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Nitric oxide is a potent inhibitor of membrane lipid peroxidation. It is unknown, however, whether nitric oxide synthase (NOS) activity increases under conditions of membrane lipid peroxidation. Importantly, cyclooxygenase (COX)-catalyzed peroxidation of arachidonic acid is well-established to be increased by lipid hydroperoxides. The results of the present study demonstrate that the COX hydroperoxide product prostaglandin G(2) (PGG(2)) greatly stimulated NOS activity in synaptosomal membrane fractions from rat brain in a dose-dependent (EC(50) = 0.2 microM) manner in the presence of ATP and the antioxidant urate. NOS activation was also produced, albeit to a lesser extent, by 15-hydroperoxyeicosatetraenoic acid (15-HPETE) but not by the corresponding hydroxy compounds PGH(2) and 15-HETE or by hydrogen peroxide. These findings demonstrate that PGG(2)-activated synaptic NOS by a hydroperoxide-mediated pathway and support the view that NOS activation may be an important physiological response to lipid peroxidation., (Copyright 2001 Academic Press.)

Previously, we have shown that prostaglandins are necessary, but not sufficient, for the stimulation of mitogenesis in BALB/c 3T3 fibroblasts by epidermal growth factor (EGF) (Nolan, R. D., Danilowicz, R. M., and Eling, T. E. (1988) Mol. Pharmacol. 33, 650-656). The purpose of this work was to extend these findings to another potent mitogen, platelet-derived growth factor (PDGF), and to determine if metabolism of arachidonic acid to prostaglandins is necessary for stimulation of expression of the protooncogene c-myc by EGF, which is an early event in the mitogenic cascade. In BALB/c 3T3 cells grown to about 70% confluence and deprived of serum for 16-24 h, PDGF stimulated [3H]thymidine uptake into DNA significantly in a concentration-dependent manner, but did not increase production of prostaglandin E2 (PGE2). The addition of indomethacin, a prostaglandin H synthase inhibitor, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, did not affect PDGF-stimulated thymidine uptake into DNA. In addition, PGE2 enhanced EGF-dependent, but not PDGF-dependent, mitogenesis. Taken together, the data support the hypothesis that prostaglandins are not involved in PDGF-dependent mitogenesis. In contrast, indomethacin (10(-6) M) and nordihydroguaiaretic acid (10(-6) M) inhibited EGF-stimulated thymidine uptake and c-myc expression by approximately 50%. Addition of PGG2 (10(-7) to 10(-5) M) in the presence of indomethacin and EGF restored the ability of EGF to elevate c-myc RNA levels and DNA synthesis. When PGF2 alpha (10(-8) to 10(-5) M) was added in the presence of EGF, c-myc RNA levels and thymidine incorporation were elevated up to 5-6-fold above levels observed with EGF alone. These data support the hypothesis that metabolism of arachidonic acid to prostaglandins is necessary for stimulation of c-myc expression by EGF in BALB/c 3T3 cells.

Purified prostaglandin endoperoxides (PGG2 and PGH2) and hydroperoxides (15-OOH-PGE2) as well as fatty acid hydroperoxides (12-OOH-20:4, 15-00H-20:4, and 13-OOH-18:2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures described (in the miniprint supplement) for the preparation, purification, and characterization of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5- to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed half-maximal stimulation was achieved in the range of 2 to 3 micrometer. However, by extending the incubation time to 90 min similar maximal increases in specific activity could be achieved with 3 or 10 micrometer PGG2 or PGH2. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or glutathione (10 to 15 mM). Na2S2O4, not known as an effective reducing agent of disulfides, prevented but was relatively ineffective in reversing activation after it had been induced by PGG2. Pretreatment of the enzyme preparation with increasing concentrations of N-ethylmaleimide in the range of 0.01 to 1.0 mM prevented activation by PGG2 without affecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast PGE2, PGF2a, hydroxy fatty acids (13-OH-18:2, 12-OH-20:4) as well as saturated (18:0) monoenoic (18:1), dienoic (18:2), and tetraenoic (20:4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 micrometer. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG2 but not 15-OOH-PGE2 (the major buffer-rearrangement product of PGG2) is most likely an activator. In the case of PGH2, a rapidly generated (30 s) metabolite of PGH2 was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH2 as an apparent activator of the enzyme. The combined effects of PGG2 and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryl groups that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.

