Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.

Reductions of gamma-aminobutyric acid and glutamate uptake and (Na+ + K+)-ATPase activity in brain slices and synaptosomes by arachidonic acid

Publisher Summary This chapter explains that free polyenoic fatty acids (PUFAs), especially arachidonic acid and docosahexaenoic acid are rapidly released following ischemia, electroconvulsive seizures, and various pathological insults. Free PUFAs and arachidonic acid in particular, have both physiological and pathological effects on cellular systems. It has been described that free arachidonic acid readily intercalates into the membrane and produces significant changes in the packing of the lipid molecules. PUFA-induced membrane fluidity has been associated with the stimulation of chloride transport in corneal epithelium; it enhanced activities of both membrane-associated adenylate cyclase, and guanylate cyclase. The molecular mechanisms of PUFA-induced cellular edema are studied further in the in vitro cortical slices system. The studies have suggested that PUFAs, especially arachidonic acid, play a key role in membrane damage and the development of edema following ischemia and injury.

Cellular and molecular effects of polyunsaturated fatty acids in brain ischemia and injury.

The effects of 15-hydroperoxyarachidonic acid (15-hpaa) on Na+, K+- and Mg + -ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+ -ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered.

Enhancement of ATPase activity by a lipid peroxide of arachidonic acid in rat brain microvessels.

Guanylate cyclase that is present in soluble and particulate fractions of homogenates from most tissues has different properties, indicating the presence of multiple forms. While the subcellular distribution of the enzyme can be altered, it is unknown whether or not the soluble and particulate activities represent similar proteins. The soluble enzyme has been purified from several tissues, including rat liver and lung, and the holoenzyme is about 150,000 Daltons. Crude and purified preparations of the enzyme can be activated with a variety of agents including azide, nitroprusside, and other nitro compounds. The common feature of this class of activators is their ability to form nitric oxide which can activate purified enzyme. Another free radical, probably hydroxyl radical, can activate the enzyme. Guanylate cyclase is also activated by fatty acids, detergents and other materials. Guanylate cyclase and cyclic GMP levels are sensitive to oxidizing and reducing agents in incubations, and hormone effects on this system may be mediated through alterations in the redox state of preparations. Increases in cyclic GMP levels in intact cells with nitro compounds are not dependent upon calcium in incubations, in contrast to the effects of acetylcholine and other hormones. Activation of guanylate cyclase results in alterations in the properties of the enzyme. Activated enzyme can utilize Mg2 + as effectively as Mn2 + for cofactor while native enzyme prefers Mn2 +. Enzyme activated with various agents can also catalyze the formation of cyclic AMP from ATP. The significance of this alternate pathway for cyclic AMP synthesis is not known. While many hormones can increase cyclic GMP levels in various tissues, these agents have failed to activate guanylate cyclase in cell-free preparations. However, a heat-stable enterotoxin from Escherichia coli increases cyclic GMP levels in intestinal mucosa and can activate particulate guanylate cyclase from intestinal mucosal homogenates from several species. The increase in cyclic GMP in this tissue may mediate the effects of the enterotoxin on fluid secretion in the intestine. While cyclic GMP may function as a second messenger in some systems, it is proposed that cyclic GMP may also act as a feedback regulator to alter the redox state of preparations.

Guanylate Cyclase: Regulation of Cyclic GMP Metabolism

Effects of psychoactive drugs on cyclic nucleotides in the central nervous system.

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Guanylate cyclase (EC 4.6.1.2) of synaptic plasma membranes of rat cerebral cortex was stimulated about 6-fold by several unsaturated fatty acids (arachidonic, linolenic, linoleic, oleic, palmitoleic and myristoleic acid). Ricinoleic acid (12-hydroxyoleic acid) was much less effective. Saturated fatty acids (C10 and C14-C20) and the methylester of linoleic acid were ineffective. Stimulation by linoleic acid was influenced by the concentration of enzyme protein. At 480 μg/ml of protein 0.6 mm-linoleic acid produced maximal activation of 6-fold_ Activity stimulated by linoleic acid examined with 1.0 mm-GTP was maximal at pH 7.8-7.9 and with 2 mm-MnCl2, whereas basal activity showed broad optimal pH and Mn2+-concentration dependence. Activation of the enzyme by linoleic acid was only partially reversed by washing. Particulate guanylate cyclase of heart, small intestine, adrenal medulla, liver and lung was also activated by linoleic acid. The extents of activation (1.5-14.7-fold) by linoleic acid and the concentrations (0.2-1.0 mat) required for maximal activation depended on the tissues.

M. Takenoshta

Papers

Activation of guanylate cyclase in synaptic plasma membranes of cerebral cortex by free fatty acids.