Evaluation of the role of the arachidonic acid cascade in anandamide's in vivo effects in mice

The pharmacological profiles of the endocannabinoid anandamide and exogenous cannabinoids (e.g., Delta9-tetrahydrocannabinol) are similar, but not exactly the same. One notable difference is that anandamide's in vivo effects in mice are not blocked by the brain cannabinoid (CB1) receptor antagonist SR141716A. The degree to which the rapid metabolism of anandamide to arachidonic acid might be involved in this unexpected lack of effect was the focus of this study. Mice were tested in a tetrad of tests sensitive to cannabinoids, consisting of spontaneous locomotion, ring immobility, rectal temperature and tail flick nociception. Anandamide and arachidonic acid produced a similar profile of effects, but neither drug was blocked by SR141716A. When hydrolysis of anandamide was inhibited by an amidase inhibitor (phenylmethyl sulfonyl fluoride; PMSF), however, SR141716A significantly attenuated anandamide's effects but did not completely block them. Similarly, the effects of the metabolically stable anandamide analog O-1812 were attenuated by SR141716A. The role of oxidative metabolism in anandamide's effects in the tetrad was also investigated through pharmacological modulation of cyclooxygenase and lipoxygenase, two major classes of enzymes that degrade arachidonic acid. Whereas the non-selective cyclooxygenase inhibitor ibuprofen blocked the in vivo effects of arachidonic acid, it did not alter anandamide's effects. Other modulators of the cyclooxygenase and lipoxygenase pathways also failed to block anandamide's effects. Together, these results offer partial support for a pharmacokinetic explanation of the failure of SR141716A to antagonize the effects of anandamide; however, they also suggest that non-CB1, non-CB2 receptors may be involved in mediation of anandamide's in vivo actions, particularly at higher doses.     

Cannabinoid pharmacology: implications for additional cannabinoid receptor subtypes

Delta(9)-Tetrahydrocannabinol (delta(9)-THC), the primary psychoactive constituent of marijuana (Cannabis sativa), is known to bind to two cannabinoid receptors: CB(1) receptors, located primarily in the brain, and CB(2) receptors, located primarily in the periphery. Recent research has suggested that other cannabinoids, including anandamide and WIN 55212-2, may also act at novel non-CB(1), non-CB(2) cannabinoid receptor(s). Anandamide produces a number of in vivo pharmacological effects in CB(1) knockout mice that are not produced by delta(9)-THC and cannot be explained by anandamide's rapid metabolism. In addition, in vitro anandamide and WIN 55212-2 stimulate [35S]GTPgammaS binding in both CB(1) knockout and wildtype mice while delta(9)-THC stimulates this binding only in wildtype mice. Although anandamide and vanilloid agonists share pharmacological effects, anandamide's actions in CB(1) knockout mice do not appear to be mediated by vanilloid VR(1) receptors. While not yet conclusive, these results suggest the possibility of additional cannabinoid receptors in the brain and periphery.     

The endocannabinoid arachidonyl ethanolamide (anandamide) increases pulmonary arterial pressure via cyclooxygenase-2 products in isolated rabbit lungs

Several cannabinoids elicit systemic vasodilation, mainly via CB1 cannabinoid and vanilloid receptors. However, effects in the pulmonary circulation are unknown. Using the isolated, ventilated, buffer-perfused rabbit lung, we have shown that the endocannabinoids arachidonyl ethanolamide (anandamide) and 2-arachidonyl glycerol (2-AG) dose-dependently increase pulmonary arterial pressure (+19.9 +/- 3.4 mmHg, 5 microM, and +39.5 +/- 10.8 mmHg, 0.4 microM, respectively). 2-AG induced lung edema. The CB1 receptor antagonist AM-251 (0.1 and 5 microM) and the VR1 vanilloid receptor antagonist capsazepine (10 microM) failed to reduce anandamide's effects. The metabolically stable anandamide and 2-AG analogs R-methanandamide and noladin ether, Delta9-tetrahydrocannabinol, and the synthetic cannabinoid HU-210, which is no arachidonic acid product, were without effect. The unspecific cyclooxygenase (COX) inhibitor aspirin (100 microM, P < 0.001) and the specific COX-2 inhibitor nimesulide (10 microM, P < 0.01) completely prevented pulmonary hypertension after 5 microM anandamide. COX-2 RNA was detected in rabbit lungs. The synthetic thromboxane receptor antagonist SQ 29,548 was without effect, but the specific EP1 prostanoid receptor antagonist SC-19220 (100 microM) inhibited the pressure increase after anandamide (P < 0.05). PCR analysis detected fatty acid amidohydrolase (FAAH), an enzyme that degrades endocannabinoids, in rabbit lung tissue. Furthermore, the specific FAAH inhibitor methyl arachidonyl fluorophosphonate (0.1 microM) blocked pressure effects of anandamide (P < 0.01). Finally, anandamide (99 +/- 55 pmol/g) and 2-AG (19.6 +/- 8.4 nmol/g) were found in native lungs. We conclude that anandamide increases pulmonary arterial pressure via COX-2 metabolites following enzymatic degradation by FAAH into arachidonic acid products.     

