scholarly journals The α1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C

1988 ◽  
Vol 253 (1) ◽  
pp. 93-102 ◽  
Author(s):  
R J Schimmel
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Neutral Lipids ◽  
Receptor Activation ◽  
Ionophore A23187 ◽  
Brown Adipocytes ◽  
Diacylglycerol Lipase

Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both of these reactions.

Oxytocin and vasopressin stimulate inositol phosphate production in human gestational myometrium and decidua cells

1986 ◽  
Vol 6 (7) ◽  
pp. 613-619 ◽  
Author(s):  
Michael P. Schrey ◽  
Alison M. Read ◽  
Philip J. Steer
Keyword(s):  
Arachidonic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Diacylglycerol Lipase ◽  
Inositol 1 ◽  
Inositol Phosphate Production ◽  
Acid Accumulation ◽  
Free Arachidonic Acid ◽  
Hydrolysis Of ◽  
Related Increase

The involvement of phosphoinositide hydrolysis in the action of oxytocin and vasopressin on the uterus was investigated in gestational myometrium and decidua cells by measuring the production of inositol phosphates. Both peptides stimulated a dose related increase in all three inositol phosphates in myometrium. This may be related to the control of sarcoplasmic Ca++ levels in the myometrium. Oxytocin and vasopressin also stimulated inositol 1-phosphate (IP) production in decidua cells. The hydrolysis of phosphatidylinositol by decidua homogenates exhibited a precursor-product relationship for diacylglycerol and arachidonic acid accumulation. Hence both peptides may mobilise free arachidonic acid, for prostaglandin biosynthesis, from decidua cell phosphoinositides by the sequential action of phospholipase C and diacylglycerol lipase.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

Angiotensin II stimulates phospholipases C and A2 in cultured rat mesangial cells

1987 ◽  
Vol 253 (1) ◽  
pp. C113-C120 ◽  
Author(s):  
D. Schlondorff ◽  
S. DeCandido ◽  
J. A. Satriano
Keyword(s):  
Arachidonic Acid ◽  
Angiotensin Ii ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Mesangial Cells ◽  
Inositol Trisphosphate ◽  
Pge2 Production ◽  
Diacylglycerol Lipase ◽  
Stimulation Of ◽  
In Cells

Angiotensin II stimulates prostaglandin (PG) E2 formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE2 are important in modulating glomerular function. We examined the mechanism for stimulation of PGE2 production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca2+-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE2 production in response to either angiotensin II or A23187. In contrast, TFP (50 microM) inhibited basal PGE2 production and stimulation by angiotensin II and A23187. TFP also decreased 14C release in response to angiotensin from cells prelabeled with [14C]arachidonic acid, which was associated with inhibition of 14C loss from phosphatidylinositol. In cells prelabeled with 32P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphospate. TFP enhanced 32P labeling of phosphatidylinositides, but did not prevent the loss of phosphatidylinositol 4,5-bisphosphate in response to angiotensin. This was verified in cells prelabeled with myo-[3H]inositol where angiotensin stimulated formation of [3H]inositol trisphosphate. TFP enhanced formation of [3H]inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in [32P]lysophospholipids, indicating activation of also phospholipase A2. This process was inhibited by TFP. Taken together, these results are consistent with stimulation of both phospholipase C and A2 by angiotensin, the latter step responsible for the release of arachidonic acid and PGE2 formation. The activation of phospholipase A2, but not that of phospholipase C, is inhibited by TFP, perhaps by interference with calmodulin-dependent steps.

Hydrolysis of phosphatidylinositol by human endometrium: modulating effects of steroids on arachidonic acid and 1,2-diacylglycerol release

1988 ◽  
Vol 117 (2) ◽  
pp. 309-314 ◽  
Author(s):  
R. C. Bonney ◽  
S. Franks
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Lipase Activity ◽  
Inositol Phosphates ◽  
Human Endometrium ◽  
Dehydroepiandrosterone Sulphate ◽  
Diacylglycerol Lipase ◽  
Fourfold Increase ◽  
Calcium Dependent ◽  
Oestrone Sulphate

