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LKB1, a unique serine/threonine kinase tumor suppressor, modulates anabolic and catabolic homeostasis, cell proliferation, and organ polarity. Chemically reactive lipids, e.g. cyclopentenone prostaglandins, formed a covalent adduct with LKB1 in MCF-7 and RKO cells. Site-directed mutagenesis implicated Cys210 in the LKB1 activation loop as the residue modified. Notably, ERK, JNK, and AKT serine/threonine kinases with leucine or methionine, instead of cysteine, in their activation loop did not form a covalent lipid adduct. 4-Hydroxy-2-nonenal, 4-oxo-2-nonenal, and cyclopentenone prostaglandin A and J, which all contain alpha,beta-unsaturated carbonyls, inhibited the AMP-kinase kinase activity of cellular LKB1. In turn, this attenuated signals throughout the LKB1 --> AMP kinase pathway and disrupted its restraint of ribosomal S6 kinases. The electrophilic beta-carbon in these lipids appears to be critical for inhibition because unreactive lipids, e.g. PGB1, PGE2, PGF2alpha, and TxB2, did not inhibit LKB1 activity (p > 0.05). Ectopic expression of cyclooxygenase-2 and endogenous biosynthesis of eicosanoids also inhibited LKB1 activity in MCF-7 cells. Our results suggested a molecular mechanism whereby chronic inflammation or oxidative stress may confer risk for hypertrophic or neoplastic diseases. Moreover, chemical inactivation of LKB1 may interfere with its physiological antagonism of signals from growth factors, insulin, and oncogenes.

Electrophilic eicosanoids of the J series, with their distinctive cross-conjugated alpha,beta-unsaturated ketone, inactivate genetically wild type tumor suppressor p53 in a manner analogous to prostaglandins of the A series. Like the prostaglandins of the A series, prostaglandins of the J series have a structural determinant (endocyclic cyclopentenone) that confers the ability to impair the conformation, the phosphorylation, and the transcriptional activity of the p53 tumor suppressor with equivalent potency and efficacy. However, J series prostaglandins have a unique structural determinant (exocyclic alpha,beta-unsaturated ketone) that confers unique efficacy as an apoptotic agonist. In seeking to understand how J series prostaglandins cause apoptosis despite their inactivation of p53, we discovered that they inhibit the ubiquitin isopeptidase activity of the proteasome pathway. In this regard, J series prostaglandins were more efficacious inhibitors than representative members of the A, B, or E series prostaglandins. Disruption of the proteasome pathway with proteasome inhibitors can cause apoptosis independently of p53. Therefore, this finding helps reconcile the p53 transcriptional independence of apoptosis caused by Delta12-prostaglandin J(2). This discovery represents a novel mechanism for proteasome pathway inhibition in intact cells. Furthermore, it identifies isopeptidases as novel targets for the development of antineoplastic agents.

Gas chromatography is the most widely used chromatographic technique. Its strength lies in the fact that three distinct operations--separation, detection, and quantitation--can be performed on all the components of mixtures within a reasonable time, and that qualitative information about the compound identity is associated with its retention time. Despite a prominent position in other areas of biochemistry, its success in the prostaglandin field has been rather limited. The technique itself is not at fault since samples with reasonable levels of prostaglandins have been analyzed without overwhelming difficulty. However, many samples demand a sensitivity exceeding the limits of either electron capture or flame ionization detectors. Shifts in research problems have contributed to its decline, but it is fair to add that these shifts were associated with the appearance of more sensitive and accurate analytical methods. Let us not despair for gas chromatography because its competitors, radioimmunoassay and gas chromatography-mass spectroscopy, also have deficiencies. In this chapter we have described several traditional gas chromatographic methods for prostaglandin analysis and outlined their strengths and weaknesses. Hopefully, this will prevent the misapplication of a useful analytical tool and also serve as an incentive to provoke contributions to this somewhat neglected technology. In conclusion, it cannot be stressed enough that all instrumental methods are at the mercy of chemistry. Chemistry can be exploited to enhance the value of a technique, or it can be disregarded to discredit the technique. The rise of sophisticated instruments has falsely diminished the value of chemistry in analytical methods development. It is imperative that we remind ourselves of its place and use it properly.

B16 malignant melanoma cell lines transform arachidonic acid and its transient metabolite, prostaglandin endoperoxide H2, into prostaglandin D2. The highly metastatic line, B16 F10, forms less prostaglandin D2 compared to the moderately metastatic parent line, B16 F1. Since platelet aggregation may be one factor involved in B16 metastasis and since prostaglandin D2 inhibits platelet aggregation, this prostaglandin could affect the outcome of platelet-tumour interactions, which may contribute ultimately to metastasis. Arachidonic acid metabolism may be another one of the intrinsic biochemical properties of tumor cells that affects their metastasis. Our results suggest that quantitative release of unusual prostaglandins must be considered in this context.

