Dynamics of Radical Intermediates in Prostaglandin H Synthase-1 Cyclooxygenase Reactions is Modulated by Multiple Factors.

Prostaglandin H synthase (PGHS) catalyzes the biosynthesis of PGG2 and PGH2, the precursor of all prostanoids, from arachidonic acid (AA). PGHS exhibits two enzymatic activities following a branched-chain radical mechanism: 1) a peroxidase activity (POX) that utilizes hydroperoxide through heme redox cycles to generate the critical Tyr385 tyrosyl radical for coupling both enzyme activities; 2) the cyclooxygenase (COX) activity inserting two oxygen molecules into AA to generate endoperoxide/hydroperoxide PGG2 through a series of radical intermediates. Upon the generation of Tyr385 radical, COX catalysis is initiated, with C13 pro-S hydrogen abstraction from AA by Tyr385 radical to generate arachidonyl substrate radical. Oxygen provides a large driving force for the subsequent fast steps leading to the formation of PGG2, including radical redistributions, ring formations, and rearrangements. On the other hand, if the supply of oxygen is severed, equilibrium between arachidonyl radical and tyrosyl radical(s) biases largely towards the latter. In this study, we demonstrate that such equilibrium is shifted by many factors, including temperature, chemical structures of fatty acid substrates and limited supply of oxygen. We also, for the first time, reveal that this equilibrium is significantly affected by co-substrates of POX. The presence of efficient POX co-substrates, which reduces heme to its ferric state, apparently biases the equilibrium towards arachidonyl radical. Therefore a dynamic interplay exists between the two activities of PGHS.

Identification of two cyclooxygenase active site residues, Leucine 384 and Glycine 526, that control carbon ring cyclization in prostaglandin biosynthesis.

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.

Determinants of the cellular specificity of acetaminophen as an inhibitor of prostaglandin H(2) synthases.

Acetaminophen has antipyretic and analgesic properties yet differs from the nonsteroidal antiinflammatory drugs and inhibitors of prostaglandin H synthase (PGHS)-2 by exhibiting little effect on platelets or inflammation. We find parallel selectivity at a cellular level; acetaminophen inhibits PGHS activity with an IC(50) of 4.3 microM in interleukin (IL)-1 alpha-stimulated human umbilical vein endothelial cells, in contrast with an IC(50) of 1,870 microM for the platelet, with 2 microM arachidonic acid as substrate. This difference is not caused by isoform selectivity, because acetaminophen inhibits purified ovine PGHS-1 and murine recombinant PGHS-2 equally. We explored the hypothesis that this difference in cellular responsiveness results from antagonism of the reductant action of acetaminophen on the PGHSs by cellular peroxides. Increasing the peroxide product of the PGHS-cyclooxygenase, prostaglandin G(2) (PGG(2)), by elevating the concentration of either enzyme or substrate reverses the inhibitory action of acetaminophen, as does the addition of PGG(2) itself. 12-Hydroperoxyeicosatetraenoic acid (0.3 microM), a major product of the platelet, completely reverses the action of acetaminophen on PGHS-1. Inhibition of PGHS activity by acetaminophen in human umbilical vein endothelial cells is abrogated by t-butyl hydroperoxide. Together these findings support the hypothesis that the clinical action of acetaminophen is mediated by inhibition of PGHS activity, and that hydroperoxide concentration contributes to its cellular selectivity.

Paradoxical effects of resveratrol on the two prostaglandin H synthases.

