calcimycin and 1-2-dioctanoylglycerol

The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Annelida; Calcimycin; Chromosomes; Colforsin; Cycloheximide; Diglycerides; Extracellular Signal-Regulated MAP Kinases; Fertilization; Flavonoids; MAP Kinase Signaling System; Meiosis; Okadaic Acid; Ovum; Parthenogenesis; Phosphorylation

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Aged; Antibodies, Monoclonal; Binding Sites; Blood Platelets; Calcimycin; Diglycerides; DNA, Complementary; Enzyme Activators; Flow Cytometry; Humans; Immunoblotting; Intercellular Signaling Peptides and Proteins; Ionophores; Macrophage-1 Antigen; Male; Myeloproliferative Disorders; Neutrophils; Peptides; Platelet Activating Factor; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Polymerase Chain Reaction; Protein Binding; Protein Kinase C; Receptors, Thrombin; RNA, Messenger; Sequence Analysis, DNA; Serotonin; Signal Transduction

The maturation of various aspects of sperm function have been demonstrated in monkey and human epididymal sperm, including the ability to undergo the acrosome reaction. The present study aimed to investigate the maturational changes in non-human primate sperm in the signal transduction mechanisms leading to the acrosome reaction involving cyclic AMP, Ca(2+) influx, protein kinase C, and protein tyrosine phosphorylation. Sperm from the caput, corpus, and cauda epididymidis of cynomolgus monkeys were incubated in a complete medium for 2.5 hr, followed by 30 min stimulation with 1 mM dibutyryl cAMP and 1 mM caffeine, 50 microM 1, 2-dioctanoyl-sn-glycerol (DOG), and 50 microM Ca(2+)-ionophore A23187. Quantitative Western blotting revealed little difference in tyrosine phosphorylated proteins among the caput, corpus, and cauda sperm without stimulation. Incubation with cAMP increased the amount of tyrosine phosphorylated proteins up to 10-fold in the corpus and cauda sperm, but to a lower extent in the caput sperm. Ca(2+)-ionophore attenuated the cAMP stimulation but had no effect on its own. Such responses in tyrosine phosphorylated proteins were in great contrast to the responses in the acrosome reaction, where A23187 was the strongest stimulant, resulting in induction of the reaction in 50 +/- 5%, 11 +/- 5%, and 8 +/- 4% cauda, corpus and caput sperm, respectively (mean +/- sem, n = 6). DOG and cAMP in combination induced acrosome reactions in about 10% of viable cells in the cauda and corpus but not caput sperm. Caput sperm responded to cAMP with increases in percentage motility without forward progression whereas cauda sperm displayed marked kinematic changes expected of hyperactivation. Comparisons of responses suggest that the major tyrosine phosphorylated proteins detected are unlikely to be involved immediately in the precipitation of the acrosome reaction, but more related to flagellar motion. Development of signal transduction pathways is part of the epididymal maturational process.

Topics: Acrosome Reaction; Animals; Biomechanical Phenomena; Blotting, Western; Calcimycin; Cyclic AMP; Diglycerides; Electrophoresis, Polyacrylamide Gel; Epididymis; Ionophores; Macaca fascicularis; Male; Phosphorylation; Signal Transduction; Sperm Motility; Spermatogenesis; Spermatozoa; Tyrosine

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Topics: Actin Depolymerizing Factors; Actins; Amino Acid Sequence; Animals; Calcimycin; Cell Membrane; Diglycerides; Diterpenes; Guinea Pigs; Humans; Hydrogen Peroxide; Immunoblotting; Marine Toxins; Microfilament Proteins; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophil Activation; Neutrophils; Okadaic Acid; Oxazoles; Oxides; Phosphorylation; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate

Defects in signal transduction mechanisms may underlie the impaired aggregation and secretion in patients with congenital platelet function defects (CPD). Both protein kinase C (PKC) induced pleckstrin phosphorylation and cytoplasmic Ca2+ mobilization play a major role in secretion. We postulated that combined platelet activation with a cell permeable direct PKC activator 1,2-dioctanoyl-sn-glycerol (DiC8) and ionophore A23187, which possibly bypass the steps involved in the intracellular synthesis of two major mediators (inositol trisphosphate, diacylglycerol), may induce normal dense granule secretion in patients with impaired receptor mediated secretion. We studied eight CPD patients with abnormal aggregation and secretion in response to several different surface receptor-mediated agonists despite the presence of normal dense granule contents. Receptor mediated Ca2+ mobilization and/or pleckstrin phosphorylation were abnormal in seven patients. Platelet activation with a combination of ADP (8 microM) with DiC8 (200 microM) or A23187 (10 microM) improved secretion in four patients. However, platelet activation with a combination of 200 microM DiC8 with 10 microM A23187, or 100 microM DiC8 with 5 microM A23187 induced normal secretion in platelet-rich plasma in all patients. These studies suggest that in such patients with CPD the ultimate process of exocytosis or secretion per se is intact and impaired secretion results from abnormalities in early signal transduction events, possibly upstream of diacylglycerol formation and calcium mobilization. Detailed studies are needed to delineate the specific abnormalities in these heterogenous patients with signal transduction defects.

