Docosahexaenoic acid and arachidonic acid release in rat brain astrocytes is mediated by two separate isoforms of phospholipase A₂ and is differently regulated by cyclic AMP and Ca²⁺

Abstract

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.

RETRACTED ARTICLE: Aging decreases rate of docosahexaenoic acid synthesis-secretion from circulating unesterified α-linolenic acid by rat liver

“…with age, whereas synthesis-secretion rates (product of conversion coefficient and unesterified plasma Introduction Docosahexaenoic acid (22:6n-3, DHA), a long-chain n-3 polyunsaturated fatty acid (PUFA), is enriched in brain and retina phospholipids, where it modulates membrane fluidity, regulates functions of membraneassociated proteins (Stubbs and Smith 1984;Salem et al 2001;Kim 2007), and participates in calciumindependent phospholipase A 2 mediated neurotransmission (Strokin et al 2003). DHA may be obtained directly from dietary sources, such as fish or fish products, or it can be synthesized from its nutritionally essential plant-derived precursor, α-linolenic acid (18:3n-3, α-LNA), through a series of elongation and desaturation steps (Sprecher 2000).…”

Chronic Lithium Administration to Rats Selectively Modifies 5-HT2A/2C Receptor-Mediated Brain Signaling Via Arachidonic Acid

“…Three major PLA 2 enzymes occur in brainFa Ca 2 þ -dependent cytosolic cPLA 2 selective for AA, a Ca 2 þ -dependent secretory sPLA 2 , and a Ca 2 þ -independent iPLA 2 selective for docosahexaenoic acid (22:6 n-3), another polyunsaturated fatty acid found usually at the sn-2 position of phospholipids (Clark et al, 1995;Dennis, 1994;Murakami et al, 1999;Strokin et al, 2003). Although we do not know which PLA 2 enzymes mediate the k* responses to arecoline and DOI, LiCl feeding to rats is reported to downregulate brain mRNA and activity levels of cPLA 2 but not of sPLA 2 or iPLA 2 (Bosetti et al, 2001;Rintala et al, 1999;Weerasinghe et al, 2004).…”

AHNAK-mediated Activation of Phospholipase C-γ1 through Protein Kinase C

“…1). It has been reported that bradykinin stimulated the release of AA (37,38). Both mechanisms for the generation of AA and activation of cPLA 2 activity and AHNAK protein would convey activation of the PLC␥ isozyme.…”