Prostaglandin H synthase (PGHS) is the primary enzyme responsible for the biosynthesis of prostaglandins and thromboxanes. Of the two isoenzymes of PGHS, PGHS-1 is constitutively expressed and PGHS-2 is inducible by mitogens or other inflammatory stimuli. Constitutive expression of PGHS-2 in neoplastic tissues has been implicated in carcinogenesis. Resveratrol, a lignan, was recently shown to be an anticarcinogen that selectively inhibits PGHS-1. In vitro experiments to resolve these seemingly paradoxical observations revealed that resveratrol is not only an inhibitor of PGHS-1 but also is an activator of PGHS-2. Resveratrol non-competitively inhibited PGHS-1 with a K1 of 26 +/- 2 microM but enhanced the PGHS-2 activity nearly twofold. Additionally, resveratrol did not serve as a reducing co-substrate for the peroxidase activities of either enzyme despite being an easily oxidizable phenolic compound. Resveratrol inhibited the peroxidase activity of PGHS-1 (IC50 = 15 microM) better than that of PGHS-2 (IC50 = > 200 microM). Inhibition of the perxidase activity but not the cyclooxygenase activity of PGHS-2 resulted in the production of PGG2 from arachidonic acid. A plausible relationship between these observation and the anticarcinogenic activity of resveratrol is discussed.

Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX.

Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostaglandins G2 and H2 from arachidonic acid. Replacement of the heme group by mangano protoporphyrin IX largely preserves the cyclooxygenase activity, but lowers the steady-state peroxidase activity by 25-fold. Thus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS. A detailed kinetic analysis of the peroxidase reaction using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), EtOOH, and other peroxides as substrates has been carried out to compare the characteristics of PGHS reconstituted with mangano protoporphyrin IX (Mn-PGHS) to those of the native heme enzyme (Fe-PGHS). The rate constant describing the reaction of Mn-PGHS with 15-HPETE to form the oxidized, Mn(IV) intermediate with absorption at 420 nm, exhibits saturable behavior as the 15-HPETE concentration is raised from 10 to 400 microM. This is most likely due to the presence of a second, earlier intermediate between the resting enzyme and the Mn(IV) species. Measurements at high substrate concentrations permitted resolution of the absorbance spectra of the two oxidized Mn-PGHS intermediates. The spectrum of the initial intermediate, assigned to a Mn(V) species, had a line shape similar to that of the later intermediate, assigned to a Mn(IV) species, suggesting that a porphyrin pi-cation radical is not generated in the peroxidase reaction of Mn-PGHS. The rate constant estimated for the formation of the earlier intermediate with 15-HPETE is 1.0 x 10(6) M-1 s-1 (20 degrees C, pH 7.3). A rate constant of 400 +/- 100 s-1 was estimated for the second step in the reaction. Thus, Mn-PGHS reacts considerably more slowly than Fe-PGHS with 15-HPETE to form the first high-valent intermediate, but the two enzymes appear to follow a similar overall reaction mechanism for generation of oxidized intermediates. The difference in rate constants explains the observed lower steady-state peroxidase activity of Mn-PGHS compared with Fe-PGHS.

Efecto gastroprotector del subcitrato de bismuto coloidal de producción cubana y del De-Nol en ratas / Gastroprotective effect of the coloidal bismuth subcitrate produced in Cuba and of De-Nol in rats

Se investigó el efecto gastroprotector del subcitrato de bismuto coloidal (SBC) producido en el Centro de Química Farmacéutica en comparación con el producido por la compañía Gist-Brocades de Holanda (De-Nol). Se determinó también la capacidad de ambos preparados de estimular la síntesis de prostaglandinas (PGE2 y 6-keto-PGF1 alfa) en la mucosa gástrica en presencia de etanol. En otra serie de experimentos se determinó el efecto del SBC y del De-Nol, administrados antes del etanol, sobre la concentración de ácido en el luen gástrico. Los resultados mostraron que ambos preparados protegieron de manera dosis-dependiente la mucosa gástrica de las ratas del daño inducido por etanol. El pretratamiento con SBC y De-Nol no influyó en la generación mucosal de prostaglandinas posterior a la administración de etanol. El SBC y el De-Nol, administrados antes del etanol, disminuyeron la concentración de ácido en el lumen gástrico de manera significativa (AU)

Efecto protector sobre la mucosa gástrica del subcitrato de bismuto coloidal y del factor de crecimiento epidermico en ratas / Protective affect of coloidal bismuth subcitrate and epidermal growth factor on gastric mucosa in rats

