher concentrations of protein kinase C activators tended to inhibit A23187- or antigen-induced preformed mediator release. All concentrations studied induced a striking mast cell hyporesponsiveness to the mediator release augmenting effect of adenosine. Agents that have been reported to block protein kinase C activity [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7) and sphingosine] demonstrated diverse responses in this system. Up to 100 microM H-7 failed to affect mast cell beta-hexosaminidase release in the presence or absence of PMA and secretagogue. Sphingosine (10 microM) was a potent inhibitor of antigen- or A23187-induced mediator release as well as adenosine responsiveness. Sphingosine also blocked the effects of PMA noted above in a dose-dependent fashion. The generation of leukotriene C4 (LTC4) by stimulated mast cells surprisingly was not affected by concentrations of diC8 that significantly inhibited granule-associated mediator release. Translocation of protein kinase C activity from the cytosol to the mast cell membrane was evident in cells briefly pretreated with A23187, adenosine alone, and diC8 in the presence of Tyrode's buffer, A23187, or adenosine. These findings lend further support to the contention that signal transduction from mast cell adenosine receptors to processes that regulate degranulation may involve protein kinase C.

Topics: Adenosine; Animals; beta-N-Acetylhexosaminidases; Calcimycin; Cells, Cultured; Cytosol; Diglycerides; Enzyme Activation; Glycerides; Mast Cells; Mice; Mice, Inbred BALB C; Protein Kinase C; SRS-A; Tetradecanoylphorbol Acetate

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.

Topics: Animals; Arachidonic Acids; Bucladesine; Calcimycin; Carcinogens; Cattle; Cholera Toxin; Cycloheximide; Dibutyryl Cyclic GMP; Diglycerides; Dinoprost; Endometrium; Female; Nucleotides, Cyclic; Oxytocin; Phorbol Esters; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate; Uterus

Membrane IL-1 (mIL-1) expression was compared with release of soluble IL-1 (sIL-1) by C3H/HeNCrl mouse peritoneal macrophages. Selective antagonists of protein kinase C (PKC) (H-7) and calmodulin (W-7) in combination inhibited LPS-induced mIL-1 and sIL-1 production, suggesting a role for these activation pathways in IL-1 induction. Low levels of A23187, when combined with OAG (a direct activator of PKC), stimulated mIL-1 expression in the absence of sIL-1 release. Induction of mIL-1 by LPS was inhibited by PGE2 and dibutyryl cAMP, but higher concentrations were required to inhibit mIL-1 expression compared with sIL-1 release. LTB4 alone did not induce mIL-1 or sIL-1 production. LTB4 did enhance LPS-induced mIL-1 expression but not sIL-1 release. These results indicate that mIL-1 expression and sIL-1 release are differentially regulated. Membrane IL-1 is induced by lower levels of certain stimuli and is less effectively inhibited than is sIL-1 release. This differential regulation is further evidence to support the existance of membrane IL-1.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antibodies; Calcimycin; Calmodulin; Cell Membrane; Diglycerides; Dinoprostone; In Vitro Techniques; Interleukin-1; Isoquinolines; L-Lactate Dehydrogenase; Leukotriene B4; Macrophages; Mice; Mice, Inbred C3H; Nucleotides, Cyclic; Piperazines; Protein Kinase C; Solubility; Sulfonamides; Thymus Gland

Glucagon at a low concentration has a stimulatory effect on Ki-ras expression, whereas, at high concentrations the hormone suppresses the level of the Ki-ras transcripts. Incubation of the hepatoma cells with 10 microM dibutyryl cyclic AMP results in suppression of Ki-ras expression but the phorbol ester, 21-O-tetradecanoylphorbol 13-acetate (TPA) causes an increase. Down regulation of protein kinase C by prolonged exposure of hepatoma cells to TPA causes a dramatic decrease in the glucagon-stimulated effect on Ki-ras expression. The presence of diacylglycerol for 2 h in the culture medium results in a significant increase in Ki-ras expression, while treatment of the cells with 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine, a potent inhibitor of protein kinase C, leads to a dramatic reduction. The calcium ionophore, A23187 is able to stimulate Ki-ras expression, whereas, addition of verapamil or EGTA results in its suppression. The present findings suggest that the inductive effect of glucagon on Ki-ras expression at low concentrations is via the activation of protein kinase C which causes phosphorylation of some regulatory proteins that may eventually affect the level of Ki-ras mRNA. The suppressive effect of glucagon at higher concentrations is via an increase in cAMP through activation of adenylate cyclase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Bucladesine; Calcimycin; Calcium; Cell Line; Diglycerides; DNA Probes; Egtazic Acid; Gene Expression Regulation, Neoplastic; Genes, ras; Glucagon; Isoquinolines; Kinetics; Liver Neoplasms, Experimental; Piperazines; Protein Kinase Inhibitors; Rats; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Verapamil

Parietal cells are a major source of gastric mucosal prostaglandins in various species. We examined cholinergic stimulation of prostaglandin E2 (PGE2) release from human parietal cells; using activators of the protein kinase C we attempted to get an indirect insight into cellular mechanisms which control PGE2 release. Gastric mucosal specimens were obtained at surgery and the cells were dispersed by collagenase and pronase E. Parietal cells were enriched to 65-80% by a Percoll gradient, and were incubated for 30 min. PGE2 release into the medium (radioimmunoassay) was 74-126 pg/10(6) cells/30 min under basal conditions and was 2.6-fold increased by carbachol (10(-5) and 10(-4) M). Similarly, PGE2 release was stimulated by phospholipase C (20-200 mU/ml, 364% above basal), 1-oleoyl-2-acetyl-sn-glycerol (10(-9)-10(-5) M, 229%), 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9)-10(-5) M, 283%) and calcium ionophore A23187 (10(-7)-10(-5) M, 219%). Simultaneous presence of A23187 and TPA synergistically induced stimulation which was slightly higher than the sum of the individual responses. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide W-7, a putative calmodulin antagonist, inhibited TPA-induced PGE2 release at concentrations regarded specific for blocking calmodulin (IC50 = 1.5 X 0(-6) M). We conclude that in human parietal cells PGE2 is released upon cholinergic stimulation and that phospholipase C and protein kinase C are involved in the control of PGE2 release. We speculate that calmodulin might interact with a protein phosphorylated by protein kinase C to cause PGE2 release.

Topics: Calcimycin; Calmodulin; Carbachol; Cell Separation; Diglycerides; Dinoprostone; Drug Synergism; Enzyme Activation; Gastric Mucosa; Humans; Parietal Cells, Gastric; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate; Type C Phospholipases

Previous studies have shown that the glyceride derivative, sn-1,2-isopropylidene-3-decanoyl-glycerol (IpOCOC9), can trigger human leukocyte histamine release. Approximately 25% of the total cellular histamine content is extruded in the presence of 206 microM of IpOCOC9; at 69 microM, however, the secretagogue action of the compound is marginal. The characteristics of the release induced by IpOCOC9 are closely similar to those reportedly recorded at hyperosmolar triggering of basophils with mannitol, and in many respects they also mimic those observed at phorbol ester-induced histamine release. The compound decanoic acid cyclopentyl methylester (DACPME), a structural analogue of IpOCOC9, fails to induce histamine release. IpOCOC9, but not DACPME, stimulates human polymorphonuclear leukocyte cytosolic Ca2+- and phospholipid-dependent histone III-S kinase activity (unpublished observations). The secretagogue action of IpOCOC9 has therefore tentatively, at least partly, been attributed to a direct protein kinase C activation. In the present studies, we examined the influence of IpOCOC9 and DACPME on histamine release triggered by an ensuing exposure to anti-IgE, the calcium ionophore A23187, formyl-methionyl-leucyl-phenylalanine (FMLP), or 4 beta-phorbol 12-myristate 13-acetate (PMA). It is shown that IpOCOC9-treatment of cells results in either enhancement or reduction of the release induced by anti-IgE or by A23187, whereas FMLP-induced release is consistently reduced and PMA-induced release consistently enhanced by such a treatment. Treatment of cells with DACPME enhances but does not reduce anti-IgE-triggered release, whereas FMLP-induced release is not affected. Pretreatment of the cells with other putative protein kinase C activators like PMA, sn-1-oleoyl-2-acetyl-glycerol (OAG), 1,2-dioctanoyl-glycerol (DiC8) or the glycerol derivative sn-1,2-diacetyl-3-decanoyl-glycerol (DiC2OCOC9) affects secretagogue-induced basophil histamine release according to specific patterns similar to but not identical with those recorded for IpOCOC9 and DACPME. Thus, e.g., DiC2OCOC9 consistently reduces but does not enhance anti-IgE-triggered release. These data show that limited structural changes of IpOCOC9 may qualitatively affect its modulating properties in the human basophil histamine release system.

