Journal of lipid mediators最新文献

We have recently cloned cDNAs for the human and mouse TXA2/PGH2 receptors and a cDNA for the mouse PGE receptor. Sequencing, homology and hydrophobicity analyses revealed that they are proteins of 343, 341 and 365 amino acid residues, respectively, and all are rhodopsin-type receptors with putative seven transmembrane domains. Homology between the human and mouse TXA2 receptors is 76% in total and that between the human TXA2 and mouse PGE receptors is 38%. The homology increases in the putative transmembrane regions to 85 and 45%, respectively, and there observed several features common to the three receptors. These results indicate that the prostanoid receptors constitute a family of receptors of similar structure. The cloned PGE receptor showed binding activity specific to so-called EP3 agonists, which verified for the first time that pharmacologically defined PGE receptor subtypes consist of different molecules. This receptor also displayed different efficiency in signal transduction to an EP3 agonist, M & B-28767, and PGE2, providing an interesting model for the analysis of receptor-G protein coupling. In addition to the above biochemical results, these studies have revealed the characteristic tissue distribution of these receptors, which will open up a new biology of these prostanoids.

We have previously shown that human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to 6,11-dihydro metabolites (Powell and Gravelle (1988) J. Biol. Chem. 263, 2170-2177). In the present study, we have shown that the first step in the formation of these dihydro metabolites is oxidation of the 5-hydroxyl group to a 5-oxo group, which is catalyzed by an NADP(+)-dependent microsomal dehydrogenase enzyme. All the dihydroxyeicosanoids we investigated which contained a 5(S)-hydroxyl group followed by a 6-trans double bond were good substrates for this reaction. However, LTB4, which contains a 6-cis double bond, was not metabolized to any detectable 5-oxo products. The preferred substrate for the dehydrogenase reaction is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE), which has a Km of about 0.2 microM, compared to approx. 0.9 microM for 12-epi-6-trans-LTB4. In contrast to 5(S)-HETE, 5(R)-HETE as well as a variety of positional isomers of 5(S)-HETE are not metabolized to significant extents by the PMNL dehydrogenase. 5-Oxo-ETE and 5-oxo-15-hydroxy-ETE, which are formed from 5(S)-HETE and 5,15-diHETE, respectively, by this pathway, are potent chemotactic agents for human neutrophils, and raise intracellular calcium levels in these cells.

The production of platelet-activating factor (PAF), an alkyl ether phospholipid, was previously shown to occur in the brain upon various stimuli, and binding sites for PAF have been reported. We have recently demonstrated the existence of functional mRNA for PAF receptor in the brain by using a Xenopus oocyte expression system (Bito et al. (1992) Neuron 9, 285-294). In this review, we have analyzed the binding characteristics of PAF receptor in the rat brain and show that PAF receptor is ubiquitously distributed in the rat central nervous system, with an emphasis to the hypothalamus, cerebral cortex, and olfactory bulb. PAF was also found to mobilize intracellular Ca2+ in rat hippocampus.

The active site sequence 385-YHWH-388 of ovine prostaglandin endoperoxide synthase-1 (PGHS-1) has residues critical for cyclooxygenase and peroxidase catalysis. Tyr385 is essential for cyclooxygenase activity, His386, for peroxidase activity, and His388, for both activities. To determine the importance of Trp387, we used site-directed mutagenesis to replace Trp387 of PGHS-1 with arginine, phenylalanine, and serine. W387R and W387S lacked significant activity. W387F retained both cyclooxygenase and peroxidase activities. Thus, we conclude that Trp387 is not essential for catalysis by PGHS-1. Purified PGHS-1 is a homodimer. There are two putative leucine zipper regions in ovine PGHS-1 involving residues 345-366 and 487-508. We tested for a role of these leucine zippers as determinants of dimer formation. Helix-breaking proline mutations were introduced at Leu359 or Leu501. Neither of these residues proved to be essential for peroxidase activity; but, mutations at each residue greatly reduced or eliminated cyclooxygenase activity. Both mutant proteins chromatographed as dimers on Sephacryl G-200. Thus, neither of these putative leucine zipper regions alone is responsible for PGHS-1 dimer formation.