Se comparan los efectos gastroprotectores del subcitrato de bismuto coloidal, registrado comercialmente como De-Nol, con los del factor de crecimiento epidérmico en lesiones agudas de la mucosa gástrica inducidas por etanol absoluto en ratas. El De-Nol administrado por vía oral protegió de manera dosis-dependiente la formación de lesiones gástricas inducidas por el etanol. El factor de crecimiento epidérmico administrado tanto tanto en forma oral como subcutánea, fue inefectivo contra las lesiones gástricas agudas inducidas por este agente necrotizante de la mucosa. El hecho de que el De-Nol no indujese cambios significativos en las alteraciones producidas por el etanol sobre los niveles mucosales de prostaglandinas (PGE2 y 6 keto-PGF1 alfa) sugiere que este fármaco puede activar algún otro mecanismo protector (AU)

Effects of various prostaglandin synthesis inhibitors on the tone of the lower oesophageal sphincter in man.

The role of endogenous prostaglandins in the modulation of lower oesophageal sphincter (LES) function has been assessed by giving three structurally unrelated cyclooxygenase inhibitors and monitoring their acute effects on LES tonus and platelet thromboxane (TX) B2 production in 20 healthy volunteers. In a double-blind, placebo-controlled, cross-over study, IV injection of soluble salts of acetylsalicylic acid and indomethacin elicited a transient increase in LES tonus of approximately 50% over baseline. A similar pattern was observed after the rectal administration of indomethacin. In contrast, indoprofen had no measurable effect on LES tones, despite comparable inhibition of platelet cyclooxygenase activity. This may have been due to the markedly different tissue distribution of the drug. The results suggest that endogenous prostaglandins physiologically exert an inhibitory influence on LES function.

Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid.

Prostaglandin H synthase catalyzes the formation of prostaglandin (PG) G2 from arachidonic acid (cyclooxygenase activity), and also the reduction of PGG2 to PGH2 (peroxidase activity). The ability of the pure synthase to accumulate the hydroperoxide, PGG2, under conditions allowing the concurrent function of both catalytic activities was investigated. The peroxidase velocity was continuously determined from the absorbance increases at 611 nm that accompanied oxidation of a peroxidase cosubstrate, N,N,N',N'-tetramethylphenylenediamine, and PGG2 concentrations were calculated from the peroxidase velocities and the peroxidase Vmax and Km values. Cyclooxygenase velocities were then calculated from the changes in PGG2. Parallel reactions monitored by the use of radiolabelled arachidonate or with a polarographic oxygen electrode were used to confirm the calculated PGG2 levels and the cyclooxygenase velocities. The concentration of PGG2 was found to follow a transient course as the reaction of the synthase progressed, rapidly rising to a maximum of 0.7 microM in the first 10 s, and then declining slowly, reaching 0.1 microM after 60 s. The maximal level of PGG2 achieved during the reaction was constant at about 0.7 microM with higher amounts of added cyclooxygenase capacity (0.3-0.6 microM PGG2/s) but was only about 0.4 microM when the added cyclooxygenase capacity was 0.1 microM PGG2/s. The peroxidase was found to lose only 30% of its activity after 90 s, a point where the cyclooxygenase was almost completely inactive. These results support the concept of a burst of catalytic action from the cyclooxygenase and a reactive, more sustained, catalytic action from the peroxidase during the reaction of the synthase with arachidonic acid.

The effect of E. coli infection on the prostaglandin synthesizing capacity of postobstructive rat kidney.

The PGE2, PGI2, PGF2 alpha and TxA2 synthesizing activities were studied in an isolated microsomal fraction of rat kidney after temporary, unilateral ureter obstruction and E. coli infection. In the early phase of regeneration the synthesis of vasodilatory PGI2 was increased, whereas that of vasoconstrictory PGF2 alpha was decreased. An increased PGE2 synthesizing activity was observed when renal obstruction was associated with infection. The role of these changes in regenerating the haemodynamics and function of postobstructive kidney is discussed.

