Effect of diarachidonin on prostaglandin E2 synthesis in rabbit kidney medulla slices

The effect of diarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. The addition of diarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. At three concentrations (10, 50 and 100 microM), increases in prostaglandin E2 formation induced by exogenous diarachidonin were 2-fold greater than those induced by exogenous arachidonic acid. Diacylglycerol or phosphatidic acid from egg lecithin had little or no effect on prostaglandin E2 production. Moreover, EGTA failed to inhibit diarachidonin-stimulated prostaglandin E2 formation, indicating that the stimulatory effect of diarachidonin is not mediated through the activation of endogenous phospholipase A2 (including phosphatidic acid-specific phospholipase A2). These results are discussed in the light of our former hypothesis that arachidonic acid release from kidney medulla phospholipids might occur through the sequential action of a phospholipase C coupled to diacylglycerol and monoacylglycerol lipases [Fujimoto, Akamatsu, Hattori & Fujita (1984) Biochem. J. 218, 69-74].

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Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices

Biochemical Journal ◽

10.1042/bj2180069 ◽

1984 ◽

Vol 218 (1) ◽

pp. 69-74 ◽

Cited By ~ 12

Author(s):

Y Fujimoto ◽

N Akamatsu ◽

A Hattori ◽

T Fujita

Keyword(s):

Fatty Acids ◽

Prostaglandin E2 ◽

Phospholipase A2 ◽

Phospholipase C ◽

Unsaturated Fatty Acids ◽

Rabbit Kidney ◽

Dependent Manner ◽

Kidney Medulla ◽

Diacylglycerol Lipase ◽

Dose Dependent

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.

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Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices

Biochemical Journal ◽

10.1042/bj1820821 ◽

1979 ◽

Vol 182 (3) ◽

pp. 821-825 ◽

Cited By ~ 17

Author(s):

A Erman ◽

A Raz

Keyword(s):

Arachidonic Acid ◽

Linoleic Acid ◽

Prostaglandin E2 ◽

Phospholipase A2 ◽

Inhibitory Effect ◽

Rabbit Kidney ◽

Dependent Manner ◽

Kidney Medulla ◽

Bivalent Cations ◽

Tissue Lipids

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3–5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.

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Prostaglandin biosynthesis and lipolysis in subcellular fractions from rabbit kidney medulla

Biochemical Journal ◽

10.1042/bj1940957 ◽

1981 ◽

Vol 194 (3) ◽

pp. 957-961 ◽

Cited By ~ 15

Author(s):

A Erman ◽

A Raz

Keyword(s):

Arachidonic Acid ◽

Linoleic Acid ◽

Prostaglandin E2 ◽

Phospholipase A2 ◽

Plasma Membranes ◽

Subcellular Fractions ◽

Rabbit Kidney ◽

Concomitant Increase ◽

Kidney Medulla ◽

Prostaglandin Biosynthesis

Three separate prostaglandin-generating activities are associated with plasma membranes, mitochondria and microsomal fractions from rabbit kidney medulla. In the plasma membranes and mitochondria, but not in microsomal fractions, Ca2+ ions stimulate the activity of phospholipase A2, yielding selective release of arachidonic acid and linoleic acid and concomitant increase in prostaglandin E2 formation.

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The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices

Biochemical Journal ◽

10.1042/bj2120167 ◽

1983 ◽

Vol 212 (1) ◽

pp. 167-171 ◽

Cited By ~ 39

Author(s):

Y Fujimoto ◽

H Tanioka ◽

I Keshi ◽

T Fujita

Keyword(s):

Lipid Peroxidation ◽

Ascorbic Acid ◽

Arachidonic Acid ◽

Prostaglandin E2 ◽

Inhibitory Effect ◽

Prostaglandin Synthesis ◽

Rabbit Kidney ◽

Kidney Medulla ◽

Prostaglandin Endoperoxide Synthase ◽

Generating System

Lipid peroxidation induced by ascorbic acid and Fe2+ was inhibited by mepacrine (phospholipase A2 inhibitor) and aspirin (prostaglandin cyclo-oxygenase inhibitor) in rabbit kidney-medulla slices. Moreover, ascorbic acid and Fe2+ potentiated the inhibitory effect on prostaglandin E2 formation by mepacrine, but they had no influence on prostaglandin E2 production decreased by aspirin. Lipid peroxidation induced by ascorbic acid and Fe2+ appears to be affecting the activity of prostaglandin endoperoxide synthase. These results suggest that lipid peroxidation is connected closely with the prostaglandin-generating system, and it has the potential to modulate the turnover of arachidonic acid and prostaglandin synthesis.

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Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid

Biochemical Journal ◽

10.1042/bj2680091 ◽

1990 ◽

Vol 268 (1) ◽

pp. 91-98 ◽

Cited By ~ 18

Author(s):

M D C Garcia ◽

S Fernandez-Gallardo ◽

M A Gijon ◽

C Garcia ◽

M L Nieto ◽

...