Effects of Menthol on Nicotine Pharmacokinetic,Pharmacology and Dependence in Mice

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine’s clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and β2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in β2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of β2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.     

The regulation of arachidonic acid metabolism in human first trimester trophoblast by cyclic AMP

Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments. db cAMP increased intracellular levels of radioactivity within 2 min, and extracellular levels of radioactivity were increased after 30 min. These changes were reflected in increased levels of arachidonic acid metabolites in both compartments, indicating that arachidonic acid was metabolised. db cAMP increased intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) within 2 min of addition to cultured cells. No changes were detected after 5-10 min, but substantial changes were found 30 min after the addition of db cAMP. The dihydroxyeicosatrienoic acid (DiHETrE) breakdown products also increased with similar kinetics. In contrast, levels of 14,15-EpETrE increased after 5-10 min.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

Hemodynamic effects of oleic acid in newborn lambs: role of arachidonic acid metabolites.

Oleic acid injection produces acute lung injury and pulmonary hypertension in adult animals. In other types of acute lung injury, such as that caused by E. coli endotoxin, metabolites of arachidonic acid are important mediators of pulmonary hypertension. In order to understand the hemodynamic response of newborn animals to oleic acid injection and the contribution of arachidonic acid metabolites to that response, we injected oleic acid into awake, chronically instrumented newborn lambs. The hemodynamic response of lambs to injections of oleic acid alone was compared to their response after pretreatment with either FPL57231, a putative leukotriene receptor antagonist, or indomethacin, a cyclooxygenase synthesis inhibitor. Oleic acid caused acute pulmonary hypertension associated with an increase in protein-rich lung lymph fluid. Systemic hemodynamic effects were variable. FPL57231 completely blocked the oleic acid-induced pulmonary hypertension while indomethacin significantly attenuated the response. Therefore, metabolites of arachidonic acid metabolism appear to be important mediators of oleic acid-induced pulmonary hypertension in newborn lambs.     

Chronic daily administration of ethyl docosahexaenoate protects against gerbil brain ischemic damage through reduction of arachidonic acid liberation and accumulation

Recently, we reported that dietary ethyl docosahexaenoate (Et-DHA) intake decreases the level of membrane arachidonic acid (AA), which reduces the generation of AA metabolites in ischemic gerbil brain. As an extended study, we further investigated the influence of the chronic administration of Et-DHA on free AA levels after ischemia. In addition, Na,K-ATPase activity, cation content, cerebral edema and brain damage were also evaluated. Weanling male gerbils were orally pretreated with either Et-DHA (200 mg/kg) or vehicle, once a day for 10 weeks, and subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. Time-course analyses revealed that pretreatment with Et-DHA, compared with pretreatment with the vehicle, significantly decreased the brain's free AA levels during ischemia (5, 15 and 30 min) and after reperfusion (5, 10, 15 and 30 min), and attenuated the decline of Na,K-ATPase activity at examined time points. Pretreatment with Et-DHA significantly prevented an increase in Na(+) concentration and a decrease in K(+) concentration after 24 h of reperfusion, which resulted in lower cerebral water content. Reduced brain infarct volume and low animal mortality were also observed in Et-DHA-treated animals. These data suggest that the reduction of ischemia-induced AA liberation and accumulation by Et-DHA pretreatment may be attributable to (a) protection against the decline of Na,K-ATPase activity, (b) postischemic cerebral edema and brain damage and (c) animal mortality.     

