Prostaglandins, arachidonic acid, and inflammation.

The enzymatic oxidation of arachidonic acid has been shown to yield potent pathological agents by two major pathways. Those of the prostaglandin (PG) pathway, particularly PGE2, have been implicated as inflammatory mediators for many years. The discovery and biological activities of thromboxane A2 and prostacyclin as well as a destructive oxygen-centered radical as additional products of this biosynthetic pathway now require these to be considered as potential inflammatory mediators. Like PGE2, their biosynthesis is prevented by nonsteroidal anti-inflammatory agents. More recently, the alternative metabolic route, the lipoxygenase pathway, has been shown to yield a new class of arachidonic acid oxygenation products, called the leukotrienes, which also appear to be important inflammatory mediators. Unlike the prostaglandins, some of which play important roles as biological regulators, the actions of the lipoxygenase products appear to be exclusively of a pathological nature.

Prostaglandin receptor site: evidence for an essential role in the action of luteinizing hormone.

A dose-response relation was established between prostaglandins and formation of adenosine 3',5'-monophosphate in the mouse ovary. The prostaglandin antagonist, 7-oxa-13-prostynoic acid, blocked the stimulatory effect of prostaglandin E(1), prostaglandin E(2), and luteinizing hormone on adenosine 3',5'-monophosphate formation in a competitive manner. Kinetic studies made it possible to suggest that there is a single luteinizing-hormone-related prostaglandin receptor in mouse ovaries, and that activation of this prostaglandin receptor is an essential requirement in the action of luteinizing hormone to stimulate adenosine 3',5'-monophosphate formation and steroidogenesis.

Phosphonomycin: structure and synthesis.

Synthesis and resolution of the antibiotic phosphonomycin are described. The structure is (-)(IR, 2S)-1,2-epoxypropylphosphonic acid.

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate.

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.

The role of macrophage secretory products in chronic inflammatory processes.

Mononuclear phagocytes participate in various stages of chronic inflammatory responses and associated diseases. Such participation is mediated by (a) direct interaction with pericellular interstitial tissue components as well as with other cell types present at sites of inflammation and (b) by secretion of soluble mediators. Several of these mediators are synthesized and secreted in increased amounts after macrophages interact with inflammatory stimuli. In this paper we pay particular attention to neutral proteinases and prostaglandins. It is shown that these 2 classes of mediators are released in significant amounts under different conditions. Prostaglandins are synthesized most readily by resident populations of mouse peritoneal macrophages responding to various model inflammatory stimuli. Mouse peritoneal macrophage populations elicited in vivo by inflammatory stimuli are less responsive in this respect. In contrast neutral proteinase secretion does not occur in resident cell populations but is observed on a continuous basis in elicited populations. Such secretion can be increased further by addition of phagocytic stimuli and initiated in resident populations by model inflammatory stimuli such as phorbol myritate acetate. Other secretory products of macrophages with possible relevance to inflammation are discussed briefly. Finally some of the effects of antiinflammatory glucocorticoids, cyclooxygenase inhibitors and dapsone on the secretory activity of macrophages are briefly summarized.

11,12-Secoprostaglandins. 5. 8-Acetyl- or 8-(1-hydroxyethyl)-12-hydroxy-13-aryloxytridecanoic acids and sulfonamide isosteres as inhibitors of platelet aggregation.

The synthesis of a series of 8-acetyl- (or 1-hydroxyethyl-) 12-hydroxy-13-aryloxytridecanoic acids and their sulfonamide isosteres is described. These compounds are formally derived from members of earlier reported series of modified secoprostaglandins by replacing the omega-butyl chain termini by substituted aryloxy groups. A number of these compounds are potent inhibitors of collagen-induced blood platelet aggregation in guinea pigs on oral administration.

Steroidal androgen biosynthesis inhibitors.

PIP: 23,17beta-acylaminoandrost-4-en-3-ones and 3 previously known nonsteroids were synthesized and screened as inhibitors of 17,20-lyase, a step in androgen synthesis from progesterone or OH-progesterone. The screening involved measuring side chain cleavage (carbon-14-acetate release) from 21-carbon-14-17alpha-OH-progesterone by rat testis microsomes. The amide, urea, guanidino and carbamate derivatives were also tested by conversion of cholesterol to pregnenolone by a bovine corpora lutea acetone powder, by conversion of corticosterone to 18-OH-corticosterone by crude adrenal mitochondria, and by feeding to male rats to check effect on adrenal weight and testis testosterone level. More than 80% inhibition was achieved with androst-4-en-3-ones having the C-17beta carbamate, formamido, acetamido and ureido groups. These compounds did not inhibit OH-corticosterone synthesis. 6-alpha methylation inhibited the lyase 50-70%. 1 compound 17-beta-ureidoandrost-1,4-dien-3-one was fed to male rats for 6 weeks at 500 mg per kg; it reduced testis testosterone but not adrenal weight. Selective inhibition of androgen synthesis would be useful for treating benign prostate hypertrophy, hirsutism, acne and androgen dependent tumors.^ieng

11,12-Secoprostaglandins. 3. 8-Alkylthio(sulfinyl and sulfonyl)-12-hydroxyalkanoic acids and related compounds.

