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Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol.

ABSTRACT: We examined the effect of diacylglycerol on Ca2+-dependent phospholipase A2 from human platelets. Phospholipase A2 was solubilized and partially purified to a stable form in the presence of n-octyl beta-D-glucopyranoside (octyl glucoside), and its enzymic activity was determined with sonicated 2.5 microM-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (arachidonoyl-PC) as substrate. Phospholipase A2 activity was increased when diacylglycerol was incorporated into the substrate arachidonoyl-PC. Stimulation was maximal in the presence of greater than or equal to 29 mol% (1 microM) diacylglycerol, and was greater than 4-fold for both 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonoylglycerol. 1-Stearoyl-2-arachidonoylglycerol at concentrations of 2-5 mol% increased phospholipase A2 activity 1.3-1.8-fold. Exogenously added 1-oleoyl-2-acetylglycerol also enhanced phospholipase A2 activity, producing a maximal stimulation of 1.6-fold at a concentration of 25 microM. Comparative studies conducted with pancreatic, bee-venom and snake-venom phospholipase A2 showed that the activity of these extracellular phospholipases towards the arachidonoyl-PC substrate was also increased by diacylglycerol, but stimulation was less than observed for platelet phospholipase A2. Our results suggest that diacylglycerol, known to be generated in stimulated platelets, may enhance Ca2+-activated phospholipase A2.

SUBMITTER: Kramer RM

PROVIDER: S-EPMC1148617 | biostudies-other | 1987 Dec

REPOSITORIES: biostudies-other

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Project description:We characterized an endocrine disruptor from ground corncob bedding material that interferes with male and female sexual behavior and ovarian cyclicity in rats and stimulates estrogen receptor (ER)-positive and ER-negative breast cancer cell proliferation. The agents were identified as an isomeric mixture of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxy-octadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid). Synthetic THF-diols inhibited rat male and female sexual behavior at oral concentrations of 0.5-1 ppm, and stimulated MCF-7 human breast cancer cell proliferation in vitro.Because THF-diols are derived from lipoxygenase and cyclooxygenase pathways, we suspected that these compounds may regulate cell proliferation by modulating specific enzymatic sites involved in linoleic acid metabolism including phospholipase A(2) (PLA2), lipoxygenases (LOX-5 and LOX-12), cyclooxygenases (COX-1 and COX-2), and closely coupled enzymes including aromatase (AROM).MCF-7 human breast cancer cells were treated with inhibitors for PLA2 (quinacrine), lipoxygenases (LOX-5 and LOX-12; baicalein, REV-5091, nordihydroguaiaretic acid), cyclooxygenases (COX-1, COX-2, indomethacin), and AROM (formestane). The effects of these enzyme inhibitors on cell proliferation in response to THF-diols or estradiol (E(2)) were assessed. THF-diol modulation of the expression (RNA and protein) of these enzymes was also evaluated by quantitative real-time PCR (QPCR) and Western blot analyses.The enzyme inhibition and gene expression (RNA and protein) studies identified PLA2, LOX-5, LOX-12, COX-2, and perhaps AROM as likely sites of THF-diol regulation in MCF-7 cells. COX-1 was not affected by THF-diol treatment.THF-diol stimulation of MCF-7 cell proliferation is mediated through effects on the expression of the PLA2, COX-2, LOX-5, and LOX-12 genes and/or their respective enzyme activities. The products of these enzymes, including prostaglandins, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecenoic acids (HODEs), are well-established mitogens in normal and malignant cells. Therefore, it is likely that these compounds are involved in the mechanism of action of THF-diols in breast cancer cells. Although the formestane inhibition studies suggested that AROM activity might be modulated by THF-diols, this was not confirmed by the gene expression studies.

Project description:We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ?2-adrenergic receptor (?2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ?2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 ?M; zint-?2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 ?M) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 ?M GW5074), phospholipase C (PLC; 0.5 ?M edelfosine), PKC (4 ?M chelerythrine) or IP3 receptor (IP3R) signaling (2 ?M 2-APB) significantly inhibited zint-?2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-?2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKC?, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-?2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-?gal. In +LMN myocytes, zint-?2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-?gal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 ?M thapsigargin failed to inhibit zint-?2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-?-cyclodextrin inhibited zint-?2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows ?2-AR stimulation to act via PKC?/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of ?-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

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Stimulation of Ca2+-activated human platelet phospholipase A2 by diacylglycerol.

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