Topics: Adolescent; Adult; Blood Platelets; Calcimycin; Calcium; Diglycerides; Female; Humans; Ionophores; Male; Middle Aged; Platelet Activation; Platelet Membrane Glycoproteins; Protein Kinase C; Receptors, Cell Surface; Signal Transduction

Treatment of metaphase II-arrested hamster eggs with activators of protein kinase C has been reported to promote resumption of the cell cycle, second polar body emission, and pronucleus formation (G.I. Gallicano, S.M. Schwarz, R.W. McGaughey, and D.G. Capco, 1993, Dev. Biol. 156, 94-106). In contrast, we have not observed these responses in mouse eggs obtained from CF-1 mice treated with these activators. In this report, we evaluated if this difference was due to differences in the technique used for PKC stimulation in the two different laboratories or due to species differences. Metaphase II-arrested hamster or mouse eggs were treated with phorbol diesters for 5 min or with a membrane-permeable diacylglycerol for 1 hr. Treatment of hamster eggs resulted in (1) the formation of "second polar body-like structures" commencing 5 min after treatment and reaching a maximum by 20-40 min; (2) a remarkable increase in the staining of filamentous actin in the region of these polar body-like structures; and (3) the disassembly of spindle microtubules. A reduction in cdc2/cyclin B1 kinase activity, as assessed by a decrease in H1 kinase activity, as well as progression from metaphase to anaphase were not observed. Treatment of mouse eggs from either CF-1 or CD-1 mice with these activators of PKC did not result in the formation of these polar body-like structures, did not cause an increase in filamentous actin, and did not result in a reduction in histone H1 kinase activity. This treatment, however, did induce disassembly of the spindle microtubules and the formation of multiple "pronucleus-like structures" that were more discernible in eggs from CD-1 mice. We conclude that the "apparent" activation of hamster eggs by activators of PKC is due to the effect of these agents on the cytoskeleton, which gives rise to structures that appear similar to polar bodies, but without any evidence of cell cycle resumption. The different responses seen in mouse and hamster eggs are mainly due to differences in the sensitivity of the cytoskeleton to rearrangements induced by these agents.

Topics: Amino Acid Sequence; Animals; Calcimycin; Cricetinae; Cytoskeleton; Diglycerides; Enzyme Activation; Female; Mesocricetus; Mice; Molecular Sequence Data; Ovum; Peptides; Phorbol Esters; Protamine Kinase; Protein Kinase C; Species Specificity; Substrate Specificity; Tetradecanoylphorbol Acetate

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.

Topics: Acrosome; Amidohydrolases; Atrial Natriuretic Factor; Calcimycin; Calcium; Carbohydrate Conformation; Carbohydrates; Diglycerides; Electrophoresis, Polyacrylamide Gel; Exocytosis; Glycoproteins; Glycosylation; Humans; Hydrogen-Ion Concentration; Male; Molecular Weight; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Semen; Spermatozoa; Tetradecanoylphorbol Acetate

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 microM) totally abolished this stimulation. Dopamine (DA; 1 microM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 microM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D2 type mode of action. The inhibition of GtH release in culture, caused by 1 microM APO, the specific DA D2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 microM) in perifusion was not reduced by DA (1 microM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca2+ ionophore A23187, however, their inhibitory effect was reversed by 10 microM ionomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Apomorphine; Arachidonic Acid; Bromocriptine; Calcimycin; Calcium; Cells, Cultured; Colforsin; Cyclic AMP; Diglycerides; Dopamine; Dopamine Agonists; Ergolines; Gonadotropin-Releasing Hormone; Gonadotropins, Pituitary; Ionomycin; Pituitary Gland; Quinpirole; Signal Transduction; Tilapia