Glycyl-glutamine inhibits the respiratory depression, but not the antinociception, produced by morphine

Glycyl-glutamine (Gly-Gln; beta-endorphin(30-31)) is an endogenous dipeptide that is synthesized through the posttranslational processing of beta-endorphin in brain stem regions that control respiration and autonomic function. This study tested the hypothesis that Gly-Gln administration to conscious rats will prevent the respiratory depression caused by morphine without affecting morphine antinociception. Rats were administered Gly-Gln (1-100 nmol) or saline (10 microl) intracerebroventricularly followed, 5 min later, by morphine (40 nmol icv). Arterial blood gases and pH were measured immediately before Gly-Gln and 30 min after morphine injection. Gly-Gln pretreatment inhibited morphine-induced hypercapnia, hypoxia, and acidosis significantly. The response was dose dependent and significant at Gly-Gln doses as low as 1 nmol. In contrast, Gly-Gln (1-300 nmol) had no effect on morphine-evoked antinociception in the paw withdrawal test. When given alone to otherwise untreated animals, Gly-Gln did not affect nociceptive latencies or blood gas values. These data indicate that Gly-Gln inhibits morphine-induced respiratory depression without compromising morphine antinociception.     

Study of Dopamine Turnover by Monitoring the Decline of Dopamine Metabolites in Rat CSF After α-Methyl-p-Tyrosine

CSF was continuously withdrawn from the third ventricle of anesthetized rats. CSF 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid concentrations were determined every 15 min by liquid chromatography coupled with electrochemical detection. Acute tyrosine hydroxylase inhibition [with alpha-methyl-p-tyrosine (alpha-MPT)] induced an exponential decline in levels of DOPAC and HVA in CSF. The decline in DOPAC and HVA concentrations was identical in CSF and forebrain but was much slower in the striatum, suggesting that CSF metabolites of 3,4-dihydroxyphenylethylamine (dopamine) reflect whole forebrain metabolites. The decay in CSF DOPAC and HVA levels after dopamine synthesis inhibition was also used as an in vivo index of forebrain dopamine turnover after various pharmacological treatments. Haloperidol pretreatment accelerated this decay, confirming the increase in brain dopamine turnover induced by neuroleptics. After reserpine pretreatment (15 h before), alpha-MPT produced a very sharp decay in levels of DOPAC and HVA. This result indicates that the residual dopamine that cannot be stored after reserpine treatment is very rapidly renewed and metabolized. Nomifensine strongly diminished the slope of DOPAC and HVA level decreases after alpha-MPT, a result which can be explained either by a slower dopamine turnover or by the involvement of storage dopamine pools. These results exemplify the use of monitoring the decay of dopamine metabolites after alpha-MPT administration in the study of the pharmacological action of drugs on the central nervous system of the rat.     

Vascular metabolism of anandamide to arachidonic acid affects myogenic constriction in response to intraluminal pressure elevation

AimsWe hypothesized that arachidonic acid produced by anandamide breakdown contributes to the vascular effects of anandamide.Main methodsIsolated, pressurized rat skeletal muscle arteries, which possess spontaneous myogenic tone, were treated with anandamide, arachidonic acid, capsaicin (vanilloid receptor agonist), WIN 55-212-2 (cannabinoid receptor agonist), URB-597 (FAAH inhibitor), baicalein (lipoxygenase inhibitor), PPOH (cytochrome P450 inhibitor), and indomethacin (cyclooxygenase inhibitor). Changes in the arteriolar diameter in response to the various treatments were measured. To assess the effect of anandamide metabolism, anandamide was applied for 20 min followed by washout for 40 min. This protocol was used to eliminate other, more direct effects of anandamide in order to reveal how anandamide metabolism may influence vasodilation.Key findingsAnandamide at a low dose (1 μM) evoked a loss of myogenic tone, while a high dose (30 μM) not only attenuated the myogenic response but also evoked acute dilation. Both of these effects were inhibited by the FAAH inhibitor URB-597 and were mimicked by arachidonic acid. The CB1 and CB2 agonist R-WIN 55-212-2 and the vanilloid receptor agonist capsaicin were without effect on the myogenic response. The inhibition of the myogenic response by anandamide was blocked by indomethacin and PPOH, but not by baicalein or removal of the endothelium. FAAH expression in the smooth muscle cells of the blood vessels was confirmed by immunohistochemistry.SignificanceAnandamide activates the arachidonic acid pathway in the microvasculature, affecting vascular autoregulation (myogenic response) and local perfusion.     

