Interactions between stimulated platelets and endothelial cells in vitro

Prostaglandins and hydroxy acids are synthesized mainly from the essential polyunsaturated fatty acid arachidonate, and these substances have been identified in almost all mammalian tissues. Prostaglandins, thromboxane A2 (TXA2) and prostacyclin (PGI2) are autocoids that appear to function in the regulation of vascular tone, cell secretion and contractile processes. So far, hydroxy acids have been found to function as chemotactic agents and in the formation of slow-reacting substances. Other actions of hydroxy acids will certainly be defined in future research. The endoperoxides PGG2 and PGH2 represent common precursors of all prostaglandin end-products. In studying the prostaglandin metabolism of a specific tissue, the total profile of endoperoxide transformation should be determined. In platelets the endoperoxides are transformed mainly into TXA2, a potent vasoconstrictor and inducer of platelet aggregation. Endothelial cells convert endoperoxides to PGI2, a vasodilator and inhibitor of platelet aggregation. In addition, endothelial cells can utilize endoperoxides from stimulated plates to form PGI2. The concept that platelets and endothelial cells can share common precursors for the production of modulating substances may be applicable to other cell types.     

The cyclooxygenase hydroperoxide product PGG(2) activates synaptic nitric oxide synthase: a possible antioxidant response to membrane lipid peroxidation

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.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Imidazole: a selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymic conversion of the endoperoxides (PGG2 and PGH2) to thromboxane A2 by platelet microsomes (IC50: 22 MICRONG/ML; DETERMINED BY BIOASSAY). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of 14C-thromboxane B2 from 14C-PGH2. Several imidazole analogues and other substances were tested but only 1-methyl-imidazole was more potent than imidazole itself. The use of imidazole to inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

Stimulation of human platelet guanylate cyclase by fatty acids.

Guanylate cyclase from human platelets was over 90% soluble, even when assayed in the presence of Triton X-100. A time-dependent increase in activity occurred when the enzyme was incubated at 37 degrees and this spontaneous activation was prevented by dithiothreitol. Arachidonic acid stimulated the soluble enzyme activity approximately 2- to 3-fold. Linear double reciprocal plots of guanylate cyclase activation as a function of arachidonic acid concentration were obtained with a Ka value of 2.1 muM. A Hill coefficient of 0.98 was obtained indicating that one fatty acid binding site is present for each catalytic site. Concentrations of arachidonic acid in excess of 10 muM caused less than maximal stimulation. Dihomo-gamma-linolenic acid and two polyunsaturated 22 carbon fatty acids stimulated the activity of guanylate cyclase to the same degree as did arachidonic acid. The methyl ester of arachidonic acid was much less effective. Diene, monoene, and saturated fatty acids of various carbon chain lengths as well as prostaglandins E1, E2, and F2alpha, had little or no effect. These data indicate that the structural determined required for stimulation by fatty acids of soluble platelet guanylate cyclase is a 1,4,7-octatriene group with its first double bond in the omega6 position. This structural group is similar to the substrate specificity determinants of fatty acid cyclooxygenase, the first enzyme of the prostaglandin synthetase complex. However, conversion of arachidonic acid to a metabolite of the cyclooxygenase pathway did not appear to be required for activation of the cyclase since activation occurred in the 105,000 X g supernatant fraction and pretreatment of this fraction with aspirin did not alter the ability of arachidonic acid to activate guanylate cyclase. Kinetic studies showed that the stimulation of guanylate cyclase by arachidonic acid is primarily an effect on maximal velocity. Arachidonic acid did not alter the concentration of free Mn2+ required for optimal activity. It is concluded that the activity of the soluble form of guanylate cyclase in cell-free preparations of human platelets can be increased by a lipid-protein interaction involving specific polyunsaturated fatty acids.     