The effects of second-messenger concentration changes on capillary diffusion capacity (permeability-surface area product [PS]) to cellular and paracellular tracers and on capillary ultrastructure were studied during countercurrent perfusion of the rete of the eel swim bladder. Cyclic nucleotide effects were investigated with isoproterenol, forskolin, and dibutyryl cAMP. Isoproterenol (5 x 10(-6) mol/L) did not modify water and solute permeability or capillary structure. Forskolin (10(-4) mol/L) immediately raised the concentrations of cAMP in the rete and produced interstitial edema but did not change permeability. The addition of dibutyryl cAMP (10(-6) mol/L) to the perfusate had rapid effects: it reduced the PS of [3H]water and oxygen and increased the PS of [125I]albumin, [14C]sucrose, and 22Na. No structural changes were observed. Phosphoinositide effects were studied with 1,2-dioctanoyl-sn-glycerol (DG) and phorbol 12-myristate 13-acetate (PMA). DG (10(-5) mol/L) had no effect on the permeability of the rete to water and solutes, while inducing cell membrane vacuolization. PMA (10(-5) mol/L) progressively reduced the PS of [3H]water. In contrast, PS values of [125I]albumin, [14C]sucrose, and 22Na rose gradually. Membrane vacuoles bulging into the lumen and in the cytoplasm were a common feature. The Ca2+ effect was investigated with the Ca2+ ionophore A23187. At 5 x 10(-6) mol/L, unsteady permeability changes and extensive cytolysis were observed. At 5 x 10(-7) mol/L, the PS of [125I]albumin, [14C]sucrose, and 22Na rapidly increased. The PS values for water were not modified. No structural changes were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Albumins; Animals; Bucladesine; Calcimycin; Calcium; Capillaries; Capillary Permeability; Colforsin; Diglycerides; Eels; Isoproterenol; Microscopy, Electron; Nucleotides, Cyclic; Phosphatidylinositols; Second Messenger Systems; Sodium; Sucrose; Tetradecanoylphorbol Acetate

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

An assay has been developed to quantitatively measure the tension and elasticity of the cytoskeleton in living plant cells. The cell optical displacement assay (CODA) uses a focused laser beam to optically trap and displace transvacuolar and cortical strands through a defined distance within the cell. Results from these experiments provide evidence for the classification of at least two rheologically distinct cytoskeletal assemblies, cortical and transvacuolar, that differ in their tension and response to both signaling molecules and reagents that perturb the cytoskeleton. It is further demonstrated that the tension of the transvacuolar strands can be significantly decreased by the addition of either linoleic acid, 1,2 dioctanoyl-sn-glycerol, or 1,3 dioctanoylglycerol. These decreases in tension could also be induced by lowering the cytoplasmic pH. In contrast, addition of Ca2+, Mg2+, or the ionophore A23187 to the cells caused a considerable increase in the tension of the transvacuolar strands. The data provides evidence that: (a) linoleic acid may be a signaling molecule in plant cells; (b) diacylglycerol functions as a signaling molecule through a protein kinase C-independent pathway mediated by PLA2; and (c) Ca2+ and pH have regulatory roles for controlling cytoskeleton tension and organization.

Topics: Calcimycin; Calcium; Cytoskeleton; Diglycerides; Fatty Acids; Glycine max; Hydrogen-Ion Concentration; Lasers; Linoleic Acid; Linoleic Acids; Lipid Metabolism; Magnesium; Microscopy, Fluorescence; Phospholipids; Signal Transduction

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

Acrosomal loss was induced in marsupial spermatozoa by an intermediate of the phosphoinositide pathway. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8; 100 mumol l-1) induced acrosomal loss in 70% of brushtail possum (Trichosurus vulpecula) spermatozoa and in 80% of tammar wallaby (Macropus eugenii) spermatozoa. The DiC8-induced acrosomal loss was not enhanced by co-incubation with calcium ionophore A23187 and occurred in Ca(2+)-free medium and in the presence of the calcium chelator EGTA (3 mmol l-1). There was no evidence of uptake of 45Ca2+ during the DiC8-induced acrosomal loss. Inhibitors of protein kinase C [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] and phospholipase A2 [dexamethasone] did not effect DiC8-induced acrosomal loss in wallaby spermatozoa. The phorbol ester, phorbol 12-myristate 13-acetate, at a concentration of 10 mumol l-1 had no effect on possum spermatozoa and induced acrosomal loss in only 6% of wallaby spermatozoa. It appears that the DiC8-induced acrosome reaction is not mediated by activation of the phosphoinositide pathway and that extracellular calcium is not required for the membrane fusion event. As acrosomal loss was seen only at relatively high concentrations of diacylglycerol (> 50 mumol l-1) and there is no evidence of involvement of other phosphoinositide intermediates or analogues, it is likely that its role is as a direct membrane fusogen.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acrosome; Animals; Calcimycin; Calcium; Cells, Cultured; Dexamethasone; Diglycerides; Exocytosis; Isoquinolines; Male; Marsupialia; Osmolar Concentration; Phorbol Esters; Piperazines; Protein Kinase Inhibitors; Semen; Spermatozoa

Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue. In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses.

Topics: Alkaline Phosphatase; Angiotensin II; Animals; Arginine Vasopressin; Calcimycin; Cells, Cultured; Culture Media; Dexamethasone; Diglycerides; Liver; Metallothionein; Phosphatidylinositols; Platelet Activating Factor; Rats; Tetradecanoylphorbol Acetate; Zinc

Activation of protein kinase C (PKC) in quiescent Swiss 3T3 cells using either the tumor promoter phorbol 12,13-dibutyrate (PDB) or diacylglycerols increased the tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) by 3.8-fold. PDB stimulation of p125FAK tyrosine phosphorylation was detected within 1 min and reached a maximum within 5 min, considerably slower than PDB stimulation of 80K/MARCKS phosphorylation which was maximal within 1 min. In sharp contrast, bombesin-induced tyrosine phosphorylation of p125FAK reached a maximum (8-fold stimulation) within 1 min after addition of the peptide and occurred with a half-maximal effect of 0.08 nM, 6-fold lower than the half-maximal effect of bombesin on 80K/MARCKS phosphorylation. Down-regulation of PKC by prolonged treatment with PDB blocked the effect of PDB on p125FAK tyrosine phosphorylation but had no effect on the response to bombesin. A selective inhibitor of PKC, GF 109203X, markedly inhibited the stimulation of p125FAK tyrosine phosphorylation by PDB but had little effect on the response to bombesin, vasopressin, and endothelin. Bombesin stimulation of tyrosine phosphorylation could also be dissociated from mobilization of Ca2+ from intracellular stores. Depletion of the intracellular Ca2+ pool by treatment with the tumor promoter thapsigargin completely blocked the ability of bombesin to transiently increase the cytosolic Ca2+ concentration but had no effect on bombesin stimulation of p125FAK tyrosine phosphorylation. In contrast, cytochalasin D, an agent which selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced p125FAK tyrosine phosphorylation. Within the same concentration range (0.3-2 microM), the drug had no effect on other early events stimulated by bombesin, including Ca2+ mobilization and activation of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathways are responsible for the rapid stimulation of p125FAK tyrosine phosphorylation by neuropeptide growth factors. Furthermore, the integrity of the actin cytoskeleton is essential for the effects of both PDB and bombesin.

Topics: 3T3 Cells; Actins; Animals; Bombesin; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Adhesion Molecules; Colchicine; Cytochalasin D; Cytoskeleton; Diglycerides; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Indoles; Intracellular Signaling Peptides and Proteins; Kinetics; Maleimides; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Phorbol 12,13-Dibutyrate; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Terpenes; Thapsigargin; Tyrosine

Preincubation of human neutrophils with 1-oleoyl-2-acetylglycerol (OAG) enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization. We have recently demonstrated that preincubation of neutrophils with OAG also reverses inhibition of A23187 stimulated [3H]arachidonate mobilization by the phospholipase A2 inhibitors, PGBx and aristolochic acid. The present study has compared the effects of 1,2-sn-dioctanoylglycerol (1,2-diC8) and 1,3-dioctanoylglycerol (1,3-diC8) on these cellular events. Dose-dependent priming (ED50 < 2.5 microM) of fMLP-stimulated [3H]arachidonate mobilization is obtained with both 1,2-diC8 and 1,3-diC8. Both diC8s also enhance fMLP-stimulated synthesis of leukotriene B4, 5-hydroxyeicosatetraenoic acid and platelet-activating factor, and generation of superoxide. Furthermore, both 1,2-diC8 and 1,3-diC8 reverse the effects of PGBx on A23187-stimulated [3H]arachidonate mobilization and platelet-activating factor synthesis. By contrast, higher concentrations (5-10 microM) of 1,2-diC8, but not 1,3-diC8, directly stimulate both [3H] arachidonate mobilization and superoxide generation. Since 1,3-diC8 does not activate protein kinase C (PKC), these results suggest that PKC is involved in direct activation of neutrophils by diacylglycerols but not in priming. Furthermore, reversal of the inhibitory effects of PGBx by diacylglycerols also appears to involve a PKC-independent mechanism.

