Amount Of Free Arachidonic Acid Research Articles

15-Hydroperoxyeicosapentaenoic acid, but not eicosapentaenoic acid, shifts arachidonic acid away from cyclooxygenase pathway into acyl-CoA synthetase pathway in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of eicosapentaenoic acid (EPA) and 15-hydroperoxyeicosapentaenoic acid (15-HPEPE) on the PG and AA-CoA formations from high and low concentrations of AA (60 and 5 μM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 μM [ 14C]-AA in 0.1 M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl 2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. EPA reduced the PG and AA-CoA formations from both 60 and 5 μM AA. In contrast, 15-HPEPE decreased the PG formation without affecting the AA-CoA formation from 60 μM AA, and increased the AA-CoA formation at the expense of PG formation when 5 μM AA was used as substrate concentration. The experiments utilizing Fe 2+ and an electron spin resonance (ESR) revealed that 15-HPEPE elicits these effects in the form of hydroperoxy adduct. These results suggest that 15-HPEPE, but not EPA, has the potential to shift AA away from COX pathway into ACS pathway at low substrate concentration (close to the physiological concentration of AA).

The regulation of formation of prostaglandins and arachidonoyl-CoA from arachidonic acid in rabbit kidney medulla microsomes by linoleic acid hydroperoxide

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of linoleic acid (LA) and 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 μM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 μM [ 14C]-AA in 0.1 M Tris–HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl 2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. LA (10–50 μM) reduced only PG formation from both 60 and 5 μM AA. 13-HPODE (10–50 μM) also reduced PG formation from 60 and 5 μM AA, but the inhibitory potency was much stronger than that by LA. Furthermore, 13-HPODE had the potential to increase the AA-CoA formation with a decrease in the PG formation from 5 μM AA. These results suggest that 13-HPODE, but not LA, may shift AA away from COX pathway into ACS pathway under low substrate concentration (near physiological concentration of AA).

Effects of endocrine disruptors on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To explore the possible actions of endocrine disruptors on the metabolic fate of free AA into these two pathways, we investigated the effects of nonylphenol (NP), bisphenol A (BPA), di- n-butyl phthalate (DBP), benzyl- n-butyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) on the formation of PG and AA-CoA from 5 μM AA (close to the physiological concentration of the substrate) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 5 μM [ 14C]-AA in 0.1 M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl 2 and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. NP (1–200 μM) strongly enhanced the AA-CoA formation with a coincident decrease in the PG formation. BPA, DBP, BBP and DEHP failed to show any effect on the PG and AA-CoA formation up to 200 μM. Experiments utilizing 60 μM AA as the substrate concentration indicated that, under a low concentration of AA, NP decreases PG formation by inhibiting the COX activity, and reduces the AA flow into the COX pathway through inhibition on the COX activity, increasing availability of the substrate for the ACS and leading to enhanced AA-CoA formation. These results firstly show that NP has the potential to disturb the balance of PG and AA-CoA formations under normal physiological conditions.

Uptake and metabolism of [3H]anandamide by rabbit platelets. Lack of transporter?

Anandamide is an endogenous ligand for cannabinoid receptor and its protein-mediated transport across cellular membranes has been demonstrated in cells derived from brain as well as in cells of the immune system. This lipid is inactivated via intracellular degradation by a fatty acid amidohydrolase (FAAH). In the present study, we report that rabbit platelets, in contrast to human platelets, do not possess a carrier-mediated mechanism for the transport of [3H]anandamide into the cell, i.e. cellular uptake was not temperature dependent and its accumulation was not saturable. This endocannabinoid appears to enter the cell by simple diffusion. Once taken up by rabbit platelets, [3H]anandamide was rapidly metabolized into compounds which were secreted into the medium. Small amounts of free arachidonic acid as well as phospholipids were amongst the metabolic products. FAAH inhibitors did not decrease anandamide uptake, whereas these compounds inhibited anandamide metabolism. In conclusion, anandamide is rapidly taken up by rabbit platelets and metabolized mainly into water-soluble metabolites. Interestingly, the present study also suggests the absence of a transporter for anandamide in these cells.