Prostaglandin G/H synthase isoenzyme 2 (PGHS-2) was identified as an immediate-early gene product induced by Rous sarcoma virus, serum, phorbol ester and a wide variety of other mitogens. Induction of PGHS-2 occurs through an increase in PGHS-2 gene transcription. Dexamethasone inhibits both the basal and induced levels of PGHS-2 mRNA. In contrast, PGHS-1 gene transcription rate, mRNA, and protein levels are unaffected by mitogens and dexamethasone. Post-transcriptional down-regulation of PGHS-2 mRNA plays a significant role in causing dexamethasone's effect. Specific cell systems have been identified which allow selective analysis of PGHS-1 and PGHS-2 at the nucleic acid and protein levels. These cell systems indicate that PGHS-1 and PGHS-2 may have different sensitivities to non-steroidal anti-inflammatory drugs. Furthermore, inhibition of PGHS activity correlates well with suppression of the transformed phenotype in an in vitro cell model.

(1) Human cartilage, both non-arthritic (N) and arthritic, is extremely sensitive to inhibition of glycosaminoglycan (GAG) synthesis by low concentrations of interleukin 1 (IL1). Local episodic synthesis and secretion of sub-nanogram concentrates of the cytokine is considered to play a significant role in the pathogenesis of osteoarthritis (OA) by preventing matrix repair. (2) The synthesis of IL1 can be controlled by prostaglandins (PGs), which may explain why the inhibitory action can be at least partially overcome by the action of the PG analogue Misoprostol in the dose range 10-100 ng/ml. It is suggested that this action is due to the suppression of a positive feedback loop for local IL1 synthesis and secretion. (3) Certain non-steroidal anti-inflammatory drugs (NSAIDs), in particular Indomethacin, Ibuprofen and Naproxen, cause inhibition of GAG synthesis, and hence may diminish the potentiality for repair in arthritic cartilage. It is suggested that these NSAIDs induce IL1 synthesis by diminishing PG levels. Misoprostol is able to reverse this effect at least partially. (4) Some cartilages in the presence of other NSAIDs, such as Diclofenac, which do not greatly inhibit chondrocyte matrix metabolism, nevertheless respond to the presence of Misoprostol by increased GAG synthetic activity. (5) The low mean matrix synthetic activity of human OA cartilages was significantly increased by Misoprostol. (6) Taken together, these studies substantiate the suggestion that Misoprostol is able to increase the repair potentiality of human OA cartilage, particularly during treatment with NSAIDs.

Biosynthesis of thromboxane A2 (TxA2) during cell-cell interactions between platelets and alveolar macrophages, eosinophils and natural killer cells from guinea pigs has been investigated. The stimulation of platelets incubated with varying numbers of macrophages or eosinophils or NK cells with 5 microM ionophore A23187 has induced an increase of TxA2 synthesis in comparison to the sum of TxA2 production by each cell population stimulated alone. These results suggest that transcellular biosynthesis of TxA2 or cell cooperation occurred between platelets and the other cell population studied, which could correspond to cell-cell interactions of type IA and/or type III, according to the classification proposed by Marcus ((1986) Prog. Hemost. Thromb. 28, 127-42). When varying concentrations of platelets were incubated with the supernatant of cells previously stimulated with the ionophore A23187, an enhancement of TxA2 release was also observed. However, the supernatant of platelets stimulated with the ionophore A23187 did not induce a significant increase of TxA2 synthesis by the other cell populations. It is suggested that the biosynthesis of TxA2 during cell-cell interactions could account for the large amount recovered in biological fluid in various pathological conditions such as inflammatory and hypersensitivity reactions.