We assessed the integrity of the prostaglandin synthetic pathway by measuring malondialdehyde (MDA) production and studied platelet aggregation responses to arachidonic acid and PGG2 in 12 patients with storage pool deficiency (SPD). Eight patients were deficient only in dense granules (delta-SPD) and four were deficient in both dense and alpha-granules (alpha delta-SPD). Production of MDA in response to arachidonic acid (AA), epinephrine, and collagen suggested that the transformation of AA to prostaglandin metabolites was normal in delta-SPD but abnormal in alpha delta-SPD and that the liberation of AA from phospholipids were abnormal in the majority of patients with SPD. Since the content of secretable adenosine diphosphate (ADP) is diminished in SPD platelets, the aggregation responses of these platelets to AA and PGG2 were studied to help answer the question whether these agents aggregate platelets directly or through release of endogenous ADP. Among patients with delta-SPD, aggregation by both AA and PGG2 was decreased in four albinos whose platelets were markedly deficient in ADP. In contrast, normal, or less strikingly abnormal, responses were observed in patients whose platelets either contained higher levels of platelet ADP or showed increased sensitivity to ADP. The more marked impaired responses to AA and PGG2 in patients with alpha delta-SPD suggest that substances derived from alpha-granules may also play a role in platelet aggregation by these agents. The aggregation responses in these patients with various types of SPD is consistent with a theory that granule-derived ADP mediates platelet aggregation by AA and PGG2.

Application of arachidonic acid or prostaglandin G(2) to the brain surface of anesthetized cats induced cerebral arteriolar damage. Scavengers of free oxygen radicals inhibited this damage. Prostaglandin H(2), prostaglandin E(2), and 11,14,17-eicosatrienoic acid did not produce arteriolar damage. It appears that increased prostaglandin synthesis produces cerebral vascular damage by generating free oxygen radicals.

Two prostaglandin endoperoxides, i. e. PGG2 and PGH2 were detected and isolated. They were unstable in aqueous medium (t1/2 at 37 degrees C about 5 min) and were converted to PGF2alpha by mild reducing agents. Human platelets as well as guinea pig lung and spleen converted the endoperoxides into thromboxane A2, an unstable (t1/2 at 37 degrees C about 30 s) oxetane/oxane derivative. Thromboxane A2 was converted into a stable hemiacetal derivative, thromboxane B2, by addition of 1 H2O. The two prostaglandin endoperoxides as well as thromboxane A2 caused platelet aggregation and the platelet release reaction. In addition they were potent stimulators of vascular and airway smooth muscle in vitro and in vivo.

To determine whether the prostaglandin endoperoxides PGG2 and PGH2 have direct effects in the kidney, PGG2 and PGH2 were administered into the renal artery of anesthetized dogs and their effects were compared to those of PGE2. Like PGE2, PGG2 and PGH2 induced a dose-related renal vasodilation. A 50% increase in the renal blood flow was observed with 0.05 microgram/kg body wt of PGE2 and with four- and sixfold higher doses of PGH2 and PGG2, respectively. Infusion of all three compounds at doses inducing a 50% increase in the renal blood flow resulted in 1) increases in blood flow to all cortical areas, with the greatest increase occurring in the juxtamedullary area, 2) diuresis with no change in the glomerular filtration rate, and 3) natriuresis and kaliuresis. In vitro incubation of PGH2, a maneuver known to result in its conversion to other prostaglandins, had no influence on its renal effects. The data indicate that PGH2 and PGG2 are biologically active when infused into the renal artery of the anesthetized dog and suggest that the endoperoxides exert their effects after bioconversion to other prostaglandins.

