Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Abstract

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2J activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEA1-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human platelet guanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatt acids. Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acid peroxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxides is attributed to oxidation of sulfhydryl residues of the enzyme protein.The role of cyclic nucleotides in platelet function has been the subject of considerable study since the report by Marcus and Zucker (1) on inhibition of platelet aggregation by adenosine 3',5'-(cyclic)monophosphate (cyclic AMP). Subsequent investigations from several laboratories revealed that platelet aggregation mediated by vanrous substances such as ADP, collagen, and epinephrine is facilitated by a decrease in the platelet cyclic AMP concentration (2, 3), and that many compounds that inhibit platelet aggregation act by increasing the cyclic AMP concentration in the platelets (3, 4). The effect on the platelet guanosine 3',5'-(cyclic)monophosphate (cyclic GMP) level of various aggregating agents, however, appears to be the opposite of that seen with cyclic AMP in that aggregating agents such as collagen (5, 6), thrombin, and epinephrine (6, 7) produce an increase in the cyclic GMP concentration in the platelets.Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity has been detected in many types of cells and the enzyme is present in both membrane-bound and soluble forms. Several hormones increase cyclic GMP levels in cells (8-14); however, it has not been demonstrated in vitro that there is a direct effect of any of these hormones on guanylate cyclase activity (12,(15)(16)(17) (pH 7.7), and an appropriate amount of the enzyme in a total volume of 0.2 ml. The reaction was started by the addition of enzyme unless otherwise indicated. After the mixture was incubated for 10 min at 300, the reaction was stopped by heating for 2 min in a boiling-water bath, following the addition of 1 M HCl (40 Ml). The radioactive cyclic GMP produced was isolated by the serial use of...

Cell Damage in the Brain: A Speculative Synthesis

“…Why, then, does not cGMP accumulate dur ing ischemia but only after oxygenation is insti tuted? A likely explanation is provided by experi ments demonstrating that guanylate cyclase is acti vated by hydroperoxide breakdown products of arachidonic acid (Hidaka and Asano, 1977;Goldberg et aI ., 1978;Murad et aI ., 1979). We can envisage a series of reactions, therefore, which start with accumulation of Ca2+ in the cytosol of cells, proceed with activation of phospholipases, and end with oxidative conversion of arachidonic acid to hydroperoxide breakdown products .…”

Lysolecithins as endothelium-dependent vascular smooth muscle relaxants that differ from endothelium-derived relaxing factor (nitric oxide)

“…Male New Zealand White rabbits weighing 2.1-3.2 kg were anesthetized with sodium pentobarbital (30 mg/kg) and heparinized with 500 international units/kg i.v. After tracheotomy, the rabbits were ventilated with a respirator (Bird Mark 10; Space Technology, Palm Springs, CA).…”

Effect of 8-substituted cyclic AMP derivatives on soluble guanylate cyclase activity from human platelets.

“…In fact, elongation of the alkyl chain of cyclic AMP derivatives make the derivatives more potent inhibitors. The soluble form of guanylate cyclase has been reported to interact with hydrophobic compounds, such as fatty acids (1,15), lysolecithin (2), fatty acid peroxides (3,4), and Triton X-1 00 (15), which activate or bind to guanylate cyclase. These data suggest that the hydrophobic region of the cyclase appears to be near the active site.…”

“…The soluble form of guanylate cyclase inter acts with hydrophobic components of cell such as fatty acids (1), lysolecithin (2), fatty acid peroxides (3,4). It was also suggested by Garbers that the soluble form of guanylate cyclase of rat lung contains bound fatty acids or other hydrophobic components of cell (5).…”