The effects of nitric oxide and peroxynitrite on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To clarify factors deciding the metabolic fate of free AA into these two pathways, we investigated the effects of a nitric oxide (NO) donor 1-hydroxyl-2-oxo-3-( N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO −) on the formation of PG and AA-CoA from high and low concentrations of AA (60 and 5 μM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 μM [ 14C]-AA in 0.1 M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced GSH and hydroquinone) and cofactors of ACS (ATP, MgCl 2 and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 μM AA was used as the substrate concentration, NOC7 stimulated the PG formation at 0.5 μM, and inhibited it at 50 and 100 μM, without affecting the AA-CoA formation. When 5 μM AA was used as the substrate concentration, NOC7 showed no effect on the PG and AA-CoA formation up to 10 μM or below, but enhanced the AA-CoA formation with a coincident decrease in the PG formation at 50 μM or over. Experiments utilizing a NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, revealed that the observed effects of NOC7 using 60 and 5 μM AA are caused by NO. On the other hand, ONOO − stimulated the PG formation from 60 μM AA, with no alteration in the AA-CoA formation at a concentration of 100 μM, but when 5 μM AA was used as the substrate concentration, it was without effect on the PG and AA-CoA formation. These findings indicate that actions of NO and ONOO − on the PG and AA-CoA formation by the kidney medulla microsomes may change depending on the substrate concentration. The effects of NO using 5 μM AA were reversed by the addition of the superoxide generating system (xanthine–xanthine oxidase plus catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO −, can be a regulator of the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that superoxide may play an important role in the action of NO.

Changes of the Children Myocardium Lipid Composition under Surgical Intervention by the Use Cold Crystalloid Cardioplegia

Changes of the children's myocardium lipid composition with congenital atrial septal defect (ASD) during a surgical intervention with the use of cold crystalloid cardioplegia were shown for the first time. At ischemia, which developed during cardioplegia, the quantities of phosphatidylinositol, phosphatidylcholine and diphosphatidylglicerol were enhanced. It was found that the quantity of short-chain fatty acids decreased almost two-fold and the content of some long-chain fatty acids esterified in phospholipids was raised. The quantity of w6-fatty acids (eicosatrienoic, arachidonic) was increased in cholesterol esters fraction. The significant accumulation of free miristoleinic, linoleic and some other free fatty acids was shown under cardioplegia. The total amount of cholesterol esters decreased by three times and the ratio of the phospholipids/cholesterol esters increased. Reperfusion caused additional changes of myocardium lipid composition. The amount of lysophosphatidylethanolamine, w6 free fatty acids (including arachidonic acid) enhanced. At the first minutes of the reperfusion, the amount of short-chain and some long-chain fatty acids esterified into phospholipids and cholesterol esters returned to initial level. We have shown that the surgery of the ASD closure at the children heart caused considerable changes of myocardium lipid composition. The amount of free arachidonic acid under reperfusion increased nearly four times.

Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [ 3H]arachidonic acid from [ 3H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K m of 3.7 μM and V max 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9–10, was abolished by phenylmethylsulfonyl fluoride and was Ca 2+- and Mg 2+-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.

The effects of fatty acyl CoA esters on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). We have previously shown that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of AA (5 μM, close to the physiological concentration of substrate). In the present study, we investigated the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)- CoAs on the formation of PG and AA-CoA from 5μM AA in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 5μM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. SA- and OA-CoAs increased AA-CoA formation with a reduction of PG formation, as well as PA-CoA. On the other hand, LA-CoA decreased formation of both PG and AA-CoA. These results suggest that fatty acyl CoA esters can be regulators of PG and AA-CoA formation in kidney medulla under physiological conditions.