Keyword(s):

Fatty Acids ◽

Arachidonic Acid ◽

Phospholipase A2 ◽

Platelet Activating Factor ◽

Inhibitory Effect ◽

Eicosatetraenoic Acid ◽

Polymorphonuclear Leucocytes ◽

Maximal Effect ◽

Dependent Manner ◽

Dose Dependent

Theophylline and 1-methyl-3-isobutylxanthine (MIX), compounds that block eicosanoid formation and modulate phospholipase A2 activity, inhibited in a dose-dependent manner the formation of both leukotriene B4 (LTB4) and platelet-activating factor (PAF) by human polymorphonuclear leucocytes (PMN) in response to ionophore A23187. Theophylline and MIX lacked any inhibitory effect on acetyl-CoA: lyso-PAF acetyltransferase activity, which is the rate-limiting step for PAF biosynthesis in PMN. The effect of theophylline and MIX on PAF formation could be reversed by incubating the cells in the presence of 1-10 microM exogenous lyso-PAF. Incubation of PMN homogenates in the presence of unsaturated non-esterified fatty acids resulted in dose-dependent inhibition of the acetyltransferase. This effect was linked to the presence of a free carboxyl group, since both arachidonic acid methyl ester and palmitoyl-arachidonoyl phosphatidylcholine lacked inhibitory activity. This inhibitory effect was also dependent on the number of double bonds, since arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) displayed maximal effect. Kinetic analysis showed that the effect of arachidonic acid was consistent with competitive inhibition, with a Ki value of about 19 microM. Oxidative metabolites of arachidonic acid showed a lesser inhibitory effect with the following order of potency: arachidonic acid greater than 15-HETE (15-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than LTB4 greater than 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than lipoxin A4. Examination of enzymes involved in CoA-dependent acylation revealed a low activity of both arachidonoyl-CoA synthetase and arachidonoyl-CoA: lyso-PAF arachidonoyltransferase. These data indicate a strong influence on PAF biosynthesis of the products of the phospholipase A2 reaction, with lyso-PAF disposal being a critical event for PAF formation, and unsaturated fatty acids acting as feed-back inhibitors. The conversion of arachidonic acid via oxidative metabolism into less active inhibitors of acetyl-CoA:lyso-PAF acetyltransferase seems to be an additional mechanism of modulation of this enzyme activity, linked to the function of lipoxygenases. Finally, the enzyme activities involved in arachidonoyl-CoA-dependent acylation of lyso-PAF show a low efficiency in capturing arachidonic acid.

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Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

Biochemical Journal ◽

10.1042/bj2010635 ◽

1982 ◽

Vol 201 (3) ◽

pp. 635-640 ◽

Cited By ~ 3

Author(s):

Arie Erman ◽

Ruth Azuri ◽

Amiram Raz

Keyword(s):

Arachidonic Acid ◽

Plasma Membrane ◽

Prostaglandin E2 ◽

Subcellular Fractions ◽

Rabbit Kidney ◽

Generation Process ◽

Kidney Medulla ◽

Prostaglandin Biosynthesis ◽

Prostaglandin Synthase ◽

Mitochondrial Fractions

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca2+-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca2+-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent Km values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (Vmax.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E2 synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca2+stimulated endogenous arachidonate deacylation and prostaglandin E2 generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca2+-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.

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Characterization of a novel inhibitor of cytosolic phospholipase A2α, pyrrophenone

Biochemical Journal ◽

10.1042/bj3630727 ◽

2002 ◽

Vol 363 (3) ◽

pp. 727-735 ◽

Cited By ~ 51

Author(s):

Takashi ONO ◽

Katsutoshi YAMADA ◽

Yukiko CHIKAZAWA ◽

Masahiko UENO ◽

Shozo NAKAMOTO ◽

...

Keyword(s):

Arachidonic Acid ◽

Ionophore A23187 ◽

Dependent Manner ◽

Arachidonic Acid Release ◽

Eicosanoid Synthesis ◽

Cytosolic Phospholipase ◽

Ic50 Value ◽

Acid Release ◽

Cytosolic Phospholipase A2α ◽

Dose Dependent

Cytosolic phospholipase A2α (cPLA2α), one of the three subtypes of cPLA2 (α, β and γ), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA2α inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA2α activity, is one of the most potent cPLA2α inhibitors reported to date. Similar inhibitory potencies for cPLA2α were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF3) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA2α and displays no characteristics of the slow-binding inhibition observed for AACOCF3. Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA2α. However, the inhibition by pyrrophenone of 14kDa secretory PLA2s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA2α. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC50 value of 0.024μM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E2 synthesis in human renal mesangial cells with an IC50 value of 0.0081μM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.

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