sq-23377 and 1-6-bis(cyclohexyloximinocarbonyl)hexane

The physiological role of IP(3)-dependent Ca(2+) release in T cell activation was in question due to the contradictory findings that [8-(Diethylamino)octyl-3,4,5-trimethoxybenzoate, HCl] (TMB-8), an inhibitor of intracellular Ca(2+) mobilization, blocked T cell proliferation, curtailing specifically the level of released Ca(2+) did not affect T cell activation and T cell line lacking IP(3) receptor was defective in IL-2 production in response to TCR/CD3 ligand. In the present study we found that TMB-8 inhibited Concanavalin A (Con A)- but not PMA/Ionomycin-induced T cell proliferation in a reversible and dose-dependent manner. The kinetic study revealed that TMB-8 exerted the inhibitory effect at a very early step of T cell activation. The Ca(2+) ionophore ionomycin augmented instead of overcoming the inhibitory effect of TMB-8, although the same doses of ionomycin alone had no effect on Con A-induced T cell proliferation. PMA the metabolically stable, but not diacylglycerol (DAG) the metabolically labile, activator of protein Kinase C (PKC) completely overcome the antiproliferative effect of TMB-8. A specific DAG lipase inhibitor RHC80267 also overcome the effect of TMB-8. Taken together, these results showed that the process of Ca(2+) release through IP(3) receptor, not the released Ca(2+), is essential for the sustained phase of PKC activation during T cell proliferation.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Division; Concanavalin A; Cyclohexanones; Diglycerides; Enzyme Activation; Gallic Acid; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ionomycin; Mice; Mice, Inbred BALB C; Naphthalenes; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; T-Lymphocytes; Tetradecanoylphorbol Acetate

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

In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cyclohexanones; Diglycerides; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrolysis; Ionomycin; Ionophores; Lipoprotein Lipase; Mice; Neuroblastoma; Phosphatidic Acids; Phospholipase D; Phosphoric Monoester Hydrolases; Prodrugs; Protease Inhibitors; Tumor Cells, Cultured; Type C Phospholipases

The association of prostaglandin D2 (PGD2) production as well as arachidonic acid release with the phospholipase D (PLD)-linked mechanism was studied in rat peritoneal mast cells. Stimulation of mast cells with cross-linking of the high-affinity Fc receptor for IgE caused increases in the release of arachidonic acid and PGD2, which are suppressed almost completely by ethanol or RHC 80267, a diacylglycerol lipase inhibitor. Ethanol did not influence inositol phosphate release in response to an antigen. An increase in diacylglycerol, that is inhibited by propranolol, was observed, with a peak within 1 min. Antigen stimulation induced little production of lysophosphatidylcholine, while ionomycin as a control markedly induced the production. However, the phospholipase A2 (PLA2) activity in the cytosol of antigen-stimulated cells increased to the level in ionomycin-stimulated cells. The addition of the ADP-ribosylation factor-containing fraction prepared from bovine brain, that is known to specifically activate PLD, to permeabilized mast cells in the presence of GTP gamma S, apparently increased arachidonic acid and PGD2 release, but not in the presence of ethanol. Furthermore, arachidonic acid release by an antigen was enhanced by melittin, that activates PLA2, but PGD2 production was not. These results suggest that antigen-stimulated PGD2 production as well as arachidonic acid release are strongly associated with the sequential PLD-linked pathway.

Topics: Adenosine Diphosphate Ribose; ADP-Ribosylation Factors; Animals; Arachidonic Acid; Brain; Cattle; Choline; Cyclohexanones; Dinitrophenols; Enzyme Activation; Enzyme Inhibitors; Ethanol; GTP-Binding Proteins; Inositol; Inositol Phosphates; Ionomycin; Kinetics; Lipoprotein Lipase; Lysophosphatidylcholines; Male; Mast Cells; Melitten; Peritoneal Cavity; Phospholipase D; Propranolol; Prostaglandin D2; Rats; Rats, Wistar; Receptors, IgE; Serum Albumin, Bovine

The protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu) induced the release of both luteinizing hormone (LH) and growth hormone (GH) from proestrous rat anterior pituitary pieces in vitro. Phorbol 12,13-dibutyrate-induced LH, but not GH release was readily inhibited by the phospholipase A2 (PLA2) inhibitors, quinacrine, aristolochic acid, ONO-RS-082 and chloracysine. Furthermore, PDBu induced release of [3H]arachidonic acid ([3H]AA) from pre-labelled anterior pituitary tissue that was prevented in the presence of quinacrine, aristolochic acid and ONO-RS-082 but not the diglyceride lipase inhibitor RHC 80267. The effect of PDBu was completely inhibited by staurosporine and the selective PKC inhibitor Ro 31-8220 but only partially by low concentrations of H7; consistent with the involvement of both H7-sensitive and H7-resistant forms of PKC in the activation of PLA2 by PDBu. The protein synthesis inhibitor cycloheximide inhibited the release of both [3H]AA and LH that had been induced by PDBu, whereas LH release induced by the PLA2 activator mellitin was cycloheximide-insensitive. These results suggest that PKC activators may induce LH but not GH release from anterior pituitary tissue by a mechanism involving activation of a PLA2, brought about by a process which is reliant on protein synthesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Aminobenzoates; Animals; Arachidonic Acid; Aristolochic Acids; Chlorobenzoates; Cinnamates; Cyclohexanones; Cycloheximide; Enzyme Activation; Female; Growth Hormone; Indoles; Ionomycin; Isoquinolines; Luteinizing Hormone; Melitten; ortho-Aminobenzoates; Phenanthrenes; Phenothiazines; Phorbol 12,13-Dibutyrate; Phospholipases A; Phospholipases A2; Piperazines; Pituitary Gland, Anterior; Proestrus; Protein Kinase C; Quinacrine; Rats; Rats, Wistar; Signal Transduction; Staurosporine

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cyclohexanes; Cyclohexanones; Diglycerides; Ethers; Female; Follicle Stimulating Hormone; Glycerides; Gonadotropin-Releasing Hormone; Ionomycin; Lipoprotein Lipase; Luteinizing Hormone; Pituitary Gland, Anterior; Rats; Tetradecanoylphorbol Acetate

We earlier showed that the diacylglycerol (DG) lipase inhibitor, RHC 80267, increased the steady-state level of DG and inhibited the release of arachidonic acid (AA) in carbamylcholine (CCh)-stimulated pancreatic minilobules (J. F. Dixon and L. E. Hokin, (1984) J. Biol. Chem. 259, 14418-14425). There was no effect on phospholipid metabolism. We have now investigated the effect of RHC 80267 on CCh-stimulated formation of inositol monophosphate formation, cGMP formation, and amylase release. CCh (10 microM) increased cGMP formation by approximately 20-fold, and this response was inhibited 55-75% by RHC 80267 (75-100 microM). RHC 80267 had no effect on either nitroprusside- or calcium ionophore-stimulated cGMP formation, arguing against a direct inhibition of guanylate cyclase by RHC 80267. Arachidonic acid, the release of which is inhibited by RHC 80267, neither stimulated cGMP formation nor reversed the effect of RHC 80267 on CCh-stimulated cGMP formation. This suggests, but does not prove, that the rise in cGMP in response to CCh is not due to an increase in AA as has been suggested. Both phorbol myristate acetate (25 nM) and the DG kinase inhibitor R 59022 (10 microM) inhibited CCh-stimulated cGMP formation by 40%. RHC 80267 also inhibited CCh-stimulated inositol phosphate accumulation and amylase release by 60 and 40%, respectively. The data suggest that the inhibition of CCh-stimulated cGMP formation and other muscarinic responses by RHC 80267 is probably the result of feedback inhibition of the cholinergic receptor via activation of protein kinase C by the elevated DG.

Topics: Amylases; Animals; Carbachol; Cyclic GMP; Cyclohexanes; Cyclohexanones; Diacylglycerol Kinase; Ethers; Guinea Pigs; Inositol Phosphates; Ionomycin; Kinetics; Lipoprotein Lipase; Nitroprusside; Pancreas; Phosphotransferases; Sugar Phosphates