Absence Of Exogenous Arachidonic Acid Research Articles

Critical role of COX‐1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone

We aimed at evaluating the relative contribution of cyclooxygenase (COX) -1 and COX-2 to the synthesis of prostacyclin in endothelial cells under static conditions in the presence or absence of exogenous arachidonic acid and/or altered intracellular redox balance. Selective inhibitors of either COX-1 (SC560 and FR122047) or COX-2 (SC236) concentration dependently (1-300 nM) reduced basal and interleukin (IL) -1beta-induced prostacyclin production in human umbilical vein endothelial cells by 70% or more; compound selectivity was confirmed using a whole-blood assay (IC(50) COX-1/COX-2: 13 nM/930 nM for SC-560; 9 microM/457 nM for SC-236). The observed concomitant formation of isoprostane appeared to be associated with COX enzyme activity, while formation of COX-1/COX-2 heterodimers was detected by immunoprecipitation. In the presence of arachidonic acid and 12-hydroperoxy-eicosatetraenoic acid, either exogenous or provided by platelet activation, or after glutathione depletion, COX-1 inhibition but not COX-2 inhibition concentration dependently decreased prostacyclin production. Both isoforms appear to contribute to basal prostacyclin production by endothelial cells, with COX-2 providing the hydroperoxide tone required for COX-1 activity. Conversely, in the case of intracellular glutathione depletion or enhanced availability of arachidonic acid and hydroperoxides, selective COX-2 inhibition did not significantly affect the production of endothelial prostacyclin. These findings contribute to a better understanding of the effects of cyclooxygenase inhibitors on prostacyclin production.

Expression of 5‐lipoxygenase (5‐LOX) in T lymphocytes

5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Anti-cyclooxygenase effects of lipid extracts from the New Zealand green-lipped mussel, Perna canaliculus

Total lipid extracts of P. canaliculus (a bivalve marine mollusc native to New Zealand, commonly called the green-lipped mussel) and Mytilus edulis (commonly called the common blue mussel) moderately inhibited ovine COX-1 and COX-2 pure enzymes in vitro. The inhibition was increased after the mussel extracts were saponified by KOH hydrolysis. Protease- and protease–lipase-hydrolysed lipid extracts of P. canaliculus exhibited similarly strong COX inhibition as the KOH-hydrolysed extract. Lyprinol® (a commercial extract from P. canaliculus) also exhibited strong inhibition of both COX isoforms, an effect that was increased 10-fold upon subsequent hydrolysis. In contrast, fish oil was not as anti-COX active as Lyprinol. The Lyprinol free fatty acid fraction, and to a lesser extent the Lyprinol triglyceride fraction, were the only lipid classes of Lyprinol to exhibit strong inhibition of the COX isoforms. The purified PUFA extracts were all bioactive, potently inhibiting COX-1 and COX-2. Incubation of Lyprinol in the absence of exogenous arachidonic acid (AA) showed the appearance of alternate prostaglandin metabolites, confirming Lyprinol PUFA as a competitive substrate inhibitor of AA metabolism.

Transcriptional induction of prostaglandin G/H synthase-2 by basic fibroblast growth factor.

In serum-free mouse osteoblastic MC3T3-E1 cells, basic fibroblastic growth factor (bFGF) induced mRNA and protein for prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in arachidonic acid (AA) conversion to prostaglandins. mRNA accumulation peaked at 1 h with bFGF 1 nM. In cells stably transfected with a 371-bp PGHS-2 promoter-luciferase reporter, bFGF stimulated luciferase activity, which peaked at 2-3 h with bFGF 1-10 nM. In the presence of exogenous AA, bFGF stimulated PGE2 production, which paralleled luciferase activity. In serum-free neonatal mouse calvarial cultures, bFGF stimulated PGE2 production in the absence of exogenous AA. bFGF stimulated PGHS-2 mRNA accumulation, which peaked at 2-4 h and then decreased; there were later mRNA elevations at 48 and 96 h that were inhibited by indomethacin. In both MC3T3-E1 cells and neonatal calvariae, bFGF produced smaller and slower increases in PGHS-1 mRNA levels than for PGHS-2. bFGF stimulated bone resorption in mouse calvariae with a maximal increase of 80% at 1 nM. Stimulation was partially inhibited by nonsteroidal anti-inflammatory drugs. We conclude that bFGF rapidly stimulates PGE2 production in osteoblasts, largely through transcriptional regulation of PGHS-2, and that prostaglandins mediate some of bFGF's effects on bone resorption.

