sq-23377 and 1-oleoyl-2-acetylglycerol

Phospholipase D (PLD), which produces the lipid messenger phosphatidic acid (PA), has been implicated in superoxide generation and degranulation in neutrophils. The basis for this conclusion is the observation that primary alcohols, which interfere with PLD-catalyzed PA production, inhibit these neutrophil functions. However, off-target effects of primary alcohols cannot be totally excluded. Here, we generated PLD(-/-) mice in order to reevaluate the involvement of PLD in and investigate the molecular mechanisms of these neutrophil functions. Surprisingly, N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced these functions in PLD(-/-) neutrophils, and these functions were suppressed by ethanol. These results indicate that PLD is dispensable for these neutrophil functions and that ethanol nonspecifically inhibits them, warning against the use of primary alcohols as specific inhibitors of PLD-mediated PA formation. The calcium ionophore ionomycin and the membrane-permeative compound 1-oleoyl-2-acetyl-sn-glycerol (OADG) synergistically induced superoxide generation. On the other hand, ionomycin alone induced degranulation, which was further augmented by OADG. These results demonstrate that conventional protein kinase C (cPKC) is crucial for superoxide generation, and a Ca(2+)-dependent signaling pathway(s) and cPKC are involved in degranulation in mouse neutrophils.

Topics: Animals; Calcium Signaling; Cell Degranulation; Diglycerides; Drug Synergism; Enzyme Activation; Ethanol; Ionomycin; Mice; Mice, Knockout; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Superoxides

Cortical granule exocytosis (CGE), following egg activation, is a secretory process that blocks polyspermy and enables successful embryonic development. CGE can be triggered independently by either a rise in intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC). The present study investigates the signal transduction pathways leading to CGE through activation of PKC or stimulation of a rise in [Ca2+]i. Using Western blot analysis, co-immunoprecipitation and immunohistochemistry, combined with various inhibitors or activators, we investigated the link between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and CGE. We were able to demonstrate translocation of MARCKS from the plasma membrane to the cortex, in fertilized as well as in parthenogenetically activated eggs. MARCKS phosphorylation was demonstrated upon PKC activation, whereas a PKC inhibitor (myrPKCpsi) prevented both MARCKS translocation and CGE in 12-O-tetradecanoyl phorbol-13-acetate (TPA)-activated eggs. We have further shown that upon egg activation the amount of phosphorylated MARCKS (p-MARCKS) and the amount of calmodulin bound to MARCKS were increased. MARCKS translocation in ionomycin activated eggs was also inhibited by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W7). These results complement other studies showing MARCKS requirement for exocytosis and imply that upon fertilization, MARCKS translocation is followed by CGE. These findings present a significant contribution to our understanding of CGE in mammalian eggs in particular, as well as cellular exocytosis in general.

Topics: Animals; Biological Transport; Calcium; Calmodulin; Cell Membrane; Cytoplasmic Granules; Diglycerides; Enzyme Activation; Exocytosis; Female; Immunoblotting; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Ionomycin; Membrane Proteins; Microscopy, Confocal; Myristoylated Alanine-Rich C Kinase Substrate; Ovum; Parthenogenesis; Pregnancy; Protein Kinase C; Rats; Rats, Wistar; Signal Transduction; Sperm-Ovum Interactions; Sulfonamides; Tetradecanoylphorbol Acetate

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.

Topics: Animals; Calcium; Cells, Cultured; Cilia; Diglycerides; Embryo, Nonmammalian; Excitatory Amino Acids; Excitatory Postsynaptic Potentials; Indoles; Ionomycin; Maleimides; Naphthalenes; Protein Kinase C; Protein Kinase Inhibitors; Serotonin; Snails

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Topics: Animals; Calcium; Cell Membrane; Diglycerides; Female; Humans; Ion Channels; Ionomycin; Jurkat Cells; Kinetics; Leukemia, Basophilic, Acute; Lymphocytes; Membrane Potentials; Oocytes; Patch-Clamp Techniques; Rats; Saccharomyces cerevisiae; Second Messenger Systems; T-Lymphocytes; Thapsigargin; Tumor Cells, Cultured; Xenopus laevis