The regulation of prostaglandin and arachidonoyl-CoA formation from arachidonic acid in rabbit kidney medulla microsomes by palmitoyl-CoA

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of palmitic acid (PA) and palmitoyl-CoA (PA-CoA) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 μM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 μM [ 14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl 2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. PA (10–100 μM) had no effect on the PG and AA-CoA formation from either 60 or 5 μM AA. PA-CoA (10–100 μM) was without effect on the PG and AA-CoA formation from 60 μM AA, whereas it markedly decreased the PG formation (6–40%) and increased the AA-CoA formation (1.1–2.3-fold) from 5 μM AA, showing that the effects of PA-CoA on the PG and AA-CoA formation change depending on the AA concentration. These results suggest that PA-CoA, but not PA, may regulate the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that this in-vitro method using 5 μM AA may be useful for clarifying the homeostatic control of the metabolic fate of AA into these two enzymatic pathways.

Simultaneous measurement of prostaglandin and arachidonoyl CoA formed from arachidonic acid in rabbit kidney medulla microsomes: the roles of Zn2+and Cu2+as modulators of formation of the two products

Under physiological conditions, small amounts of free arachidonic acid (AA) is released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) act competitively on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To date, there is no information about the factors deciding the metabolic fate of free AA into these two pathways. In this study, we tried to establish a method for the simultaneous measurement of PG and AA-CoA synthesis from exogenous AA in microsomes from rabbit kidney medulla. The kidney medulla microsomes were incubated with [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. When 60μM AA was used as the substrate, indomethacin (an inhibitor of COX) and triacsin C (an inhibitor of ACS) reduced only PG and AA-CoA formation, respectively. On the other hand, when 5μM AA was used as the substrate, indomethacin and triacsin C came to increase significantly the AA-CoA and PG formation, respectively. Thus, the experiments utilizing indomethacin and triacsin C revealed that the incubation using 60μM AA can simultaneously detect the changes in the activities of COX and ACS caused by drugs, while the incubation using 5μM AA can detect the changes in the product formation elicited by the resulting shunt of AA. Further, using these incubation conditions, the effects of Zn2+and Cu2+on the PG and AA-CoA formation were examined. Zn2+inhibited the AA-CoA synthesis from 60μM AA without affecting the PG synthesis. In contrast, when 5μM AA was used as the substrate, a significant increase in the PG formation was observed in the presence of this ion, indicating that drug actions on the PG formation from AA by the kidney medulla microsomes may change depending on the substrate concentration. On the other hand, Cu2+increased PG synthesis and inhibited AA-CoA synthesis from both 60 and 5μM AA. These results suggest that the simultaneous measurements of PG and AA-CoA formation by the kidney medulla microsomes under high (60μM) and low (5μM) substrate concentrations can investigate the direct and indirect actions of drugs on the COX and ACS activities, and are useful for clarifying the haemostatic control of the metabolic fate of AA into the two enzymatic pathways. Furthermore, this study showed that Zn2+and Cu2+can modulate PG and AA-CoA formation by affecting COX activity, ACS activity, and/or the AA flow into the two enzymatic pathways.

Mass spectrometric analysis of arachidonyl-containing phospholipids in human U937 cells.