Topics: Arachidonic Acid; Calcimycin; Diglycerides; Dose-Response Relationship, Drug; Humans; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Polymers; Prostaglandins B; Protein Kinase C; Signal Transduction; Superoxides

We investigated the signal transduction pathways that mediate activation of Syrian hamster eggs. Under conditions in which the concentration of intracellular free calcium ([Ca2+]i) is clamped low, activation of protein kinase C (PKC) can induce second polar body formation, reformation of the nuclear envelope, and decondensation of chromatin, as well as golgi reformation. However, calcium is necessary for normal transition from meiotic metaphase II to anaphase II. Conversely, under conditions in which the level of PKC activity is clamped low, induction of a rise in [Ca2+]i, using the calcium ionophore A23187, does not induce egg activation. These results strongly suggest that PKC acts after the calcium signal as a proximal inducer of egg activation. This suggestion is supported by the kinetics of egg activation; PKC stimulators activate the eggs at a significantly enhanced rate (P < 0.01) compared with activation by calcium ionophore. We show here that PKC stimulators induce emission of the second polar body, but that subsequently, with longer culture, the emitted polar body is absorbed. Our results suggest that the rise in [Ca2+]i serves two functions, to activate PKC and to induce the transition from metaphase II to anaphase II. PKC, once activated, mediates several other events of egg activation.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Cricetinae; Diglycerides; Egtazic Acid; Enzyme Activation; Female; Kinetics; Mesocricetus; Ovum; Phorbol Esters; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Carcinogens; Cells, Cultured; Cyclic GMP; Diglycerides; Enzyme Induction; Kinetics; Lipopolysaccharides; Liver; Nitric Oxide Synthase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rats; Tetradecanoylphorbol Acetate

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Alkaloids; Animals; Biological Transport; Calcimycin; Calcium; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Fluorescence; Fura-2; Goldfish; Gonadotropin-Releasing Hormone; Pituitary Gland; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Verapamil

We have examined the effects on X-ray induced malignant transformation in vitro of a number of activators and inhibitors of protein kinase C (PKC). Several of these substances were found to enhance or inhibit transformation, and the extent of the effects on transformation were found to be consistent with the potencies of the substances in activating or inhibiting PKC. Additionally, the observed transformation enhancement was found to be reversed by the presence of the anticarcinogenic protease inhibitors antipain or the Bowman-Birk inhibitor. These results suggest that activation of protein kinase C may be involved in the mechanism of in vitro X-ray induced malignant transformation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antipain; Calcimycin; Cell Transformation, Neoplastic; Diglycerides; Enzyme Activation; Isoquinolines; Mice; Mice, Inbred C3H; Neoplasms, Radiation-Induced; Piperazines; Protein Kinase C; Sulfonamides; Trifluoperazine; Trypsin Inhibitor, Bowman-Birk Soybean

The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.

Topics: Apolipoprotein A-I; Apolipoproteins A; Biological Transport; Calcimycin; Carrier Proteins; Cell Membrane; Cells, Cultured; Cholesterol; Diglycerides; Down-Regulation; Enzyme Activation; Humans; Lipoproteins, HDL; Protein Kinase C; Receptors, Cell Surface; Receptors, Lipoprotein; RNA-Binding Proteins; Skin; Sphingosine; Tetradecanoylphorbol Acetate

RNA transcription from the BamHI Z and BamHI R and HindIII G regions of the Epstein-Barr virus (EBV) genome was studied after treatment of Akata cells with anti-immunoglobulin G (IgG), with second messenger agonists or antagonists to determine how latent EBV activation is regulated by B cell second messengers. Northern gel analysis demonstrated that BZLF1, BZLF1 + BRLF1, and BMLF1 + BSLF2 transcripts were induced at 2 hr and increased in concentration at 4 hr after induction with anti-IgG; transcripts from BRRF1, BaRF1, BMLF1, and BMRF1 were initiated at 4 hr; a transcript from BRRF2 appeared at 6 hr. The patterns of transcription from these genes after repeated stimulations with calcium ionophore A23187 + dioctanoylglycerol paralleled those with anti-IgG except that times of initiation were delayed by about 2 hr. Nuclear run-off assay of BZLF1 gene showed rapid increases in their transcriptions from 30 to 60 min after anti-IgG treatment. The protein kinase C antagonist, staurosporine, completely blocked the appearance of these transcripts, while 8-bromo cAMP + theophylline suppressed the transcription by about 40%. The regulation of EBV activation in Akata cells with anti-IgG or with second messenger agonists or antagonists can be explained by regulation at the level of transcription of immediate-early genes of EBV.

