Antiinflammatory and neuroprotective actions of COX2 inhibitors in the injured brain

Overexpression of COX2 appears to be both a marker and an effector of neural damage after a variety of acquired brain injuries, and in natural or pathological aging of the brain. COX2 inhibitors may be neuroprotective in the brain by reducing prostanoid and free radical synthesis, or by directing arachidonic acid down alternate metabolic pathways. The arachidonic acid shunting hypothesis proposes that COX2 inhibitors' neuroprotective effects may be mediated by increased formation of potentially beneficial eicosanoids. Under conditions where COX2 activity is inhibited, arachidonic acid accumulates or is converted to eicosanoids via lipoxygenases and cytochrome P450 (CYP) epoxygenases. Several P450 eicosanoids have been demonstrated to have beneficial effects in the brain and/or periphery. We suspect that arachidonic acid shunting may be as important to functional recovery after brain injuries as altered prostanoid formation per se. Thus, COX2 inhibition and arachidonic acid shunting have therapeutic implications beyond the suppression of prostaglandin synthesis and free radical formation.     

Deacylation and reacylation of neural membrane glycerophospholipids

The deacylation-reacylation cycle is an important mechanism responsible for the introduction of polyunsaturated fatty acids into neural membrane glycerophospholipids. It involves four enzymes, namely acyl-CoA synthetase, acyl-CoA hydrolase, acyl-CoA: lysophospholipid acyltransferase, and phospholipase A₂. All of these enzymes have been purified and characterized from brain tissue. Under normal conditions, the stimulation of neural membrane receptors by neurotransmitters and growth factors results in the release of arachidonic acid from neural membrane glycerophospholipids. The released arachidonic acid acts as a second messenger itself. It can be further metabolized to eicosanoids, a group of second messengers involved in a variety of neurochemical functions. A lysophospholipid, the second product of reactions catalyzed by phospholipase A₂, is rapidly acylated with acyl-CoA, resulting in the maintenance of the normal and essential neural membrane glycerophospholipid composition. However, under pathological situations (ischemia), the overstimulation of phospholipase A₂ results in a rapid generation and accumulation of free fatty acids including arachidonic acid, eicosanoids, and lipid peroxides. This results in neural inflammation, oxidative stress, and neurodegeneration. In neural membranes, the deacylation-reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. This is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes.     

Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly.

Phospholipase A2 (PLA2) generates arachidonic acid, docosahexaenoic acid, and lysophospholipids from neural membrane phospholipids. These metabolites have a variety of physiological effects by themselves and also are substrates for the synthesis of more potent lipid mediators such as eicosanoids, platelet activating factor, and 4-hydroxynonenal (4-HNE). At low concentrations, these mediators act as second messengers. They affect and modulate several cell functions, including signal transduction, gene expression, and cell proliferation, but at high concentrations, these lipid mediators cause neurotoxicity. Among the metabolites generated by PLA2, 4-HNE is the most cytotoxic metabolite and is associated with the apoptotic type of neural cell death. Levels of 4-HNE are markedly increased in neurological disorders such as Alzheimer disease, Parkinson disease, ischemia, spinal cord trauma, and head injury. The purpose of this review is to summarize and integrate the vast literature on metabolites generated by PLA2 for a wider audience. The authors hope that this discussion will jump-start more studies not only on the involvement of PLA2 in neurological disorders but also on the importance of PLA2-generated lipid mediators in physiological and pathological processes.     

Role of Ca-independent phospholipase A2 and n−3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A₂ and cyclooxygenases. These enzymes release the prostaglandin precursor, the n−6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H₂. Docosahexaenoic acid, which belongs to the n−3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca²⁺ signaling, which results in changes of activity of Ca²⁺-dependent phospholipase A₂, hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A₂, i.e. Ca²⁺-dependent phospholipase A₂ and Ca²⁺-independent phospholipase A₂, respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca²⁺- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca²⁺-independent phospholipase A₂ and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.     