The human histiocytic lymphoma U937 cell line contains a rich source of the 85 kDa cytosolic phospholipase A2 (cPLA2). DMSO-differentiated U937 cells were used as a model to investigate the free arachidonic acid release, the arachidonate distribution and the phospholipid source of arachidonate upon Ca2+ ionophore stimulation. A combination of several chromatographic and mass spectrometric techniques was employed in this study. The amount of free arachidonic acid (AA) released upon stimulation, the arachidonate content in total lipids and in each of the phospholipid classes were determined by gas chromatography/mass spectrometry (GC/MS). Glycerophosphoethanolamine (GPE) was found to be the major pool of arachidonate in differentiated human U937 cells (55%) and glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) contributed 22 and 8%, respectively. Upon Ca2+ ionophore stimulation, GPE class lost the largest amount of arachidonate, followed by GPC class. GPI class, however, gained a substantial amount of arachidonate. Most of the arachidonate depleted from GPE and GPC was recovered as free AA, some of which was rapidly esterified into GPI species. GC/MS with electron capture negative chemical ionization provided excellent sensitivity for the measurement of arachidonic acid which was derivatized to its pentafluorobenzyl ester. Intact phospholipid molecular species including the arachidonyl-containing phospholipid species were identified using capillary high-performance liquid chromatography/continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS). No specificity was found for releasing free AA among the arachidonyl-containing GPE and GPC species upon Ca2+ ionophore stimulation. CF-LSIMS provided a sensitive and effective means of detecting intact phospholipid species.

Purification of a Lysophospholipase from Bovine Brain That Selectively Deacylates Arachidonoyl-substituted Lysophosphatidylcholine

A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.

Activation of phospholipase A2 and acylation of lysophospholipids: the major regulators for platelet activating factor production in rat neutrophils.

Rat inflammatory neutrophils induced arachidonic acid release and platelet-activating factor (PAF) production in response to opsonized zymosan (OPZ) dose-dependently. Phospholipase A2 activity also increased dose-dependently, paralleling the increases in arachidonic acid and PAF. The time courses of the activities of phospholipase A2 and acetyltransferase, and the amounts of free arachidonic acid, lyso-PAF, and PAF demonstrated that activation of the enzymes in the remodeling pathway could be required for PAF production in rat neutrophils, which agrees with the documented fact for macrophages. Phospholipase A2 could be a rate-limiting enzyme for PAF production, since an increased lyso-PAF amount or addition of exogenous lyso-PAF reflected the increase in PAF formation in the cells. This phospholipase A2 activity in rat neutrophils could be attributed to cytosolic type phospholipase A2, because the activity was mostly suppressed by a specific antibody to cytosolic phospholipase A2. As previously reported, pretreatment of neutrophils with the acyl-CoA synthetase inhibitor, triacsin C, or the acyltransferase inhibitor, merthiolate, enhanced PAF production as well as arachidonic acid release by the cells in response to OPZ. Triacsin C inhibited arachidonoyl-CoA production and merthiolate suppressed the transacylation of lyso-PAF to 1-alkyl-2-arachidonoyl-GPC. These results suggest that these inhibitors of acylation of lyso-PAF caused accumulation of lyso-PAF, which resulted in enhancement of PAF production when phospholipase A2 and acetyltransferase were activated by OPZ. Thus the activation of cytosolic phospholipase A2 and the acylation of lyso-PAF by such as arachidonic acid could be regulating factors for PAF production in stimulated rat neutrophils.

Differences Related to the Production of Arachidonic Acid between Collagen- and Thrombin-Stimulated Human Platelets

The effects of collagen and thrombin on the liberation of free arachidonic acid were investigated in human platelets by fluorometric high-performance liquid chromatography. Collagen induced a concentration-dependent increase in the extent of platelet aggregation, as well as an accumulation of arachidonic acid in human platelets. By contrast, thrombin effectively provoked a potent aggregation at relatively low concentration without any accumulation of free arachidonic acid, although the accumulation of arachidonic acid was detected at a high concentration of thrombin (> 0.1 U/ml) that induced full aggregation. The selective liberation of arachidonic acid was found in thrombin-stimulated platelets. Non-selective liberation of fatty acids occurred in platelets that had been stimulated with a high concentration of collagen (10 micrograms/ml), as well as in platelets stimulated with A23187. The net amount of free arachidonic acid in collagen-stimulated platelets was estimated by use of eicosatetraenoic acid (ETYA), which is an inhibitor of both cyclooxygenase and lipoxygenase. ETYA markedly potentiated the accumulation of free arachidonic acid in collagen-stimulated platelets without changing the amounts of other fatty acids in the cell.