A calcium sequestering platelet membrane fraction was prepared and the effect of arachidonic acid, PGG2 and PGH2 on calcium content evaluated. At 4 degrees C, 6.7--16.7 micrometers arachidonic acid caused significant release of calcium from preloaded vesicles. Such release was completely inhibited by aspirin pretreating the platelets from which the membrane fraction was prepared. Gamma-linolenic acid, not a substrate for prostaglandin synthesis, did not cause calcium release. At 37 degrees C, after a 5 minute calcium loading of the membrane vesicles, arachidonic acid, PGG2, and PGH2 caused release of calcium. Calcium release by the PGG2 and PGH2 was only slightly inhibited by aspirin. Imidazole, which prevented conversion of the prostaglandin endoperoxides to thromboxanes, also only slightly inhibited calcium release. Other prostaglandins including PGD2, PGE1, PGE2 and PGD2 had no effect on the calcium content of the vesicles. These studies suggest that PGG2 and PGH2 may exert their effects on platelets by mobilizing calcium from an internal membrane store to make it available to promote platelet activation.

A labile prostaglandin was isolated as one of the products generated from [1-14C] eicosatetraenoic acid incubated with sheep vesicular gland microsomes. The eicosatetraenoic acid metabolite amounted to ca. 16% of the total radiolabeled products. Formation of this new prostaglandin was prevented when heat-denatured microsomes were employed or when incubation mixtures were supplemented with indomethacin or phenol. However, incubation of prostaglandin G2 (PGG2) with hematin in the presence or absence of catalytically active or heat-inactivated microsomes led to production of approximately the same quantity of the new prostaglandin. These results indicated that the new prostaglandin can be formed nonenzymically. The new prostaglandin was conclusively identified by gas liquid chromatography-mass spectrometry analysis as 15-keto-9,11-peroxidoprosta-5,13-dienoic acid (15-keto-PGG2) after chemical conversion to known prostaglandins. The effects of 15-keto-PGG2 and PGG2 were similar on canine lateral saphenous vein; both promoted contraction followed by prolonged relaxation, but 15-keto-PGG2 appeared to be 1/50 as potent as PGG2.

Effect of Ticlopidine, 5-(2-chlorobenzyl)-4, 5, 6, 7-tetrahydro[3,2-C]pyridine hydro-chloride, on platelet aggregation was studied in the rat. Ticlopidine was found to be a potent, long-lasting inhibitor of platelet aggregation. It inhibited the aggregation induced by any of ADP, collagen, thrombin, arachidonic acid and prostaglandin endoperoxides and/or thromboxane A2-like substancee. Ticlopidine was effective at doses as low as 30 mg/kg when orally given to rats, and the effect lasted as long as the life span of the circulating platelets (half time: about 48 hours). Ticlopidine inhibited also nucleotide release from and prostaglandin synthesis in the platelets, but did not significantly affect platelet adhesiveness to glass, platelet factor 3 availability and clot retraction.

In this study the ability of prostaglandin E1 (PGE1), Misoprostol (a stable analog of PGE1), and 16,16-dimethyl PGE2 (a stable analog of PGE2) to suppress immune responses in vitro and in vivo was determined. All of the compounds caused a titratable (10(-6) to 10(-9) M) suppression of Con A blastogenesis and the mixed lymphocyte response whereas there was only slight inhibition of the LPS response. When either 16,16-dimethyl PGE2 (30 ug/mouse) or Misoprostol (60 ug/mouse) was administered daily in vivo, there was a significant suppression of splenomegaly in F1 mice (C57Bl/6 x CBA) which had been injected with parental (C57Bl/6) spleen cells. We conclude that prostaglandins of the E series can function as immunosuppressive reagents both in vitro and in vivo. In the future they may serve to augment existing forms of immunosuppressive therapy.

The effect of various prostaglandins (PGs) and a thromboxane A2 (TXA2)-generating system on the contractility of rabbit splenic capsular smooth muscle has been investigated. PGI2 as well as higher concentrations of PGE1 and 5, 6 beta-dihydro-PGI2 inhibit noradrenaline-induced contractions of the smooth muscle preparation. The TXA2-generating system and high concentrations of PGD2, on the other hand, increase the contractions. The results support the concept that the hypothetical PGI2 receptor on the smooth muscle of the rabbit splenic capsule resembles, in its specificity, the PGI2 receptor on platelets.

The conversion of PGG2 to 15-hydroperoxythromboxane A2 by purified thromboxane synthase from human platelets was recently reported (Hammarström, S. (1980) J. Biol. Chem.255, 518). The hydroperoxythromboxane is approximately 25 times more potent than PGG2 in causing aggregation of human platelet rich plasma and 30 times more potent than PGG2 as an inducer of contractions of rabbit aorta.