Localization of cyclooxygenase and production of prostaglandins in bovine spermatozoa

Ejaculated bovine spermatozoa were examined for their capacity to synthesize prostaglandins E2 and F2 alpha (PGE2, PGF2 alpha). It was found that in the absence of exogenous substrate, arachidonic acid, basal PGF2 alpha production was less than that of PGE2. However, addition of 61 mumol arachidonic acid I-1 resulted in at least a twofold increase in PGE2 and PGF2 alpha above control values (1.3 ng and 0.3 ng per 10(8) spermatozoa, respectively). Addition of calcium and the calcium ionophore A23187 to the incubation medium did not cause a significant increase in the production of either PG. The presence of indomethacin (100-200 micrograms ml-1) caused a 50-70% inhibition of the production of both PGs. Activity of cyclooxygenase was determined by western blot analysis, using a specific polyclonal antiserum, and by fluorescence immunohistochemistry using a monoclonal antibody. The western blot displayed a clear signal for the presence of cyclooxygenase in ejaculated and epididymal spermatozoa. The immunohistochemical studies showed that the enzyme is localized in the apical region of the head, the post-acrosomal region and the mid-piece of the tail. Since the synthesis of PGs in the absence of exogenous arachidonic acid is low, the effect of melittin, a known phospholipase A2 activator, on PG production was examined. Incubation of spermatozoa with melittin produced a threefold increase in PGE2 and a sixfold increase in PGF2 alpha. Staurosporine, a protein kinase C inhibitor, inhibited the effect of melittin indicating that activation of phospholipase A2 by protein kinase C is an obligatory step in PG synthesis by bovine spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Pasteurella haemolytica leukotoxin enhances production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by bovine polymorphonuclear leukocytes.

The influence of the leukotoxin of Pasteurella haemolytica on the generation of arachidonic acid metabolites by bovine polymorphonuclear leukocytes (PMNs) was investigated. PMNs released 5-, 12-, and 15-hydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) and leukotriene B4 (LTB4) upon stimulation with arachidonic acid. The leukotoxin preparations dose dependently enhanced the release of the 5-lipoxygenase products 5-HETE and LTB4 in arachidonic acid-stimulated PMNs, whereas the release of 12- and 15-HETE was not affected. The enhanced release of LTB4 and 5-HETE was not due to a decreased cellular retention of the 5-lipoxygenase products. In addition, leukotoxin preparations by themselves were also able to induce LTB4 and 5-HETE production in the absence of exogenous arachidonic acid. Generation of 5-lipoxygenase products by PMNs stimulated by leukotoxin may represent an important cellular event that occurs during infections with P. haemolytica.

Leukotriene B4 levels from stimulated neutrophils from healthy and allergic subjects: effect of platelets and exogenous arachidonic acid.

Leukotriene B4 (LTB4) levels were measured in peripheral blood neutrophils from allergic and healthy donors after stimulation by calcium ionophore A 23187. This level was higher in neutrophils from allergic subjects than in neutrophils from healthy subjects in the presence as well as in the absence of exogenous arachidonic acid. Platelets from allergics increased LTB4 levels from neutrophils from allergics but not levels in those from healthy donors. Moreover, platelets from healthy subjects reduced LTB4 in neutrophils from both groups. These results suggest that biochemical differences exist in neutrophils and platelets from allergics which contribute to changes in arachidonic acid metabolism via the 5-lipoxygenase pathway. In addition, they support the concept that platelets may play an important role in the regulation of neutrophil LTB4 levels, possibly by affecting the 5-lipoxygenase activity during the course of allergic inflammatory reactions.

In vitro effects of arachidonic acid on the prostaglandin synthesizing system in gastric mucosa

Self-destruction of the prostaglandin cyclooxygenase has been suggested to be an important factor in the regulation of endogenous prostaglandin synthesis. The present study was done in order to define the role of this substrate-induced inactivation in the regulation of prostaglandin synthesis in gastric mucosa. In tissue homogenate, the prostaglandin synthesizing capacity is rapidly inactivated at 37°C, even in the absence of exogenous arachidonic acid. It was shown that this inactivation can be prevented both by EDTA as a chelator of calcium-ions and by tetracaine, a specific inhibitor of the phospholipase A2. Additional exogenous arachiodonic acid again inactivated prostaglandin synthesis in a dose dependent manner. In contrast, prostaglandin synthesizing capacity in organ cultured mucosal biopsies is well preserved, although the release of endogenous substrate was activated by extracellular calcium and Ca-ionophore A23187. Furthermore, even at high concentrations of exogenous arachidonic acid present in the culture medium, the synthesizing capacity in intact biopsies was only slighly and reversibly reduced. These large differences between intact biopsies and cell free tissue preparations point to very efficient mechanisms controlling the substrate availability for the cyclooxygenase system both from endogenous and exogenous sources in intact gastric mucosa.

Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels.

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.

Regulation of intracellular free arachidonic acid inAplysia nervous system

We have studied the regulation of arachidonic acid (AA) uptake, metabolism, and release in Aplysia nervous system. Following uptake of [3H]AA, the distribution of radioactivity in intracellular and extracellular lipid pools was measured as a function of time in the presence or absence of exogenous AA. The greatest amount of AA was esterified into phosphatidylinositol (relative to pool size). We found that the intracellular free AA pool underwent rapid turnover, and that radioactive free AA and eicosanoids were released at a rapid rate into the extracellular medium, both in the presence and absence of exogenous AA. Most of the released radioactivity originated from phosphatidylinositol. Two pharmacological agents were found to modulate AA metabolism in Aplysia ganglia. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, stimulated liberation of AA from phosphatidylinositol and phosphatidylcholine. This resulted in an increase in free internal and secreted AA, an increase in conversion of AA to eicosanoids, and an increase in esterification of AA into triacylglycerol. The half maximal dose for TPA-stimulated AA turnover was 15 nM, and the stimulation was dependent on the presence of extracellular calcium. 4-bromophenacylbromide inhibited the redistribution of radioactivity from phospholipid into triacylglycerol, indicating BPB was acting as a phospholipase inhibitor in Aplysia as it does in other systems. These pharmacological agents, in addition to providing information about the regulation of AA metabolism and release, are useful tools for investigating the physiological function of the rapid turnover of AA in Aplysia nervous system.

Inhibitory effects of MK‐886 on arachidonic acid metabolism in human phagocytes

1. We have investigated the inhibitory activity of compound MK-886 (formerly L-663,536), an indole derivative, on 5-lipoxygenase product synthesis in various human phagocytes stimulated with either the ionophore A23187, in the presence and absence of exogenous arachidonic acid, or platelet-activating factor (PAF). The lipoxygenase products were analysed by reversed-phase h.p.l.c. 2. MK-886 inhibited the formation of 5-hydroxy-eicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), its omega-oxidation products and 6-trans-isomers with an IC50 value of 10-14 nM in A23187-stimulated neutrophils. In the same system, nordihydroguaiaretic acid (NDGA), AA-861 and L-655,240 showed IC50 values of 250-510, 110-420 nM and 1.7-3.9 microM, respectively. 3. MK-886 inhibited 5-lipoxygenase product synthesis in A23187-stimulated blood eosinophils and monocytes, and in neutrophils primed with granulocyte-macrophage colony-stimulating factor and stimulated with PAF with IC50 values of 1-13 nM. 4. The inhibitory activity of MK-886 was not reversed by addition of 10 microM arachidonic acid to A23187-stimulated neutrophils. 5. Compound MK-886 had no effect on 15-lipoxygenase product synthesis in blood eosinophils and neutrophils up to a concentration of 1 microM. 6. At 100 nM compound MK-886 had no significant effects on calcium ion mobilization, superoxide anion production and actin polymerization in neutrophils. 7. In conclusion, MK-886 is a very potent and specific inhibitor of both LTB4 and LTC4 synthesis in various types of human phagocytes.

Antidiuretic hormone reduces the high PGE2 synthesis in papillary collecting duct of DI rats.

PGE2 synthesis was measured along the nephron of Brattleboro (DI) rats, lacking ADH, and control LE rats, using an enzyme immunoassay. Experiments were performed in vitro, in the absence of exogenous arachidonic acid, using microdissected tubular segments. The effect of a chronic treatment of dDAVP was tested on three ADH sensitive tubular segments, medullary thick ascending limb (MTAL), medullary collecting tubule (OMCD) and papillary collecting duct (IMCD). No difference in PGE2 synthesis was present between LE and DI in glomerulus and tubular segments up to OMCD. In both strains, values were low in the proximal tubule and the loop of Henle, and gradually increased along the collecting tubule. In IMCD, PGE2 synthesis was much higher in DI (12.8 +/- 2.0 pg per 30 min per mm tubular length) than in LE (3.8 +/- 0.5, LE vs. DI p less than 0.001). In MTAL and OMCD, dDAVP treatment did not affect PGE2 synthesis. In IMCD, dDAVP reduced PGE2 synthesis to values (5.3 +/- 0.8 pg per 30 min per mm tubular length), which were not significantly different from those of LE. Neither oxytocin, which has been shown to be elevated in DI rats, nor furosemide, that reduced papillary osmolarity to values comparable to those of DI rats, were able to increase PGE2 synthesis in IMCD of LE rats. The mechanism of the increase in PGE2 synthesis in IMCD of DI rats, and of the inhibitory effect of dDAVP is yet unknown; it may participate to compensate for the lack of ADH in the Brattleboro rat.