ABSTRACT Phospholipase C and 1,2-diacylglycerol lipase activities were demonstrated in human endometrium using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylinositol as substrate. Phosphatidylinositol is hydrolysed by phospholipase C to inositol phosphates and to 1,2-diacylglycerol which is then further metabolized by 1,2-diacylglycerol lipase to release free arachidonic acid. In the present study the radiolabelled products formed (1,2-diacylglycerol and arachidonic acid) were measured following chloroform/methanol extraction and thin-layer chromatography. Phospholipase C activity was calcium dependent and optimal at pH 5·0–5·5 and 7·5; 1,2-diacylglycerol lipase activity was also calcium dependent, with an optimum pH of 5·5. A significant increase in 1,2-diacylglycerol production was stimulated by steroid sulphates. Pregnenolone sulphate, oestrone sulphate, testosterone sulphate and dehydroepiandrosterone sulphate stimulated 4, 3·2-1·8- and 2·6-fold increases in release respectively. Oestradiol sulphate stimulated a 25% increase in diacylglycerol release which was not significantly different from the control value. Progesterone stimulated a fourfold increase but other free steroids had no effect. Arachidonic acid release was increased in the presence of oestradiol sulphate, oestrone and oestradiol but reduced by oestrone sulphate, dehydroepiandrosterone sulphate, progesterone, dehydroepiandrosterone and, to a lesser extent, by pregnenolone sulphate and testosterone sulphate. 5-Androstene-3β, 17β-diol had no effect on the liberation of either product. This study demonstrates a potential route for the liberation of arachidonic acid from phosphatidylinositol in human endometrium. The opposing effects of steroids on phospholipase C and 1,2-diacylglycerol lipase activity could be important in regulating the release of arachidonic acid by this pathway. J. Endocr. (1988) 117, 309–314

EVIDENCE FOR INVOLVEMENT OF GTP-BINDING PROTEIN IN ARACKIDONIC ACID RELEASE BY PHOSPHOLIPASE A2 IN PERMEABILIZED HUMAN PLATELETS

1987 ◽  
Author(s):  
S Nakashima ◽  
T Tohmatsu ◽  
H Hattori ◽  
A Suganuma ◽  
Y Nozawa
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Binding Protein ◽  
Human Platelets ◽  
Arachidonic Acid Release ◽  
Gtp Binding Protein ◽  
Diacylglycerol Lipase ◽  
Gtp Binding ◽  
Acid Release

Platelet activation is accompanied by the active metabolism of membrane phospholipids. Phosphoinositide breakdown by phospholipase C generates second messengers; inositol trisphosphate and diacylglycerol. Recently, it is suggested that GTP-binding protein is linked to the activation of phospholipase C as is true for adenylate cyclase. Although it is known that the receptor stimulation by agonists leads to generation of arachidonic acid, its molecular mechanism has not yet been clear. However, several studies in neutrophils and mast cells using pertussis toxin, have shown the possibility that a GTP-binding protein may act as an intermediary unit component between the receptor and phospholipase A2. The present study was therefore designed to examine the effect of GTP and its analogue GTPγS on the arachidonic acid release in saponin-permeabilized human platelets. GTP or GTPγS alone caused a small but significant liberation of arachidonic acid in permeabilized cells but not in intact cells. GTP or GTPγS was found to enhance thrombin-induced [3H]arachidonic acid release in saponi n-permeabi li zed human platelets. The release of arachidonic acid has been ascribed to activity of phospholipase A2 and/or to sequential action of phospholipase C and diacylglycerol lipase. Inhibitors of phospholipase C (neomycin)/ diacylglycerol lipase (RHC 80267) pathway of arachidonate liberation did not reduce the level of the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost complementary to the increment of released [3H]arachidonic acid, suggesting thrombin-induced hydrolysis of phosphatidylcholine by phospholipase A2. Although phospholipase A2 usually are described as having a requirement for calcium, the effect of GTPγS was more evident at lower calcium concentrations (buffer>0.1 mM>1.0 mM). These data thus indicate that release of arachidonic acid by phospholipase A2 in saponin-treated platelets is closely linked to GTP-binding protein which may decrease the calcium requirement for phospholipase A2 activation.

scholarly journals Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4−

1989 ◽  
Vol 264 (3) ◽  
pp. 703-711 ◽  
Author(s):  
M K Magnússon ◽  
H Halldórsson ◽  
M Kjeld ◽  
G Thorgeirsson
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Ionophore A23187 ◽  
Kinase C

In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release.

scholarly journals Phospholipid turnover in isolated rat pancreatic acini. Consideration of the relative roles of phospholipase A2 and phospholipase C

