Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to "bypass Sec14p" and inositol auxotrophy.
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SacIp dysfunction results in bypass of the requirement for phosphatidylinositol transfer protein (Sec14p) function in yeast Golgi processes. This effect is accompanied by alterations in inositol phospholipid metabolism and inositol auxotrophy. Elucidation of how sac1 mutants effect "bypass Sec14p" will provide insights into Sec14p function in vivo. We now report that, in addition to a dramatic accumulation of phosphatidylinositol-4-phosphate, sac1 mutants also exhibit a specific acceleration of phosphatidylcholine biosynthesis via the CDP-choline pathway. This phosphatidylcholine metabolic phenotype is sensitive to the two physiological challenges that abolish bypass Sec14p in sac1 strains; i.e. phospholipase D inactivation and expression of bacterial diacylglycerol (DAG) kinase. Moreover, we demonstrate that accumulation of phosphatidylinositol-4-phosphate in sac1 mutants is insufficient to effect bypass Sec14p. These data support a model in which phospholipase D activity contributes to generation of DAG that, in turn, effects bypass Sec14p. A significant fate for this DAG is consumption by the CDP-choline pathway. Finally, we determine that CDP-choline pathway activity contributes to the inositol auxotrophy of sac1 strains in a novel manner that does not involve obvious defects in transcriptional expression of the INO1 gene. (+info)
Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.
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This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors. (+info)
Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats - effects of feeding.
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The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes. (+info)
Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.
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The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation. (+info)
Effect of protein kinase C and phospholipase A2 inhibitors on the impaired ability of human diabetic platelets to cause vasodilation.
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1. The aim of this study was to examine the mechanism of impaired platelet-mediated endothelium-dependent vasodilation in diabetes. Exposure of human platelets to high glucose in vivo or in vitro impairs their ability to cause endothelium-dependent vasodilation. While previous data suggest that the mechanism for this involves increased activity of the cyclo-oxygenase pathway, the signal transduction pathway mediating this effect is unknown. 2. Platelets from diabetic patients as well as normal platelets and normal platelets exposed to high glucose concentrations were used to determine the role of the polyol pathway, diacylglycerol (DAG) production, protein kinase C (PKC) activity and phospholipase A2 (PLA2) activity on vasodilation in rabbit carotid arteries. 3. We found that two aldose-reductase inhibitors, tolrestat and sorbinil, caused only a modest improvement in the impairment of vasodilation by glucose exposed platelets. However, sorbitol and fructose could not be detected in the platelets, at either normal or hyperglycaemic conditions. We found that incubation in 17 mM glucose caused a significant increase in DAG levels in platelets. Furthermore, the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused significant impairment of platelet-mediated vasodilation. The PKC inhibitors calphostin C and H7 as well as inhibitors of PLA2 activity normalized the ability of platelets from diabetic patients to cause vasodilation and prevented glucose-induced impairment of platelet-mediated vasodilation in vitro. 4. These results suggest that the impairment of platelet-mediated vasodilation caused by high glucose concentrations is mediated by increased DAG levels and stimulation of PKC and PLA2 activity. (+info)
Alpha 1-adrenergic receptor-mediated increase in the mass of phosphatidic acid and 1,2-diacylglycerol in ischemic rat heart.
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OBJECTIVE: 1,2-Diacylglycerol (1,2-DAG) and phosphatidic acid (PA) are produced by phospholipase C and D activity and play a key role as second messengers in receptor-mediated signal transduction. So far, little is known about alterations of endogenous 1,2-DAG and PA production during myocardial ischemia. METHODS: Rat isolated perfused hearts were subjected to global ischemia, total lipids were extracted, and separated by thin-layer chromatography. The mass of PA and 1,2-DAG were quantified using laserdensitometric analysis of visualized lipids. RESULTS: Compared to normoxic control values (1,2-DAG 713 +/- 45 ng/mg protein, PA 171 +/- 11 ng/mg protein), the myocardial content of 1,2-DAG and PA was unaltered after 10 min of ischemia. Prolonged myocardial ischemia (20 min), however, which was accompanied by marked overflow of endogenous norepinephrine, significantly increased the mass of both second messengers (1,2-DAG 1062 +/- 100 ng/mg protein, PA 340 +/- 29 ng/mg protein). The increase in PA and 1,2-DAG in response to ischemia was abolished by inhibition of ischemia-induced norepinephrine release as well as by alpha1-adrenergic blockade but unaffected by beta-adrenergic blockade. While inhibition of diacylglycerol kinase did not affect ischemia-induced increase in PA and 1,2-DAG, inhibition of phosphatidylinositol-specific phospholipase C activity significantly suppressed ischemia-induced increase in 1,2-DAG but did not affect endogenous production of PA indicating phospholipase C-independent formation of PA and activation of both, phospholipase C and D, in the ischemic heart. CONCLUSIONS: Ischemia elicits an alpha1-adrenergic receptor-mediated increase in the mass of myocardial PA and 1,2-DAG. The increase in endogenous PA is suggested to be due to the activation of myocardial phospholipase D, whereas 1,2-DAG is formed predominantly by activation of phospholipase C in the ischemic heart. (+info)
Surfactant secretagogue activation of protein kinase C isoforms in cultured rat type II cells.
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Several lung surfactant secretagogues are known to activate protein kinase C (PKC) in type II cells. Such agents include 12-O-tetradecanoylphorbol 13-acetate (TPA) and cell-permeable diacylglycerols that directly activate PKC. Other agents include ATP and UTP, which act at P2Y(2) receptors coupled to phosphoinositide-specific phospholipase C, activation of which leads to formation of diacylglycerols and consequent activation of PKC. Activation of PKC is associated with redistribution of enzyme from a cytosolic to a membrane fraction of the cell. We examined the PKC isomers that are translocated by ATP, UTP, TPA, and dioctanoylglycerol in cultured type II cells isolated from adult rats. PKC isoforms were identified by Western blotting using isoform-specific antibodies. Treatment of type II cells with ATP, UTP, TPA, and dioctanoylglycerol resulted in a significant redistribution of PKC-mu from cytosol to membrane. TPA and dioctanoylglycerol also activated PKC-alpha, -betaI, -betaII, -delta, and -eta, but those isoforms were not activated by ATP or UTP. The effects of TPA and dioctanoylglycerol on PKC-mu were more pronounced than those of the P2Y(2) agonists, and the effect of TPA was also more rapid than that of ATP. The data show that direct activators and agents that generate endogenous diacylglycerols have different PKC activation patterns. Because it is activated by different types of secretagogues, PKC-mu may have an important role in the physiological regulation of surfactant secretion. (+info)
Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine.
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The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl-sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl-sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10(-7) mol/l of the PKC-specific inhibitor bisindolylmaleimide. The alpha(1)-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion alpha-ketoglutarate or to impaired alpha-ketoglutarate recycling, because it was unchanged in the continuous presence of alpha-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT. (+info)