We monitored the chemical stability of prostaglandin I2 in the presence of different plasma proteins. PGI2 hydrolyzes by first order reaction kinetics, dependent on the albumin concentration in aqueous buffers. Chemical analysis shows that vertebrate albumins prolong the half-life of prostaglandin I2. The degree of stabilization varies quantitatively among different species. The results indicate that prostaglandin I2 may persist in its active form longer than previously supposed in some experimental conditions.

Antigen challenge of passively sensitized chopped human lung resulted in the generation of several arachidonic acid cyclooxygenase metabolites (AACM): thromboxane A2 (TxA2) as measured by its stable metabolite TxB2, prostaglandin D2 (PgD2), prostacyclin (PgI2) as measured by its stable metabolite 6-keto-PgF1 alpha, prostaglandin F2 alpha (PgF2 alpha), and prostaglandin E (PgE). The kinetics of AACM release after antigen challenge paralleled histamine release. All AACM were released in an antigen dose-dependent manner and reached maximal release at antigen concentrations lower than those required for maximal histamine release. Quantitatively, of the AACM measured, PgD2 and PgI2 were found to predominate in anaphylactic reactions of human lung parenchyma. Generation of PgD2 and PgI2 were 3- to 7-fold greater than that of other AACM measured. Thromboxane B2 was generated in quantities comparable to PgE and PgF2 alpha. Studies were designed to test the hypothesis that lung smooth muscle contraction per se can account for the generated AACM that are released during anaphylaxis of the lung. The studies compared antigen-induced AACM generation with methacholine-induced (10(-4) M) AACM generation. The failure to confirm this hypothesis was especially evident for PgD2 where release was dependent on mast cell activation. Thromboxane A2, PgD2, and PgI2 have been reported to have potent effects on smooth muscle. Our data suggested that these AACM are generated in such sufficient quantities that they may function in important aspects of the modulation of hypersensitivity responses in human lungs.

Glass capillary gas chromatography of the prostaglandins was performed on a system including an all-glass, solventless injector; thermostable methylphenyl-polysiloxane glass capillary columns; and a conventional electron-capture detector fitted with a make-up gas tee. The principal stable metabolites of prostaglandin endoperoxide were separated as perfluorinated derivatives in 35 min. Detection limits equal or exceed those obtained for packed column separations and electron capture detection. Prostaglandin endoperoxide metabolic profiles from mammalian cell cultures were obtained using this system. These profiling studies are not possible with other chromatographic methods because of inferior resolution and sensitivity.

The synthetic prostaglandin analog 9,11-azoprosta-5,13-dienoic acid (azo analog I) has been found to be a potent inhibitor of human platelet thromboxane synthetase by three independent analytical methods: electron-capture gas chromatography, radioisotopic thin-layer chromatography, and radioimmunoassay. In the presence of azo analog I, human platelet aggregation induced by either the prostaglandin endoperoxide PGH2 or arachidonic acid was antagonized. The addition of azo analog I shifted the transformation of endoperoxides away from thromboxane synthesis and toward prostaglandin E2 synthesis. The specificity of azo analog I is demonstrated by its selective inhibition of the second wave of either ADP- or epinephrine-induced platelet aggregation. These data indicate that PGH2 must be converted to thromboxane A2 in order to induce human platelet aggregation.

Dinoprost, dinoprostone, and prostaglandin E1 were each separated from their major 15-keto metabolites by high-performance liquid chromatography on a microparticulate, bonded, reversed-phase column after conversion to their p-bromophenacyl esters. Detection and simultaneous quantitation of prostaglandins in 1.0 ml of a 5-muM solution is possible. The method was applied to monitor prostaglandins formed during in vitro incubations with prostaglandin 15-dehydrogenase from monkey lungs. The advantages of this technique for assessing enzyme purity and activity are discussed.

Addition of the calcium inophore, A 23187, and cysteine to isolated mononuclear cells from rat peritoneal washings causes a marked increase in the formation of thromboxane B2 (TxB2) along with the formation of leukotrienes C and D (LT's). The formation of LT's in this system was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1, U-60,257, or its methyl ester, U-56,467, (ID50 4.6 and 0.31 microM, respectively). There was no inhibition of TxB2 formation. By contrast, two structurally-related compounds, PGI2 and its stable analog, 6-beta-PGI1, did not affect the formation of either LT's or TxB2. The inhibition of LT formation by U-60,257 was rapidly reversed after removal of this compound from the cells. U-60,257 did not inhibit the cyclooxygenase of human polymorphonuclear leukocytes. Nor did it inhibit formation of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) in human platelets. On the other hand, U-60,257 inhibited glutathione S-transferase activity of rat basophil leukemia cells (ID50, 37 microM), suggesting that this compound may inhibit the last step in LTC biosynthesis. In addition to inhibiting LT synthesis, U-60,257 also appears to be a competitive inhibitor of the action of LT on the guinea pig ileum, although this inhibition requires a higher drug concentration than those ordinarily encountered during assay for LT's in U-60,257-treated incubations.