Topics: Calcimycin; Cyclopentanes; Decanoic Acids; Diglycerides; Glyceryl Ethers; Histamine Release; Humans; Immunoglobulin E; Leukocytes; N-Formylmethionine Leucyl-Phenylalanine; Tetradecanoylphorbol Acetate; Triglycerides

The effect of native prostaglandins and their methylated analogues on pancreatic enzyme secretion remains unclear, with previous studies reporting inconsistent results. To determine whether the E series prostaglandins directly influence pancreatic secretion, we studied the effect of rioprostil, a prostaglandin E1 analogue, and 16,16-dimethyl prostaglandin E2 (DMPGE2), a prostaglandin E2 analogue, on enzyme release from dispersed guinea pig pancreatic acini. Basal amylase release (4.3 +/- 0.6% of total acinar content) was not altered by either analogue (10(-10)-10(-5) M). A 50% inhibition of maximal cholecystokinin stimulation (10(-9) M; 28.8 +/- 1.2%) was seen with rioprostil (10(-7) M; 14.6 +/- 1.3%) and DMPGE2 (10(-6) M; 15.9 +/- 0.7%) (both p less than 0.005). Prostaglandin inhibition of carbachol-stimulated amylase was less pronounced. The most effective inhibitory dosage with maximal carbachol (10(-5) M; 30.2 +/- 1.9%) was 10(-6) M for both rioprostil (19.2 +/- 1.6%) and DMPGE2 (22.4 +/- 1.7%) (both p less than 0.005). Incubation of acini with A23187, phorbol ester, and 1-oleoyl-2-acetyl-glycerol resulted in a dose-dependent increase in amylase release that was not altered by maximal concentrations of either prostaglandin analogue. Our results indicate that rioprostil and DMPGE2 can directly inhibit pancreatic acinar secretion. This effect appears to occur before activation of the inositol phospholipid system.

Topics: 16,16-Dimethylprostaglandin E2; Amylases; Animals; Anti-Ulcer Agents; Calcimycin; Carbachol; Cholecystokinin; Diglycerides; Dose-Response Relationship, Drug; Female; Guinea Pigs; Pancreas; Prostaglandins E; Prostaglandins E, Synthetic; Rioprostil; Tetradecanoylphorbol Acetate

Stimulation of human polymorphonuclear leukocytes (PMN) may result in the metabolism of phospholipids other than phosphoinositides to generate second-messenger intermediary metabolites. We investigated agonist-induced breakdown of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acyl-GPC), which constitutes almost half the diradyl-GPC fraction in human PMN (Mueller, H. W., O'Flaherty, J. T., Green, D. G., Samuel, M. P., and Wykle, R. L. (1984) J. Lipid Res. 25: 383-388), in cells prelabeled with 1-O-[3H] alkyl-2-acyl-GPC. We also utilized normal-phase high pressure liquid chromatography to quantitate the accumulation of diradylglycerols (1-O-alkyl-2-acylglycerols and diacylglycerols) in stimulated PMN. Phorbol-12-myristate-13-acetate (PMA), 1-oleoyl-2-acetyl-sn-glycerol-, calcium ionophore A23187-, and f-methionyl-leucyl-phenylalanine (fMLP) stimulation of PMN resulted in a time- and concentration-dependent hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA) and 1-O-[3H]alkyl-2-acylglycerol. In all cases formation of 1-O-[3H]alkyl-2-acyl-PA preceded that of 1-O-[3H]alkyl-2-acylglycerol. The times between addition of stimulus and appearance of 1-O-[3H] alkyl-2-acylglycerol varied for PMA (40 s at 1.6 microM), A23187 (5 min at 5 microM), and fMLP (30 sec at 1 microM). Preincubation of cells with 1 microgram/ml pertussis toxin (PT) inhibited the breakdown of 1-O-[3H]alkyl-2-acyl-GPC in cells stimulated with 1 microM fMLP, indicating a role for a PT-sensitive G protein with this stimulus. Quantitation of diglycerides as diradylglycerobenzoates in PMN stimulated with PMA (10 min), A23187 (10 min), or fMLP demonstrated marked accumulation of both 1-O-alkyl-2-acylglycerols and diacylglycerols. The highest increases over controls were observed for fMLP (33-fold for 1-O-alkyl-2-acylglycerols and 17-fold for diacylglycerols). In stimulated PMN prelabeled with 1-O-[3H]hexadecyl-2-acyl-GPC and 1-O-alkyl-2-acyl-sn-glycero-3-[32P]phosphocholine, the ratio of 3H to 32P in 1-O-alkyl-2-acyl-PA compared to 1-O-alkyl-2-acyl-GPC suggested the involvement of a phospholipase D in the hydrolysis of 1-O-[3H]-alkyl-2-acyl-GPC. Thus, stimulation of human PMN results in the hydrolysis of 1-O-[3H]alkyl-2-acyl-GPC to yield 1-O-[3H] alkyl-2-acyl-PA and 1-O-[3H]alkyl-2-acylglycerol possibly initiated by activation of a phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Calcimycin; Choline; Chromatography, High Pressure Liquid; Diglycerides; Glycerides; Glycerophosphates; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Phosphatidic Acids; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Exocytosis; Oocytes; Ovum; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin; Xenopus laevis

Topics: Animals; Blood Platelets; Calcimycin; Cell Membrane Permeability; Diglycerides; Drug Synergism; Enzyme Activation; Glycerides; Kinetics; Protein Kinase C; Rabbits

It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.

Topics: Calcimycin; Cell Line; Diglycerides; Enzyme Activation; Glycerides; Granulocytes; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Phorbol 12,13-Dibutyrate; Phospholipase D; Phospholipases; Protein Kinase C; Tetradecanoylphorbol Acetate

The release of free arachidonic acid (AA) in cultured intestinal epithelial cells (INT 407) was investigated. INT-407 cells were first incubated overnight with radiolabeled 14C-AA, and most of the incorporated 14C-AA esterified into phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol. Labeled cells were then exposed to different stimulating agents and the release of free 14C-AA determined. The calcium ionophore A23187 caused a dose-dependent AA release that was preceded by a rapid uptake and a subsequent efflux of 45Ca2+. By contrast, phospholipase C from Clostridium perfringens caused a great AA release that was accompanied by an apparent uptake and a sustained intracellular accumulation of 45Ca2+. The cells alos released AA when exposed to the protein kinase C activator, 4 beta-phorbol-12-myristate-13-acetate (PMA), and this agent, like the diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol, significantly potentiated the AA release caused by A23187. Not only A23187-mediated but also phospholipase C- and PMA-mediated AA release was inhibited by 4-bromophenacyl bromide, a known phospholipase A2 inhibitor. These findings, taken together, indicate that AA release in intestinal epithelial cells can be caused by (i) Ca2+-mediated phospholipase activation, (ii) products of phospholipase C activity, and (iii) stimulation of protein kinase C. It is suggested, therefore, that AA release in intestinal epithelial cells is governed by intracellular Ca2+, protein kinase C-mediated protein phosphorylation, and activation of phospholipase A2.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Diglycerides; Enzyme Activation; Epithelial Cells; Epithelium; Humans; Intestinal Mucosa; Phospholipases; Tetradecanoylphorbol Acetate; Type C Phospholipases

We demonstrated a unique myoelectric response to phorbol esters and a diacylglycerol, 1-oleoyl-2-acetyl-snglycerol, in rabbit ileal loops. This retrograde activity, the orad migrating complex (OMC), was devoid of slow-wave distortion or alterations in slow-wave frequency. The OMC was not inhibited by the cyclooxygenase inhibitor indomethacin or enhanced by addition of the calcium ionophore A23187. The OMC occurred after 4 beta-phorbol 12-myristate 13-acetate, a phorbol ester known to activate protein kinase C, and after 4 alpha-phorbol 12,13-didecanoate, one that does not. Therefore, these studies provide initial evidence that the myoelectric activity induced by phorbol esters and a diacylglycerol is most likely in addition to the activation of protein kinase C.

