sq-23377 and phosphatidylethanol

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.

Topics: Adenosine Triphosphate; Arachidonic Acid; Bucladesine; Cell Differentiation; Cytosol; Diglycerides; Electroporation; Ethanol; Glycerophospholipids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ionomycin; Ionophores; N-Formylmethionine Leucyl-Phenylalanine; Phospholipase D; Phospholipases A; Phospholipases A2; Signal Transduction; Solvents; U937 Cells

The production of platelet-activating factor (PAF) and the release of [3H]arachidonate were studied in human polymorphonuclear leukocytes (PMN) stimulated with thapsigargin, an inhibitor of endomembrane Ca(2+)-ATPase. Concentrations of thapsigargin as low as 10-25 nM primed PMN for both PAF production and [3H]arachidonate release in response to the chemotactic peptide (fMLP), whereas concentrations in the range 25-200 nM induced a time- and dose-dependent production of PAF, which occurred in the absence of both [3H]arachidonate release and [3H]phosphatidylethanol formation. Studies in fura-2/AM-loaded cells showed that concentrations of thapsigargin that elicited PAF production induced a protracted and long lasting elevation of cytosolic free calcium concentration ([Ca2+]i) between 200 and 700 nM. The lower concentrations primed the cells for a late [Ca2+]i elevation in response to fMLP similar to that elicited by cytochalasin B or ionomycin. PAF production showed a good correlation with the increase of [Ca2+]i (r = 0.91) irrespective of the procedure used to grade [Ca2+]i. In contrast, phorbol 12,13-dibutyrate failed to induce both PAF production and elevation of [Ca2+]i, but it was a very effective stimulator of [3H]arachidonate release and [3H]phosphatidylethanol production. These data indicate that PAF production and [3H]arachidonate release in PMN differ in both biochemical pathway and modulatory mechanisms. Whereas PAF production seems extremely sensitive to changes in [Ca2+]i, which seems to exert its modulatory effect at the lyso-PAF:acetyl-CoA acetyltransferase step, [3H]arachidonate release seems tightly modulated by protein kinase C-dependent mechanisms and is coincidental with activation of phospholipase D.

Topics: Arachidonic Acid; Calcium; Calcium-Transporting ATPases; Cell Membrane; Glycerophospholipids; Humans; In Vitro Techniques; Ionomycin; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidic Acids; Platelet Activating Factor; Terpenes; Thapsigargin

In the present study, we examined the ability of the phosphatase inhibitors p-nitrophenyl phosphate and 2,3-diphosphoglycerate (DPG) to inhibit phospholipase D (PLD) activation in the rabbit peritoneal neutrophil. Also assessed were choline, a product of PLD-catalyzed hydrolysis of phosphatidylcholine, and its metabolite phosphocholine. PLD activity was determined by measuring the accumulation, in the presence of ethanol, of [3H]phosphatidylethanol ([3H]PEt) in neutrophils prelabeled with 1-O-[3H]octadecyl-2-lyso-snglycero-3-phosphocholine. Of the compounds tested, only DPG interfered with PLD activation by N-formyl-Met-Leu-Phe (fMLP) in a dose- and time-dependent manner. In contrast, it augmented fMLP-stimulated levels of [3H]inositol phosphates in myo-[3H]inositol-labeled neutrophils. DPG also prevented PLD activation by the calcium ionophore ionomycin and by phorbol 12-myristate 13-acetate. The suppression of PLD activation by DPG appeared to arise from direct interaction with the enzyme, as evidenced by a DPG competitive pattern of inhibition (Ki = 9.0 +/- 1.5 mM) for PLD from Streptomyces chromofuscus. These results suggest that DPG may be a useful tool for investigating the role of PLD in physiological function in a wide variety of cell types. Interestingly, DPG inhibited fMLP-induced N-acetyl-beta-glucosaminidase release and O2- generation by the cytochalasin B-primed neutrophils in a dose-dependent manner, whereas it had minimal effect (at concentrations up to 5 mM) on O2- generation induced by fMLP in nonprimed cells. These results suggest that PLD plays an important role in fMLP stimulation of both N-acetyl-beta-glucosaminidase release and O2- generation in the primed neutrophils, but that a PLD-independent pathway plays the primary role in O2- generation by the nonprimed neutrophils.

Topics: 2,3-Diphosphoglycerate; Acetylglucosaminidase; Animals; Diphosphoglyceric Acids; Dose-Response Relationship, Drug; Enzyme Activation; Glycerophospholipids; In Vitro Techniques; Ionomycin; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitrophenols; Organophosphorus Compounds; Phosphatidic Acids; Phospholipase D; Rabbits; Streptomyces; Superoxides; Tetradecanoylphorbol Acetate

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.