Metabolism of anandamide in cerebral microvascular endothelial cells

Anandamide (N-arachidonoylethanolamine, AEA), an endogenous cannabinoid receptor agonist, causes potent vasodilation in the cerebral circulation through an endothelial-dependent or -independent mechanism. We have investigated the processing of [3H]AEA in cultured mouse cerebral microvascular endothelial cells (MEC) in order to better understand its mechanism of action in the cerebral vasculature. These cells took up anandamide very quickly, reaching a maximum value in 5 min and remaining at that level for at least 8 h. Analysis of the cell lipids demonstrated that, in addition to free anandamide, radioactivity was incorporated into phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) in a time-dependent manner. Analysis of the hydrolyzed cell lipids indicated that anandamide was converted to arachidonic acid, a process that was inhibited by the selective fatty acid amide hydrolase inhibitor oleyl trifluoromethyl ketone (OTMK). Phospholipase A2 (PLA2) hydrolysis of the PC, PI, and PE fractions indicated that the arachidonic acid formed from anandamide was esterified predominately into sn-2 position of the endothelial phospholipids. Furthermore, anandamide and arachidonic acid were released when the cells were incubated with A23187. These results suggest that the biological activity of anandamide might be regulated by its rapid uptake and calcium-dependent release in endothelial cells, and conversion of anandamide to arachidonic acid might serve as an inactivation process in the cerebral microcirculation.     

Effects of endotoxin and tumor necrosis factor alpha on regional brain neurotransmitters in mice

Alterations in regional brain concentration of dopamine (DA), norepinephrine (NE), serotonin (5-HT) and their metabolites were investigated in male BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS, 2 mg kg(-1)) or recombinant murine tumor necrosis factor alpha (TNFalpha, 0.1 mg kg(-1)) at 2, 6, 12 and 24 h after the injection. At 2 h post-injection the LPS administration resulted in hypothermia, which was not apparent at later time points. No consistent effects were observed by either LPS or TNFalpha on peripheral leukocyte counts or plasma transaminase levels. Both LPS and TNFalpha slightly elevated NE metabolism in the striatum at 2-12 h. Concentrations of DA and its metabolites were significantly elevated only in the hypothalamus following TNFalpha at 24 h. Tumor necrosis factor alpha exerted pronounced effects on 5-HT metabolism in most brain regions at 2 h. Results suggest that the effect of LPS is more complex compared with TNFalpha because of the endogenous production of other cytokines including the TNFalpha.     

Reversal of reflex pulmonary vasoconstriction induced by main pulmonary arterial distension

Distension of the main pulmonary artery (MPA) induces pulmonary hypertension, most probably by neurogenic reflex pulmonary vasoconstriction, although constriction of the pulmonary vessels has not actually been demonstrated. In previous studies in dogs with increased pulmonary vascular resistance produced by airway hypoxia, exogenous arachidonic acid has led to the production of pulmonary vasodilator prostaglandins. Hence, in the present study, we investigated the effect of arachidonic acid in seven intact anesthetized dogs after pulmonary vascular resistance was increased by MPA distention. After steady-state pulmonary hypertension was established, arachidonic acid (1.0 mg/min) was infused into the right ventricle for 16 min; 15-20 min later a 16-mg bolus of arachidonic acid was injected. MPA distension was maintained throughout the study. Although the infusion of arachidonic acid significantly lowered the elevated pulmonary vascular resistance induced by MPA distension, the pulmonary vascular resistance returned to control levels only after the bolus injection of arachidonic acid. Notably, the bolus injection caused a biphasic response which first increased the pulmonary vascular resistance transiently before lowering it to control levels. In dogs with resting levels of pulmonary vascular resistance, administration of arachidonic acid in the same manner did not alter the pulmonary vascular resistance. It is concluded that MPA distension does indeed cause reflex pulmonary vasoconstriction which can be reversed by vasodilator metabolites of arachidonic acid. Even though this reflex may help maintain high pulmonary vascular resistance in the fetus, its function in the adult is obscure.     

Fatty Acid-binding Protein 5 (FABP5) Regulates Cognitive Function Both by Decreasing Anandamide Levels and by Activating the Nuclear Receptor Peroxisome Proliferator-activated Receptor β/δ (PPARβ/δ) in the Brain

Endocannabinoids modulate multiple behaviors, including learning and memory. We show that the endocannabinoid anandamide (AEA) can alter neuronal cell function both through its established role in activation of the G-protein-coupled receptor CB1, and by serving as a precursor for a potent agonist of the nuclear receptor PPARβ/δ, in turn up-regulating multiple cognition-associated genes. We show further that the fatty acid-binding protein FABP5 controls both of these functions in vivo. FABP5 both promotes the hydrolysis of AEA into arachidonic acid and thus reduces brain endocannabinoid levels, and directly shuttles arachidonic acid to the nucleus where it delivers it to PPARβ/δ, enabling its activation. In accordance, ablation of FABP5 in mice results in excess accumulation of AEA, abolishes PPARβ/δ activation in the brain, and markedly impairs hippocampus-based learning and memory. The data indicate that, by controlling anandamide disposition and activities, FABP5 plays a key role in regulating hippocampal cognitive function.     