At fertilization of the mammalian egg, resumption of the cell cycle and the cortical reaction are two events of egg activation, correlated with an increase in intracellular Ca2+ concentration and activation of protein kinase C. To evaluate the pathways leading to both events, rat eggs were parthenogenetically activated by the calcium ionophore ionomycin, or by the protein kinase C activators 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG). Cortical granule exudate was visualized by the lectin Lens culinaris and Texas Red streptavidin, using a confocal microscope. Resumption of meiosis was detected by Hoechst dye, and intracellular Ca2+ concentration by fura-2. Ionomycin triggered both a cortical reaction and resumption of meiosis, while chelation of intracellular Ca2+ rise by BAPTA-AM (1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) revealed a segregation between these two events. A low Ca2+ transient (approximately 150 nM) induced a partial cortical reaction in half of the eggs, but the meiotic status was not affected. TPA triggered a cortical reaction with neither resumption of meiosis nor intracellular Ca2+ rise, while OAG induced both aspects of activation, as well as a significant intracellular Ca2+ rise. We conclude that in the cascade of events leading to egg activation, the initial Ca2+ rise is followed by a segregation in the pathway. A relatively low Ca2+ rise is sufficient to induce a partial cortical reaction. However, a higher level of Ca2+ is required to complete the cortical reaction and resumption of meiosis. The activation of the cell cycle is Ca2+-dependent, but protein kinase C-independent.

Topics: Animals; Calcium; Cell Cycle; Chelating Agents; Diglycerides; Egtazic Acid; Enzyme Activation; Female; Ionomycin; Ionophores; Lectins; Meiosis; Oocytes; Plant Lectins; Protein Kinase C; Rats; Rats, Wistar; Tetradecanoylphorbol Acetate; Xanthenes

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs.

Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cell Membrane; Diglycerides; Embryo, Mammalian; Enzyme Activation; Epidermal Growth Factor; Fibroblasts; Gene Targeting; Inositol Phosphates; Ionomycin; Isoenzymes; Mice; Mice, Knockout; Microscopy, Fluorescence; Phospholipase C gamma; Phospholipase D; Phosphorylation; Platelet-Derived Growth Factor; Signal Transduction; Type C Phospholipases

Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca(2+)-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca2+ ionophore ionomycin. The Ca2+ dependence of this effect was established, as removal of Ca2+ eliminated evoked release and the lipid-dependent potentiation. Also, Ca2+ channel blockers attenuated ionomycin- and KCl-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca2+, it may not involve an enhancement of evoked Ca2+ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca2+ accumulation. Also, the facilitation may not depend on inhibition of K+ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCl- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca2+ and involves multiple presynaptic effects, some of which depend on protein kinase C.

Topics: 4-Aminopyridine; Action Potentials; Animals; Arachidonic Acid; Calcium; Calcium Channel Blockers; Cholinergic Agents; Cytochalasin D; Cytoskeleton; Diazoxide; Diglycerides; Drug Synergism; Excitatory Amino Acid Agents; Exocytosis; Glutamic Acid; Hippocampus; Ionomycin; Ionophores; Nerve Endings; Potassium; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Stimulation, Chemical; Synaptic Transmission

K+ conductance species in a human intestinal epithelial cell line (Intestine 407) were studied in connection with their sensitivities to an intestinal secretagogue, histamine, using the tight-seal whole-cell patch-clamp technique. Applications of positive command pulses rapidly induced outward K+ currents. The conductance became progressively larger with increasing command voltages, exhibiting an outwardly rectifying current voltage relation. Inward K+ currents were also rapidly activated upon applications of hyperpolarizing pulses at potentials negative to the equilibrium potential of K+ (EK), and the conductance inwardly rectified. Application of a Ca2+ ionophore, ionomycin, brought about activation of additional K+ currents. An inhibitor of protein kinase C, polymyxin B, did not affect the ionomycin-induced response. Histamine (10-200 microM) also activated a similar K+ current which was abolished by cytosolic Ca2+ chelation. Under conditions where Ca2+ mobilization was minimized, histamine was found to significantly augment inwardly rectifying K+, but suppress outwardly rectifying K+, currents. Polymyxin B blocked these effects of histamine. An activator of protein kinase C, 1-oleoyl-2-acetylglycerol, mimicked the histamine effects. It is concluded that the intestinal epithelial cell has three distinct types of K+ conductance and that histamine modulates not only Ca(2+)-activated K+ conductance via Ca2+ mobilization, but also inward- and outward-rectifier K+ conductances via activation of protein kinase C.

Topics: Calcium; Cell Line; Diglycerides; Electric Conductivity; Epithelium; Histamine; Humans; Intestine, Small; Ionomycin; Polymyxin B; Potassium; Protein Kinase C

Parathyroid hormone-related protein (PTHrP) was discovered as a hypercalcemia-inducing product of malignant cells and has since been demonstrated to be a product of many tissues. Although it is robustly expressed in fetal lung, PTHrP expression has not been assigned to alveolar epithelial cells in adult lung. We have shown that PTHrP is expressed in the adult rat lung and by cultured rat alveolar type II epithelial cells with sensitive and specific immunoassays and immunohistochemical techniques. Immunoassay of cell extracts demonstrated that freshly isolated type II cells contained PTHrP (136 pg/10(7) cells), whereas freshly isolated alveolar macrophages and cultured macrophages did not express PTHrP. Cultured type II cells secreted PTHrP into medium, 202 +/- 11 fg PTHrP/micrograms cell protein in 24 h. Basal secretion remained stable up to 7 days in culture. Treatment with phorbol myristate acetate or 1-oleoyl-2-acetyl-sn-glycerol produced a dose-related, 2- to 4-fold increase in PTHrP secretion. However, forskolin, ionomycin, ATP, phenylephrine, capsaicin, and bradykinin had no effect. Thus, PTHrP secretion appeared to be regulated by a protein kinase C-dependent pathway. PTHrP could also be demonstrated in pulmonary lavage fluid. Although the function of PTHrP in the adult lung is unknown, it could involve control of cell growth and differentiation or control of surfactant lipid secretion. Further studies are necessary to elucidate the function of PTHrP in the lung.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cells, Cultured; Diglycerides; Epithelial Cells; Epithelium; Ionomycin; Lung; Male; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Protein Kinase C; Proteins; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Specific Pathogen-Free Organisms; Tetradecanoylphorbol Acetate

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

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium. The permeable cAMP analog 8-Br-cAMP (100 microM) and the calcium ionophore ionomycin (1 microM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12-myristate 13-acetate (100 nM) or with the diacylglycerol analog 1-oleoyl-2-acetyl-glycerol (100 microM): (i) down-modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8-Br-cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP-dependent chloride conductance in T84 cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Biological Transport; Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diglycerides; Down-Regulation; Electric Conductivity; Fluorescence; Fluorescent Dyes; Humans; Ionomycin; Membrane Proteins; Protein Kinase C; Protein Kinases; Quinolinium Compounds; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the alpha-subunits of the stimulatory and inhibitory G-proteins of AC (Gs alpha and G(i)-2 alpha) in cultured prolactin-producing rat pituitary adenoma cells (GH3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6-48 h) with ionomycin (1 microM) or 1-oleoyl-2-acetylglycerol (OAG; 1 microM) showed that ionomycin regulated Gs alpha levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered Gs alpha levels by more than 50% at all time-points. G(i)-2 alpha levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane Gs protein alpha-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of 'cross-talk' between the PLC- and AC-dependent signalling pathways.

Topics: Adenylyl Cyclases; Animals; Cell Line; Diglycerides; GTP-Binding Proteins; Ionomycin; Pituitary Gland; Rats; Signal Transduction; Thyrotropin-Releasing Hormone; Type C Phospholipases; Vasoactive Intestinal Peptide

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.

Topics: Adenosine Triphosphate; Blood Platelets; Cyclic GMP; Diglycerides; Ionomycin; Nitroprusside; Phosphorylation; Platelet Activation; Tetradecanoylphorbol Acetate

Endothelial relaxing factor has been identified as nitric oxide, formed from L-arginine by the soluble enzyme nitric oxide synthase. Nitric oxide inhibits platelet aggregation and adhesion by stimulating a soluble guanylate cyclase and increasing the intracellular concentration of cyclic GMP. Nitrovasodilators, such as sodium nitroprusside, release the active moiety, nitric oxide. In the present study, we have investigated the effect of sodium nitroprusside and of a permeable cGMP derivative on the aggregation and ATP secretion of human platelets stimulated with the protein kinase C activators 1-oleoyl-2-acetylglycerol or 4 beta-phorbol-12- myristate-13-acetate. Human platelets were treated with lysine acetylsalicylate, washed and resuspended in Tyrode-buffered solution. ATP secretion was evaluated by luciferin-luciferase luminescence. Nitroprusside (4-40 microM) or 8-Br-cGMP (0.1-2.4 mM) inhibited both platelet aggregation and ATP secretion evoked by 1-oleoyl-2-acetylglycerol (40 microM) or 4 beta-phorbol-12-myristate-13- acetate (4 nM) in a dose-dependent manner, in the presence of the selective inhibitor of cGMP phosphodiesterase, M&B 22948 (5 microM). The inhibitory effect of nitroprusside was reversed by hemoglobin, known to bind and inactivate nitric oxide. To study the calcium-dependent pathway, we treated platelets with the ionophore ionomycin. The ensuing aggregation and ATP secretion were rapid and were dependent on agonist concentration. Nitroprusside (4-40 microM) inhibited the aggregation evoked by ionomycin (0.4 microM) as well as ATP release, in a dose-dependent manner. We conclude that cGMP is able to inhibit both the protein kinase C-dependent and the calcium-dependent pathways leading to platelet activation.

