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Biomedical subjects

W E Lands

Publications and source records attributed to W E Lands.

At least 73 records · Page 4Linked to original sources
PubMed1984

Prostaglandin H synthase. Stoichiometry of heme cofactor.

The stoichiometry of heme interaction with prostaglandin H synthase was determined by titration of the apoenzyme purified from sheep seminal vesicles. Maximal cyclooxygenase activity was reached when 0.53 +/- 0.11 (n = 6) heme/70,000-Da subunit had been added. Spectrophotometric titrations at 411 nm showed a transition when 0.53 +/- 0.04 (n = 5) heme/subunit had been added. The results from the titration end points were corroborated by comparison of the specific cyclooxygenase activity based on subunit concentration with the specific activity/mol of heme (calculated from the incremental increases in activity during the titration). The value based on subunit was approximately half (0.58 +/- 0.11; n = 6) that based on heme, consistent with one heme/two subunits. Analysis of synthase holoenzyme after chromatography on DEAE-cellulose provided validation for the concept that only one subunit needs to bind heme to give a catalytically active synthase dimer. Binding of some heme to the second subunit appears to be only coincidentally associated with complete saturation of the active subunit. Titrations of the synthase with Mn-protoporphyrin IX gave results which confirmed the presence of two high affinity metalloporphyrin sites/dimer. Unlike heme, two Mn-protoporphyrin IX need be bound per dimer to obtain full catalytic activity. Prostaglandin H synthase appears to have two high affinity binding sites for metalloporphyrins. The two sites have slightly different affinities for heme. The synthase dimer is capable of cyclooxygenase catalysis when the site with higher affinity is occupied by heme. The two subunits of the enzyme are thus not completely identical.

Animals↗
PubMed1984

Biological consequences of fatty acid oxygenase reaction mechanisms.

Fatty acid oxygenases catalyze the insertion of molecular oxygen into polyunsaturated fatty acids. The enzymic reactions that have been studied in detail exhibit a continuous requirement for a hydroperoxide activator and appear to proceed by a free radical chain reaction. The self-limiting nature of fatty acid oxygenase-catalyzed reactions appears to be due to enzyme self-inactivation during the reaction rather than to product inhibition. Thus "suicide" substrates are potential regulators of overall enzyme activity, although linoleate is a much weaker inactivator than the highly unsaturated fatty acids. Inhibition by added glutathione peroxidase has demonstrated the need for hydroperoxide activator in the cyclooxygenase reaction catalyzed by prostaglandin H synthase and the lipoxygenase reactions catalyzed by lung, leukocyte, and soybean enzyme preparations. The regulation of cellular hydroperoxide levels may influence the formation of prostaglandins and other autacoids by fatty acid oxygenases.

Animals↗
PubMed1984

The effect of flurbiprofen, a potent inhibitor of prostaglandin synthesis, on insulin and glucagon release from isolated rat pancreas.

The influence of different levels of inhibition of prostaglandin (PG) synthesis on the release of insulin and glucagon was investigated in the basal state (5.6 mM glucose) and in response to 30-min perfusion of 16.7 mM glucose using the isolated perfused rat pancreas model. Flurbiprofen (FLR), a potent and selective inhibitor of PG synthesis, was present in the perfusate during the entire experimental period at a concentration of 10(-8), 5 X 10(-8), or 10(-6) M; control experiments were performed without the drug. Levels of immunoreactive PGE2, PGF2 alpha, insulin, and glucagon were measured in the portal venous effluent. FLR inhibited PG synthesis in a dose-related manner; PGE2 was inhibited more than PGF2 alpha. Basal and glucose-induced secretion of insulin was augmented by FLR at 5 X 10(-8) M, but was inhibited at 10(-6) M. At 10(-6) M FLR, basal glucagon secretion was inhibited; glucose-induced suppression still occurred without any potentiation. We conclude that 1) endogenous PGs modulate the secretion of insulin and glucagon; 2) divergence of the effects of low and high levels of inhibition of PG biosynthesis on insulin release may be due to altered tissue proportions of various PGs and related autacoids; and 3) the predominant effect of endogenous PGs on glucagon release is tonic stimulation.

Animals↗
PubMed1984

Arachidonic acid induced release of insulin and glucagon: role of endogenous prostaglandins in pancreatic hormone secretion.

The involvement of prostaglandins in the effects of arachidonic acid (20:4n-6) on insulin and glucagon release was investigated, using the isolated, perfused rat pancreas model. 20:4n-6, the substrate for dienoic prostaglandins, or 20:3n-3, a fatty acid that cannot be metabolized to prostaglandins were perfused over 55 min. 20: 4n-6 evoked triphasic insulin release: early and late phase during, and "off-response" following the perfusion. With 20:3n-3 the early phase of insulin release was 57% of that with 20:4n-6. 20:4n-6 stimulated only an early phase release of glucagon; 20:3n-3 had no effect. Indomethacin (10 microM, a cyclooxygenase inhibitor) inhibited by 50% the early phase of insulin and glucagon release induced by 20:4n-6, but did not modify insulin release during the early phase with 20:3n-3, or the late phase or off-response with either 20:4n-6 or 20:3n-3. We conclude that 1) the early phase release of insulin and glucagon which occurs with arachidonic acid is due in part to pancreatic biosynthesis of prostaglandins; and 2) in the other phases of insulin release evoked by the fatty acids, alternate "nonspecific" mechanisms may be involved.

8,11,14-Eicosatrienoic Acid↗
PubMed1984

Pathophysiologic modulation of arachidonate metabolism.

Low concentrations of lipid hydroperoxides are necessary to activate the biosynthesis of prostaglandins and other autacoids from arachidonate. When the concentration is too low (less than 10(-9) M) that biosynthesis is suppressed. However, lipid peroxides at concentrations higher than micromolar can inactivate prostacyclin synthase and, at higher levels, even prostaglandin H synthase. Thus, lipid hydroperoxides may be important regulators of biological processes, and their reliable quantitation is important in interpreting the physiologic status of a tissue. The plasma levels of lipid hydroperoxide that are indicated by the thiobarbituric acid (TBA) assay of normal human plasma (26-63 microM) would be high enough to totally inactivate prostacyclin synthase and thus they seem incompatible with a healthy vascular system. To resolve this paradox, we have developed a new assay which directly measures the level of lipid hydroperoxide. Controlled studies showed that the TBA-positive response of normal human plasma gave values for hydroperoxide that had no clear relationship to the actual level of hydroperoxide that was present and were 50-100 times greater than the directly observed values. The new direct assay indicates that the normal level of plasma lipid hydroperoxide may be approximately 0.5 microM. Such a level may provide chronic stress on the ability of vascular endothelial cells to provide sufficient amounts of the antithrombotic autacoid, prostacyclin. Our results indicate that relatively slight elevations from the normal circulating hydroperoxide levels might be expected to have undesirable effects, and that a reliable monitoring of plasma hydroperoxide levels may be useful.

Arachidonic Acid↗
PubMed1983

Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids.

Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.

Fatty Acids, Unsaturated↗
PubMed1983

Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase.

Purified prostaglandin H synthase contains cyclooxygenase activity that forms the hydroperoxide, prostaglandin G, and peroxidase activity which removes hydroperoxides. Since hydroperoxides are necessary activators of cyclooxygenase activity, the paradoxical presence of two apparently opposing activities requires careful interpretation. Kinetic studies indicate that the concentration of hydroperoxide needed for full cyclooxygenase activity is much less than that which gives 50 percent effectiveness with the peroxidase. Thus, the peroxidase activity of the synthase is very ineffective in decreasing the hydroperoxide concentration below levels that still permit rapid cyclooxygenase action.

Animals↗
PubMed1982

Selective incorporation of polyunsaturated fatty acids into phosphatidylcholine by rat liver microsomes.

The various polyunsaturated fatty acids found in cellular lipids are transferred from their coenzyme A thiol esters to phospholipid acceptors with greatly different maximal velocities. The very low apparent Km values for the thiol esters in acylating 1-acylglycerol-3-phosphocholine could not be directly measured, but their values could be estimated relative to that for arachidonate. The competitive effectiveness of various polyunsaturated acyl-CoAs was estimated by measuring the equivalent concentrations which allow incorporations equal to arachidonyl-CoA. These values help predict the way in which various polyunsaturated acyl-CoAs may be selectively esterified to membrane lipids by the 1-acylglyceol 3-phosphocholine (1-acyl-GPC) acyltransferase system of liver microsomes. The acyl-CoA esters of saturated acids, as well as those for 22:1, 22:2, and 22:3, had negligible ability to compete for the active sites of the 1-acyl-GPC acyltransferase system. The acyl-CoA esters of arachidonate (20:4n-6), eicosatrienoate (20:3n-6), eicosapentaenoate (20:5n-3), and both isomers of linolenate (18:3n-6 and n-3) were handled preferentially by the 1-acyl-GPC acyltransferases. The system from liver has a high selectivity for unsaturated acids but does not appear to discriminate among the polyunsaturated acids of the n-6 and n-3 series that serve as precursors of prostaglandins and leukotrienes.

Acyl Coenzyme A↗
PubMed1982

Distinctive selectivity for docosatetraenoic acid incorporation by Ehrlich ascites tumor cells.

Three polyunsaturated fatty acids were incorporated into the lipids of Ehrlich ascites cells to examine the selectivity of the synthetic enzymes for chain length and the number of double bonds. Arachidonic acid (20:4n-6) and timnodonic acid (20:5n-6) showed approximately similar patterns of incorporation into neutral acylglycerols, choline glycerophospholipids and ethanolamine glycerophospholipids. Further division of the lipids into diacyl and alkylacyl categories established that 20:4n-6 was incorporated at relatively lower amounts in the triacylglycerols and in the alkylacyl and alkenylacyl forms of the glycerophospholipids. The pattern of incorporation of radioactive 20:5n-3 into the phospholipids was similar to that for 20:4n-6, but there was no evidence for a selectively restrained incorporation into the triacylglycerols. The 22-carbon acid (22:4n-6) differed greatly from its shorter homolog (20:4n-6) in being incorporated in much greater amounts into triacylglycerols than into phospholipids. It did not appear to be present at an appreciably higher relative amount in either the alkyl diacylglycerols or in the ether-containing choline glycerophospholipids. Nevertheless, it occurred with exceptionally high specific activities in the alkylacyl and alkenylacyl forms of ethanolamine glycerophospholipids in a manner that suggests marked discrimination by the cytidine-mediated ethanolamine phosphotransferase. This distinctive selectivity provides evidence for special role for a 22-carbon acid in lipid metabolism.

Animals↗
PubMed1982

Modification of anti-inflammatory drug effectiveness by ambient lipid peroxides.

When the steady-state concentrations of peroxide in prostaglandin H synthase assay systems was lowered by added glutathione peroxidase, several agents (meclofenamic acid, mefenamic acid, acetamidophenol and phenylbutazone) became more potent inhibitors of the prostaglandin-forming cyclooxygenase reaction. Paradoxically, these agents stimulated oxygen incorporation in the absence of added peroxidase. On the other hand, dithiothreitol, ibuprofen, flurbiprofen and indomethacin all inhibited the reaction in a dose-dependent manner, and their inhibitory potencies were unaffected by the action of glutathione peroxidase. Aspirin, dl-gamma-tocopherol and salicylic acid were not inhibitory (without preincubation) under the assay conditions employed in this study. The findings demonstrate that the potencies of some anti-inflammatory agents may be diminished by high local peroxide concentrations.

Animals↗
PubMed1982

Phenolic anticyclooxygenase agents in antiinflammatory and analgesic therapy.

This report demonstrates that a non-steroidal antiinflammatory drug, MK 447, and phenol act similarly to inhibit cyclooxygenase activity in vitro in a dose-dependent manner when the concentration of peroxide activators is decreased by glutathione peroxidase. Increasing the rate of peroxide removal with higher amounts of glutathione peroxidase increases the inhibitory potency of the phenolic agents. The results support ascribing a vital role to tissue peroxides in facilitating prostaglandin biosynthesis in vivo. They also resolve the paradoxical problem of predicting antiinflammatory activity when an agent appears to stimulate prostaglandin formation. A corollary concept is that tissues with general hyperalgesic conditions may have lower levels of hydroperoxide than are found in inflammatory conditions, and thus be more amenable to therapy with phenolic agents like MK 447.

Anti-Inflammatory Agents↗