THE RELEASE OF BRAIN FREE FATTY ACIDS DURING ISCHAEMIA IN ESSENTIAL FATTY ACID-DEFICIENT RATS

—The release of free fatty acids and especially of free arachidonic acid occurring in rat brain during ischaemia has been studied in essential fatty acid-deficient animals. Free fatty acid levels are lower in essential fatty acid-deficient brains before and especially after 5 min of ischaemia. The percentage of arachidonic acid, in respect of total free fatty acids, is similar in both control and essential fatty acid-deficient brains before ischaemia (0 min), but is greatly reduced in deficient brains in respect of controls after ischaemia (5 min). Total levels of free arachidonic acid are thus greatly reduced after ischaemia in the brain of essential fatty acid-deficient rats. Focussed microwave irradiation of the brain, a technique which instantaneously kills the animals, and which was used for the determination of brain basal levels of free fatty acids and free arachidonic acid, does not per se modify brain lipid and fatty acid composition.     

Mobilization of arachidonic acid in thrombin-stimulated human platelets

In the present work we investigated the effect of serine esterase inhibitors such as 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and phenylmethylsulfonyl fluoride (PMSF), as well as the effect of mepacrine on thrombin-induced mobilization of arachidonic acid (AA) in human platelets. The inhibitor NCDC (0.6 mM) completely abolished the thrombin-induced activation of phospholipase C, phospholipase A2, and transacylase enzymes, whereas the pretreatment of platelets with PMSF (2 mM) resulted in a highly selective inhibition of phospholipase A2 and transacylase activities, with no marked effect on thrombin-induced activation of phospholipase C. The thrombin-induced release of [3H]AA from phosphatidylcholine and phosphatidylinositol was reduced by 90 and 56%, respectively, in the presence of PMSF. This inhibitor also caused a parallel inhibition in the accumulation of [3H]AA (85%) with little effect on thrombin-induced formation of [3H]phosphatidic acid (5%), whereas mepacrine (0.4 mM) caused a selective inhibition of phospholipase A2 and transacylase activities with concomitant stimulation of [3H]phosphatidic acid formation in intact human platelets. These results demonstrate that NCDC and PMSF (serine esterase inhibitors) do not affect agonist-induced activation of phospholipases that mobilize arachidonic acid through a common site. Our results further demonstrate that the inhibition of [3H]AA release observed in the presence of NCDC, PMSF, and mepacrine is primarily due to their direct effects on enzyme activities, rather than due to their indirect effects through formation of complexes between inhibitors and membrane phospholipids. Based upon these results, we also conclude that the combined hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 serves as a major source for eicosanoid biosynthesis in thrombin-stimulated human platelets.     

Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis

Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids.     

Influence of dietary fatty acids on endocannabinoid and N-acylethanolamine levels in rat brain, liver and small intestine

Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biological functions including food intake. We investigated short-term effects of feeding rats five different dietary fats (palm oil (PO), olive oil (OA), safflower oil (LA), fish oil (FO) and arachidonic acid (AA)) on tissue levels of 2-arachidonoylglycerol, anandamide, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide, linoleoylethanolamide, eicosapentaenoylethanolamide, docosahexaenoylethanolamide and tissue fatty acid composition. The LA-diet increased linoleoylethanolamide and linoleic acid in brain, jejunum and liver. The OA-diet increased brain levels of anandamide and oleoylethanolamide (not 2-arachidonoylglycerol) without changing tissue fatty acid composition. The same diet increased oleoylethanolamide in liver. All five dietary fats decreased oleoylethanolamide in jejunum without changing levels of anandamide, suggesting that dietary fat may have an orexigenic effect. The AA-diet increased anandamide and 2-arachidonoylglycerol in jejunum without effect on liver. The FO-diet decreased liver levels of all N-acylethanolamines (except eicosapentaenoylethanolamide and docosahexaenoylethanolamide) with similar changes in precursor lipids. The AA-diet and FO-diet had no effect on N-acylethanolamines, endocannabinoids or precursor lipids in brain. All N-acylethanolamines activated PPAR-alpha. In conclusion, short-term feeding of diets resembling human diets (Mediterranean diet high in monounsaturated fat, diet high in saturated fat, or diet high in polyunsaturated fat) can affect tissue levels of endocannabinoids and N-acylethanolamines.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.