Antibodies were prepared against 9-deoxy-6,9-epoxy-PGF1alpha, the 5,6-dihydro analog of prostacyclin (PGI2). By using as the hapten, this structurally similar, stable analog, an antibody population was developed which recognized PGI2 and reversed its influence on platelet aggregation. The antibodies also opposed the normal effect of PGI2 on the cAMP and thromboxane B2 levels during aggregation. By anticipating the cross reaction between the analog and PGI2 and by considering it beneficial, the problem of raising antibodies against an unstable compound has been circumvented.

We determined the effect of vertebrate albumins on the stability of several physiologically relevant prostaglandins. All naturally occurring prostaglandins with beta-hydroxy ketone group decomposed by first-order kinetics, dependent on the albumin concentration in 0.1 M, pH 7.4, buffer at 37 degrees C. Even subphysiological levels of albumin (1-20 mg/mL) significantly reduced the stability of these compounds in vitro. The prostaglandins with a beta-hydroxy ketone responded to albumin in the order of their intrinsic stability; namely, less stable compounds were more susceptible. The destructive effect of albumin was nearly maximal at a 1:1 mole ratio of albumin (20 mg/mL):prostaglandin (100 micrograms/mL). Albumin had no destructive effect on prostaglandins without a beta-hydroxy ketone. Albumins from different vertebrates varied in destructive severity, but all were effective. Near neutrality, in the absence of albumin, decomposition of E-type prostaglandins was practically suspended at the dehydration stage. In the presence of albumin, dehydration was accompanied by rapid isomerization reactions (e.g., PGA1 leads to PGB1) that occur only at an elevated pH. The results suggest that albumin sequesters prostaglandins to one principal binding site and exposes them to its associated highly alkaline microenvironment. This results in a uniform and predictable influence on prostaglandin stability. Our proposed model system successfully reconciles apparently anomalous or contradictory reports regarding the effect of albumin on prostaglandin stability.

Nonspecific resistance to infectious and neoplastic disease can be enhanced by administration of "immunomodulators". The levels of enhancement can be monitored by following in vitro function of cells of the lympho-reticuloendothelial system. To gain a better understanding of the physiological and biochemical nature of this enhancement, the metabolism of prostaglandin endoperoxide PGH2 was followed in mouse peritoneal cells (PCs). Homogenates of PCs from normal, unstimulated mice yielded primarily prostacyclin (PGI2) when incubated with PGH2. Homogenates of PCs from mice injected with the immunomodulators C. parvum, levamisole HCl, pyran copolymer, or thioglycollate yielded less PGI2. Reductions ranged from 73% for C. parvum to 32% for levamisole. A statistically significant inverse correlation existed between the level of macrophage "activation" and ability of cellular homogenates to produce prostacyclin. The results suggest that prostacyclin may be involved in modulation of nonspecific resistance.

Addition of the 3-series fatty acid precursor (icosapentaenoic acid, IPA), its endoperoxide [prostaglandin (PG)H(3)], or thromboxane A(3) to human platelet-rich plasma (PRP) does not result in aggregation of the platelets. In fact, preincubation of human PRP with exogenous PGH(3) actually inhibited aggregation by increasing platelet cyclic AMP concentrations. PGH(3) undergoes rapid spontaneous degradation to PGD(3) in human PRP. The PGD(3) so formed is adequate to account for the increase of platelet cAMP and inhibition of aggregation. Furthermore, addition of PGD-specific antisera to human PRP blocked the platelet inhibitory activity of exogenous PGH(3). PGD(3) has considerable potential as a circulating antithrombotic agent. Pretreatment of human PRP with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocked the increase of platelet cyclic AMP and the inhibition of aggregation normally produced by PGI(2), PGE(1), PGD(2), PGH(3), and PGD(3). Furthermore, the dideoxyadenosine unmasked a direct but moderate reversible aggregatory effect in response to the subsequent addition of PGH(3). Similarly, the dideoxyadenosine markedly enhanced the aggregation produced by exogenous PGH(2). IPA is readily incorporated into tissue lipids but proved to be a poor substrate for kidney, blood vessel, or heart cyclooxygenase. IPA was previously shown to be a poor substrate for platelet cyclooxygenase. IPA is readily deacylated from the renal phospholipid pool in response to bradykinin, a substance that also stimulates the release of arachidonic acid. A diet that relies primarily on cold-water fish, as in the case of the Greenland Eskimos, lowers endogenous arachidonic acid and markedly increases the IPA content of tissue lipids. Thus, because IPA has the potential to act as an antagonist with arachidonic acid for platelet cyclooxygenase and lipoxygenase, the simultaneous release of IPA could suppress any residual arachidonic acid conversion to its aggregatory metabolites.