sq-23377 and mastoparan

Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium Chloride; Capsaicin; Dinoprost; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Activators; Extracellular Space; Intercellular Signaling Peptides and Proteins; Ionomycin; Ionophores; PC12 Cells; Peptides; Phospholipases A; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, Drug; Wasp Venoms

Both activation of phospholipase A2 causing arachidonic acid release and tyrosine phosphorylation have been proposed to be involved in neuronal functions. Previously, we reported that orthovanadate (Na3VO4), an inhibitor of tyrosine phosphatases, stimulated tyrosine phosphorylation in proteins and enhanced Ca2+-induced noradrenaline release in rat pheochromocytoma PC12 cells. However, the role of tyrosine phosphorylation on phospholipase A2 activity and/or arachidonic acid release in neuronal cells has not been well established. The effects of Na3VO4 on arachidonic acid release and prostaglandin F(2alpha) formation were investigated in two types of neuronal cell lines. In PC12 cells, addition of Na3VO4 stimulated [3H]arachidonic acid release and prostaglandin F(2alpha) formation in a concentration-dependent manner. Co-addition of 5 mM Na3VO4 enhanced ionomycin-stimulated [3H]arachidonic acid release. Na3VO4 also enhanced ionomycin-stimulated [3H]arachidonic acid release from GH3 cells, a clonal strain from rat anterior pituitary. These findings suggest that the tyrosine phosphorylation pathway regulates arachidonic acid release by phospholipase A2 and prostaglandin F(2alpha) formation in neuronal cells.

Topics: Animals; Arachidonic Acid; Chelating Agents; Cytosol; Dinoprost; Dose-Response Relationship, Drug; Drug Synergism; Egtazic Acid; Hydrogen Peroxide; Intercellular Signaling Peptides and Proteins; Ionomycin; PC12 Cells; Peptides; Phospholipases A; Phospholipases A2; Rats; Tumor Cells, Cultured; Vanadates; Wasp Venoms

Previously, we proposed that S-nitroso-cysteine (SNC) was incorporated via the L-type-like amino acid transporters in rat brain slices. In PC12 cells (rat neuronal cell line), SNC inhibited [(3)H]arachidonic acid (AA) release induced by mastoparan (wasp venom peptide). We investigated the involvement of amino acid transporters on SNC-induced inhibition of [(3)H]AA release in PC12 cells. SNC inhibited mastoparan-stimulated [(3)H]AA release in a concentration-dependent manner in normal Na(+)- and low Na(+)-containing buffer. The inhibitory effect of 0.6 mM SNC in low Na(+) buffer decreased by 10 mM L-leucine, L-phenylalanine, L-methionine and L-cysteine. In contrast, L-alanine, L-threonine, L-valine or L-isoleucine showed very limited effects. Addition of L-leucine and L-phenylalanine, but not L-alanine or L-valine, also decreased the inhibitory effect of SNC on ionomycin/Na(3)VO(4)-stimulated [(3)H]AA release in normal Na(+) buffer. These findings suggest that SNC is incorporated via the amino acid transporters and inhibits AA release in PC12 cells.

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Arachidonic Acid; Cysteine; Intercellular Signaling Peptides and Proteins; Ionomycin; Ionophores; Neurons; Nitric Oxide Donors; PC12 Cells; Peptides; Rats; S-Nitrosothiols; Tritium; Vanadates; Wasp Venoms

Addition of arachidonic acid or stimulation of arachidonic acid production by secretory phospholipase A2 selectively upregulated apical endocytosis of ricin in MDCK cells without affecting basolateral endocytosis. Electron microscopic studies revealed that MDCK cells treated with secretory phospholipase A2 and incubated with horseradish peroxidase had an increased number of normal appearing peroxidase-labeled endosomes and no sign of membrane ruffling. Moreover, inhibition of basal arachidonic acid release, either by decreasing the cytosolic phospholipase A(2) activity or the diacylglycerol lipase activity, reduced the rate of apical endocytosis. Furthermore, indomethacin, an inhibitor of the cyclooxygenase pathway, counteracted the stimulation of endocytosis seen with both secretory phospholipase A2 and arachidonic acid, suggesting that formation of eicosanoids such as prostaglandins could be essential for the regulation. This idea was supported by the finding that prostaglandin E2, the predominant prostaglandin formed in kidney, also upregulated ricin uptake. The regulatory effect of the cyclooxygenase pathway on apical endocytosis of ricin was found to be independent of protein kinases A and C, which are known to selectively control apical clathrin-independent endocytosis in polarized cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Cell Polarity; Cyclic AMP-Dependent Protein Kinases; Cyclohexanones; Cyclooxygenase Inhibitors; Dogs; Endocytosis; Enzyme Activation; Enzyme Inhibitors; Heterotrimeric GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Ionomycin; Lipoprotein Lipase; Lipoxygenase Inhibitors; Organophosphonates; Peptides; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Kinase C; Ricin; Signal Transduction; Tetradecanoylphorbol Acetate; Wasp Venoms

