1 Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2 Primary astrocytes were cultured under standard conditions and prelabeled with [ 14 C]DHA or with [ 3 H]AA. Adenosine 5 0 -triphosphate (ATP) (20 mm applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca 2 þ -independent phospholipase A 2 (iPLA 2 ), since it is blocked by the selective iPLA 2 inhibitor 4-bromoenol lactone (BEL, 5 mm) and is not affected either by removal of Ca 2 þ from extracellular medium or by suppression of intracellular Ca 2 þ release through PLC inhibitor (U73122, 5 mm). 3 AA release, on the other hand, which is stimulated by ATP, is attributed to Ca 2 þ -dependent cytosolic PLA 2 (cPLA 2 ). AA release is abolished by U73122 and, by removal of extracellular Ca 2 þ , is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 mm), a general inhibitor of intracellular PLA 2 s. 4 Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA 2 and 40 kDa protein reactive to iPLA 2 antibodies. 5 The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 mm), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mm). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 mm) and inhibited by 34% by H89 (10 mm), inhibitors of adenylyl cyclase and PKA, respectively. 6 Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7 Different sensitivities of iPLA 2 and cPLA 2 to Ca 2 þ and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

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.

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Infantile neuroaxonal dystrophy (INAD; OMIM #no. 256600) is an inherited degenerative nervous system disorder characterized by nerve abnormalities in brain, spinal cord and peripheral nerves. About 85% of INAD patients carry mutations in the PLA2G6 gene that encodes for a Ca(2+)-independent phospholipase A(2) (VIA iPLA(2)), but how these mutations lead to disease is unknown. Besides regulating phospholipid homeostasis, VIA iPLA(2) is emerging with additional non-canonical functions, such as modulating store-regulated Ca(2+) entry into cells, and mitochondrial functions. In turn, defective Ca(2+) regulation could contribute to the development of INAD. Here, we studied possible changes in ATP-induced Ca(2+) signaling in astrocytes derived from two mutant strains of mice. The first strain carries a hypomorphic allele of the Pla2g6 that reduces transcript levels to 5-10% of that observed in wild-type mice. The second strain carries a point mutation in Pla2g6 that results in inactive VIA iPLA(2) protein with postulated gain in toxicity. Homozygous mice from both strains develop pathology analogous to that observed in INAD patients. The nucleotide ATP is the most important transmitter inducing Ca(2+) signals in astroglial networks. We demonstrate here a severe disturbance in Ca(2+) responses to ATP in astrocytes derived from both mutant mouse strains. The duration of the Ca(2+) responses in mutant astrocytes was significantly reduced when compared with values observed in control cells. We also show that the reduced Ca(2+) responses are probably due to a reduction in capacitative Ca(2+) entry (2.3-fold). Results suggest that altered Ca(2+) signaling could be a central mechanism in the development of INAD pathology.

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