Topics: Adenosine Triphosphate; Blood Platelets; Cyclic GMP; Diglycerides; Humans; In Vitro Techniques; Ionomycin; Nitroprusside; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Tetradecanoylphorbol Acetate

The ability of Ca2(+)-mobilizing hormones to promote changes in the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes. In recently isolated cells the distribution of PKC between the soluble and particulate fractions was 47 and 53% respectively. Exposure of the hepatocytes to 100 nM-vasopressin produced an increased phosphoinositide turnover, as reflected by the changes in the concentrations of inositol trisphosphate and Ca2+, and in glycogen phosphorylase a activity. However, the distribution of both PKC activity and [3H]phorbol dibutyrate binding between the cytosol and the membranes remained unchanged under these conditions. To determine the threshold values of the concentrations of Ca2+ and diacylglycerol required to produce a redistribution of PKC, the hepatocytes were treated with the Ca2+ ionophore ionomycin, and with permeant diacylglycerol derivatives. Hepatocytes incubated in the presence of 100 nM-vasopressin required concentrations of Ca2+ 2.5 times those produced physiologically by the hormone to produce translocation of PKC from the cytosol to the membranes. These studies suggest that, at least in hepatocytes, activation of PKC in response to Ca2(+)-mobilizing hormones involves only the pre-existent membrane-bound enzyme without affecting the soluble enzyme.

Topics: Animals; Calcium; Cell Membrane; Cytosol; Diglycerides; Ionomycin; Kinetics; Liver; Male; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphorylase a; Protein Kinase C; Rats; Rats, Inbred Strains; Vasopressins

The relationship between free cytoplasmic calcium, activation of protein kinase C (PKC) and expression of high-affinity interleukin 2 receptors (HA-IL-2R) on human T lymphocytes was studied. Induction of HA-IL-2R by phytohaemagglutinin (PHA) was associated with an increase in free cytoplasmic calcium and a transient increase in membrane-associated PKC. However, whereas addition of EGTA inhibited induction of receptors by PHA, addition of the PKC-inhibitor H7 did not. 12-o-tetradecanoyl-phorbol-13-acetate (PMA) and 1-oleoyl-2-acetyl-rac-glycerol (OAG) were both found to activate and translocate PKC. However, only PMA induced expression of HA-IL-2R. Not surprisingly, the effect of PMA was independent of extracellular calcium, but was inhibited by H7. Furthermore, a correlation between the number of HA-IL-2R and free cytoplasmic calcium upon stimulation with ionomycin was observed. Associated with the rise in intracellular calcium, the ionophore caused a slight increase in membrane-associated PKC. Also, addition of H7 inhibited expression of HA-IL-2R. Finally, OAG and ionomycin acted synergistically on expression of HA-IL-2R. In conclusion, induction of HA-IL-2R requires at least two different signals and neither activation of PKC nor an increase in free cytoplasmic calcium is sufficient. However, these two signals may act synergistically. There is evidence for both a PKC- and calcium-independent pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Diglycerides; Egtazic Acid; Ethers; Humans; Interleukin-2; Ionomycin; Isoquinolines; Phytohemagglutinins; Piperazines; Protein Kinase C; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate

Leukocyte adherence to endothelial cells (EC) is an important early event in inflammatory responses, which are often characterized by a predominance of either neutrophils (PMN) or monocytes. However, there is little information concerning the molecular events important in leukocyte adherence to EC. Intracellular activation of protein kinase C and the calcium-second messenger system leads to the stimulation of a number of important functions in PMN and monocytes. We compared the effects of members of these pathways on human PMN and monocyte adherence to cultured bovine aortic EC. We observed that phorbol myristate acetate, phorbol, 12,13-dibutyrate, L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol, and ionomycin each induced significant dose-dependent increases in PMN adherence to EC monolayers. In contrast, similar concentrations of each of these agents induced significant decreases in EC adherence of monocytes enriched by countercurrent centrifugal elutriation. Separate experiments determined that the differences in PMN and monocyte adherence to EC were not related to differences in oxidant production because 1) phorbol myristate acetate and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol caused similar marked increases in both PMN and monocyte superoxide anion and hydrogen peroxide production and 2) ionomycin, which had opposing effects on PMN and monocyte adherence, had no effect on PMN and monocyte superoxide anion or hydrogen peroxide release. We conclude that activators of protein kinase C and the Ca-second messenger pathway have opposite effects on PMN and monocyte adherence to EC and that these effects are mediated by O2 radical-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cattle; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydrogen Peroxide; Immune Adherence Reaction; Ionomycin; Monocytes; Neutrophils; Phorbol 12,13-Dibutyrate; Phorbols; Protein Kinase C; Second Messenger Systems; Superoxides