Topics: Animals; Calcimycin; Diglycerides; Electric Conductivity; Electrophysiology; Ileum; Indomethacin; Male; Muscle, Smooth; Phorbol Esters; Rabbits; Tetradecanoylphorbol Acetate

Na+ "self-inhibition" in tight epithelia describes the reduction in apical Na+ permeability observed with increasing luminal Na+ concentration. Patch clamp was used to examine regulation of self-inhibition at the level of single Na+ channels. After cell-attached patches (pipette solution, 129 mM NaCl) were obtained on amphibian distal nephron cells (A6), the 129 mM NaCl (high Na+) apical bath outside of the patch was replaced with 3 mM NaCl (low Na+). Within minutes there was an increase in open channel probability (Po) and the appearance of one to five "new" channels in patch membranes. A similar increase occurred when apical Na+ entry was blocked by luminal amiloride (10 microM). A23187 (1 microM), a calcium ionophore, added after low Na+ exchange, abolished the rise in channel activity. Increased Po and new channels, induced by either luminal Na+ or amiloride, were also reversed by either 4B-phorbol 12-myristate 13-acetate (PMA; 0.1 microM) or 1-oleyl-2-acetyl glycerol (OAG; 10 microM) over 15-30 min. 4 alpha-Phorbol (0.1 microM), an inactive phorbol, did not reduce channel activity. D-Sphingosine (100 microM), a protein kinase C (PKC) inhibitor, increased Po and new channels.. 1) modulation of apical Na+ permeability by luminal Na+ does not require direct interaction of Na+ with the channel protein but, rather, appears to involve an intracellular regulatory pathway, 2) relieving self-inhibition alters both the number and kinetics of single Na+ channels, 3) the effect of low Na+ must be modulated via decreased apical Na+ entry and intracellular Na+, since amiloride yielded similar results, 4) changes in intracellular Na+ probably affect Na+ channel activity via cytosolic Ca2+, 5) the effects of decreasing luminal Na+ are reversed by PKC activators and mimicked by PKC inhibitors suggesting a possible role for PKC in Na+ self-inhibition.

Topics: Amiloride; Animals; Calcimycin; Cell Line; Diglycerides; Electric Conductivity; Homeostasis; Kidney; Kinetics; Protein Kinase C; Sodium; Sodium Channels; Sphingosine; Tetradecanoylphorbol Acetate

Generation of superoxide anion (O2-) and mobilization of intracellular Ca2+ in human neutrophils upon exposure to stimuli such as N-formylmethionylleucylphenylalanine (fMLP) were inhibited by the antibiotic cerulenin, which inhibits fatty acid and cholesterol biosynthesis. The inhibition of O2- generation and Ca2+-mobilization required certain periods of incubation with cerulenin and both abilities of the cells were gradually lost following a similar time-course. In contrast, significant Ca2+-influx from the medium was observed in cerulenin-treated cells as well as untreated cells. The results suggest that an event which coincides with the Ca2+-mobilization and not Ca2+ per se is important for the induction of O2- generation in the fMLP-stimulated cells and that this step is blocked in cerulenin-treated cells. Phorbol myristate acetate or synthetic 1-oleoyl-2-acetylglycerol were able to bypass the block and induced O2- generation in cerulenin-treated cells.

Topics: Antifungal Agents; Bacterial Toxins; Calcimycin; Calcium; Cerulenin; Diglycerides; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen; Phagocytes; Phagocytosis; Sodium Fluoride; Superoxides; Tetradecanoylphorbol Acetate

1. Inhibition of human polymorphonuclear leucocyte (PMN) function by adenosine was studied with respect to effects of adenosine on intracellular cyclic AMP and calcium during the PMN respiratory burst. 2. The adenosine analogue 5'-N-ethylcarboxamide-adenosine (NECA) and L-N6-phenyl-isopropyl-adenosine (L-PIA) inhibited PMN oxygen metabolite generation with relative potencies (NECA greater than adenosine greater than L-PIA) characteristic of an A2 receptor. 3. The respiratory burst was inhibited by adenosine when PMN were activated by calcium ionophore or chemotactic peptide but not when cells where activated by oleoyl-acetyl-glycerol (OAG). 4. Adenosine increased intracellular cyclic AMP during the PMN respiratory burst regardless of whether cells were stimulated by ionophore, chemotactic peptide or OAG. 5. To determine whether the differences in cell inhibition by adenosine were related to differences in intracellular calcium mobilization by each activating agent, calcium was evaluated with the fluorescent probe, indo-1. Adenosine suppressed the increase in intracellular calcium following PMN activation by calcium ionophore or chemotactic peptide. In contrast, calcium did not increase in PMN activated by OAG and adenosine did not affect intracellular calcium changes following this stimulus. 6. These results demonstrate that physiological concentrations of adenosine inhibit the PMN respiratory burst in association with an increase in intracellular cyclic AMP and reduction of intracellular calcium.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adult; Calcimycin; Calcium; Cyclic AMP; Diglycerides; Humans; In Vitro Techniques; Luminescent Measurements; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen Consumption; Phenylisopropyladenosine

The addition of 1-oleoyl-2-acetylglycerol (OAG), or phorbol-12-myristate-13-acetate (PMA) to platelets induced the phosphorylation of a 47,000 dalton protein (47 Kd), fusion of granule membranes with membranes of the surface connected canalicular system, the formation of large vesicles and the secretion of serotonin. 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7), and sphingosine, inhibitors of protein kinase C, significantly inhibited the ultrastructural changes and the phosphorylation of 47 Kd. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), structurally similar to H7, but a weaker inhibitor of protein kinase C, did not attenuate these responses to OAG or to PMA. H7, but not HA1004, also markedly inhibited secretion induced by the synergistic combination of OAG and the calcium ionophore A23187. Amiloride and 5-(N,N dimethyl)-amiloride, inhibitors of the Na+/H+ transporter, did not inhibit the ultrastructural response and the protein phosphorylation induced by OAG, or the secretion induced by the combination of A23187 and OAG. The results link the activation of protein kinase C by diglycerides to the labilization and fusion of granule membranes important for secretion.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amiloride; Blood Platelets; Calcimycin; Carrier Proteins; Cytoplasmic Granules; Diglycerides; Humans; In Vitro Techniques; Isoquinolines; Membrane Fusion; Membrane Potentials; Phosphoproteins; Piperazines; Platelet Aggregation; Protein Kinase C; Sodium-Hydrogen Exchangers; Sphingosine; Tetradecanoylphorbol Acetate

Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.

Topics: Animals; Calcimycin; Calcium; Cell Survival; Diglycerides; Enzyme Activation; HeLa Cells; Humans; L Cells; Mice; Protein Kinase C; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

The phosphorylation of 46K protein(s), which was generally observed in parallel with an activation of NADPH oxidase of guinea pig polymorphonuclear leukocytes (PMNL) in our previous studies (N. Okamura et al. (1984) Arch. Biochem. Biophys. 228, 270-277; T. Ohtsuka et al. (1987) J. Biochem. 101, 897-903), was increased by treatment with 1-oleoyl-2-acetylglycerol (OAG), even at low concentrations at which both superoxide anion (O2-) production and arachidonate release were little induced. On the other hand, exposure of PMNL to low concentrations of a calcium ionophore, A23187, stimulated arachidonate release without causing substantial O2- production and increase in phosphorylation of 46K protein(s). Simultaneous addition of the above-mentioned suboptimal concentrations of both OAG and A23187 markedly enhanced O2- production in PMNL. This enhancing effect may be ascribable to the increase in the phosphorylation of 46K protein(s) and arachidonate release, which are induced by the addition of OAG and A23187, respectively. Another arachidonate-releasing agent, N-formyl-methionylleucyl-phenylalanine (FMLP), also stimulated O2- production in accordance with an increase in the arachidonate release and protein phosphorylation. Simultaneous addition of OAG significantly enhanced the FMLP-induced O2- production, presumably by maintaining the 46K protein phosphorylation which would facilitate the effect of arachidonate released by FMLP. Thus, the present results suggest that phosphorylation of 46K protein(s) and arachidonate release synergistically induce O2- production in PMNL, although either of them alone hardly induces the production.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Diglycerides; Female; Guinea Pigs; N-Formylmethionine Leucyl-Phenylalanine; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophils; Phosphorylation; Proteins; Superoxides

Protein kinase C activity was studied in superoxide-producing human polymorphonuclear leukocytes. Using equivalent cell concentrations, superoxide production and particulate fraction-associated protein kinase C activity increased in parallel in phorbol 12-myristate 13-acetate (PMA), oleoyl-acetyl-glycerol (OAG), opsonized zymosan, and A23187-activated leukocytes. Also, an increase in particulate fraction-associated phospholipid-independent kinase activity was observed upon stimulation with these activators. In contrast, in formyl-methionyl-leucine-phenylalanine (FMLP)-activated cells the increase in superoxide production was only accompanied by an increase in particulate fraction-associated protein kinase C activity if the cells were pretreated with cytochalasin B. Purified protein kinase C activity was stimulated by OAG and PMA, whereas no stimulation was observed using A23187 or opsonized zymosan. It is suggested that the activation induced in human neutrophils by PMA, OAG, opsonized zymosan, and A23187 involves a tight membrane association of phospholipid-dependent and -independent protein kinase activity. This contrasts to FMLP-activated neutrophils, in which a membrane-bound form is only observed after pretreatment with cytochalasin B.

