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.

High-affinity CCK receptors are coupled to phospholipase A2 pathways to mediate pancreatic amylase secretion

1995 ◽  
Vol 269 (3) ◽  
pp. G435-G444 ◽  
Author(s):  
Y. Tsunoda ◽  
C. Owyang
Keyword(s):  
Phospholipase A2 ◽  
Acinar Cells ◽  
Receptor Activation ◽  
Pancreatic Acinar Cells ◽  
Rabbit Serum ◽  
Amylase Secretion ◽  
Pancreatic Acini ◽  
Cck Receptors ◽  
Permeabilized Cells ◽  
High Affinity

It is well recognized that JMV-180, a cholecystokinin (CCK) analogue, acts as an agonist on the high-affinity CCK receptor in pancreatic acinar cells. It caused Ca2+ oscillations and amylase secretion in a manner independent of the phospholipase C-inositol 1,4,5-trisphosphate (IP3) pathway. We investigated the mechanism by which the high-affinity CCK receptor utilizes IP3-independent Ca2+ signal transduction to mediate amylase secretion. JMV-180 (1-1,000 nM)-stimulated Ca2+ oscillations and amylase secretion were significantly inhibited by the phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (10 microM). Using streptolysin O-permeabilized cells, we showed that a porcine pancreatic anti-PLA2 antibody from rabbit serum (250 ng/ml) inhibited JMV-180-stimulated amylase secretion. In contrast to CCK octapeptide, JMV-180 (1 nM-10 microM) had no effect on intracellular IP3 levels. These concentrations of JMV-180 did, however, increase intracellular levels of arachidonic acid (AA) metabolite by 2.5-fold in a biphasic manner. Application of exogenous AA (10 microM) released 60% of ATP-incorporated 45Ca2+ from permeabilized pancreatic acini within 3 min in a transient manner. We also showed that active phorbol ester (100 nM) inhibited Ca2+ oscillations and amylase secretion stimulated by JMV-180 (10 nM) or CCK-OPE (100 nM). Application of Mn2+ (2 mM) to superfused acini resulted in a rapid quench of fura 2 fluorescence during 10 nM JMV-180 stimulation, suggesting an involvement of extracellular Ca2+ influx. However, the major source of Ca2+ utilized for oscillations during high-affinity CCK receptor activation was intracellular. In conclusion, we have demonstrated that the high-affinity CCK receptors are coupled to PLA2 pathways to produce AA, which mediates cytosolic Ca2+ oscillation and monophasic amylase secretion, in rat pancreatic acinar cells.

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.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

scholarly journals Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho

1997 ◽  
Vol 327 (2) ◽  
pp. 461-472 ◽  
Author(s):  
J. Luis GARCÍA ◽  
A. Juan ROSADO ◽  
Antonio GONZÁLEZ ◽  
T. Robert JENSEN
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphate ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Significant Inhibition ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
Pkc Activation

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125FAK and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21rho activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125FAK and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125FAK and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125FAK and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21rho, caused significant inhibition of CCK-8-stimulated p125FAK and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125FAK and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21rho.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

2001 ◽  
Vol 85 (05) ◽  
pp. 882-889 ◽  
Author(s):  
Leslie Lockhart ◽  
Caroline Pampolina ◽  
Brent Nickolaychuk ◽  
Archibald McNicol
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Cytosolic Phospholipase A2 ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Low Concentrations ◽  
Acid Release ◽  
Induced Aggregation

SummaryThe release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

scholarly journals Permissive stimulation of Ca(2+)-induced phospholipase A2 by an adenosine receptor agonist in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells: a new ‘cross-talk’ mechanism in Ca2+ signalling.

1994 ◽  
Vol 299 (3) ◽  
pp. 845-851 ◽  
Author(s):  
S Shimegi ◽  
F Okajima ◽  
Y Kondo
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Arachidonic Acid Release ◽  
Thyroid Cells ◽  
Kinase C ◽  
Adenosine Receptor Agonist ◽  
Acid Release