The effects of PGG2 and PGH2 on platelets are mimicked by synthetic PG analogues in which the nature and specificity of the substituents on carbons 11 and 15 (or 16) are important determinants of reactivity. Arachidonic acid and stimulatory PGs induce secretion of platelet dense granule and alpha granule constituents, but not lysomal constituents, although arachidonate metabolism is necessary for collagen-induced release of lysosomal enzymes. NO164 acts on platelets as an endoperoxide antagonist: Trimethoquinol acts as an endoperoxide and TxA2 antagonist. PGs induce platelet aggregation by combining with a specific (endoperoxide) receptor.

Acute, severe increases in arterial blood pressure cause sustained cerebral arteriolar dilation, abnormal reactivity to carbon dioxide and to changes in blood pressure, abolition of endothelium-dependent dilation from acetylcholine, discrete morphological lesions of the endothelium and vascular smooth muscle, and breakdown of the blood-brain barrier to plasma proteins. The dilation, abnormal reactivity, and morphological abnormalities are inhibited by pretreatment with cyclooxygenase inhibitors or with free radical scavengers. Superoxide dismutase-inhibitable reduction of nitroblue tetrazolium applied to the brain surface was detectable both during hypertension and one hour after hypertension subsided. Nitroblue tetrazolium reduction is also reduced by inhibitors of the anion channel. The abnormalities seen after hypertension are reproduced by topical application of arachidonate. The results are consistent with the view that acute hypertension induces generation of superoxide anion radical in association with accelerated arachidonate metabolism via cyclooxygenase. This radical enters cerebral extracellular space via the anion channel and gives rise to hydrogen peroxide and hydroxyl radical. All three radicals are capable of causing vasodilation by relaxation of cerebral vascular smooth muscle. The hydroxyl radical is the most likely candidate for vascular wall damage. The significance of this mechanism in chronic experimental hypertension or its relevance to human disease is not known.

To determine the possible role that endogenously produced prostaglandins may play in the regulation of cerebral blood flow, the responses of cerebral precapillary vessels to prostaglandins (PG) D2, E2, G2, and I2 (8.1 X 10(-8) to 2.7 X 10(-5) M) were studied in cats equipped with cranial windows for direct observation of the microvasculature. Local application of PGs induced a dose-dependent dilation of large (greater than or equal to 100 microns) and small (less than 100 microns) arterioles with no effect on arterial blood pressure. The relative vasodilator potency was PGG2 greater than PGE2 greater than PGI2 greater than PGD2. With all PGs, except D2, the percent dilation of small arterioles was greater than the dilation of large arterioles. After application of prostaglandins in a concentration of 2.7 X 10(-5) M, the mean +/- standard error of the percent dilation of large and small arterioles was, respectively, 47.6 +/- 2.7 and 65.3 +/- 6.1 for G2, 34.1 +/- 2.0, and 53.6 +/- 5.5 for E2, 25.4 +/- 1.8, and 40.2 +/- 4.6 for I2, and 20.3 +/- 2.5 and 11.0 +/- 2.2 for D2. Because brain arterioles are strongly responsive to prostaglandins and the brain can synthesize prostaglandins from its large endogenous pool of prostaglandin precursor, prostaglandins may be important mediators of changes in cerebral blood flow under normal and abnormal conditions.

We examined effects of arachidonic acid (AA) on eicosapentaenoic acid (EPA) metabolism in washed human platelets. Although human platelets had been considered to metabolize scarcely EPA, a simultaneous addition of EPA and AA to washed platelet suspensions stimulated markedly EPA metabolism. In addition, the stimulatory effect was more potent over the formation of thromboxane (TX) B3 than that of 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (HEPE). The stimulation by AA can be due to AA itself and/or AA metabolites. Indomethacin decreased the stimulatory effect of AA on the HEPE formation, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE formation. Among the metabolites of AA investigated, prostaglandin (PG)G2 and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid had the stimulatory effect on both TXB2 and HEPE formations, whereas PGH2, PGD2, TXB2 and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid were ineffective.