Topics: Alkaloids; Antibodies, Anti-Idiotypic; B-Lymphocytes; Blotting, Northern; Calcimycin; Cell Line; Diglycerides; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Immunoglobulin G; Lymphocyte Activation; Second Messenger Systems; Staurosporine; Theophylline; Transcription, Genetic; Virus Activation

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

These studies examined the cellular basis for the inhibitory effects of triiodothyronine (T3) on growth hormone-releasing factor (GRF)-evoked growth hormone (GH) release from chicken anterior pituitary cells in vitro. A primary monolayer culture of anterior pituitaries from 4- to 8-week-old White Leghorn cockerels was performed as previously described by this laboratory. Following a 72-hr preincubation period, cells were washed and incubated (2 hr) with either secretagogues or media alone (control). T3 (20 ng/ml) or vehicle was added to cells during both the preincubation (48-72 hr) and incubation (2 hr period. Triiodothyronine reduced (P less than 0.05) GH release (ng/ml) in response to (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) the cAMP analog and protein kinase A activator, 8-bromo cAMP; and (4) the phorbol ester and protein kinase C activator, phorbol 12-myristate 13-acetate. Triiodothyronine reduced (P less than 0.05) the intracellular content of GH and total GH (released GH and intracellular GH) irrespectively of whether secretagogues were also present. When GH release was expressed as a percentage of total GH [released GH/(intracellular GH + released GH)], percentage GH released in response to GRF, or the protein kinase A, protein kinase C, or calcium pathway activators was not as great in T3-treated versus non-T3-treated cells. These data indicate that T3 inhibits GRF-evoked GH release by reducing the availability of intracellular stores of GH and by also inhibiting second messenger-stimulated GH release pathways.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Analysis of Variance; Animals; Calcimycin; Cells, Cultured; Chickens; Colforsin; Diglycerides; Dose-Response Relationship, Drug; Growth Hormone; Growth Hormone-Releasing Hormone; In Vitro Techniques; Male; Pituitary Gland, Anterior; Radioimmunoassay; Second Messenger Systems; Tetradecanoylphorbol Acetate; Triiodothyronine

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine; Adrenal Medulla; Animals; Biological Transport; Calcimycin; Caprylates; Cells, Cultured; Diglycerides; Dinucleoside Phosphates; Enzyme Activation; Ethers, Cyclic; Isoquinolines; Kinetics; Okadaic Acid; Phorbol 12,13-Dibutyrate; Piperazines; Protein Kinase C; Tetradecanoylphorbol Acetate; Triglycerides

Human peripheral blood monocytes synthesize the potent lipid autacoid platelet-activating factor (PAF) following appropriate stimulation. We examined the role of protein kinase C (PKC) in regulating the synthesis of PAF by stimulated monocytes. 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1,2-dioctanoyl-sn-glycerol, which directly activate PKC, stimulated the synthesis of PAF. Sphingosine, a long-chain amine that inhibits PKC, blocked both the binding of phorbol esters to monocytes and the synthesis of PAF in response to PMA (half-maximal inhibition at 5 to 10 microM and complete inhibition at 10 to 30 microM sphingosine). Thus, the activation of PKC was necessary and sufficient for PAF synthesis in response to phorbol ester. Sphingosine also blocked PAF synthesis in response to the calcium ionophore A23187 and opsonized zymosan particles by specific inhibition of PKC. Two other PKC inhibitors, stearylamine and staurosporine, also blocked PAF synthesis following A23187 or opsonized zymosan stimulation. These experiments demonstrated that PKC activation was required for PAF synthesis in response to the calcium signal generated by A23187 or a receptor-mediated agonist, opsonized zymosan. The synthesis of PAF and leukotriene B4 were temporally coupled following cell stimulation. Further, production of these two lipid mediators, and the release of arachidonic acid, were inhibited in parallel by sphingosine. Thus, PKC regulate the synthesis of both PAF and leukotriene B4 at a common step, probably phospholipase A2.

