The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

New class of biphenylene dibenzazocinones as potent ligands for the human EP1 prostanoid receptor.

A new class of potent and selective ligands for the human EP1 prostanoid receptor is described. SAR studies reported herein allowed the identification of several potent dibenzazocinones bearing an acylsulfonamide side chain. The binding affinity of these compounds on all eight human prostanoid receptors is reported.

Cytosolic phospholipase A2, cyclo-oxygenases and arachidonate in human stomach tumours.

Human stomach tumours usually form more prostaglandins (PGs) than their associated normal mucosa/submucosa, but the mechanisms are not fully understood. The key enzymes are cytosolic phospholipase A2 (cPLA2, Mr 85,000) and the cyclo-oxygenases (COXs) which exist in constitutive (COX-1) and inducible forms (COX-2). In human stomach tumours and associated macroscopically normal tissues, we determined the fatty acid composition by gas chromatography, amounts of cPLA2, COX-1 and COX-2 by immunoblotting with specific antibodies and cPLA2 enzyme activity using a tritiated substrate. Although compared to normal mucosa there was less arachidonate in tumours (P < 0.05), the arachidonate/total fatty acid ratio was higher. Mean amounts of cPLA2 and COX-1 and cPLA2 activity were similar in tumours and normal mucosa. However, substantial amounts of COX-2 were found in the tumours but not in the mucosa, which may explain why many gastric tumours form increased amounts of PGs.

A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.

Human group II phospholipase A2 expressed in Trichoplusia ni larvae--isolation and kinetic properties of the enzyme.

Human secreted synovial fluid/platelet-type group II phospholipase A2 (sPLA2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 micrograms/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA2 showed a preference for substrate vesicles with a net negative charge. The baculovirus expression system provided active sPLA2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic phospholipase A2 from U937 cells: size of the functional enzyme by radiation inactivation.

We have studied the cytosolic phospholipase A2 (cPLA2) of human U937 cells by radiation inactivation in order to characterize the functional form of the native enzyme by a method that was independent of the discrepancies observed by SDS-PAGE and cDNA cloning. The Radiation Inactivation Size of cPLA2 was reproducible and gave a value of 76,800-80,100 daltons. We eluted the active enzyme from polyacrylamide-gradient gel electrophoresis at a molecular weight of 77,000, confirming the irradiation result. We conclude that cPLA2 is active as the monomeric enzyme and is composed of a single major functional domain that is sensitive to irradiation.

Apolipoprotein D--an atypical apolipoprotein.

The structure of ApoD and its sites of synthesis have been discovered. These characteristics differ from those of the other apolipoproteins. The role of ApoD in the plasma lipoprotein system remains to be discovered, but the recent, rapid increase in our knowledge of this protein suggests that it plays an important role in the homeostasis or housekeeping of probably all organs. One of its functions is likely to be the transport of a hydrophobic ligand (a lipid) in a one-to-one molar ratio with itself. This transport is likely to occur unidirectionally between neighboring cells in an organ, and between perivascular cells and the blood circulation. The chemical structure of the natural ligand, or ligands, of ApoD in normal cells in vivo or in culture is not known, but ApoD has been shown to bind some steroids and bilirubin. Remarkable upregulation of synthesis of ApoD has been observed during regeneration of injured peripheral nerves. Perhaps the physiologic role of ApoD will prove to be more interesting and of equal importance in biology to the roles of the other apolipoproteins in cardiovascular disease.

Molecular characterization and differential mRNA tissue distribution of rabbit apolipoprotein D.

We report for the first time the quantification of relative apolipoprotein D (apoD) mRNA concentrations in a wide selection of organs and a detailed characterization of the rabbit protein. ApoD cDNA clones were isolated from a rabbit testis cDNA library by screening with a human apoD cDNA-derived RNA probe. The 912 nucleotide sequence of rabbit apoD cDNA contains a unique reading frame coding for a protein sharing 80% homology with human apoD. The two sequences have two potential asparagine-linked glycosylation sites at the same positions, almost superimposable hydrophobicity plot, and the antigenic proteins show similar charge polymorphism, Mr, and lipoprotein distribution. This high degree of similarity shows that the rabbit system can be used as a model for apoD studies. Moreover, the two consensus sequences of the hydrophobic ligand carrier (alpha 2-microglobulin) family present in human apoD are also found in the rabbit protein and these sequences coincide with the most conserved regions. The distribution of apoD mRNA among rabbit organs was determined by Northern blot and quantitative dot blot analysis. The highest levels of mRNA were found in spleen, adrenal glands, lungs, brain, testis, and kidneys. Moderate or low concentrations were detected in all the other organs tested including liver and small intestine. Thus, our results show that the apoD gene is expressed mainly in peripheral organs, with levels as high as 59-fold that of the liver, unlike other apolipoproteins. We suggest that apoD exerts its main function locally in peripheral organs.