A series of 8-alkylthio(sulfinyl and sulfonyl)-12-hydroxyalkanoic acids which embody structural features of 11,12-secoprostaglandins was synthesized and evaluated for their ability to mimic the E series prostaglandins in stimulating cAMP formation in the mouse ovary and in binding to the rat lipocyte prostaglandin receptor. A key member of the series, 8-methylsulfonyl-12-hydroxyheptadecanoic acid, markedly stimulated cAMP formation at reasonable pharmacological concentrations, shows significant affinity for a prostaglandin receptor, and effectively inhibits antigen-induced lymphocyte transformation. In contrast, this compound is not a substrate for 15-hydroxyprostaglandin dehydrogenase, the major prostaglandin-metabolizing enzyme.

11,12-Secoprostaglandins. 2. N-acyl-N-alkyl-7-aminoheptanoic acids.

A series of N-acyl-N-alkyl-7-aminoheptanoic acids has been prepared and evaluated for their ability to mimic the natural prostaglandins in certain biological systems. These compounds can be regarded as 8-aza-11,12-secoprostaglandins and, indeed, most of them stimulate cAMP formation in the mouse ovary assay, just as is observed with the natural prostaglandins. Selected compounds from this series also have been studied and shown to have prostaglandin-like effects in vivo.

Pharmacological effects of non-steroidal antiinflammatory agents on prostaglandin and leukotriene synthesis in mouse peritoneal macrophages.

Resident mouse peritoneal macrophages, exposed to zymosan, synthesized and released products of both the cyclooxygenase and lipoxygenase pathways. The effects of various non-steroidal antiinflammatory agents were evaluated for their abilities to inhibit zymosan-stimulated prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) synthesis. The order of potencies to inhibit PGE2 synthesis and release was: indomethacin greater than or equal to sulindac sulfide greater than ibuprofen greater than or equal to aspirin greater than 3-amino-1-[3-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C) greater than benoxaprofen greater than or equal to nordihydroguaiaretic acid (NDGA) greater than 5,8,11-eicosatriynoic acid (ETYA). BW755C and ETYA also inhibited zymosan-stimulated LTC4 production. None of the compounds tested showed selective inhibition of lipoxygenase products.

Biologically active derivatives of fatty acids: prostaglandins, thromboxanes, and endoperoxides.

The causal role assigned to the E and F prostaglandins in inflammatory processes, implied by the antiinflammatory action of prostaglandin synthetase inhibitors, is not consistent with the findings reported here that a compound (MK-447) capable of increasing levels of these prostaglandins is antiinflammatory in classical animal models of acute inflammation. That both MK-447 and prostaglandin synthetase inhibitors depress the enzymatic formation of PGG2 from arachidonic acid suggests that this endoperoxide plays a pivotal role in acute inflammation. However, in view of the intermediate nature of PGG2, it seems likely that such a pivotal role for this substance is a function of its ability to be converted to other inflammatory mediators. Possible candidates for a causal role are thromboxane A2 (TXA2) prostacyclin (PGI2), both of which derive from PGG2. However, direct evidence is presented to show that an oxygen equivalent released in the enzymatic conversion of PGG2 to PGH2 is a prime factor in inflammation.

Direct evidence for a prostaglandin receptor and its application to prostaglandin measurements (rat-adipocytes-antagonists-analogues-mouse ovary assay).

A prostaglandin receptor is present in rat adipocytes, as shown by competition studies with [(3)H]-prostaglandin E(1). The affinity of the E prostaglandins for this receptor preparation is greater than that of A and F prostaglandins, an observation consistent with their relative potencies in stimulating cyclic AMP formation in isolated mouse ovaries and various other organs. In the protocol described, binding constants of 3-8 nM were obtained with prostaglandins E(2) and E(1). A sensitivity of 1 pmole makes this method readily applicable for measurement of tissue concentrations of the E prostaglandins.

Mechanism for irreversible self-deactivation of prostaglandin synthetase.

It has been shown that prostaglandin (PG) cyclooxygenase is irreversibly self-deactivated during the oxygenation of arachidonic acid (Smith, W. L., and Lands, W.E.M. (1972) Biochemistry II, 3273-3285). Using several experimental approaches and an enzyme preparation which was highly active without artificial stimulation, we have extensively investigated the mechanism of this deactivation process. During the generation of PGH2 from arachidonic acid, oxidizing equivalents were released and the reductive breakdown of PGG2 was found to deactivate the cyclooxygenase. The cyclooxygenase-catalyzed metabolism of both arachidonic acid and PGG2 generated radicals which were scavenged by phenol. Both phenol and methional (scavengers of oxygen-centered radicals) promoted the formation of PGH2 at the expense of PGG2 and increased the initial rate and the extent of reaction prior to deactivation of the cyclooxygenase. Hence, it appears that the cyclooxygenase was irreversibly self-deactivated during the formation of PGH2 from arachidonic acid, due to its oxidation by oxygen-centered radicals formed as a result of the reductive breakdown of the hydroperoxide on PGG2. Some experiments with dithiothereitol and N-ethylmaleimide suggested that the enzyme may contain a disulfide at the active site. A mechanism has been devised which accounts for the self-deactivation phenomenon, the effect of phenol and methional, the disulfide at the active site, and the pathway of substrate oxygenation.