Topics: Animals; Calcium; Endothelins; Enzyme Activation; Glycerophospholipids; Ionomycin; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Tetradecanoylphorbol Acetate

We have investigated phospholipase D activity in rat brain cortical slices prelabeled with [32P]orthophosphoric acid. In the presence of ethanol (170 mM), norepinephrine stimulated, in a dose-dependent manner (EC50 = 2.2 microM), the accumulation of [32P]phosphatidylethanol as a result of phospholipase D activity. Norepinephrine-stimulated phospholipase D activity was completely inhibited by prazosin, a specific alpha 1-adrenergic antagonist (Ki = 2.8 nM). However, no accumulation of phosphatidylethanol was observed in the presence of the muscarinic agonist carbachol. The Ca2+ ionophore ionomycin and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also stimulated [32P]phosphatidylethanol accumulation in cortical slices, in a dose- and time-dependent manner, whereas the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate, did not stimulate phospholipase D activity. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, two potent inhibitors of protein kinase C, inhibited PMA and ionomycin stimulation of phospholipase D activity, but did not affect the response to norepinephrine. Furthermore, the effects of PMA and norepinephrine were additive. Differences between PMA and norepinephrine stimulation of phospholipase D activity were also found with regard to the extracellular Ca2+ requirement and time course of phosphatidylethanol accumulation. No stimulation of phospholipase D activity by norepinephrine was observed in slices from cerebellum, a brain area with a low density of alpha 1-adrenergic receptors, while the effect of PMA was greater in the cerebellum than in cortical or hippocampal slices. These results strongly suggest that activation of phospholipase D in cortical slices by norepinephrine and PMA involve different mechanisms.

Topics: Animals; Calcium; Carbachol; Cerebral Cortex; Chromatography, Thin Layer; Drug Antagonism; Enzyme Activation; Glycerophospholipids; Ionomycin; Male; Norepinephrine; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase D; Phosphoric Diester Hydrolases; Prazosin; Protein Kinase C; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Tetradecanoylphorbol Acetate

This study was performed to determine the effects of phorbol esters and ionomycin on phospholipase D (PLD) activity in bovine corneal epithelial cells (BCEC). The cells were prelabeled with [3H]myristic acid and incubated for specific time intervals with various test agents in the presence and absence of ethanol. The PLD activity was assayed by monitoring the formation of labeled phosphatidylethanol ([3H]PEt) in [3H]myristate labeled cells. In the absence of ethanol, 1 microM phorbol 12-myristate 13-acetate (PMA) increased the formation of labeled phosphatidic acid ([3H]PA) with no significant effect on the radioactivity of [3H]PEt. In the presence of 85 mM ethanol, whereas there was only a small further increase in [3H]PA, the formation of [3H]PEt was increased by several-fold, demonstrating activation of PLD by the phorbol ester. The effects of PMA were time- and dose-dependent, and were mimicked by phorbol 12,13-dibutyrate. The inactive phorbol derivatives, 4-alpha-phorbol, 4-alpha-phorbol 12,13-didecanoate, 4-alpha-phorbol 12-myristate 13-acetate and 4-alpha-phorbol 12,13-dibutyrate, were without effect. Short-time (30 min) incubation of BCEC with staurosporine or H-7, or prolonged (20 hours) incubation with PMA rendered the cells less sensitive to subsequent treatment with PMA, suggesting that activation of PLD in the cells is mediated by protein kinase C (PKC). Addition of 20 microM ionomycin in the presence of ethanol also increased the formation of [3H]PA and [3H]PEt in a time- and dose-dependent manner. Co-presence of ionomycin and PMA at submaximal concentrations in the incubation medium resulted in increased formation of [3H]PA and [3H]PEt which was less than their individual effects combined, indicating a lack of synergism between Ca2+ and PMA in activating PLD. Incubation of BCEC with staurosporine resulted in significant inhibition of ionomycin-induced production of [3H]PEt, suggesting that in addition to direct activation of PLD by Ca2+, the enzyme is probably stimulated by sequential activation of PLC (producing diacylglycerol) and PKC following the ionomycin addition. We conclude that BCEC possess PLD which is stimulated by PKC as well as elevated intracellular Ca2+.

Topics: Animals; Cattle; Cornea; Enzyme Activation; Epithelium; Ethanol; Glycerophospholipids; Ionomycin; Myristic Acid; Myristic Acids; Phorbol Esters; Phosphatidic Acids; Phospholipase D; Phospholipids; Protein Kinase C; Protein Kinase Inhibitors