Topics: Calcimycin; Cell Membrane; Cells, Cultured; Diglycerides; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Superoxides; Tetradecanoylphorbol Acetate

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.

Topics: Calcimycin; Calcium; Cell Membrane; Diglycerides; Enzyme Activation; Humans; In Vitro Techniques; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Superoxides; Tetradecanoylphorbol Acetate

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cytochalasin B; Diglycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2

To clarify the possible role of protein kinase C in the control of parathyroid hormone (PTH)-degrading activity (PTHDA) in a PTH-responsive opossum kidney (OK) cell line, we investigated the effects of protein kinase C activators, 12-O-tetradecanoyl phorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 4 beta-phorbol 12, 13-didecanoate (4 beta-PDD). TPA, OAG, and 4 beta-PDD enhanced PTHDA in a dose-dependent fashion (10-50 ng/ml, 10-100 microgram/ml, and 10-50 nM, respectively), whereas 4 alpha-PDD, a non-activator of protein kinase C, did not affect it. HPLC analysis of TPA-treated samples revealed increase of all immunoreactive PTH fragments produced by OK cells. These findings suggested that activation of protein kinase C in OK cells would augment PTHDA in the cells.

Topics: Animals; Bucladesine; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Colforsin; Diglycerides; Enzyme Activation; Humans; Kidney; Opossums; Parathyroid Hormone; Peptide Fragments; Phorbol Esters; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h. These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction.

Topics: Adenosylmethionine Decarboxylase; Animals; Calcimycin; Carboxy-Lyases; Cholera Toxin; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Induction; Glycerides; Guinea Pigs; Lymphocytes; Protein Kinase C

Electron probe X-ray microanalysis revealed that cytoplasmic Ca2+ concentration increased in the restricted apical cytoplasm during stimulation of isolated guinea pig parietal cells with gastrin. Furthermore, this study, using 45Ca2+, aequorin and fura-2, revealed the mechanism involved in intracellular Ca2+ shifts caused by gastrin and the involvements of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol in producing those shifts. The gastrin-mediated and IP3-sensitive Ca2+ pool was located in the smooth-surfaced membrane-enriched areas and released Ca2+ in the initial phase. Gastrin-mediated Ca2+ mobilization was also evoked by diacylglycerol, comprising an intracellular Ca2+ mobilization followed by a late, sustained and more localised Ca2+ entry from the extracellular space.

Topics: Adenosine Triphosphate; Aequorin; Animals; Benzofurans; Calcimycin; Calcium; Calcium Radioisotopes; Cytoplasm; Diglycerides; Egtazic Acid; Electron Probe Microanalysis; Fluorescent Dyes; Fura-2; Gastrins; Glycerides; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Parietal Cells, Gastric; Sugar Phosphates; Tetradecanoylphorbol Acetate

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aminoquinolines; Animals; Calcimycin; Diglycerides; Enzyme Activation; Ethanol; Fluorescent Dyes; Isoquinolines; Liver; Male; Phenylephrine; Piperazines; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Type C Phospholipases

The effects of 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore A23187 on the proliferation of Schwann cells stimulated with either a myelin-enriched membrane fraction (MEF) or an axolemma-enriched membrane fraction (AEF) have been examined. Using incorporation of [3H]thymidine as an index of proliferation, 16% of the cells became labeled after incubation with MEF (20 micrograms protein/ml) and AEF (40 micrograms protein/ml) for 72 h. Only 0.5% of the cells became labeled in cultures which were not exposed to the membrane fractions. Addition of OAG (10-500 microM) or A23187 (1.9-190 nM) in the absence of the membrane mitogens had no effect on the proliferative response of quiescent cultures of Schwann cells. When added simultaneously, however, OAG and A23187 were able to induce proliferation of the cells, although the response was only 30% of the response achieved with maximal doses of either AEF or MEF. Both OAG and A23187 were able to potentiate the mitogenicity of AEF or MEF, but only when AEF and MEF were added at submaximal concentrations. When Schwann cells were prelabeled with [3H]glycerol and then stimulated to proliferate with AEF or MEF, the amount of [3H]diacylglycerol was increased two- to threefold above that in control cultures for time periods up to 1 h. These results suggest that the proliferation of Schwann cells induced by either AEF or MEF is partially mediated through the combined effects of diacylglycerol and an increase in intracellular calcium.

Topics: Animals; Axons; Calcimycin; Cell Division; Diglycerides; DNA; Glycerides; Myelin Sheath; Rats; Schwann Cells; Sciatic Nerve

Activation of cell phospholipase, release of arachidonic acid and stimulation of prostaglandin synthesis were studied in a newly described human tumor cell line (Lu-65). In the Lu-65 tumor cell line, the calcium ionophore A23187 (2 microM) caused a 100% increase in the release of 3H-arachidonic acid and a 7-fold increase in the synthesis of prostaglandin E2. 1-oleoyl, -2-acetyl-glycerol (100 microM) increased arachidonate release and prostaglandin E2 synthesis by 100%. A23187 and the protein kinase C activators, 1,2-dioctanoyl-glycerol and 1-oleoyl, -2-acetyl-glycerol, decreased the specific radioactivity of 3H-arachidonate in phosphatidylinositol by 37% and 57%, respectively. The effects of A23187 were blocked in Ca2+-free media or in the presence of the phospholipase A2 inhibitor, p-bromophenacyl bromide, while those of 1-oleoyl, -2-acetyl-glycerol were not. The data provide evidence in a human tumor cell line for calcium/phospholipase A2-dependent and independent pathways for arachidonic acid release, both of which preferentially hydrolyze phosphatidylinositol.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Diglycerides; Dinoprostone; Enzyme Activation; Humans; Lung Neoplasms; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Protein Kinase C; Tumor Cells, Cultured

1-oleoyl-2-acetyl glycerol (OAG), a potent activator of protein kinase C, inhibited the binding of 125I-labelled epidermal growth factor (EGF) in isolated rat pancreatic acini. Unlike cholecystokinin-octapeptide (CCK8) and the C-kinase activator 12-O-tetradecanoyl phorbol-13-acetate (TPA), two inhibitors of 125I-EGF endocytosis in the pancreas, OAG had no effect on the distribution of bound ligand between the cell surface and intracellular compartments. Unlike TPA, OAG failed to potentiate the inhibitory effects of the calcium ionophore A23187 on 125I-EGF cell-associated radioactivity and had no effect on either basal or carbachol-stimulated amylase release in acini. These data suggest that the actions of the synthetic diacyl-glycerol OAG are not fully equivalent with the action of other known activators of protein kinase C in the pancreatic acinar cell.

Topics: Amylases; Animals; Biological Transport; Calcimycin; Carbachol; Diglycerides; Enzyme Activation; ErbB Receptors; Glycerides; In Vitro Techniques; Male; Pancreas; Protein Kinase C; Rats; Rats, Inbred Strains; Sincalide; Tetradecanoylphorbol Acetate

1 Indomethacin (10(-4)M) causes marked augmentation of O-2 release from human neutrophils when these are stimulated by either 1,oleoyl-2,acetylglycerol or the divalent cation ionophore, A23187, the concentration-response curve for each agent being shifted to the left and the maximum response to each increased. 2 The diacylglycerol kinase inhibitor, R59022 (10(-5)M) has effects very similar to those of indomethacin on both the 1,oleoyl-2,acetylglycerol-induced and the A23187-induced concentration-response curves for O-2 generation. 3 The diacylglycerol lipase inhibitor, RHC80267 (10(-5 M) on the other hand, has a similar effect to indomethacin on 1,oleoyl-2,acetylglycerol-induced O2- generation but, unlike indomethacin, has no effect on A23187-induced O2- generation. Comparison of the effects of these three agents provides a clue to the locus of the action of indomethacin in increasing superoxide release, suggesting that it may act as a diacylglycerol kinase inhibitor. A component of diacylglycerol lipase inhibition may also be present. It is suggested that these results could have relevance for the use of indomethacin as an anti-inflammatory agent in chronic rheumatoid diseases.