Immunocytochemical localization of cPLA2 in rat and monkey spinal cord

Rat spinal cord contains a high level of calcium-dependent cytosolic phospholipase A₂ (PLA₂) activity. A dense immunoreactivity is present in motor neurons from cervical, thoracic, lumbar, and sacral regions of rat spinal cord. Under normal conditions, this enzyme liberates arachidonic acid, a polyunsaturated fatty acid that is a second messenger itself, and a precursor for eicosanoids. However, under pathological conditions during spinal cord injury, intracellular calcium increases so the cytosolic PLA₂ may also be involved in the release and accumulation of arachidonic acid, eicosanoids, and lipid peroxides.     

Inhibitors of Cyclooxygenases produce Amnesia for a Passive Avoidance Task in the Chick

Arachidonic acid is a putative messenger in synaptic transmission which presumably plays a role in learning and memory. Previous experiments showed that inhibitors of phospholipase A₂-dependent release of arachidonic acid cause amnesia in a one-trial passive avoidance task in the chick. To test if arachidonic acid is metabolized to other messengers, the effects of inhibitors of enzymes which metabolize arachidonic acid were tested in the same task. The cyclooxygenase inhibitors indomethacin, naproxen and ibuprofen caused amnestic effects at all concentrations tested when injected intracerebrally before training. Injections were 5 μl of 5–20 mmolar solutions per hemisphere. The onset of amnestic effects was always 2 h after training, independently of drug type, concentration, and injection time before training. The delay of 2 h after training suggests that the drugs prevent induction of cyclooxygenase synthesis. Post-training injections had no effect. Control tests showed little effect of the drugs on motor control and motivation. Caffeic acid and esculetin, inhibitors of lipoxygenases, and sodium furegrelate, a thromboxane synthase inhibitor, had no effect on performance of chicks in the task at all concentrations or time points tested. The results indicate that cyclooxygenase products, but not lipoxygenase or thromboxane synthase products, play a role in memory consolidation in the chick when learning this task.     

Nordihydroguaiaretic acid and RHC 80267 potentiate astroglial injury during combined glucose-oxygen deprivation

Membrane phospholipid degradation has been proposed to play a key role in hypoxic-ischemic brain injury. We tested the hypotheses that both nordihydroguaiaretic acid, a phospholipase A₂ and lipoxygenase inhibitor, and RHC 80267, a diacylglycerol lipase inhibitor, would decrease the release of [³H]arachidonic acid metabolites from prelabeled cultures of astroglia subjected to combined glucose-oxygen deprivation and that these inhibitors would also decrease astroglial injury during combined glucose-oxygen deprivation. Both nordi-hydroguaiaretic acid and RHC 80267 significantly inhibited the release of [³H]arachidonic acid metabolites during combined glucose-oxygen deprivation. This suggests that two separate enzymic pathways, the phospholipase A₂ pathway and the phospholipase C/diacylglycerol lipase pathway, contribute to the release of astroglial [³H]arachidonic acid metabolites during combined glucose-oxygen deprivation. However, both of these lipase inhibitors increased astroglial cell death during combined glucose-oxygen deprivation, probably due to inhibition of arachidonic acid release. We speculate that arachidonic acid release may be a mechanism of astroglial self-preservation during combined glucose-oxygen deprivation.     

Role of Ca2+-independent phospholipase A2 and n-3 polyunsaturated fatty acid docosahexaenoic acid in prostanoid production in brain: perspectives for protection in neuroinflammation.

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A(2) and cyclooxygenases. These enzymes release the prostaglandin precursor, the n-6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H(2). Docosahexaenoic acid, which belongs to the n-3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca(2+) signaling, which results in changes of activity of Ca(2+)-dependent phospholipase A(2), hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A(2), i.e. Ca(2+)-dependent phospholipase A(2) and Ca(2+)-independent phospholipase A(2), respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca(2+)- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.     

