guanosine-triphosphate and 1-oleoyl-2-acetylglycerol

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclase Inhibitors; Animals; Aristolochic Acids; Bridged-Ring Compounds; Caffeine; Calcium; Carbachol; Diglycerides; Estrenes; Guanosine Triphosphate; Guinea Pigs; Ileum; Indoles; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Isoenzymes; Isoproterenol; Male; Membrane Potentials; Muscle, Smooth; Norbornanes; Patch-Clamp Techniques; Phospholipase C beta; Phospholipases A; Pyrrolidinones; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Ryanodine; Thiocarbamates; Thiones; Type C Phospholipases

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19-31)], and staurosporine] on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cell Membrane Permeability; Cytosol; Diglycerides; Dogs; Enzyme Inhibitors; Escin; Female; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Receptors, Muscarinic; Staurosporine; Trachea

Intracellular loading of nonhydrolyzable GTP analogs into innervated muscle cells in Xenopus cultures led to a marked increase in the frequency of spontaneous synaptic currents (SSCs), while extracellular application of the drugs at the same concentration was without effect. The increase in SSC frequency appeared to be unrelated to changes in the muscle membrane sensitivity toward acetylcholine (ACh), but resulted from an elevated spontaneous ACh secretion from the presynaptic nerve terminal. Postsynaptic loading of arachidonic acid (AA) produced a similar effect as the GTP analogs, and the potentiation effect of both GTP analogs and AA was reversed by an inhibitor of AA metabolism, AA861. Further studies indicate that a lipoxygenase metabolite, 5-HPETE, appears to be a likely candidate for the retrograde factor involved in modulating ACh secretion. These results suggest that G protein activation of the AA cascade in the postsynaptic cell could produce a retrograde signal to modulate transmitter secretion from the presynaptic nerve terminal at developing synapses.

Topics: Acetylcholine; Animals; Arachidonic Acid; Benzoquinones; Cyclic AMP; Diglycerides; Electric Conductivity; Embryo, Nonmammalian; Guanosine Triphosphate; Leukotrienes; Neuromuscular Junction; Organ Culture Techniques; Synapses; Xenopus

The major mortality and morbidity resulting from influenza virus infections are due to secondary bacterial infections which occur in association with virus-induced inhibition of polymorphonuclear leukocyte (PMNL) function. The present study was undertaken to determine if compounds which prime PMNL function to subsequent stimulation with N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) can overcome influenza A virus (IAV)-induced inhibition of the PMNL chemiluminescence response to these stimuli. Granulocyte-macrophage colony stimulating factor (GM-CSF), guanosine triphosphate (GTP), and 1-oleoyl-2-acetylglycerol (OAG) were able to prime the PMNL response to FMLP and/or PMA and totally or partially overcome IAV-induced PMNL dysfunction in cells stimulated with FMLP or PMA. A direct correlation was found between the extent of PMNL priming due to GM-CSF, GTP, and OAG and the capacity of these compounds to overcome virus-induced PMNL dysfunction. The implications of these findings in regard to the mechanism by which priming agents overcome IAV-induced cell dysfunction and the potential of these compounds as therapeutic agents to treat secondary bacterial infections are discussed.

Topics: Diglycerides; Granulocyte-Macrophage Colony-Stimulating Factor; Guanosine Triphosphate; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins, Viral; Humans; In Vitro Techniques; Influenza A virus; Luminescent Measurements; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Recombinant Proteins; Tetradecanoylphorbol Acetate; Viral Envelope Proteins

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.

Topics: Biological Factors; Bryostatins; Cell Line; Cell Nucleus; Cytokines; Diglycerides; Enzyme Activation; Guanosine Triphosphate; HIV; Humans; Lactones; Leukemia, Promyelocytic, Acute; Macrolides; Mitogens; Protein Kinase C; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Viral Proteins; Virus Replication

The hydrolysis of [3H]phosphatidylinositol (PI) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) by cytosolic inositide phosphodiesterase (phospholipase C) from Ehrlich ascites tumour cells was determined. Cytosolic fractions were prepared from tumour cells that had been cultivated for two days at low serum level (2%) in the presence of 1-oleoyl-2-acetyl-sn-glycerol (OAG). Cytosols from unstimulated cells (2% serum without OAG) were used for comparison. Phospholipase C acting on PI and PIP2 was significantly inhibited in the cytosol of OAG-stimulated cells. The suppressed enzyme was activated by Ca2+ and also by the guanine nucleotide GTP in a concentration-dependent manner independently of calcium ions. In the presence of Ca2+, GTP exerted a synergistic stimulatory effect. In contrast, GTP and GTP gamma S showed no effect on the phospholipase C activity of unstimulated cells. It is suggested that the suppressed PI- and PIP2-specific enzyme activity can be modulated by its susceptibility to Ca2+ ions and GTP probably via the GTP-binding protein.

Topics: Animals; Calcium Chloride; Carcinoma, Ehrlich Tumor; Diglycerides; Enzyme Activation; Glycerides; Guanosine Triphosphate; Kinetics; Mice; Type C Phospholipases

Addition of GTP markedly enhances the ability of thrombin to cause a leftward shift in the Ca2+ dose/response curve for 5-hydroxytryptamine secretion from permeabilised human platelets. Little effect is observed on addition of GTP in the absence of thrombin. Neither ADP nor adrenaline, in the presence or absence of GTP, causes such a shift, whereas 5-hydroxytryptamine does so to a small extent but only in the presence of GTP. The leftward shift in the Ca2+ dose/response curve induced by 12-O-tetradecanoyl-phorbol-13-acetate or 1-oleyl-2-acetylglycerol is not enhanced by addition of GTP. The thrombin concentration required for half-maximal enhancement of the response to Ca2+ is markedly reduced by addition of GTP. The results support the postulate that the effects of excitatory agonists in this system correlate with their ability to activate phospholipase C and provide further evidence for a role for GTP in signal transduction between the receptor and phospholipase C.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Blood Platelets; Calcium; Cell Membrane Permeability; Diglycerides; Dose-Response Relationship, Drug; Epinephrine; Guanosine Triphosphate; Humans; Serotonin; Tetradecanoylphorbol Acetate; Thrombin

Exposure of 'leaky' adrenal medullary cells to GTP-y-S inhibits Ca-dependent exocytosis in bovine cells, but stimulates exocytosis in chicken cells. The inhibitory action on bovine cells persists in the presence of TPA suggesting that in this tissue an inhibitory GTP-binding protein may modulate the action of protein kinase C on exocytosis.

Topics: Adrenal Glands; Animals; Calcium; Cattle; Chickens; Diglycerides; Exocytosis; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Tetradecanoylphorbol Acetate; Thionucleotides