Topics: Angiotensin-Converting Enzyme Inhibitors; Calcimycin; Cyclohexanes; Cyclohexanones; Diglycerides; Glycerides; Humans; In Vitro Techniques; Indomethacin; Kinetics; Neutrophils; Platelet Activating Factor; Pyrimidinones; Superoxides; Thiazoles

The regulation of pineal phospholipase A2 activity was studied indirectly by measuring the release of [3H]arachidonic acid from [3H]arachidonic acid-labeled tissue in organ culture and the formation of radiolabeled lysophosphatidylcholine by glands labeled with 32Pi or [14C]choline. Glands were transferred sequentially through a series of 10-min incubations in label-free medium. Norepinephrine (10(-5) M) stimulated [3H]arachidonic acid release by 2-fold; release peaked during the first 10 min and returned to basal levels during the third incubation period. Studies with selective alpha 1-, alpha 2-, and beta-adrenergic agents indicated that norepinephrine was acting through alpha 1-adrenergic receptors. Ca2+ appears to play a critical role because the effects of norepinephrine were mimicked by treatment with the Ca2+ ionophore A23187 and inhibited by inorganic Ca2+ channel blockers or EGTA; other [Ca2+]i elevating treatments also stimulated [3H]arachidonic acid release. The possibility that protein kinase C may be involved was studied because it is activated by the alpha 1-adrenergic agonist phenylephrine in the pineal gland (Sugden, D., Vanecek, J., Klein, D. C., Thomas, T. P., and Anderson, W. B. (1985) Nature 314, 359-361). Three protein kinase C activators stimulated [3H]arachidonic acid release with the same relative potency as that established for activation of protein kinase C (4 beta-phorbol 12-myristate 13-acetate greater than 4 beta-phorbol 12,13-dibutyrate greater than 1-oleoyl 2-acetylglycerol). The effects of norepinephrine, A23187, and protein kinase C activators appear to be mediated by phospholipase A2 because the effects of these compounds on [3H]arachidonic acid release are blocked by an established inhibitor of this enzyme, mepacrine, and because these compounds stimulate the formation of 32P- and 14C-labeled lysophosphatidylcholine by glands incubated with 32Pi or [14C]choline. In addition, an inhibitor of diacylglycerol lipase, another enzyme which generates arachidonic acid, did not inhibit the stimulation of [3H]arachidonic acid release by norepinephrine, A23187, or a phorbol ester. Cyclic nucleotides do not appear to play an important role in the regulation of phospholipase A2 activity because dibutyryl cyclic AMP does not alter [3H]arachidonic acid release and also because the amounts of cAMP and cGMP in the culture medium are not consistently associated with [3H]arachidonic acid release. These findings suggest that pineal phosphol

Topics: Adrenergic alpha-Agonists; Animals; Arachidonic Acid; Arachidonic Acids; Bucladesine; Calcimycin; Calcium; Diglycerides; Lysophosphatidylcholines; Male; Norepinephrine; Organ Culture Techniques; Ouabain; Ovariectomy; Phenylephrine; Phospholipases; Phospholipases A; Phospholipases A2; Pineal Gland; Potassium; Protein Kinase C; Quinacrine; Rats; Tetradecanoylphorbol Acetate

The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3-4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3',5'-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins

Topics: Animals; Calcimycin; Cells, Cultured; Cyclic AMP; Diglycerides; Enzyme Activation; Leydig Cells; Male; Protein Kinase C; Receptors, LH; Swine; Testosterone; Tetradecanoylphorbol Acetate

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetylglycerol (OAG) and the ionophore A23187 for 8 h, [3H]-thymidine incorporation into the acid-insoluble fraction of the cells was stimulated synergistically. Further addition of dibutyryl cAMP caused a biphasic effect on the synergistic stimulation. Dibutyryl cAMP augmented the synergistic stimulation when A23187 was at the concentration of 0.075 micrograms/ml, but inhibited it when the ionophore was at 0.25 micrograms/ml. At the higher concentration of A23187, dibutyryl cAMP stimulated the [3H]thymidine incorporation when culture was for 4 h, but inhibited it when culture was for 8 h. The results were the same when 12-0-tetradecanoylphorbol-13-acetate (TPA) was used instead of OAG. Butyrate could replace dibutyryl cAMP for stimulation of [3H]thymidine incorporation in combination with TPA and A23187, but not with OAG and A23187 at the lower ionophore concentration. Dibutyryl cAMP but not butyrate stimulated ornithine decarboxylase induction caused by TPA and A23187. These results suggest that the effect of dibutyryl cAMP on DNA synthesis induced by OAG and A23187 was biphasic and depended on the concentration of A23187 and on the time of culture, and that the stimulation mechanism of butyrate is different from that of dibutyryl cAMP.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Cells, Cultured; Diglycerides; DNA; Glycerides; Guinea Pigs; Lymphocytes; Ornithine Decarboxylase; Tetradecanoylphorbol Acetate

Newborn rat keratinocytes, the NBR cell line, synthesized the cyclooxygenase metabolic products, prostaglandins E2 and F2 alpha, and the lipoxygenase metabolic product, hydroxyeicosatetraenoic acid. This metabolism was stimulated by incubation of the cells with the Ca++ ionophore, A23187; melittin; bradykinin; recombinant human f-met epidermal growth factor; the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate; and the synthetic analog of diacylglycerol, 1-oleoyl-2-acetyl glycerol. Production of the cyclooxygenase products was inhibited by the synthetic glucocorticoid, dexamethasone. The stimulation appeared to be modulated by deesterification of arachidonic acid from the cellular lipids, presumably by phospholipase A2. Increased intracellular levels of Ca++ and phosphorylating activities that result from polyphosphoinositol turnover as well as phosphorylating activities independent of phosphatidylinositol turnover appear to be regulating phospholipase A2 hydrolysis of phospholipids.

Topics: Animals; Animals, Newborn; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cell Line; Dexamethasone; Diglycerides; Epidermal Growth Factor; Melitten; Prostaglandin-Endoperoxide Synthases; Rats; Skin; Tetradecanoylphorbol Acetate

Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.

Topics: Animals; Calcimycin; Capillaries; Cats; Cattle; Cells, Cultured; Diglycerides; DNA; Dose-Response Relationship, Drug; Drug Synergism; Glycerides; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mitosis; Pigment Epithelium of Eye; Retinal Vessels; Sugar Phosphates; Thymidine

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.

Topics: Animals; Calcimycin; Cells, Cultured; Diglycerides; Kidney; Kidney Tubules, Proximal; Kinetics; Opossums; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphates

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.

Topics: Calcimycin; Cell Differentiation; Cell Line; Diglycerides; Diterpenes; Enzyme Activation; Humans; Leukemia, Myeloid, Acute; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate

In rats, prostaglandins (PGs) have an essential role in the decidual cell reaction (DCR), but their mechanism of action at the cellular level within the endometrium is at present uncertain. To test the hypothesis that both protein kinase C activation and calcium mobilization mediate the action of PGs within the endometrium during decidualization, the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG), activators of protein kinase C in vitro, and the calcium ionophore A23187, which causes calcium mobilization, were infused, alone or combined, into the uterine lumen of rats sensitized for the DCR. The results obtained indicate that both PMA and OAG have an inhibitory effect on the DCR in rats. The calcium ionophore A23187, although having no apparent effect by itself, had a synergistic effect with PMA, but not with OAG, in inhibiting the DCR. The intrauterine infusion of PMA and/or A23187 had no effect on the increase in endometrial vascular permeability (EVP), which precedes the DCR. The inhibitory effect of PMA or PMA plus A23187 on decidualization is probably not mediated by a decrease in uterine PG synthesis, as assessed by the measurement of uterine prostaglandin E concentrations at various times during the intraluminal infusion. These data suggest that activation of protein kinase C can modulate the DCR.