Retrograde messengers, long-term potentiation and memory

Long-term potentiation has been involved in certain forms of learning and memory. In the CA₁ region of the hippocampus, long-term potentiation (LTP) is triggered postsinaptically and, at least in part, it expression depends on presynaptic mechanisms. Therefore, a retrograde messenger that is released from the postsynaptic dendrite and diffuses back across the synapse to increase neurotransmitter release has been proposed. Several candidates including lipid mediators such as arachidonic acid and platelet-activating factor, and gases such as nitric oxide and carbon monoxide, have recently attracted much interest. The involvement of these intercellular messengers in LTP, the relation between LTP and memory and the role of these candidate retrograde messengers in the acquisition and consolidation of memories are discussed. Evidence for the involvement of NO, CO and PAF in the early stages of memory processing will be presented.     

Arachidonate Metabolism in the Anterior Pituitary: Effect of Arachidonate Inhibitors on Basal and Stimulated Secretion of Prolactin, Growth Hormone and Luteinizing Hormone. I. Hormone Release from Incubated or Perifused Pituitary Fragments

The potential involvement of arachidonic acid metabolites in the regulation of adenohypophyseal secretion was analysed on pituitary glands from male rats incubated in the presence of various inhibitors with different mechanisms of action: two inhibitors of phospholipase A₂ (parabromophenacylbromide, PB and compound CB 874), an inhibitor of cyclooxygenase- and lipoxygenase-catalysed pathways (5, 8, 11, 14-eicosatetraynoic acid, ETYA) and an inhibitor of cyclooxygenase (ε-lysyl acetylsalicylate, ASP). Under conditions which minimize side effects of the drugs, all inhibitors reduced prostaglandin synthesis and release, without affecting the metabolic integrity of the tissues (assessed by their intracellular adenosine triphosphate levels). All agents tested (PB, ETYA, ASP) suppressed prolactin secretion induced either by thyrotropin-releasing hormone or vasoactive intestinal peptide. Basal prolactin secretion was sensitive to phospholipase A₂ inhibitors. Similar inhibitions were obtained with ETYA and CB 874 on growth hormone secretion under basal conditions as well as after stimulation by growth hormone-releasing factor, thyrotropin-releasing hormone, or vasoactive intestinal peptide. In contrast, luteinizing hormone secretion, stimulated or not by gonadotropin-releasing hormone, was not sensitive to any of the agents used. It is concluded that, in intact male hemipituitaries, arachidonic acid metabolism is involved in the stimulation of prolactin and growth hormone secretion by neuropeptides. In contrast, luteinizing hormone release does not seem to depend on that mechanism. It has been verified that the inhibitors of arachidonic acid metabolism do not directly interfere with adenylate cyclase, or with the activation of protein kinase C, two enzymes which are involved in the regulation of secretory mechanisms.     

Arachidonic acid metabolites do not mediate modulation of neurotransmitter release by adenosine in rat hippocampus or striatum

The possible involvement of arachidonic acid metabolites as mediators of the modulation of neurotransmitter release by adenosine, acetylcholine, and GABA was examined in brain slices of rat hippocampus and striatum. The synaptic modulatory effects of these 3 agents on excitatory transmission in the CA1 region of hippocampus were completely unaffected by a phospholipase inhibitor (p-bromophenacyl bromide, BPB; 10-50 microM), a lipoxygenase inhibitor (nordihydroguaiaretic acid; 5-50 microM), the cyclooxygenase inhibitor indomethacin (10-20 microM), and a cyclooxygenase/lipoxygenase inhibitor (U53059; 5-10 microM). BPB was also found to be ineffective in altering the modulation of transmission by adenosine in the perforant path, and the adenosine inhibition of electrically stimulated release of endogenous dopamine from striatal slices. Arachidonic acid itself also had no effect on synaptic transmission. While these experiments do not rule out such a role for arachidonic acid or its metabolites in mammalian brain, they suggest that in a number of systems the inhibition of transmitter release must occur through an entirely independent mechanism.     