1982 ◽  
Vol 208 (3) ◽  
pp. 713-721 ◽  
Author(s):  
Stephen P. Halenda ◽  
Ronald P. Rubin
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Calcium Ionophore ◽  
Receptor Activation ◽  
Amylase Secretion ◽  
Stimulatory Action ◽  
Pancreatic Acini ◽  
Phospholipases A2 ◽  
Isolated Rat

The purpose of the present study was to explore the interaction of phosphatidylinositol breakdown and the turnover of arachidonic acid in isolated rat pancreatic acini by using receptor agonists and the calcium ionophore ionomycin. Acini prelabelled with myo-[3H]inositol in vivo responded to carbachol with a rapid breakdown of phosphatidylinositol. In the presence of [32P]Pi, carbachol increased labelling of phosphatidic acid and phosphatidylinositol within 1 and 5 min respectively. Carbachol also rapidly stimulated the incorporation of [14C]arachidonic acid into phosphatidylinositol within 2 min, and the peptidergic secretagogue caerulein caused the loss of radioactivity from phospholipids prelabelled with arachidonic acid. Ca2+ deprivation partially impaired the stimulatory action of carbachol on arachidonic acid turnover. In contrast with its stimulatory effects on [32P]Pi and [14C]arachidonate incorporation, carbachol inhibited the incorporation of the saturated fatty acid stearic acid into phosphatidylinositol. Whereas ionomycin stimulation of phosphatidylinositol breakdown and [32P]Pi labelling of phospholipids was slower in onset and less effective than carbachol stimulation, the ionophore effectively promoted (arachidonyl) phosphatidylinositol turnover within 2 min. These results implicate two separate pathways for stimulated phosphatidylinositol degradation in the exocrine pancreas, involving phospholipases A2 and C. Whereas mobilization of cellular Ca2+ appears sufficient to cause activation of phospholipase A2 and amylase secretion, additional events triggered by receptor activation may be required to act in concert with Ca2+ to optimally stimulate phospholipase C. The nature of the interaction between phospholipases A2 and C and their specific physiological roles in pancreatic secretion remain to be elucidated.

scholarly journals Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate

1984 ◽  
Vol 219 (2) ◽  
pp. 655-659 ◽  
Author(s):  
R P Rubin ◽  
P P Godfrey ◽  
D A Chapman ◽  
J W Putney
Keyword(s):  
Phospholipase C ◽  
Exocrine Pancreas ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Receptor Activation ◽  
Early Step ◽  
Pancreatic Acini ◽  
Rat Exocrine Pancreas ◽  
Stimulus Secretion Coupling

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.

PHOSPHOLIPASE A2 ACTIVATION BY A MECHANISM SEPARATE TO THAT RESPONSIBLE FOR PHOSPHOLIPASE C STIMULATION IN ALPHA-THROMBIN-STIMULATED HUMAN PLATELETS

1987 ◽  
Author(s):  
Michael F Crouch ◽  
Eduardo G Lapetina
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Control Mechanisms ◽  
Induced Aggregation ◽  
Stimulation Of

The ability of cell surface receptor occupation to increase the activity of phospholipase A2 has been thought to be due to the prior activation of phospholipase C and an increase in the intracellular Ca2+ concentration. However, recent evidence from our and other laboratories has suggested that this may not be the case, but rather stimulation of phospholipase A2 may be under the control of separate receptor-activated events. We have investigated this further by comparing the ability of prostacyclin (PGI2) and epinephrine to alter platelet responses to thrombin and examining the resulting phospholipase A2 activities.Alpha-thrombin stimulated aggregation of human platelets, the formation of inositol phosphates and phosphatidic acid, mobilizaton of Ca2+ from internal stores and Ca2+ influx, protein phosporylation (47 kDa and 20 kDa) and arachidonic acid (AA) release. Each of these responses was partially inhibited by prostacyclin (PGI2) except that of AA release, which was abolished. In combination with epinephrine and PGI2, alpha-thrombin-induced aggregation, phosphatidic acid formation and protein phosphorylation were restored, but the release of AA only reached 50% of its control value. Epinephrine alone had no effect on any of these responses, either in the presence or absence of PGI2. Thus, alpha-thrombin-induced activation of phospholipase A2 is more sensitive to the effects of PGI2 than is phospholipase C, and supports the possibility that there are distinct control mechanisms for receptor activation of these enzymes. We are presently examining the role of Gs in the inhibition by PGI2 of platelet phospholipase A2 and of Gi in the thrombin stimulation of this enzyme

scholarly journals Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP

1984 ◽  
Vol 222 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S E Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Free Arachidonic Acid ◽  
Stimulation Of

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.

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