The role of selenium-dependent and selenium-independent glutathione peroxidases in the formation of prostaglandin F2 alpha

In recent years, growing evidence suggests that glutathione peroxidases (GSH-Pxs), both selenium-dependent GSH-Px (Se-GSH-Px) and selenium-independent GSH-Px (non-Se-GSH-Px) play an important role in the biosynthesis of prostaglandins and leukotrienes and in the regulation of key enzymes associated with the arachidonic acid cascade. The precise nature of their involvement in eicosanoid metabolism, however, is not yet completely understood. In the study reported here, we have systematically determined the catalytic efficiencies of Se-GSH-Px and non-Se-GSH-Px toward prostaglandin (PG) G2 (PGG2) and PGH2. Se-GSH-Px exhibited high catalytic activity for the reduction of PGG2 as indicated by Km and Vmax values of 12 microM and 78 mumol/min/mg, respectively, whereas PGH2 was found to be a poor substrate, an indication that Se-GSH-Px reduces the hydroperoxide moiety but not the endoperoxide moiety of PGG2. The kinetic constants of Se-GSH-Px toward PGG2 were comparable to those determined for such classical substrates as H2O2 and cumene hydroperoxide. In contrast to Se-GSH-Px, non-Se-GSH-Px associated with cationic isozyme II of glutathione S-transferases (GSTs) from sheep lung cytosol was very active in the conversion of PGH2 to PGF2 alpha with a Vmax of 960 nmol/min/mg and a Km of 77 microM. This study shows that PGF2 alpha formation by non-Se-GSH-Px occurred in a GSH-dependent reduction of either PGG2 or PGH2. When PGG2 was used as the substrate for non-Se-GSH-Px, a novel intermediate compound appeared and was later identified by several methods of structural analysis as 15-hydroperoxy PGF2 alpha. Thus, the reductive cleavage of the endoperoxide occurs faster than the 15-hydroperoxide reduction allowing 15-hydroperoxy PGF2 alpha to accumulate briefly. A study of GSTs from several different tissues and species indicated that the transformation of PG endoperoxides to PGF2 alpha is catalyzed specifically by GST isozymes, which contain Ya size subunits. This specificity of GST isozymes in PG biosynthesis, coupled with their tissue-specific expression, may be a mechanism by which the body modulates the type of PGs produced in these tissues. Also, these results suggest a possible interaction of Se-GSH-Px and non-Se-GSH-Px in the biosynthesis of PGF2 alpha.     

Kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase. Effect of electron donors on the cyclooxygenase reaction

Prostaglandin-H-synthase (PGHS, EC 1.14.99.1) catalyzes the first committed step in biosynthesis of all prostaglandins, thromboxanes, and prostacyclins by converting arachidonic acid to prostaglandin H(2) (PGH(2)). PGHS exhibits two enzymatic activities: cyclooxygenase activity converting arachidonic acid to prostaglandin G(2) (PGG(2)) and peroxidase activity reducing the hydroperoxide PGG(2) to the corresponding alcohol, PGH(2). Despite the many investigations of the kinetics of PGHS, many features such as the absence of competition and mutual activation between the cyclooxygenase and peroxidase activities cannot be explained in terms of existing schemes. In this work we have studied the influence of different electron donors (N,N,N ,N -tetramethyl-p-phenylenediamine, L-epinephrine, 2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), potassium ferrocyanide) on the PGHS activities. The proposed scheme describes independent but interconnected cyclooxygenase and peroxidase activities of PGHS. It also explains the experimental data obtained in the present work and known from the literature.     

Thromboxane synthetase inhibitors as pharmacological tools: differential biochemical and biological effects on platelet suspensions

The comparative effects of three so called "thromboxane-synthetase-inhibitors" (imidazole, N-0164, and U-51605) on arachidonate metabolism and on platelet aggregation were studied. All three compounds blocked platelet microsomal thromboxane synthesis from prostaglandin endoperoxides without affecting platelet adenyl cyclase. Imidazole, blocked thromboxane synthesis in intact platelets either from arachidonic acid or PGH2, without affecting aggregation. U-51605 simultaneously inhibited thromboxane synthesis and platelet suspension aggregation. N-0164 inhibited aggregation probably at extracellular sites, at concentrations that did not alter arachidonate or PGH2 metabolism. High concentrations of N-0164 simultaneously inhibited PG cyclo-oxygenase and thromboxane synthetase. The lack of specificity of these compounds requires that other actions of these compound must be considered when they are used as pharmacological tools to inhibit thromboxane synthetase.     

Identification of the major urinary metabolite of thromboxane B2 in the monkey.

Thromboxane B2 (TxB2) was biosynthesized from prostaglandin endoperoxides (PGG2, PGH2) using guinea pig lung microsomes and infused into an unanesthetized monkey. Urine was collected and TxB2 metabolites were isolated by reversed phase partition chromatography and high performance liquid chromatography. A major metabolite (TxB2-M) was found to be excreted in greater than two-fold abundance relative to other metabolites. Its structure was determined by gas chromatography-mass spectrometry to be dinor-thromboxane B2. In vitro incubation of TxB2 with rat liver mitochondria yielded a C18 derivative with a mass spectrum identical to that of TxB2-M, substantiating that the major urinary metabolite of TxB2 in the monkey is a product of a single step of beta-oxidation.     

Activation of microsomal guanylate cyclase by a cytotoxic polypeptide: melittin.

Melittin, a 26 amino acid polypeptide, activated membrane-associated guanylate cyclase in a manner suggesting that membrane phospholipids play an important role in regulating the enzyme activity. Melittin was unique in that it activated only the particulate activity in a dose-dependent manner in the concentration range 15 to 200 μg/ml, and, unlike other known activators, solubilization of enzyme did not occur. The effects of melittin on guanylate cyclase showed a lag of two minutes and were not blocked by inhibitors of prostaglandin synthetase or phospholipase suggesting that the effect was not mediated by prostaglandin endoperoxides or phospholipase products but may be due to the ability of melittin to alter membrane lipid properties.     

Reaction of hematin with allylic fatty acid hydroperoxides: identification of products and implications for pathways of hydroperoxide-dependent epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene

Reaction of 10-hydroperoxyoctadec-8-enoic acid (10-OOH-18:1) (50 microM) with hematin (0.5 microM) in sodium phosphate buffer containing Tween 20 (200 microM) generates 10-oxooctadec-8-enoic acid, 10-oxodec-8-enoic acid (10-oxo-10:1), and 10-hydroxyoctadec-8-enoic acid in relative yields of 79, 4, and 17%, respectively. The product profile and relative distribution are unaffected by 1 mM butylated hydroxyanisole. Approximately 5% of the hydroperoxide isomerizes from the 10- to the 8-position. 10-Oxo-10:1 most likely arises via beta-scission of an intermediate alkoxyl radical to the aldehyde and the n-octyl radical. To test this, 10-hydroperoxyoctadeca-8,12-dienoic acid was reacted with hematin under identical conditions. 10-Oxooctadeca-8,12-dienoic acid, 10-oxodec-8-enoic acid, and 10-hydroxyoctadeca-8,12-dienoic acid are formed in relative yields of 50, 45, and 5%, respectively. The product ratios are constant with time and hydroperoxide to catalyst ratio and unaffected by inclusion of phenolic antioxidants. The higher yield of 10-oxo-10:1 from 10-OOH-18:2 compared to 10-OOH-18:1 is due to the higher rate of beta-scission of the intermediate alkoxyl radical from the former to the resonance-stabilized octenyl radical. Two products of reaction of the 2-octenyl radical with O2, octenal and octenol, were detected in 10% yield relative to 10-oxo-10:1. Inclusion of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) led to epoxidation by both 10-OOH-18:1 and 10-OOH-18:2. Studies with isotopically labeled hydroperoxide or O2 indicated approximately 65% of the epoxide oxygen was derived from O2 and 35% from hydroperoxide oxygen, consistent with the involvement of peroxyl free radicals as the oxidizing agents. The available evidence indicates that hematin reduces the fatty acid hydroperoxides homolytically to alkoxyl radicals that are oxidized to ketones, reduced to alcohols, or undergo beta-scission to aldehydes. Carbon radicals generated during these reactions couple to O2, generating peroxyl free radicals that epoxidize BP-7,8-diol. The smaller percentage of epoxidation that results from hydroperoxide oxygen may arise from oxidation of the hydroperoxide group to peroxyl radicals or from heterolytic cleavage of the hydroperoxide to alcohol and an iron-oxo complex.     