Phospholipase A2 and Somatostatin Release are Activated in Response to N-Methyl-D-Aspartate Receptor Stimulation in Hypothalamic Neurons in Primary Culture

We have recently shown that glutamate primarily induces somatostatin release in hypothalamic neurons through N-methyl-D-aspartate (NMDA)-type receptor sites. Here we report that glutamate and NMDA also stimulate the release of [³H]arachidonic acid in a dose-dependent manner. The NMDA-induced effects (arachidonic acid release and somatostatin secretion) were both inhibited by MK-801, an NMDA receptor-type antagonist, or mepacrine, a phospholipase A₂ inhibitor. In addition, mepacrine was able to inhibit A23187-stimulated arachidonic acid release and somatostatin secretion. p-Bromophenacylbromide, another phospholipase A₂ inhibitor, also blocked NMDA-induced secretion of somatostatin. However, responses to NMDA were unaffected by H7 (inhibitor of protein kinase C), nordihydroguaiaretic acid or indomethacin (inhibitors of lipoxygenase and cyclooxygenase). Melittin, a phospholipase A₂ activator, was found to stimulate both responses, but omission of extracellular Ca²⁺ from the incubation media strongly reduced melittin-induced somatostatin release. Six-h pertussis toxin pretreatment did not significantly reduce the action of NMDA on either of the two parameters studied. High-performance liquid chromatography analysis of [₃H]metabolites released in the medium after NMDA stimulation revealed that [₃H]arachidonic acid was the only detectable metabolite. External addition of arachidonic acid increased the release of somatostatin, whereas E² and F²α prostaglandins had no effect. Our results show a close correlation between arachidonic acid release and somatostatin secretion, the two parameters we investigated.     

Interactions between neural membrane glycerophospholipid and sphingolipid mediators: a recipe for neural cell survival or suicide

The neural membranes contain phospholipids, sphingolipids, cholesterol, and proteins. Glycerophospholipids and sphingolipids are precursors for lipid mediators involved in signal transduction processes. Degradation of glycerophospholipids by phospholipase A(2) (PLA(2)) generates arachidonic acid (AA) and docosahexaenoic acids (DHA). Arachidonic acid is metabolized to eicosanoids and DHA is metabolized to docosanoids. The catabolism of glycosphingolipids generates ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. These metabolites modulate PLA(2) activity. Arachidonic acid, a product derived from glycerophospholipid catabolism by PLA(2), modulates sphingomyelinase (SMase), the enzyme that generates ceramide and phosphocholine. Furthermore, sphingosine 1-phosphate modulates cyclooxygenase, an enzyme responsible for eicosanoid production in brain. This suggests that an interplay and cross talk occurs between lipid mediators of glycerophospholipid and glycosphingolipid metabolism in brain tissue. This interplay between metabolites of glycerophospholipid and sphingolipid metabolism may play an important role in initiation and maintenance of oxidative stress associated with neurologic disorders as well as in neural cell proliferation, differentiation, and apoptosis. Recent studies indicate that PLA(2) and SMase inhibitors can be used as neuroprotective and anti-apoptotic agents. Development of novel inhibitors of PLA(2) and SMase may be useful for the treatment of oxidative stress, and apoptosis associated with neurologic disorders such as stroke, Alzheimer disease, Parkinson disease, and head and spinal cord injuries.     

A potentially critical role of phospholipases in central nervous system ischemic, traumatic, and neurodegenerative disorders

Phospholipases are a diverse group of enzymes whose activation may be responsible for the development of injury following insult to the brain. Amongst the numerous isoforms of phospholipase proteins expressed in mammals are 19 different phospholipase A₂'s (PLA₂s), classified functionally as either secretory, calcium dependent, or calcium independent, 11 isozymes belonging to three structural groups of PLC, and 3 PLD gene products. Many of these phospholipases have been identified in selected brain regions. Under normal conditions, these enzymes regulate the turnover of free fatty acids (FFAs) in membrane phospholipids affecting membrane stability, fluidity, and transport processes. The measurement of free fatty acids thus provides a convenient method to follow phospholipase activity and their regulation. Phospholipase activity is also responsible for the generation of an extensive list of intracellular messengers including arachidonic acid metabolites. Phospholipases are regulated by many factors including selective phosphorylation, intracellular calcium and pH. However, under abnormal conditions, excessive phospholipase activation, along with a decreased ability to resynthesize membrane phospholipids, can lead to the generation of free radicals, excitotoxicity, mitochondrial dysfunction, and apoptosis/necrosis. This review evaluates the critical contribution of the various phospholipases to brain injury following ischemia and trauma and in neurodegenerative diseases.     