Analysis of lipids in the salivary glands of Amblyomma americanum (L.): detection of a high level of arachidonic acid.

Analysis of lipids in salivary glands of the lone star tick, Amblyomma americanum, demonstrated that arachidonic acid (20:4, n-6) comprises 8% of all fatty acids identified by gas chromatography. The occurrence of arachidonic acid and other C20 polyunsaturated fatty acids in tick salivary glands was confirmed by gas chromatography-mass spectrometry. Arachidonate is located entirely in the phospholipid fraction and is associated exclusively with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Salivary glands stored and frozen for several months had a similar lipid composition as freshly dissected salivary glands, with the exception of a small amount of free arachidonic acid and an increase in lysophosphatidylcholine. Incubation of salivary gland homogenates with snake venom phospholipase A2 showed that most saturated fatty acids are esterified in the sn-1 position of PC and PE, with the unsaturated fatty acids in the sn-2 position. Approximately 75% of arachidonic acid is in the sn-2 position of PC and PE, adding support to the hypothesis that arachidonic acid is released into the cytoplasm after activation of a phospholipase A2 for subsequent metabolism to prostaglandins and/or other eicosanoids.

Cellular mechanism of synergistic stimulation of PGE 2 production by phorbol diester and Ca 2+ ionophore A23187 in cultured madin-darby canine kidney cells

The synergistic stimulation of arachidonic acid release and prostaglandin (PG) E 2 production was observed when quiescent Madin-Darby canine kidney (MDCK) cells were exposed to phorbol 12-myristate 13-acetate (PMA) and Ca 2+ ionophore, A23187. Addition of PMA or A23187 by itself was not effective. When cells were treated with 50 n m PMA and 0.1 μ m A23187 at a suboptimal concentration, there was a marked increase in the production of PGE 2. In the presence of higher concentrations of A23187 (5–10 μ m), 50 n m PMA was only synergistic in potentiating the liberation of free arachidonic acid, but failed to stimulate the PGE 2 production. The amount of free arachidonic acid liberated by 50 n m PMA and 10 μ m A23187 reached a maximum level within several hours, whereas PGE 2 synthesis induced by 50 n m PMA and 0.1 μ m A23187 proceeded with a slower process requiring more than 24 h to reach a maximum. The stimulated PGE 2 synthesis was blocked by transcription and translation inhibitors. The addition of 50 n m PMA alone or the mixture of 50 n m PMA and 0.1 μ m A23187 was found similarly to increase the cellular PG endoperoxide synthase activity, suggesting that PMA was responsible for the increased enzyme activity. However, these agents failed to enhance the activities of phospholipases and PGE 2 synthase from PGH 2. Northern blot analysis confirmed the increased level of PG endoperoxide synthase mRNA in the cells treated with PMA. The effect of PMA was mimicked by other protein kinase C activators. The pretreatment with PMA caused a down-regulation in the PGE 2 production. The stimulated PGE 2 production was abolished in the presence of selective protein kinase C inhibitors such as staurosporine and H-7. In addition, sphingosine, dihyrosphingosine, and psychosine, recently found to be protein kinase C inhibitors, blocked the effect of PMA in intact MDCK cells. Thus, the results indicate that the synergistically stimulated PGE 2 production with phorbol diesters and 0.1 μ m A23187 occurred principally through the de novo synthesis of PG endoperoxide synthase, also implying a role for protein kinase C.

Fusion of neurotransmitter vesicles with target membrane is calcium-independent in a cell-free system

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.

Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system.

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A 2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5–10 μM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca 2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30–50 μM). However, much higher concentrations than 80 μM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B 2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 μM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [ 3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [ 3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide /thromboxane A 2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.

Enhancement of eicosanoid synthesis in mouse peritoneal macrophages by the organic mercury compound thimerosal.

Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. Körner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins 32:373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C4 was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C4 synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C4 levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.