On the mechanism of prostacyclin and thromboxane A2 biosynthesis

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.     

A possible role of thromboxane A2 (TXA2) and prostacyclin (PGI2) in circulation.

Recently two local hormones, thromboxane A2 (TXA2) and prostacyclin (PGI2) have been discovered. These hormones are labile metabolites of arachidonic acid. TXA2 is generated by blood platelets, while PGI2 is produced by vascular endothelium. TXA2 is a potent vasoconstrictor. It also initiates the release reaction, followed by platelet aggregation. PGI2 is a vasodilator, especially potent in coronary circulation. It also inhibits platelet aggregation by virtue of stimulation of platelet adenyl cyclase. Common precursors for both hormones are cyclic endoperoxides PGG2 and PGH2, being formed by cyclooxygenation of arachidonic acid. This last enzymic reaction is more efficient in platelets than in vascular endothelium, and therefore the generation of PGI2 by vasuclar wall is accelerated by an interaction between platelets and endothelial cells. During this interaction platelets supply the endothelial PGI2 synthetase with their cyclic endoperoxides. The newly formed PGI2 repels the platelets from the intima. When PGI2 synthetase is irreversibly inactivated by low concentration of lipid peroxides, then the platelets are not rejected but stick to the endothelium, generate TXA2 and mature thrombi are formed. A balance between formation and release of PGI2, TXA2 and/or cyclic endoperoxides in circulation is of utmost importance for the control of intra-arterial thrombi formation and possibly plays a role in the pathogenesis of atherosclerosis.     

Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of prostaglandin endoperoxides

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates. Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation. U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer. PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2). CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2). 18-Hydroxy metabolites were also formed (approximately 17%). PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8. (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.     

Activation of guanyl cyclase during lipid peroxidation of biomembranes

The intensity of lipid peroxidation in the microsomal membranes of rat liver influences the activity of "soluble" guanylate cyclase preparations. The increased production of lipid peroxidation products after addition of Fe(II) results in a rise the guanylate cyclase activity; alpha-tocopherol causes a decrease of this activity. An addition of fatty acids hydroperoxides at concentrations above 10(-6) M activates both the membrane-bound and "soluble" guanylate cyclase. It was shown that the hydroperoxide degradation products--carbonyl derivatives responsible for the activation, at concentrations above 10(-9) M provide for activation of the enzyme. The blocking of the SH-groups in "soluble" enzyme preparations by N-ethylmaleimide completely prevents the enzyme activation by carbonyl.     

Activation of soluble guanylate cyclase by arachidonic acid and 15-lipoxygenase products

The activity of soluble guanylate cyclase can be increased by exposure of the enzyme to arachidonic acid or to some oxidized metabolites of the fatty acid. We have tried to determine whether activation of the enzyme by arachidonate requires that the fatty acid be converted to an oxidized metabolite, either by a possible trace contaminant of a lipoxygenase or by guanylate cyclase itself, which contains a heme moiety. Soluble guanylate cyclase purified from bovine lung was activated 4-6-fold by arachidonic acid. This activation was not dependent on the presence of oxygen in the incubation medium. No detectable metabolites of arachidonic acid were formed during incubation with soluble guanylate cyclase. Addition of soybean lipoxygenase to the incubation did not increase activation by arachidonic acid. The inhibitors of lipoxygenase activity, nordihydroguaiaretic acid and eicosatetraynoic acid, had direct effects on soluble guanylate cyclase and interfered with its activation by arachidonate, whereas another lipoxygenase inhibitor, BW 755 C, did not. The data suggest that arachidonic acid increases the activity of guanylate cyclase by direct interaction with the enzyme rather than by being converted to an active metabolite.     