Prostacyclin synthesis and deacylation of phospholipids in human endothelial cells: comparison of thrombin, histamine and ionophore A23187

Thrombin, histamine and ionophore A23187 stimulated human endothelial cells to release arachidonic acid and synthesize prostaglandins. To compare the activation of arachidonic acid release by these three stimuli in endothelial cells, we examined the intracellular lipid metabolism by prelabeling the cells with [14C]stearic acid and [3H]arachidonic acid. Thrombin stimulated the loss of 3H and 14C label from intracellular phospholipids. At the same time [3H]arachidonic acid and prostaglandins were released into the incubation medium. Thin layer chromatography analysis indicated that prostacyclin is the major metabolite formed followed by PGF2 alpha, PGE2, HHT and PGD2. In addition, several intracellular lipid metabolites were accumulated. These include: phosphatidic acid and 1,2-diacylglycerol detected by increase of both 14C and 3H radioactivity; lysophosphatidylinositol, lysophosphatidylethanolamine, and to a smaller extent lysophosphatidylcholine and lysophosphatidylserine detected by increase of 14C radioactivity. Like thrombin, both histamine and ionophore A23187 also stimulated release of arachidonic acid and synthesis of prostaglandins. Despite the different nature of the agonists, the type and the relative amount of prostaglandins synthesized in response to histamine and A23187 were similar to that stimulated by thrombin. The relative extents of hydrolysis of phospholipids and the accumulation of phosphatidic acid, 1,2-diacylglycerol and lysophospholipids are similar to that of 3H radioactivity and prostacyclin released into the medium and follow the order: ionophore A23187 greater than thrombin greater than histamine. These results suggest that in human endothelial cells, histamine, thrombin and ionophore A23187 directly or indirectly activated both phospholipase C and phospholipase A2 and these activations most likely involve mobilization of Ca2+.     

Evidence for a role for phospholipase C, but not phospholipase A2, in platelet activation in response to low concentrations of collagen

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 microg/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 microg/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 Ro31-8220 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 Cgamma2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Ro31-8220. 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 Cgamma2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.     

Metabolites of arachidonic acid in the nervous system of Aplysia: possible mediators of synaptic modulation

Release of arachidonic acid from membrane phospholipids is receptor-mediated and might generate second messengers in neurons. We tested this idea using the simple nervous system of the marine mollusk, Aplysia californica. Aplysia neural components metabolize arachidonic acid through lipoxygenase and cyclo-oxygenase pathways. We identified 2 major lipoxygenase products, 12- and 5-hydroxyeicosatetraenoic acids (12-HETE and 5-HETE), and 2 cyclo-oxygenase products, PGE2 and PGF2 alpha. These metabolites of arachidonic acid are formed in synaptosomes, as well as in identified nerve cell bodies, indicating that both lipoxygenase and cyclo-oxygenase pathways are active within neurons. Application of the modulatory neurotransmitter histamine to cerebral ganglia that had been labeled with 3H-arachidonic acid induced the formation of 3H-12-HETE. This response was inhibited by the histamine antagonist cimetidine. Furthermore, release of radioactive 5-HETE and 12-HETE was observed after intracellular stimulation of the histaminergic cell C2 in cerebral ganglia labeled with 3H-arachidonic acid. Cimetidine also inhibited this response. Application of serotonin or stimulation of the giant serotonergic cell (GCN) in the cerebral ganglion did not cause detectable amounts of the labeled eicosanoids to be released. We found that intracellular stimulation of putative histaminergic neurons in the L32 cluster of the abdominal ganglion, which produces presynaptic inhibition in L10 neurons, also elicited the release of 3H-12-HETE and 3H-PGE2. Thus, for the first time we provide evidence that synaptic stimulation promotes turnover of arachidonic acid in neurons. We suggest that metabolites of arachidonic acid are likely to participate in some postsynaptic responses to histamine and may be second messengers for presynaptic inhibition.     