Topics: Binding Sites; Calcimycin; Diglycerides; Enzyme Activation; Humans; Kinetics; Leukotriene B4; Monocytes; Opsonin Proteins; Phagocytosis; Platelet Activating Factor; Protein Kinase C; Sphingosine; Tetradecanoylphorbol Acetate; Zymosan

Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33 degrees C, or room temperature when long-term sperm survival (greater than 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.

Topics: Acrosome; Animals; Bucladesine; Calcimycin; Cell Membrane; Cells, Cultured; Dibutyryl Cyclic GMP; Diglycerides; Lysophosphatidylcholines; Macropodidae; Male; Marsupialia; Microscopy, Electron; Sperm Capacitation; Spermatozoa; Tetradecanoylphorbol Acetate; Trypsin

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

Dispersed estradiol-treated rat pituitary cells were used to characterize progesterone (P) modulation of luteinizing hormone (LH) secretion in response to a variety of pharmacologic secretagogues which influence cell biochemistry. Acute (less than 3 h) and chronic (24 h) exposures to P prior to secretagogue challenge respectively enhanced and inhibited Ca2+ ionophore (A23187)-stimulated and gonadotropin-releasing hormone (GnRH)-stimulated LH release in similar quantitative fashion without any effect on concurrent prolactin release. Similar responses were also noted with cholera toxin-stimulated secretion. However, when protein kinase C activators such as phorbol esters and dioctanoylglycerol were used to trigger LH release, chronic exposure to P did not inhibit, but rather enhanced, LH release. Again, P had no effect on prolactin release. 'Washout' studies indicated that chronic treatments with P would suppress LH secretion stimulated by these compounds, but only when the steroid was cleared from the cells 4 h beforehand. These studies provide further evidence that P specifically modulates gonadotroph secretory function via mechanisms which bypass GnRH receptors. Moreover, they suggest that P exerts many different actions within the gonadotroph and question the role of protein kinase C in GnRH action.

Topics: Animals; Calcimycin; Cells, Cultured; Cholera Toxin; Cyclic AMP; Diglycerides; Estradiol; Female; Luteinizing Hormone; Pituitary Gland, Anterior; Pituitary Hormone-Releasing Hormones; Progesterone; Prolactin; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector e

Topics: Binding, Competitive; Caenorhabditis elegans Proteins; Calcimycin; Calcium; Carrier Proteins; Cell Membrane; Cytosol; Diglycerides; Humans; Ionomycin; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phorbol 12,13-Dibutyrate; Phospholipids; Protein Kinase C; Receptors, Drug; Sphingosine

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.

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

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 intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.

Topics: Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Membrane; Cytoskeletal Proteins; Cytosol; Desmoplakins; Desmosomes; Diglycerides; Dose-Response Relationship, Drug; Humans; Phorbol 12,13-Dibutyrate; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.

Topics: Animals; Calcimycin; Clone Cells; Diglycerides; Dose-Response Relationship, Immunologic; Drug Combinations; Enzyme Activation; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Protein Kinase C; Receptors, Antigen, T-Cell; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tetradecanoylphorbol Acetate; Time Factors

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.

Topics: Antigens, Surface; Calcimycin; Cell Adhesion Molecules; Diglycerides; Humans; Membrane Glycoproteins; Nerve Growth Factors; Pheochromocytoma; Phorbol Esters; Protein Kinase C; Thy-1 Antigens; Tumor Cells, Cultured

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

Cheek pouches of male Syrian golden hamsters were topically treated with a single dose of TPA (.5 microgram), calcium ionophore A23187 (75 micrograms) or Sn-1,2-dioctanoylglycerol (DiC8) (500 micrograms) dissolved in 0.25 ml acetone. Acetone-treated animals served as controls. After 48 h the mitotic index for the control group was 1.1 +/- 0.1 per 1 mm of the basement membrane length. All the test congeners exhibited higher mitotic indices than controls: TPA (4.8 +/- 0.4), A23187 (3.9 +/- 0.3), DiC8 (2.1 +/- 0.2). All groups exhibited an increase in the epithelial thickness manifested by cellular hyperplasia. The treatment of the pouches with the anti-inflammatory agent fluocinolone acetonide inhibited the mitogenic and hyperplasiogenic affects on the epithelium induced by the various test chemicals. These studies indicate a possible role of calcium-phospholipid dependent protein kinase (protein kinase C) in the mediation of oral epithelial cell proliferation.