Tyrosine phosphorylation has been shown to participate in the signal cascade after receptor stimulation with neurotransmitters and neurotrophins. However, the role of tyrosine phosphorylation in the process(es) of neurotransmitter release has not been well established. The effects of orthovanadate (Na3VO4), an inhibitor of protein-tyrosine phosphatases, on cytosolic free Ca2+ concentrations ([Ca2+]i), phosphotyrosine accumulation and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. Addition of Na3VO4 enhanced ionomycin-stimulated [3H]NA release in a concentration-dependent manner, although Na3VO4 alone had no effect. Na3VO4 also enhanced [3H]NA release induced by P2 receptor stimulation with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or by depolarization with 50 mM KCl, which stimulated a [Ca2+]i increase. A cell permeable inhibitor of protein-tyrosine phosphatases, L-p-bromotetramisole oxalate, at 0.3 mM enhanced ionomycin-stimulated [3H]NA release, although pervanadate had no effect. Addition of 5 mM Na3VO4 stimulated phosphotyrosine accumulation in several protein bands such as p130cas, but did not increase [Ca2+]i in PC12 cells. These findings suggest that the tyrosine phosphorylation pathway regulates Ca2+-stimulated NA release without changes of [Ca2+]i in PC12 cells.

Topics: Animals; Calcium; Intercellular Signaling Peptides and Proteins; Intracellular Membranes; Ionomycin; Ionophores; Norepinephrine; Osmolar Concentration; PC12 Cells; Peptides; Phosphorylation; Rats; Tetradecanoylphorbol Acetate; Tetramisole; Tyrosine; Vanadates; Wasp Venoms

Differentiation with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line toward a monocyte/granulocyte-like cell results in the cell acquiring an ability to release arachidonate upon stimulation. In contrast, the calcium ionophore ionomycin was able to stimulate phospholipase C, as measured by inositol 1,4,5-trisphosphate formation, to equal extents in both undifferentiated and dBcAMP-differentiated U937 cells. The role and regulation of cytosolic phospholipase A2 (cPLA2) in the production of arachidonate in these cells when either the chemotactic peptide fMLP or ionomycin are used as stimulus were investigated. The ionomycin- and fMLP-stimulated release of arachidonate were sensitive to the cPLA2 inhibitor arachidonyl trifluoromethylketone (IC50 values of 32 and 18 microM, respectively), but were not inhibited by E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2 H-pyran-2-one, a bromoenol lactone inhibitor of the calcium-independent phospholipase A2. These results, coupled with the inhibition of ionomycin-induced arachidonate production by electroporation of differentiated cells to introduce an anti-cPLA2, demonstrate that the cPLA2 is the enzyme responsible for arachidonate release in differentiated cells. SDS-PAGE and immunoblot analysis of differentiated cells showed the cells to contain both phosphorylated and unphosphorylated forms of cPLA2 (ratio of about 2: 3). Surprisingly, undifferentiated cells contain 30% more enzyme than differentiated cells and contain a higher percentage (approximately 75%) of the phosphorylated in the absence of stimulation. The inability of undifferentiated cells to produce arachidonate is not due to insufficient intracellular calcium concentrations since ionomycin induces large (820-940 nM) influxes of intracellular calcium in both differentiated and undifferentiated cells. This demonstrates that phosphorylation of cPLA2 andan influx of intracellular calcium are not sufficient to activate the enzyme to produce arachidonate. Instead, activation of a pertussis toxin-sensitive Gi alpha-type G-protein is required as evidenced by the production of arachidonate in undifferentiated cells stimulated with mastoparan, an activator of Gi alpha subunits, in combination with ionomycin. This activation of a Gi alpha-type G-protein is independent of modulations of adenylyl cyclase activity since cellular cAMP levels were not modulated upon treatment with mastoparan and ionomycin.

Topics: Acyltransferases; Adenylate Cyclase Toxin; Antibodies, Monoclonal; Arachidonic Acids; Blotting, Western; Bucladesine; Calcium; Cell Differentiation; Cyclic AMP; Cytosol; Electroporation; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Inositol 1,4,5-Trisphosphate; Intercellular Signaling Peptides and Proteins; Ionomycin; Ionophores; N-Formylmethionine Leucyl-Phenylalanine; Peptides; Pertussis Toxin; Phospholipases A; Phospholipases A2; Phosphorylation; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms