Group Of Prostaglandins Research Articles

Control of Prostaglandin Stereochemistry at the 15-Carbon by Cyclooxygenases-1 and -2: A CRITICAL ROLE FOR SERINE 530 AND VALINE 349

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.

C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.

Adrenal and sex steroid receptor evolution: environmental implications.

The nuclear receptor family responds to a diverse group of ligands, including steroids, retinoids, thyroid hormone, prostaglandins and fatty acids. Previous sequence analyses of adrenal and sex steroid receptors indicate that they form a clade separate from other nuclear receptors. However, the relationships of adrenal and sex steroid receptors to each other and to their ancestors are not fully understood. We have used new information from androgen, estrogen, mineralocorticoid and progesterone receptors in fish to better resolve the phylogeny of adrenal and sex steroid receptors. Sequence divergence between fish and mammalian steroid receptors correlates with differences in steroid specificity, suggesting that phylogeny needs to be considered in evaluating the endocrine effects of xenobiotics. Among the vertebrate steroid receptors, the most ancient is the estrogen receptor. The phylogeny indicates that adrenal and sex steroid receptors arose in a jawless fish or a protochordate and that changes in the sequence of the hormone-binding domain have slowed considerably in land vertebrates. The retinoid X receptor clade is closest to the adrenal and sex steroid receptor clade. Retinoid X receptor is noteworthy for its ability to form dimers with other nuclear receptors, an important mechanism for regulating the action of retinoid X receptor and its dimerization partners. In contrast, the adrenal and sex steroid receptors bind to DNA as homodimers. Moreover, unliganded adrenal and sex steroid receptors form complexes with heat shock protein 90. Thus, the evolution of adrenal and sex steroid receptors involved changes in protein-protein interactions as well as ligand recognition.

Replacement of the carboxylic acid group of prostaglandin f(2alpha) with a hydroxyl or methoxy substituent provides biologically unique compounds.

Replacement of the carboxylic acid group of PGF(2alpha) with the non-acidic substituents hydroxyl (-OH) or methoxy (-OCH(3)) resulted in an unexpected activity profile. Although PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) exhibited potent contractile effects similar to 17-phenyl PGF(2alpha) in the cat lung parenchymal preparation, they were approximately 1000 times less potent than 17-phenyl PGF(2alpha) in stimulating recombinant feline and human FP receptors. In human dermal fibroblasts and Swiss 3T3 cells PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) produced no Ca(2+) signal until a 1 microM concentration was exceeded. Pretreatment of Swiss 3T3 cells with either 1 microM PGF(2alpha) 1-OH or PGF(2alpha) 1-OCH(3) did not attenuate Ca(2+) signal responses produced by PGF(2alpha) or fluprostenol. In the rat uterus, PGF(2alpha) 1-OH was about two orders of magnitude less potent than 17-phenyl PGF(2alpha) whereas PGF(2alpha) 1-OCH(3) produced only a minimal effect. Radioligand binding studies on cat lung parenchymal plasma membrane preparations suggested that the cat lung parenchyma does not contain a homogeneous population of receptors that equally respond to PGF(2alpha)1-OH, PGF(2alpha)1-OCH(3), and classical FP receptor agonists. Studies on smooth muscle preparations and cells containing DP, EP(1), EP(2), EP(3), EP(4), IP, and TP receptors indicated that the activity of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) could not be ascribed to interaction with these receptors. The potent effects of PGF(2alpha) 1-OH and PGF(2alpha) 1-OCH(3) on the cat lung parenchyma are difficult to describe in terms of interaction with the FP or any other known prostanoid receptor.

Clinical aspects of COX-2 inhibitors.

Inflammation is the body's response to tissue damage. The signs of inflammation are pain, swelling and edema. One of the major chemical mediators involved in the inflammatory response is the group of prostaglandins (PGs) synthesized by the enzyme cyclooxygenase (COX). There are two isoforms of COX, the constitutive enzyme COX-1, which is responsible for the production of PGs with general housekeeping functions such as maintenance of renal perfusion and a protective effect on the gastric mucosa against ulceration; and the inducible enzyme COX-2, which is responsible for the production of proinflammatory PGs. Non-steroidal antiinflammatory drugs (NSAIDs), by inhibiting the COX enzyme, are able to reduce the pain and swelling associated with inflammation. In theory, new molecules that preferentially inhibit the inducible enzyme alone would not affect the housekeeping function of COX-1 but would retain the antiinflammatory action. Currently, there are a number of molecules available which claim to have some selectivity for the COX-2 enzyme. These have been labeled COX-2 preferential drugs. Highly selective COX-2 enzyme inhibitors are being assessed in phase II and phase III clinical studies. What are the issues these drugs will create and will they be successful? The first question is whether they are truly COX-2-selective. The balance of evidence suggests that they are. There is less gastric ulceration by the preferential drugs and there is very little evidence of gastric damage with the COX-2-selective drugs. All these drugs seem to maintain renal perfusion better than other NSAIDs and platelet aggregation is not affected. The second question is whether these drugs are as effective as conventional NSAIDs. From the preliminary data available it appears that the inhibition of COX-2 is effective in reducing inflammation. There are some doubts from studies in animal models as to whether some inhibition of COX-1 is also required, but this has not yet been shown in humans. There are concerns regarding the cerebral function of COX-2 and the expression of COX-2 in the kidney and gut. Thirdly, will new benefits arise from the use of these drugs? It seems likely that selective COX-2 inhibition will have the same beneficial effects on Alzheimer's disease and colonic cancer as nonselective drugs. In fact, these may even become new indications. What are the likely hazards? There will probably be individual problems with particular drugs but these cannot be predicted. The crucial issue is that patients who benefit most from NSAIDs are those who will be most likely to have side effects from the nonselective COX-1 and COX-2 inhibitors. Therefore, once there is good evidence of their efficacy and safety they will become widely used drugs.

Biosynthesis of the endogenous cyclic adenosine monophosphate (AMP) antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), from prostaglandin E and activated inositol polyphosphate in rat liver plasma membranes.

The endogenous cyclic adenosine monophosphate (AMP) antagonist, cyclic PIP, has been identified as a prostaglandylinositol cyclic phosphate. It inhibits protein kinase A 100% and activates protein serine phosphatase about sevenfold. It is biosynthesized by an enzyme of the plasma membrane when the assay mixture contains adenosine triphosphate (ATP), Mg2+, prostaglandin E and a novel inositol polyphosphate, which cannot be substituted by commercially available inositol phosphates. This novel inositol polyphosphate is a very labile compound. On anion exchange chromatography it elutes in the range of ATP, which may indicate the presence of three phosphate groups. It adsorbs on charcoal, which suggests the presence of a hydrophobic component, possibly a guanosine. Pyrophosphates obtained from inositol 1,4- and inositol 2,4-bisphosphate are accepted by cyclic PIP synthetase for the synthesis of cyclic PIP. The biosynthesis is characterized by enzyme kinetic parameters like dependence on time, enzyme and substrate concentration. The pH optimum of the enzyme is in the range 7.5-8. The enzyme functions optimally with prostaglandin E and poorly with prostaglandin A as the substrate. The presence of fluoride in the assay causes a three- to fourfold increase in cyclic PIP synthesis, which may be correlated with activation via G proteins. These data support previous reports on the chemical structure and action of cyclic PIP. With respect to the possible isomers of cyclic PIP, these indicate that it is most likely the C4-hydroxyl group of the inositol which binds the C15-hydroxyl group of prostaglandin E. A model of hormone-stimulated synthesis of cyclic PIP is proposed: phospholipase A2 and phospholipase C, activated by G proteins upon alpha-adrenergic stimulation, liberate either unsaturated fatty acids or inositol phosphates, which are transformed to prostaglandins and to novel inositol polyphosphate with an energy-rich bond. The cyclic PIP synthetase combines these two substrates to cyclic PIP.

Differential activation of G i and G S proteins by E- and I-type prostaglandins in membranes from the human erythroleukaemia cell line, HEL

The group of prostaglandin (PG) E 2- and prostacyclin receptors consists of different subtypes, which exibit different affinities for prostaglandins and synthetic analogues. PGE 2 activates the E-type PG receptor subtypes EP 1, EP 2 and EP 3, whereas the PGE 2 analogue, sulprostone, binds only to the EP 1 and EP 3 receptor subtypes. The stable PGI 2 analogues, iloprost and cicaprost, both activate the PGI 2 receptor (IP) and iloprost, additionally, bind to the EP 1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with G s and G i-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE 2, iloprost and cicaprost were only partially inhibited by PTX. PGE 2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified G i protein α subunits. Furthermore, PGE 2, iloprost and cicaprost enhanced GTP azidoanilide-labelling of G s protein α subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP 1, and EP 3 receptor subtypes activate G i proteins, that (2) the EP 2 receptor subtype activates G s proteins and that (3) the IP receptor activates both G i and G s proteins.

Bacterial expression and site-directed mutagenesis of two critical residues (tyrosine-151 and lysine-155) of human placental NAD + -dependent 15-hydroxyprostaglandin dehydrogenase

NAD +-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and che recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of K m and V ax values for a number of the prostaglandins and NAD + indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine-151 and lysine-155 are required for 15-PGDH activity.

Chemistry and biology of heme. Effect of metal salts, organometals, and metalloporphyrins on heme synthesis and catabolism, with special reference to clinical implications and interactions with cytochrome P-450.

Although free porphyrins occur in nature in small quantities, no known function has been assigned to them. In contrast, heme and cobalamin, which are Fe and Co chelates of porphyrins or porphyrin derivatives, respectively, carry out crucial biological functions. Heme is the prosthetic group for a number of hemoproteins. These include myoglobin and hemoglobin, which carry out oxygen binding or transport; mitochondrial cytochromes aa3, b, c, and c3, which are important in transferring electrons; microsomal cytochrome P-450, which catalyzes mixed-function oxidations; catalase, which decomposes H2O2; peroxidase, which activates H2O2; and tryptophan pyrrolase, which catalyzes the oxidation of tryptophan. Recently, heme has also been shown to be the prosthetic group of prostaglandin and peroxide synthetase and indoleamine dioxygenase. The elegant studies of the biochemical pathway for the formation of heme demonstrated the arrangement in the porphyrin macrocycle of the carbon and nitrogen atoms originating from the eight glycine and the succinic acid molecule that are the precursors of porphyrins. There are eight enzymes involved in the synthesis of heme. The first and last three of these enzymes are localized in mitochondria, while the intermediate enzymes are localized in cytosol. The catalytic site of HMOX recognizes metalloporphyrins with central metal atoms other than iron; it favors some of these metalloporphyrins over heme as a potential substrate, sometimes by a large factor, permitting the synthetic heme analogue to serve as a potent competitive inhibitor of HMOX reaction. Since these synthetic metalloporphyrins do not bind molecular oxygen, they are not metabolically degraded by ring rupture and do not add to the body pool of bile pigment. One possible consequence of this competitive inhibition of heme degradation is suppression of bile pigment formation to such a degree that excessive plasma levels of bilirubin may be diminished. The studies of Drummond and Kappas (1981) and later studies in rats, mice, monkeys, and man, and also our studies have proved the latter phenomenon. The compound does not appear to affect the metabolic disposition of preformed bilirubin but inhibits biliary bilirubin excretion derived from the metabolism of endogenous or exogenous heme. Whether some of the effect of Sn-PP on naturally occurring or experimentally induced jaundice in animals reflects diversion of heme to nonheme to oxygenase-dependent pathways of heme metabolism, or whether a pathway which is normally latent becomes activated concurrent with HMOX inhibition is not known.(ABSTRACT TRUNCATED AT 400 WORDS)

NADP-dependent 15-hydroxyprostaglandin dehydrogenase is homologous to NAD-dependent 15-hydroxyprostaglandin dehydrogenase and other short-chain alcohol dehydrogenases

Human tissues contain two enzymes that catalyze the oxidation of the 15-hydroxy group of prostaglandins: NAD-dependent 15-hydroxyprostaglandin dehydrogenase which is fairly specific for prostaglandins and NADP-dependent 15-hydroxyprostaglandin dehydrogenase with low specificity for prostaglandins. The recent determination of the amino acid sequence of the NAD-dependent enzyme has revealed similarities with a number of oxidoreductases with distinct specificities that constitute the short-chain alcohol dehydrogenase family. Comparison of the amino acid sequence of NADP-dependent 15-hydroxyprostaglandin dehydrogenase with the sequences of the NAD-dependent enzyme and other short-chain alcohol dehydrogenases revealed the same similarities, identifying the NADP-dependent enzyme as a short-chain alcohol dehydrogenase. The degree of homology between the two 15-hydroxyprostaglandin dehydrogenases is similar to the one between each of the two enzymes and other members of the family indicating an early evolutionary divergence of the two proteins.

Investigation of the prostaglandin E (EP-) receptor subtype mediating relaxation of the rabbit jugular vein.

1. Prostaglandin E2 (PGE2) relaxes circular smooth muscle of the rabbit isolated jugular vein at very low concentrations (mean pIC50 against histamine-induced contraction = 9.34). This effect is not blocked by the EP1-receptor antagonist, AH 6809 (2 microM). 2. From a group of prostaglandin E analogues examined, 16,16-dimethyl PGE2, misoprostol, 11-deoxy PGE2-1-alcohol and 11-deoxy PGE1 were highly potent relaxant agents, whereas 17-phenyl-omega-trinor PGE2, MB 28767 and butaprost had low potency and sulprostone and oxoprostol were virtually inactive. 3. Comparison of the jugular vein data with published data for inhibitory agonist potencies on the cat trachea (EP2 preparation) and the field-stimulated guinea-pig vas deferens (EP3) indicates that the EP-receptor in the rabbit jugular vein is closest to the EP2 subtype. However, the correlation is not entirely convincing. For example, butaprost, 16,16-dimethyl PGE2 and 11-deoxy PGE1 are of similar potency on the cat trachea, whereas butaprost is about 300 times less potent than the other two analogues on the jugular vein. The existence of more than one EP2-receptor appears possible. 4. It was felt that the activity of butaprost required further investigation in view of the claim that it is a specific EP2-receptor agonist. We have shown that butaprost has very low inhibitory activity on the guinea-pig vas deferens, a very sensitive EP3-receptor containing preparation. However, on the chick ileum, the original EP3 preparation, butaprost showed potent contractile activity (pEC25 approximately 8.0).In addition, its maximum response was lower than that of PGE2; lower maxima were also found for sulprostone, MB 28767 and oxoprostol, but not for ICI 80205, 16,16-dimethyl PGE2 and 17-phenyl-omega-trinor PGE2. The maximal response to a combination of either sulprostone and butaprost or sulprostone and PGE2 was similar to that achieved by PGE2 alone. Analysis of the interaction between sulprostone and PGE2 appears to exclude a partial agonist action for sulprostone. Furthermore neither sulprostone nor butaprost appear to have inhibitory activity on the ileum. AH 6809 at 2 pM produced only a small shift of the PGE2 log concentration-response curve.5. It is likely that contraction of the longitudinal smooth muscle of the chick ileum is mediated by (at least) two EP-receptor subtypes; activation of only one receptor system does not induce the maximum response (i.e. the acetylcholine maximum) of the preparation. One receptor could be an EP3 subtype, at which sulprostone exerts a selective agonist action. The other receptor is unlikely to be an EP, subtype, because of the high agonist potency of butaprost, the low agonist potency of iloprost, and the low antagonist potency of AH 6809. An alternative hypothesis is that the chick ileum contains a novel EP-receptor subtype in addition to an EP3-receptor.

High-performance liquid chromatography—thermospray mass spectrometry of prostaglandin and thromboxane acetyl derivatives

A quantitative analysis of prostaglandin-related substances has been developed. Hydroxyl groups of prostaglandin and thromboxane were acetylated by acetic anhydride, the mixture was partially purified on a Sep-Pak C 18 cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, twenty kinds of prostaglandin derivative could be detected simultaneously within 11 min on a selected-ion monitoring detection chromatogram without a gradient system. Generally, the base ion, [M + H − n(60)] +, is produced through elimination of acetic acid ( n = number of the hydroxyl group of prostaglandin or thromboxane). The detection limit for these derivatives was ca. 0.2 pmol at the levels of prostaglandin-related substances prior to derivatization. They could be analysed in the range 0.5–10 pmol. The assay was successfully applied to prostaglandin-related substances in human seminal fluid and rat brain.

Essential fatty acids and immune response

The implication that essential fatty acids (EFA) can affect immune response was based on the observation that EFA deficiency can accentuate or improve symptoms of certain autoimmune diseases in animals, and that supplementation of linoleic acid to animals reversed such effects. Furthermore, treatment of animals with cyclooxygenase inhibitors abrogated the effect of linoleic acid. Administration of cyclooxygenase inhibitors to animals enhanced both cell-mediated and humoral immune responses. In vitro studies have shown that prostaglandin E (PGE) group inhibits both T and B lymphocyte functions; it is suggested that effects of EFA on immune response are, in part, mediated through eicosanoids. Growing evidence now suggests that the PGE group of prostaglandins can serve as a negative feedback modulator of immune response. However, in vitro effects of other cyclooxygenase-derived products, such as PGI2 and thromboxane A2 (TXA2) have not been well established, perhaps because of their instability in aqueous media. Unlike the PGE group, some of lipoxygenase-derived products of arachidonic acid have shown immunostimulatory effects, as assessed by lymphokine production in vitro. Whether such effects can be seen in vivo remains to be determined. Some lipoxygenase-derived products with strong chemotactic action may indirectly influence immune response by modulating the population of antigen-presenting macrophages in tissues. Thus, the net effect of eicosanoids synthesized in macrophages on modulating immune response may depend on relative amounts of cyclooxygenase-derived products as compared with lipoxygenase-derived products. Macrophages are the major source of eicosanoids among immunocompetent cells. The profile of eicosanoids, produced in vitro by macrophages, varies with type of stimuli and anatomical sites. It can also be affected by the fatty acid composition of tissue lipids, which in turn can be modified by the composition of dietary EFA. Whether manipulating dietary EFA can modulate immune response in normal humans and animals needs to be determined.

Derivatization of prostaglandis and related compounds to (methoxime) alkyl ester alkyl ether derivatives for gas chromatographic analysis

Rapid and convenient methods are described for the exhaustive derivatization of carbonyl, carboxyl and hydroxyl groups of prostaglandins and related compounds to methoxime, alkyl ester and alkyl ether compounds respectively. Optimal reaction conditions were established for each group. The reactions were carried out in polar aprotic solvents. Alkyl ester alkyl ether derivatives were obtained quantitatively and rapidly in one step with n-alkyl (C 1C 4) halides in the presence of sodium hydroxide. Methyl ester methyl ether derivatives have the highest volatility, but propyl ester propyl ether derivatives improved the separation of complex mixtures. The cabonyl group sometimes induced side-products, so the carbonyl group was converted into methoxime. Methoximation was achieved quantitatively by using methoxylamine in the presence of hydrochloric acid or sodium hydroxide, followed by alkylation in same reaction medium. Methoximation gave syn- and anti-isomers, which were separated chromatographically, decreasing the resolution for complex samples.

The invention of radical reactions part XVIII. A convenient solution to the 1-carbon problem (R-CO 2H → R- 13CO 2H)

Radicals generated by photolysis (W light) of esters derived from N -hydroxy-2-thiopyridone react with electrophilic isocyanides 2a and (in the presence of trifluoracetic acid) 2b to give adducts of type 3 . Convenient reaction procedures have been worked out to hydrolyse the adducts to amides of type 5 , from which the originial acid can be regenerated under mild conditions. The three important acids oleic, Iinoleic and arachidonic have all given smooth reactions. In suitable examples, quantitative evolution of carbon dioxide and incorporation of 13C without dilution have been demonstrated. This reaction sequence will be useful for the labelling in the carboxyl group of prostaglandins, leukotrienes, and the side chain carboxyls of peptides.

Oxidation of 15-hydroxyeicosatetraenoic acid and other hydroxy fatty acids by lung prostaglandin dehydrogenase

The oxidation of the 15-hydroxy group of prostaglandins of the A, E, and F series by the NAD +-dependent prostaglandin dehydrogenase (PGDH) has been well documented. In addition to prostaglandins, we have observed that the purified lung PGDH also will oxidize 15-HETE to a novel metabolite that was isolated by reverse-phase HPLC and identified by gas chromatography-mass spectrometry as the 15-keto-5,8,11- cis-13- trans-eicosatetraenoic acid (15-KETE). The K m for 15-HETE was 16 μ m, which was 2.5 times lower than the value obtained for PGE 1. In addition to 15-HETE, 5,15-diHETE and 8,15-diHETE also were substrates for the lung PGDH with K m values of 138 and 178 μ m, respectively. Other hydroxy derivatives of eicosatetraenoic acid that did not have a hydroxy group at carbon atom 15 did not support the PGDH-mediated reduction of NAD +. In addition to the 15-hydroxy derivatives of eicosatetraenoic acid, 12-HHT also was a substrate for the lung enzyme with a K m of 12 μ m. These data indicate that ω6-hydroxy fatty acids, in addition to prostaglandins, are also substrates of the lung NAD +-dependent PGDH and that the enzyme does not require the cyclopentane ring of prostaglandins.

Detection of prostaglandins by high-performance liquid chromatography after conversion to p-(9-anthroyloxy)phenacyl esters

p-(9-Anthroyloxy)phenacyl bromide (panacyl bromide) undergoes rapid reaction with the carboxyl group of prostaglandins in the presence of N,N-diisopropylethylamine in acetonitriletetrahydrofuran (4:1). The resulting prostaglandin panacyl esters are strongly uv absorbing with a λ max at 253 nm and an ϵ of 174,280 in acetonitrile. The lower limit of detection of prostaglandins was approximately 200 pg with uv detection (254 nm) and about 30 pg with fluorescent detection (exitation 253 and emission 445 nm) using normal-phase HPLC. The reactivity of panacyl bromide with 23 prostaglandins as well as prostaglandins released by human lung tissues was investigated.

Concentration of prostaglandins in seminal fluid of fertile men.

Semen samples from 31 men, all of whom had fathered a child within the preceding year, were analysed for sperm characteristics and for the content of prostaglandins (PGs). Mean concentrations (mg/l) for the four main groups of PGs were 67.1 for PGE, 3.2 for PGF, 245.7 for 19-hydroxy-PGE and 13.3 for 19-hydroxy-PGF. The individual values were distributed over a relatively wide range but the extremely wide ranges reported by previous authors were not confirmed. Information is also presented concerning the relative proportions of the four isomers of PGF, as well as those of 19-hydroxy-PGF. Only sperm density was related to PG concentration in fertile men. Polyzoospermia was associated with a low PGE concentration.

Absence of prostaglandins in semen of men with cystic fibrosis is an indication of the contribution of the seminal vesicles

Five men with cystic fibrosis delivered semen samples which were analysed for the content of the four main groups of prostaglandins (PGs). No PGs could be found in any of the specimens. The results of the present study provide further evidence that the seminal vesicles constitute the dominant site of production of PGs in human semen.