Effects of aqueous extracts of onion, garlic and ginger on platelet aggregation and metabolism of arachidonic acid in the blood vascular system: in vitro study.

Aqueous extracts of onion, garlic and ginger were found to inhibit aggregation induced by ADP, epinephrine, collagen and arachidonate in a dose-dependent manner in vitro. In the case of onion and garlic extracts relatively much higher volumes were need to bring about even a modest inhibition (by ca. 13-18%) of thromboxane synthesis in washed platelets from labelled AA. On the other hand a good correlation was found between the amounts of ginger extract needed to inhibit platelet aggregation and those to inhibit platelet thromboxane synthesis. Ginger extract reduced also platelet prostaglandin-endoperoxides. A dose-related inhibition of platelet thromboxane- and prostaglandin (PGF2 alpha, PGE2 and PGD2) synthesis was affected by ginger extract. Extracts of onion, garlic and ginger inhibited biosynthesis of prostacyclin in rat aorta from labelled AA. Ginger extract mildly inhibited the synthesis of prostacyclin from endogenous pool of AA in rat aorta; the other two extracts were without effect.

Influence of asbestos fibers on collagen and prostaglandin production in fibroblast and macrophage co-cultures.

The interaction of fibroblasts, macrophages, and crocidolite asbestos fibers was studied in cell culture. We determined the effects of co-cultivation and asbestos fibers on collagen, total protein, and PG production. The co-cultivation of guinea pig alveolar macrophages and fetal lung fibroblasts resulted in a 155% increase in protein and a 31% increase in collagen production above fibroblast controls. The collagen was derived exclusively from the fibroblasts. Although total protein production was derived predominantly from the fibroblasts, the macrophages in co-culture also contributed to the protein levels. The addition of asbestos fibers to fibroblast cultures resulted in a decrease in collagen and total protein production. The addition of asbestos fibers to fibroblast and macrophage co-cultures prevented the enhancement of collagen production and limited the increase in protein production above fibroblast controls. PGE2, PGI2, and TXA2 were measured in macrophage and fibroblast cultures. Very low, almost undetectable PG production was observed under basal conditions by either cell type alone or in co-culture. Bradykinin induced release of these PGs in fibroblast but not macrophage cultures. This release was enhanced in co-cultures. Asbestos fibers, when added to the co-cultures caused a significant increase in the release of PGs, particularly PGE2. PGE2 is known to inhibit collagen and total protein production by fibroblasts. The increased release of PGs in asbestos-treated co-cultures may have contributed to the described asbestos suppression of collagen production

Prostaglandins, antidiuretic hormone and renin angiotensin system.

Current knowledge on prostaglandin biosynthesis is reviewed, centering on how PGs participate in the regulation of vascular tone and the prevention of platelet deposition on endothelial surfaces. Discussion includes review of the vasoactivity of the PG endoperoxides, thromboxane, prostacyclin, and prostaglandin E2; prostaglandin-catabolizing enzymes; polyunsaturated fatty acid precursors; nutritional factors; antidiuretic hormone; and interrelations of PGs with the renin-angiotensin system.

Sensitivity of fatty acid cyclooxygenase from human aorta to acetylation by aspirin.

The rate of acetylation of fatty acid cyclooxygenase (prostaglandin synthase, EC 1.14.99.1) by [acetyl-3H]-aspirin was measured in microsomes from human aortas and coronary arteries and intact and disrupted human platelets. We also measured the inhibition by aspirin of prostacyclin generation from exogenous arachidonic acid in shredded human aorta. Cyclooxygenase in human aorta and coronary artery microsomes is approximately 1/250th as sensitive to aspirin as enzyme in intact platelets, and 1/60th as sensitive to aspirin as enzyme measured in a platelet microsomal preparation. On the basis of the in vitro data presented, we predict that small oral doses of aspirin are sufficient to inhibit platelet prostaglandin production but are not sufficient to substantially affect aorta or coronary artery prostaglandin production.