The subcellular localization of protein kinase C in unstimulated human neutrophils and neutrophils stimulated by phorbol-myristate-acetate (PMA), 1-oleoyl-2-acetyl-rac-glycerol (OAG), and ionomycin was investigated in subcellular fractions obtained by nitrogen cavitation and Percoll density gradient centrifugation. Protein kinase C was found to be localized mainly in the cytosol in unstimulated cells, whereas significant translocation to fractions containing the plasma membrane was observed after stimulation by PMA, OAG, and ionomycin. At the same time, phospholipid-insensitive protein kinase activity appeared in the cytosol and the plasma membrane fractions. To determine whether binding of protein kinase C occurred to the plasma membrane or to intracellular membranes that had translocated to the plasma membrane, we investigated the ability of isolated azurophil, specific and secretory granules, and plasma membrane vesicles to bind protein kinase C in response to addition of PMA and OAG. Only fractions containing plasma membranes and secretory granules were able to bind protein kinase C. The observation explains the selective activation of plasma membrane structures by protein kinase C.

Topics: Binding Sites; Cytoplasmic Granules; Cytosol; Diglycerides; Enzyme Activation; Ethers; Exocytosis; Humans; Ionomycin; Neutrophils; Protein Kinase C; Subcellular Fractions; Tetradecanoylphorbol Acetate

1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro) and 1-oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets. 2. Pre-treatment (10-300 s) with Oco2Gro (15-60 microM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62-125 microM) had no effect on thrombin-induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3. Collagen (20 micrograms/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10-300 s) with Oco2Gro (15-60 microM), PMA (16 nM) or OleAcGro (62 microM) before collagen addition or addition of these agents 30-60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2--2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations (less than 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5-15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 microM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with less than 2-min incubations) at sub-maximal agonist concentrations. 5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while Oco2Gro (60 microM) and PMA (16 nM) caused a 4--5-fold increase

Topics: Blood Platelets; Blood Proteins; Calcium; Collagen; Diglycerides; Ethers; Glycerides; Humans; Ionomycin; Kinetics; Male; Molecular Weight; Phosphorylation; Tetradecanoylphorbol Acetate; Thrombin; Thromboxane B2

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

Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete absence of added extracellular Ca (2.5-20 mM EDTA). The order of activity of phorbol esters and related compounds was that of their effect on protein kinase C (phorbol myristate acetate, phorbol dibutyrate greater than phorbol diacetate much greater than phorbol, 4 alpha-phorbol). 1-Oleyl, 2-acetylglycerol a synthetic activator of protein kinase C, also showed synergy with ionomycin. Phorbol esters and 1-oleyl, 2-acetylglycerol acted in synergy with ionomycin to liberate membrane Ca as detected by decreased fluorescence of chlortetracycline in prelabeled cells. Moreover, urushiol, the toxic principle of poison ivy, but not pentadecanylcatechol, its inert analogue, showed synergy with ionomycin. Synergistic aggregation was inhibited by calmidazolium (10 microM), piroxicam (20-100 microM), and pertussis toxin (20 micrograms/ml). The data not only confirm that marine sponge cell aggregation follows the general sequence of stimulus-response coupling in the cells of higher organisms but also support, in this most ancient of multicellular creatures, the hypothesis that mobilization of intracellular Ca and activation of protein kinase C provide the twin signals for cell activation in the absence of added extracellular Ca.

Topics: Animals; Calcium; Catechols; Cell Adhesion Molecules; Cell Aggregation; Chlortetracycline; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Ethers; Fluorescence; Glycerides; Ionomycin; Ionophores; Phorbol Esters; Piroxicam; Porifera; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate; Thiazines

Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl-2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells.

Topics: B-Lymphocytes; Binding, Competitive; Cell Division; Diglycerides; Drug Synergism; Enzyme Activation; Ethers; Glycerides; Humans; Ionomycin; Lymphocyte Activation; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Protein Kinase C; Protein Kinases; Receptors, Antigen, B-Cell; Staphylococcus aureus

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