Prostaglandin endoperoxide/thromboxane A2 receptor desensitization. Cross-talk with adenylate cyclase in human platelets.

Platelet activation by the prostaglandin endoperoxide (PGH2)/thromboxane (Tx) A2 analog, U46619, involves stimulation of phospholipase (PL) C and an increase in intracellular calcium via distinct receptor subtypes. Agents which stimulate adenylate cyclase inhibit platelet function. We demonstrate that PGH2/TxA2 receptor desensitization is associated with enhanced stimulation of platelet cyclic AMP by the prostacyclin analog, iloprost and by forskolin. Sensitization of adenylate cyclase is mediated via the PGH2/TxA2 receptor subtype which activates PLC, as it is blocked by the specific antagonist, GR32191 (Takahara, K., Murray, R., FitzGerald, G. A., and Fitzgerald, D. J. (1990) J. Biol. Chem. 265, 6838-6844). This effect is not observed in platelets desensitized with thrombin or platelet activating factor and is not mediated by protein kinase C. Prior exposure of platelets to platelet activating factor results in much greater desensitization of PGH2/TxA2-induced aggregation (mean 64%) compared with calcium stimulation (mean 18%), consistent with selective heterologous desensitization of the PLC-linked PGH2/TxA2 receptor subtype. Platelet activation by PGH2/TxA2 is a tightly regulated process, involving both homologous desensitization of at least two receptor subtypes and sensitization of the platelet adenylase cyclase system.     

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2.

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.     

Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme

The mechanism by which arachidonic acid activates soluble guanylate cyclase purified from bovine lung is partially elucidated. Unlike enzyme activation by nitric oxide (NO), which required the presence of enzyme-bound heme, enzyme activation by arachidonic acid was inhibited by heme. Human but not bovine serum albumin in the presence of NaF abolished activation of heme-containing guanylate cyclase by NO and nitroso compounds, whereas enzyme activation by arachidonic acid was markedly enhanced. Addition of heme to enzyme reaction mixtures restored enzyme activation by NO but inhibited enzyme activation by arachidonic acid. Whereas heme-containing guanylate cyclase was activated only 4- to 5-fold by arachidonic or linoleic acid, both heme-deficient and albumin-treated heme-containing enzymes were activated over 20-fold. Spectrophotometric analysis showed that human serum albumin promoted the reversible dissociation of heme from guanylate cyclase. Arachidonic acid appeared to bind to the hydrophobic heme-binding site on guanylate cyclase but the mechanism of enzyme activation was dissimilar to that for NO or protoporphyrin IX. Enzyme activation by arachidonic acid was insensitive to Methylene blue or KCN, was inhibited competitively by metalloporphyrins, and was abolished by lipoxygenase. Whereas NO and protoporphyrin IX lowered the apparent Km and Ki for MgGTP and uncomplexed Mg2+, arachidonic and linoleic acids failed to alter these kinetic parameters. Thus, human serum albumin can promote the reversible dissociation of heme from soluble guanylate cyclase and thereby abolish enzyme activation by NO but markedly enhance activation by polyunsaturated fatty acids. Arachidonic acid activates soluble guanylate cyclase by heme-independent mechanisms that are dissimilar to the mechanism of enzyme activation caused by protoporphyrin IX.     

Regulation of synthesis of guanosine 3'':5''-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.