We have described the pertussis toxin (PTX)-sensitive potentiation of P2-purinergic agonist-induced phospholipase C activation, Ca2+ mobilization and arachidonic acid release by an adenosine receptor agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which alone cannot influence any of these cellular activities [Okajima, Sato, Nazarea, Sho and Kondo (1989) J. Biol. Chem. 264, 13029-13037]. In the present study we have found that arachidonic acid release was associated with lysophosphatidylcholine production, and conclude that arachidonic acid is produced by phospholipase A2 in FRTL-5 thyroid cells. This led us to assume that PIA augments P2-purinergic arachidonic acid release by increasing [Ca2+]i which, in turn, activates Ca(2+)-sensitive phospholipase A2. The arachidonic acid-releasing response to PIA was, however, always considerably higher (3.1-fold increase) than the Ca2+ response (1.3-fold increase) to the adenosine derivative. In addition, arachidonic acid release induced by the [Ca2+]i increase caused by thapsigargin, an endoplasmic-reticulum Ca(2+)-ATPase inhibitor, or calcium ionophores was also potentiated by PIA without any effect on [Ca2+]i and phospholipase C activity. This action of PIA was also PTX-sensitive, but not affected by the forskolin- or cholera toxin-induced increase in the cellular cyclic AMP (cAMP), suggesting that a PTX-sensitive G-protein(s) and not cAMP mediates the PIA-induced potentiation of Ca(2+)-generated phospholipase A2 activation. Although acute phorbol ester activation of protein kinase C induced arachidonic acid release, P2-purinergic and alpha 1-adrenergic stimulation of arachidonic acid release was markedly increased by the protein kinase C down-regulation caused by the phorbol ester. This suggests a suppressive role for protein kinase C in the agonist-induced activation of arachidonic acid release. We conclude that PIA (and perhaps any of the G1-activating agonists) augments an agonist (maybe any of the Ca(2+)-mobilizing agents)-induced arachidonic acid release by activation of Ca(2+)-dependent phospholipase A2 in addition to enhancement of agonist-induced phospholipase C followed by an increase in [Ca2+]i.

Glycine protection against hypoxic but not phospholipase A2-induced injury in rat proximal tubules

1993 ◽  
Vol 264 (1) ◽  
pp. F94-F99 ◽  
Author(s):  
J. F. Wetzels ◽  
X. Wang ◽  
P. E. Gengaro ◽  
R. A. Nemenoff ◽  
T. J. Burke ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Lactate Dehydrogenase ◽  
Phospholipase A2 ◽  
Cell Injury ◽  
Proximal Tubules ◽  
Exogenous Arachidonic Acid ◽  
Induced Changes ◽  
Phospholipid Degradation ◽  
Isolated Rat

We studied the effects of glycine (2 mM) on hypoxia-induced changes in phospholipids and fatty acids in isolated rat proximal tubules. In this preparation, 25 min of hypoxia caused cell injury, as reflected by the release of lactate dehydrogenase (LDH) (13.1 +/- 0.8 vs. 43.5 +/- 3.2%; P < 0.01). Hypoxia caused increases in fatty acids and in lysophospholipids. Glycine prevented the hypoxia-induced cell injury (LDH 13.1 +/- 0.8 vs. 11 +/- 0.7%; not significant) but did not attenuate the increases in fatty acids or lysophospholipids. In additional experiments, the effects of glycine on phospholipid changes and cell injury induced by exogenous phospholipase A2 (PLA2) were studied. PLA2 caused dramatic increases in fatty acids and lysophospholipids and mild cell injury; these effects were not influenced by glycine. In contrast, glycine attenuated increases in LDH release induced by exposing the tubules to exogenous arachidonic acid. In conclusion, glycine does not prevent the phospholipid degradation induced by either exogenous PLA2 or hypoxia in isolated proximal tubules and yet affords protection against hypoxia and exogenous arachidonic acid.

Platelet Activation : Role of Exogenous and Endogenous Phospholipases

1979 ◽  
Author(s):  
B. Perret ◽  
G. Mauco ◽  
M.F. Simon ◽  
H. Chap ◽  
L. Douste-Blazy
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Alternative Pathway ◽  
Hydrolysis Product ◽  
Calcium Ionophore ◽  
Human Platelets ◽  
Platelet Response ◽  
Phospholipid Hydrolysis ◽  
Outer Leaflet

Phosoholipase A2 from bee venom induces aggregation of human platelets, provided that phospholipid hydrolysis is enabled by simultaneous incubation with sphingomyelinase C. Inhibition of the platelet response by indomethacin indicates that aggregation is due to arachidonic acid release. On another hand, this model allows to describe an asymmetrie distribution of arachidonic acid, whose only 6% is located in the outer leaflet of the plasma membrane.During platelet aggregation by phospholipase C, the diacylglycerol and its hydrolysis product 2-acyl-glycerol are phosphorylated into phosphatide and lysophosphatidic acids, respectively. As the same kinds of changes occur in the presence of thrombin, a unifying hypothesis for platelet activation is proposed, involving the stimulation of an endogenous phospholipase C, whose some properties will be reported (neutral optimal pH, Ca-requlrement, phosphatidylinositol specificity and cytosol-localization). This model can be related to the recent finding that phosphatide acid behaves as a calcium-ionophore (Gerrard, J.M. et al., Prostaglandins Med., 1978, 1, 387) and provides an alternative pathway for arachidonic acid mobilization.

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