Topics: Animals; Calcimycin; Calcium; Decidua; Diglycerides; Enzyme Activation; Female; Glycerides; Indomethacin; Prostaglandins; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Aggregation and serotonin secretion were studied in washed rat platelets after oral administration of ticlopidine or its more potent analog PCR 4099. Besides a complete suppression of the ADP-induced aggregation, the two drugs significantly inhibited aggregation and secretion induced by three protein kinase C activators (1-oleoyl-2-acetyl-sn-glycerol, OAG; 12-0-tetradecanoyl phorbol-13-acetate, TPA; phospholipase C), by the calcium ionophore A 23187 and by thrombin. The highest inhibition was observed at low stimuli concentrations but could be partly or almost completely overcome by increasing their concentrations. The combination of aspirin (ASA) with the ADP scavenging system, creatine phosphate/creatine phosphokinase (CP/CPK) in vitro resulted in an inhibition similar to that observed ex vivo after ticlopidine or PCR 4099 treatment. Moreover, these in vitro and ex vivo treatments were not additive. As identical results were obtained with CP/CPK alone but not with ASA, it is concluded that ticlopidine and PCR 4099 do not interfere with protein kinase C or calcium movements but specifically inhibit the effects of released ADP, which might explain the broad spectrum anti-platelet activity of these drugs.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Calcimycin; Clopidogrel; Diglycerides; Female; Platelet Aggregation Inhibitors; Rats; Serotonin; Tetradecanoylphorbol Acetate; Thrombin; Ticlopidine; Type C Phospholipases

This study aimed at investigating the mechanisms by which stimulation of human platelets results in activation of Na+/H+ exchange. Platelets were suspended in a slightly buffered medium and the stimulus-induced, amiloride-sensitive H+ release, reflecting Na+/H+ exchange, was estimated from changes in the medium pH. H+ release could be evoked by thrombin and by activators of protein kinase C such as 1-oleoyl-2-acetylglycerol (OAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the thrombin-and the OAG-induced Na+/H+ exchange could be blocked by trifluoperazine, a protein kinase C inhibitor. The thrombin-induced H+ release was also sensitive to increased intracellular cAMP levels, probably due to inhibition of phospholipase C activation, whereas the OAG-induced activation of Na+/H+ exchange was unaffected. Our data suggest that activation of Na+/H+ exchange is mediated by protein kinase C.

Topics: Alprostadil; Biological Transport; Blood Platelets; Bucladesine; Calcimycin; Carrier Proteins; Diglycerides; Epoprostenol; Glycerides; Humans; In Vitro Techniques; Protein Kinase C; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Thrombin; Trifluoperazine

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.

Topics: Animals; Calcimycin; Calcium; Cell Line; Diglycerides; Enzyme Activation; Ethers; Fibroblast Growth Factors; Fibroblasts; Gene Expression Regulation; Ionomycin; Mice; Oncogenes; Phorbol 12,13-Dibutyrate; Phorbol Esters; Platelet-Derived Growth Factor; Protein Kinase C; Tetradecanoylphorbol Acetate

The effects of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and a diacylglyceride, 1-oleoyl-2-acetyl-glycerol (OAG) on the secretion of two major exocrine products by dispersed rat submandibular cells were investigated. TPA stimulated the release of acinar cell mucin and ductal cell protease (arginine esterase) in a dose- and time-dependent manner. Mucin secretion was also provoked by OAG, which, however, had no effect on arginine esterase release. The unsaturated diacylglycerol, 1,2-diolein, elicited a greater mucosecretory response than did OAG at the same concentration, while the saturated 1,2-distearin produced a smaller response. Mucin and enzyme secretion caused by TPA or OAG in the rat submandibular model was not inhibited by either of two putative antagonists, the antipsychotic drug, fluphenazine, and the antibiotic, polymyxin B. The involvement of extracellular Ca2+ in TPA-induced secretion was examined by comparing responses of cells maintained in normal or Ca2+-free medium, or in medium containing the ionophore A23187. Although extracellular Ca2+ was not an absolute requirement for a secretory response, the results indicate a synergistic relationship between TPA and Ca2+ in stimulating the release of both mucin and arginine esterase. These results suggest a role for the Ca2+-, phospholipid-dependent enzyme, protein kinase C in the secretory mechanism of mucous and serous cells in the submandibular gland. This is consistent with the proposal that receptor-mediated hydrolysis of membrane phosphoinositides is an initial event in stimulus-response coupling in exocrine cells.

Topics: Animals; Calcimycin; Calcium; Carboxylic Ester Hydrolases; Diglycerides; Fluphenazine; Glycerides; Male; Mucins; Phorbols; Polymyxin B; Rats; Rats, Inbred Strains; Submandibular Gland; Tetradecanoylphorbol Acetate

A 37-year-old female who suffered from SLE had a bleeding disorder. At the time of initial evaluation, the main disease demonstrated was a delta-storage pool deficiency. After this improved, a marked decrease of aggregation still remained, when induced by either ADP, epinephrine, collagen, A23187, thrombin, or PAF-acether. Although arachidonate-induced aggregation was slightly decreased, thromboxane B2 was produced normally in response to exogenous arachidonate. The patient's endoperoxides and/or thromboxane A2 aggregated aspirin-treated platelets, though her platelets were themselves unresponsive. Impaired aggregability induced by TPA (12-0-tetradecanoylphorbol-13-acetate) or OAG (1-oleoyl-2-acetyl-glycerol) was also found. However, the phosphorylation of P43 and P20 induced by several stimulators including CA++ ionophore was normal, using 32P-labelled platelets. It is suggested that TPA or OAG-induced platelet aggregation requires not only the phosphorylation of those proteins, but also another unknown mechanism after the phosphorylation, and that the platelet dysfunction of this patient was due to a defect of some mechanism involving Ca++ uptake or mobilization of cytoplasmic Ca++ from intracellular storage sites.

Topics: Adenosine Diphosphate; Adult; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Blood Proteins; Calcimycin; Collagen; Diglycerides; Epinephrine; Female; Humans; Lupus Erythematosus, Systemic; Phorbols; Phosphorylation; Platelet Activating Factor; Platelet Aggregation; Tetradecanoylphorbol Acetate; Thrombin

The effects of phorbol esters and diacylglycerol on phosphate accumulation in the cultured mouse kidney cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on the renal phosphate handling. 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated phosphate accumulation dose-dependently. TPA-induced phosphate accumulation was synergistically enhanced with A23187. 4 alpha-phorbol 12,13-didecanoate did not stimulate the phosphate accumulation, while 4 beta-phorbol 12,13-didecanoate stimulated it. Additionally, 1-oleoyl-2-acetyl-glycerol exhibited a stimulatory effect on phosphate accumulation. These data indicated that protein kinase C is one of possible regulators of phosphate transport at the renal tubules.

Topics: Animals; Calcimycin; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Isomerism; Kidney Tubules; Mice; Phorbol Esters; Phosphates; Protein Kinase C; Tetradecanoylphorbol Acetate

Phosphorylation of a 36,000-dalton (36k-Da) protein of rat basophilic leukemia (RBL-2H3) cell membranes was investigated. This phosphoprotein has been suggested to be the beta-subunit protein of the immunogloblin E (IgE) receptor of RBL-2H3 cells [Teshima et al., Biochem. biophys. Res. Commun. 125, 867-874 (1984)]. Phospholipids such as phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine, which are known to be activators of protein kinase C, enhanced the phosphorylation of the 36K-Da protein. In contrast, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) which has been identified as a potent inhibitor of protein kinase C in vitro decreased incorporation of radioactive phosphate from [gamma-32P]ATP into this protein. These results indicate that the phosphorylation of the 36K-Da protein of RBL-2H3 cell membranes is catalyzed by protein kinase C. H-7 also inhibited the release of serotonin from RBL-2H3 cells stimulated with an antigen or calcium ionophore A23187 and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Treatment of the antigen-stimulated cells with TPA caused a synergistic effect on the serotonin release. A similar effect was obtained by treatment of A23187-stimulated cells with TPA or 1-oleoyl-2-acetyl glycerol.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Basophils; Calcimycin; Cell Membrane; Diglycerides; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Isoquinolines; Leukemia, Experimental; Molecular Weight; Phosphoproteins; Phosphorylation; Piperazines; Rats; Serotonin; Tetradecanoylphorbol Acetate

Calcium antagonists inhibit platelet aggregation, but whether this action is due to inhibition of the effect of agonists on cytoplasmic ionized calcium concentration is unknown. We studied this problem by loading gel-filtered platelets with either quin2 or aequorin and stimulating them with epinephrine, arachidonate, thrombin, the calcium ionophore A23187, 1-oleoyl-2-acetyl glycerol, or adenosine diphosphate in media with or without extracellular calcium. In response to all of these agonists, aequorin indicated an increase in cytoplasmic calcium that accompanied or preceded platelet aggregation. In calcium-containing media, verapamil, nifedipine, and diltiazem inhibited these effects in a concentration-dependent fashion, except for those produced by thrombin and A23187. Removal of extracellular calcium with EGTA reduced the calcium response to arachidonate, adenosine diphosphate, and 1-oleoyl-2-acetyl glycerol, and the calcium response and aggregation were further inhibited by the calcium antagonists. In general, strong inhibition of the aequorin cytoplasmic calcium signal by approximately 100 microM concentrations of nifedipine, verapamil, and diltiazem was correlated with inhibition of platelet aggregation, but high concentrations of the inhibitors were required. Since inhibition by the calcium antagonists of the cytoplasmic calcium response and aggregation exceeded the effect of simple removal of extracellular calcium, these drugs may affect internal redistribution of calcium in human platelets.