Topics: Animals; Calcimycin; Cell Division; Cheek; Connective Tissue; Connective Tissue Cells; Cricetinae; Diglycerides; Epithelial Cells; Epithelium; Fluocinolone Acetonide; Glycerides; Hyperplasia; Male; Mesocricetus; Mitosis; Mouth Mucosa; Tetradecanoylphorbol Acetate

The release of histamine and other inflammatory mediators from human basophils is triggered by numerous stimuli, including chemical, physical and receptor-mediated activators. Several mechanisms of cell activation including protein kinase C activation have been proposed to operate in these cells. We used phorbol ester and DiC8 to induce histamine release from human basophils and the protein kinase C inhibitors H-7 and H-9 to inhibit this release. Both DiC8 and TPA induced histamine release were inhibited by H-7 (ID 50 = 37 mcM) and H-9 (IC 50 = 20 mcM). However, anti-IgE, fmlp and A23187-induced histamine release were unaffected. In contrast, the calmodulin antagonists W-7 and perphenazine effectively inhibited histamine release by all five stimuli. Therefore, different biochemical pathways appear to be critical for basophil activation depending on the nature of the stimulus used.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Basophils; Calcimycin; Calmodulin; Diglycerides; Glycerides; Histamine Release; Humans; Immunoglobulin E; In Vitro Techniques; Isoquinolines; Kinetics; Perphenazine; Piperazines; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate

We studied the regulation of thromboxane (TX) synthesis in promyelocytic leukemia cells during macrophage differentiation. Cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) showed rates of TXB2 synthesis from exogenous arachidonic acid that exceeded that of control cells by a factor of up to 81. Cells treated with sn-1,2-dioctanoylglycerol (diC8) showed similarly high TXB2 synthesis rates when diC8 was added concomitantly with a subthreshold concentration of TPA or when given in multiple doses. These activities depended on de novo synthesis of prostaglandin H (PGH) synthase because: microsomal PGH synthase activity showed large increases in Vmax values, and mass measurements of PGH synthase revealed the presence of PGH synthase in differentiating cells whereas the enzyme was undetectable in control cells. These results indicate that macrophage differentiation is associated with stimulation of TXB2 synthesis that requires both activation of protein kinase C and de novo synthesis of PGH synthase.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Differentiation; Cell Line; Diglycerides; Glycerides; Humans; Kinetics; Leukemia, Myeloid; Macrophages; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Tetradecanoylphorbol Acetate; Thromboxane B2; Trifluoperazine

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

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

The mechanism by which thrombin increases platelet fructose 2,6-bisphosphate content was investigated. The action of thrombin was mimicked by phorbol 12 myristate 13-acetate and 1,2-dioctanoylglycerol. Ca2+ with A23187 potentiated the action of both these compounds. The action of thrombin required mobilization of intracellular and extracellular Ca2+ and was not decreased by indomethacin. This study suggests that protein kinase C activation and Ca2+ mobilization are both involved in the activation of glycolysis by thrombin.

Topics: Blood Platelets; Calcimycin; Calcium; Diglycerides; Egtazic Acid; Fructosediphosphates; Glycolysis; Hexosediphosphates; Humans; Indomethacin; Tetradecanoylphorbol Acetate; Thrombin

GnRH stimulates LH release from pituitary gonadotropes. Prolonged exposure of these cells to GnRH results in decreased sensitivity to further stimulation by the releasing hormone both in vivo and in vitro. Chelation of extracellular Ca++ with EGTA blocks GnRH-stimulated LH release but does not prevent subsequent desensitization. Desensitization occurs when cells are preincubated in EGTA containing 10(-7) M GnRH for a variety of times (20 min to 12 h) or when cells are preincubated for 3 h in EGTA with 10(-10), 10(-9), or 10(-8) M GnRH. A GnRH antagonist does not cause desensitization to GnRH and blocks desensitization in response to GnRH in the Ca++-free medium. Preincubation in EGTA containing 10(-7) M GnRH for 3 h did not alter sensitivity of cells to sn 1,2 dioctanoylglycerol (a protein kinase C activator), Ca++ ionophore A23187, or veratridine (an activator of endogenous ion channels). These results suggest that desensitization results from occupancy of the GnRH receptor by an agonist and may be uncoupled from LH release.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Drug Tolerance; Egtazic Acid; Female; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Pituitary Gland, Anterior; Rats; Veratridine