Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase.

Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes. The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min. No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon. The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+. Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols. In hepatocytes prelabelled with [14C]arachidonic acid, vasopressin increased the amount of [14C]diacylglycerol. The effects of vasopressin on the total concentration of diacylglycerols and [14C]diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens. The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.

Download Full-text

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

Reproduction ◽

10.1530/reprod/118.1.57 ◽

2000 ◽

pp. 57-68 ◽

Cited By ~ 11

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.

Download Full-text

Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44408-5 ◽

1983 ◽

Vol 258 (18) ◽

pp. 11236-11242 ◽

Cited By ~ 21

Author(s):

W Siess ◽

F L Siegel ◽

E G Lapetina

Keyword(s):

Arachidonic Acid ◽

Phosphatidic Acid ◽

Protein Phosphorylation ◽

Phospholipase C ◽

Shape Change ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Shape

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615763 ◽

2001 ◽

Vol 85 (05) ◽

pp. 882-889 ◽

Cited By ~ 21

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.

Download Full-text

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

Bioscience Reports ◽

10.1007/bf01362507 ◽

1987 ◽

Vol 7 (5) ◽

pp. 443-454 ◽

Cited By ~ 37

Author(s):

Claes B. Wollheim ◽

Susanne Ullrich ◽

Paolo Meda ◽

Lucia Vallar

Keyword(s):

Arachidonic Acid ◽

Insulin Secretion ◽

Cyclic Amp ◽

Phospholipase C ◽

Guanine Nucleotide ◽

Rinm5f Cells ◽

Enzyme Systems ◽

Enhanced Secretion ◽

Insulin Secreting Cells ◽

High Voltage Discharge

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 μM). The stable GTP analogue GTPγS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 μM) the effect of GTPγS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPβS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.

Download Full-text

Relationship of GTP-binding proteins, phospholipase C, and PGE2 synthesis in rat glomerular mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.1.f171 ◽

1989 ◽

Vol 256 (1) ◽

pp. F171-F178 ◽

Cited By ~ 2

Author(s):

D. Schlondorff ◽

P. Singhal ◽

A. Hassid ◽

J. A. Satriano ◽

S. DeCandido

Keyword(s):

Arachidonic Acid ◽

Phospholipase C ◽

G Protein ◽

Binding Proteins ◽

Mesangial Cells ◽

Cdna Probe ◽

Ang Ii ◽

Glomerular Mesangial Cells ◽

Gtp Binding Proteins ◽

Gtp Binding

We evaluated the role of GTP-binding proteins in the activation of phospholipase C, release of arachidonic acid, and synthesis of prostaglandin (PG) E2 in response to platelet-activating factor (PAF) and angiotensin II (ANG II) in cultured rat mesangial cells. Pretreatment with pertussis toxin (PT) decreased PGE2 formation and arachidonic acid release in response to PAF and ANG II but not that to A 23187. PT pretreatment also inhibited formation of inositol trisphosphate (IP3) in response to ANG II or PAF but did not significantly alter the rise in intracellular calcium detected by fura-2. PT catalyzed ADP ribosylation of two proteins of molecular mass approximately 40 and 41 kDa. Further evidence for involvement of GTP-binding protein in phospholipase C activation was that GTP-gamma S stimulated IP3 generation. Immunoblots with antibodies directed against different inhibitory alpha subunits of GTP-binding proteins showed that the major 40-kDa PT substrate reacted with an antibody directed against a decapeptide of the G protein subunit alpha i2 that is also found in leukocytes. This was further confirmed by Northern blot that showed the existence of mRNA in mesangial cells that hybridized with a cDNA probe for G alpha i2. In addition lesser amounts of mRNA hybridized with a restriction fragment cDNA probe for G alpha i3, which corresponds to the 41-kDa substrate for PT ribosylation. These results show that phospholipase C activation by PAF and ANG II in mesangial cells involves a specific G protein, most likely G alpha i2.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text