Topics: Adenosine Diphosphate; Aequorin; Aminoquinolines; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Calcium; Calcium Channel Blockers; Culture Media; Cytoplasm; Diglycerides; Diltiazem; Epinephrine; Fluorescent Dyes; Humans; Luminescent Proteins; Nifedipine; Platelet Aggregation; Thrombin; Verapamil

Interleukin 2 (IL 2) induces specific mRNA synthesis and secretion of an important immunoregulatory molecule, interferon-gamma (IFN-gamma). We have observed that treatment of an IL 2 independent murine T cell line, BUD-27, with IL 2, calcium ionophore A23187, or agents that activate phospholipid/Ca2+-dependent protein kinase C results in increased IFN-gamma mRNA transcription and release of anti-viral activity. These same agents each induced the subcellular redistribution of protein kinase C from cytosol to plasma membrane in both the BUD-27 cell line and its IL 2-dependent parent, CT6. Ionophore concentrations greater than 1 micron exhibited the most significant induction of IFN-gamma mRNA, which also correlated with the dose of ionophore, inducing translocation of protein kinase C. This correlation between increased mRNA levels and protein kinase C translocation suggests that a calcium-dependent event is involved in induction of IFN-gamma mRNA synthesis. Furthermore, the magnitude of the translocation of protein kinase C from cytosol to plasma membrane corresponded to the physiologic IL 2 dose-response for IFN-gamma secretion. The data suggest that the activation of protein kinase C and/or coordinate elevation of intracellular calcium may provide at least one mechanism of signal transduction for the regulation of IFN-gamma gene transcription.

Topics: Animals; Calcimycin; Cell Line; Cell Membrane; Cytosol; Diglycerides; Enzyme Activation; Interferon-gamma; Interleukin-2; Mice; Protein Kinase C; RNA, Messenger; Stimulation, Chemical; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.

Topics: Calcimycin; Calcium; Calcium Channel Blockers; Complement Activation; Diglycerides; Drug Synergism; Gallic Acid; Glycerides; Humans; Neutrophils; Phagocytosis; Phorbol Esters; Receptors, Complement; Receptors, Complement 3b; Type C Phospholipases

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.

Topics: Alprostadil; Calcimycin; Diglycerides; Dinoprostone; Enzyme Activation; Glycerides; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Prostaglandins E; Protein Kinase C; Protein Kinases; Superoxides; Tetradecanoylphorbol Acetate

Indomethacin at a concentration (10(-4) M) which depressed the effect on O2- generation by fMet-Leu-Phe, markedly enhanced O-2 generation by both 1-oleoyl-2-acetylglycerol and the calcium ionophore, A23187. These results are explicable in terms of the hypothesis that synergism between cytosolic calcium and protein kinase C is involved in signal transduction for the respiratory burst in the human neutrophil.

Topics: Calcimycin; Diglycerides; Glycerides; Humans; Indomethacin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Cholera Toxin; Diglycerides; Drug Synergism; Enzyme Induction; Glycerides; Guinea Pigs; Lymphocytes; Ornithine Decarboxylase; Protein Kinase C; Protein Kinases; Time Factors

The mechanism of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on calcitonin secretion was studied in a rat C-cell line, rMTC 6-23. TPA stimulated calcitonin secretion at the concentration of 16nM. This effect was synergistically enhanced with calcium ionophore, A23187. Synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), also showed a synergism with A23187 on calcitonin secretion. When dibutyryl cyclic AMP was added with TPA, an additive effect was obtained. These data suggest that C-kinase might be a possible regulator of calcitonin secretion in addition to the cyclic AMP-mediated pathway.

Topics: Animals; Bucladesine; Calcimycin; Calcitonin; Cell Line; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Phorbols; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.

Topics: Bucladesine; Calcimycin; Calcium; Calmodulin; Cytoplasmic Granules; Dibutyryl Cyclic GMP; Diglycerides; Drug Interactions; Egtazic Acid; Enzyme Activation; Glucuronidase; Lactoferrin; Muramidase; Neutrophils; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate; Vacuoles

For mitogenic response of macrophage-depleted human peripheral lymphocytes, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG) and Ca2+ ionophore are both needed in addition to a small quantity of plant lectin, phytohemagglutinin (PHA). PHA alone is not sufficient to produce the cellular response. The addition of TPA or OAG to these cells induces the activation of protein kinase C as assayed by the phosphorylation of its endogenous substrates. Apparently, TPA or OAG and A23187 together substitute for macrophages and act synergistically to potentiate the DNA synthesis of this lymphocyte preparation. The results suggest that protein kinase C activation and Ca2+ mobilization are essential and that additional receptor occupation by PHA is necessary for producing cell proliferation.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Lymphocyte Activation; Lymphocytes; Macrophages; Mitosis; Phosphoproteins; Phosphorylation; Phytohemagglutinins; Protein Kinase C; Protein Kinases; Rats; Tetradecanoylphorbol Acetate

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.

Topics: Biological Transport, Active; Calcimycin; Cytochalasin B; Deoxyglucose; Diglycerides; Diterpenes; Hexoses; Humans; Isomerism; Neutrophils; Protein Kinase C; Protein Kinases; Terpenes; Tetradecanoylphorbol Acetate; Trifluoperazine

1-Oleoyl-2-acetyl-glycerol induced a rise in ornithine decarboxylase activity in isolated epidermal cells in a concentration-dependent manner. The time course of the induction of ornithine decarboxylase by 1-oleoyl-2-acetyl-glycerol was similar to that by 12-O-tetradecanoylphorbol-13-acetate. A23187 did not enhance the enzyme induction caused by 1-oleoyl-2-acetyl-glycerol. Palmitoyl-DL-carnitine prevented the induction of the enzyme either by 1-oleoyl-2-acetyl-glycerol or 12-O-tetradecanoyl-phorbol-13-acetate. These results suggest that the activation of protein kinase C is an initial and essential event in the process of ornithine decarboxylase induction caused by 12-O-tetradecanoyl-phorbol-13-acetate.

Topics: Animals; Calcimycin; Cell Survival; Diglycerides; Enzyme Induction; Epidermis; Female; Glycerides; Mice; Oleic Acid; Oleic Acids; Ornithine Decarboxylase; Pregnancy; Protein Kinase C; Protein Kinases; Tetradecanoylphorbol Acetate; Time Factors

The in vitro effect of synthetic diacylglycerol (DG) and phorbol myristate acetate (PMA), potent stimulators of protein kinase C, was studied on prolactin release. These substances increased, in a concentration-dependent manner, prolactin release from primary cultures of anterior pituitary cells. Similarly, exposure of pituitary cells to phospholipase C, which liberates endogenous DG from various substrates, also enhanced prolactin release. The effect of Ca2+ mobilization on PMA-, synthetic DG- or phospholipase C-induced prolactin release was examined. A23187 at 400 nM or 2 ng/ml maitotoxin, a Ca2+ channel activator, did not affect prolactin release by themselves, but enhanced the release of prolactin induced by DG, PMA or phospholipase C. The stimulatory effects of DG, PMA and phospholipase C on prolactin release were reduced by co-incubation with dopamine. These results suggest that the presumed activation of protein kinase C by DG and mobilization of Ca2+ may be synergistically involved in the regulation of prolactin release. Dopamine appears to inhibit prolactin release at a point distal to the DG-enhanced stimulation of the process.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Diglycerides; Dopamine; Enzyme Activation; Female; Marine Toxins; Oxocins; Pituitary Gland, Anterior; Prolactin; Protein Kinase C; Protein Kinases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Type C Phospholipases

When cultured pituitary cells were stimulated with synthetic diacylglycerol such as 1-oleoyl-2-acetylglycerol (OAG), or with a potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which are known stimulators of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), enhanced release of luteinizing hormone (LH) was observed. Similarly, LH release was also stimulated by the Ca2+-ionophore, A23187. Simultaneous presence of A23187 and OAG or TPA resulted in a synergistic response that mimicked the full physiological response to gonadotropin releasing hormone (GnRH). Removal of extracellular Ca2+ only slightly affected the stimulatory action of TPA and OAG on LH release, but completely blocked the effect of GnRH. The results suggest that the stimulatory effect of GnRH on LH release may be mediated by two intracellular pathways involving Ca2+ and diacylglycerol as second messengers.