Inhibition of GABA-gated chloride channel function by arachidonic acid

The effects of arachidonic acid and its metabolites on gamma-aminobutyric acid (GABAA) receptor function were determined in rat cerebral cortical synaptoneurosomes. Incubation of synaptoneurosomes with phospholipase A2 decreased muscimol-induced 36Cl- uptake. Arachidonic acid, the major unsaturated fatty acid released by phospholipase A2, also inhibited muscimol-induced 36Cl uptake. Similar inhibition was obtained with other unsaturated fatty acids (docosahexaenoic, oleic) but not with saturated fatty acids (stearic, palmitic). The effect of arachidonic acid on muscimol responses was inhibited by bovine serum albumin (BSA), and BSA enhanced muscimol responses directly, indicating the generation of endogenous arachidonic acid in the synaptoneurosome preparation. The generation of endogenous arachidonic acid was also indicated by the ability of 2 inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguaiaretic acid (NDGA), to inhibit muscimol-induced 36Cl uptake. We conclude that arachidonic acid probably has both direct and indirect actions on muscimol responses since both enzyme inhibitors inhibited muscimol responses but did not prevent the effect of exogenously added arachidonic acid. In additional experiments, arachidonic acid metabolites generated by cyclooxygenase, prostaglandins D2, E2 and F2 alpha, each decreased muscimol responses; prostaglandins F2 alpha was the most potent inhibitor. Since the unsaturated fatty acids and their metabolites are most susceptible to peroxidation, a generating system of superoxide radicals was tested on muscimol responses. A combination of xanthine and xanthine oxidase inhibited muscimol-induced 36Cl uptake in a concentration-dependent manner. We propose that the inhibition of GABAA neurotransmission by arachidonic acid and its metabolites can lead to increased neuronal excitability. This mechanism may play an important role in the development of neuronal damage following seizures or cerebral ischemia.     

Chronic carbamazepine selectively downregulates cytosolic phospholipase A2 expression and cyclooxygenase activity in rat brain.

BACKGROUND: Carbamazepine is a mood stabilizer used as monotherapy or as an adjunct to lithium in the treatment of acute mania or the prophylaxis of bipolar disorder. Based on evidence that lithium and valproate, other mood stabilizers, reduce brain arachidonic acid turnover and its conversion via cyclooxygenase to prostaglandin E(2) in rat brain, one possibility is that carbamazepine also targets the arachidonic acid cascade. METHODS: To test this hypothesis, carbamazepine was administered to rats by intraperitoneal injection at a daily dose of 25 mg/kg for 30 days. RESULTS: Carbamazepine decreased brain phospholipase A(2) activity and cytosolic phospholipase A(2) protein and messenger RNA levels without changing significantly protein and activity levels of calcium-independent phospholipase A(2) or secretory phospholipase A(2). Cyclooxygenase activity was decreased in carbamazepine-treated rats without any change in cyclooxygenase-1 or cyclooxygenase-2 protein levels. Brain prostaglandin E(2) concentration also was reduced. The protein levels of other arachidonic acid metabolizing enzymes, 5-lipoxygenase and cytochrome P450 epoxygenase, were not significantly changed nor was the brain concentration of the 5-lipoxygenase product leukotriene B(4). CONCLUSIONS: Carbamazepine downregulates cytosolic phospholipase A(2)-mediated release of arachidonic acid and its subsequent conversion to prostaglandin E(2) by cyclooxygenase. These effects may contribute to its therapeutic actions in bipolar disorder.