Topics: Animals; Calcimycin; Diglycerides; Drug Synergism; Enzyme Activation; Female; Luteinizing Hormone; Pituitary Gland, Anterior; Protein Kinase C; Protein Kinases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.

Topics: Aminoquinolines; Animals; Calcimycin; Calcium; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Ethers; Ionomycin; Liver; Male; Phenylephrine; Phorbols; Phosphorylases; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Vasopressins

Intercellular gap-junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate lyase-deficient human fibroblasts and argininosuccinate synthetase-deficient Chinese Hamster V79 cells. As previously shown, in this system junctional communication is completely inhibited by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the absence of extracellular calcium, TPA inhibition was less pronounced. However, synergism with calcium ionophore A23187 could not be demonstrated. Chlorpromazine, trifluoperazine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester partially antagonised the effect of TPA. No antagonism was demonstrable between calmidazolium and TPA. Treatment of co-cultures with exogenous phospholipase C or 1-oleoyl-2-acetylglycerol (OAG) resulted in communication inhibition, suggesting that protein kinase C activation is involved in the mechanism of phorbol ester-mediated communication inhibition. However co-cultures which had been made refractory to TPA by prolonged exposure to high concentrations remained sensitive to inhibition by phospholipase C and OAG. These results suggest either that diacylglycerol can produce other effects independent of protein kinase C activation, or that refractoriness to phorbol esters is not simply due to a decrease in the amount of protein kinase C.

Topics: Animals; Calcimycin; Cell Communication; Cells, Cultured; Chlorpromazine; Cricetinae; Cricetulus; Diglycerides; Gallic Acid; Humans; Imidazoles; Phorbols; Tetradecanoylphorbol Acetate; Type C Phospholipases

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.1 +/- 0.5-fold in eight experiments). This effect of PMA was mimicked by other stimulators of protein kinase C, such as phorbol dibutyrate and oleoyl acetoyl glycerol, but not by 4-alpha-phorbol 12,13-didecanoate, which does not stimulate protein kinase C. However, phosphorylation of the cytosolic 47-kDa protein, the major substrate for protein kinase C in platelets, was produced at lower concentrations of PMA and at a much higher rate than enhancement of arachidonic acid release by PMA, suggesting that 47-kDa protein phosphorylation is not directly involved in mobilization of the fatty acid. PMA also potentiated arachidonic acid release when stimulation of phospholipase C by the ionophore (which is due to thromboxane A2 and/or secreted ADP) was blocked by aspirin plus ADP scavengers, i.e. apyrase or creatine phosphate/creatine phosphokinase. Increased release of arachidonic acid was attributable to loss of [14C]arachidonic acid primarily from phosphatidylcholine (79%) with lesser amounts derived from phosphatidylinositol (12%) and phosphatidylethanolamine (8%). Phosphatidic acid, whose production is a sensitive indicator of phospholipase C activation, was not formed. Thus, the potentiation of arachidonic acid release by PMA appeared to be due to phospholipase A2 activity. These results suggest that diacylglycerol formed in response to stimulation of platelet receptors by agonists may cooperatively promote release of arachidonic acid via a Ca2+/phospholipase A2-dependent pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Diglycerides; Enzyme Activation; Fatty Acids, Unsaturated; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Phorbols; Phospholipases A; Phospholipases A2; Phospholipids; Phosphoproteins; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.

Topics: Acid Phosphatase; Animals; Brain; Calcimycin; Cell Adhesion; Cell Differentiation; Cell Line; Diglycerides; Enzyme Activation; Glycerides; Humans; Leukemia, Myeloid; Mice; Molecular Weight; Phorbols; Phosphoproteins; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Type C Phospholipases

Concentrations of the calcium ionophore A23187 and 1-oleoyl-2-acetyl-glycerol, which on their own are minimally effective in stimulating superoxide release from human neutrophils, show marked mutual potentiation when given simultaneously. The potentiating effect of the diacylglycerol can be shown to be dose-related. These results support the hypothesis that synergism between cytosolic calcium and protein kinase C is involved in signal transduction for the respiratory burst in the human neutrophil.

Topics: Calcimycin; Diglycerides; Drug Synergism; Glycerides; Humans; Neutrophils; Superoxides

Topics: Acetylglucosaminidase; Animals; Blood Platelets; Blood Proteins; Calcimycin; Diglycerides; Drug Synergism; Enzyme Activation; Exocytosis; Glycerides; In Vitro Techniques; Neutrophils; Protein Kinase C; Protein Kinases; Rabbits; Rats; Receptors, Cell Surface

When rat mast cells were stimulated by concanavalin A, phosphatidylinositol turnover was induced. Concurrently, several cytosol proteins were phosphorylated and histamine was released. 1-Oleoyl-2-acetyl-glycerol ( OAG ) and 12-O-tetradecanoylphorbol-13-acetate (TPA), known as direct activators of protein kinase C in intact cells, induced phosphorylation of the same spectrum of proteins. These proteins also served in vitro as preferable substrates for protein kinase C. The simultaneous addition of OAG or TPA and A23187 synergistically enhanced histamine release from mast cells. It is suggestive that protein kinase C activation and Ca2+ mobilization play essential roles for this cellular response.

Topics: Animals; Ascitic Fluid; Calcimycin; Calcium; Concanavalin A; Cytosol; Diglycerides; Drug Synergism; Histamine Release; In Vitro Techniques; Mast Cells; Phosphoproteins; Protein Kinase C; Protein Kinases; Rats; Tetradecanoylphorbol Acetate

When added independently to bovine adrenocortical fasciculata cell suspensions, 12 tetradecanoyl-phorbol-13 acetate (TPA) and the Ca2+ ionophore A23187 activated net cortisol production in a time and dose-dependent manner during one hour incubation. When added together (each at 1 microM concentration), the drugs appeared synergistic and mimicked the steroidogenic effect of suboptimal concentration of angiotensin II or acetylcholine on these cells, with no detectable variation of cellular cyclic nucleotide levels. In addition, the drug mixture markedly enhanced the steroidogenic effect of acetylcholine. These observations suggest that Ca2+-activated, phospholipid dependent protein kinase, which is present in adrenal cortex, might be considered as a possible target in the mechanism of action of steroidogenic agents such as angiotensin and acetylcholine, acting in adrenocortical cell through cyclic AMP independent processes.

Topics: Adrenal Cortex; Animals; Calcimycin; Cattle; Diglycerides; Hydrocortisone; In Vitro Techniques; Phorbol Esters; Phorbols; Protein Kinase C; Protein Kinases; Tetradecanoylphorbol Acetate

When human platelets were stimulated by synthetic diacylglycerol such as 1-oleoyl-2-acetyl-glycerol, which was a potent activator in vitro of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) (Mori, T., Takai, Y., Yu, B., Takahashi, J., Nishizuka, Y., and Fujikura, T. (1982) J. Biochem. (Tokyo) 91, 427-431), a protein having Mr approximately 40,000 (40-kilodalton protein) was rapidly phosphorylated, just as it was by natural extracellular messengers such as thrombin. Fingerprint analysis appeared to indicate that protein kinase C was indeed responsible for this 40-kilodalton protein phosphorylation in intact platelets. Under these conditions, neither inositol phospholipid breakdown nor endogenous diacylglycerol formation was observed, indicating that the synthetic diacylglycerol intercalated into the membrane and directly activated protein kinase C without interaction with cell surface receptors. During this process, the diacylglycerol was converted in situ to the corresponding phosphatidate, 1-oleoyl-2-acetyl-glyceryl-3-phosphoric acid. Experiments with the synthetic diacylglycerol and Ca2+ ionophore A23187 suggested that the protein phosphorylation catalyzed by protein kinase C was a prerequisite requirement for the release of serotonin, and that the receptor-linked protein phosphorylation and Ca2+ mobilization acted synergistically to elicit the full physiological cellular response.

Topics: Anti-Bacterial Agents; Blood Platelets; Blood Proteins; Calcimycin; Calcium; Diglycerides; Drug Synergism; Glycerides; Humans; Kinetics; Phosphorylation; Platelet Aggregation; Protein Kinases; Thrombin