Mast cell mediators stimulate synthesis of arachidonic acid metabolites in macrophages.

Mast cells and macrophages were isolated from human lung tissues by using density gradient centrifugation, cell sorter, and adherence techniques. Passively sensitized mast cells in the absence of exogenous arachidonic acid (AA) released leukotriene (LT)C4, LTD4, PGD2, and thromboxane-B2 when challenged with Ag, and in the presence of AA, released 5-hydroxyeicosatetraenoic acid (HETE) and 15-HETE in addition to the above metabolites. Passively sensitized macrophages did not release significant amounts of AA metabolites when challenged with Ag. However, these cells released LTB4, LTC4, LTD4, LTE4, 5-HETE, PGE2 and 6-keto-PGF1 alpha when co-incubated with activated mast cells. During co-incubation, mast cells also generated greater amount of AA metabolites than when they were activated alone. The stimulatory action of mast cells on macrophages was shown to be due to the extracellular factor(s) present in the supernatant of the activated mast cells. Both heat and trypsin inhibited the biologic activity of mast cell-derived stimulatory factor. In addition, extraction of mast cells' materials with chloroform or ether showed no activity associated with the organic phase, suggesting it possibly possesses a protein nature, such as peptides, protease, or peptidase. These results suggest that mast cell-macrophage interaction might be important in the generation of multiple mediators in the airways during immediate hypersensitivity reactions.

Prostaglandin synthesis by isolated cells of rat uterus: Production rates, and effects of indomethacin and histamine

Luminal epithelial and residual cells (mainly of the endometrial stromal tissue) of proestrous rat uteri have been isolated and cultured in defined medium. The prostaglandins produced during a short-term incubation (2 h) in the presence of 10 μM arachidonic acid (to optimize PG production) were determined by direct assay of the culture medium. For the epithelial cells, PGF 2α was produced in greatest amounts, followed by 6-keto PGF 1α and PGE, while low levels were synthesized by the residual cells. The synthesis of PGF 2α by the epithelial cells was inhibited by incorporating indometahcin into the medium and an IC 50 value of 2.3 μM was obtained. Incubations performed with histamine in the absence of exogenous arachidonic acid indicated that the pathways for the production of individual prostaglandins were followed to different relative extents, with the production of 6-keto PGF 1α being enhanced for both groups of cells when compared to incubations with arachidonic acid.

A rapid method for isolating rabbit renal microvessels.

A technique has been described to isolate rabbit renal preglomerular microvessels using an infusion of a magnetized iron oxide suspension into the renal artery. An initial separation of the vascular tissue was performed with a magnet and a final separation was performed using a nylon sieve allowing nonvascular, tubular, and glomerular contaminants to be filtered free from the microvessels. Light and electron microscopy showed morphology consistent with interlobular arteries and afferent arterioles. The purity of the preparation was greater than 95%. In the absence of exogenous arachidonic acid, the microvessels were able to synthesize prostacyclin (7.5 +/- 1.2 ng.mg protein-1.min-1) in excess of prostaglandin E2 (2.2 +/- 0.7 ng.mg protein-1.min-1). The tissue exhibited a time-dependent increase in prostanoid biosynthesis up to 60 min and responded to stimulators (e.g., melittin and arachidonic acid) and inhibitors (e.g., meclofenamate and indomethacin) of prostanoid biosynthesis. These studies indicate that a high-yielding and versatile preparation of renal preglomerular microvessels can be rapidly obtained. The microvessels are viable and may represent an excellent model for studying a variety of biochemical interactions under in vitro conditions.

A possible requirement for arachidonic acid lipoxygenation in the mechanism of phagocytic degranulation by human neutrophils stimulated with aggregated immunoglobulin G

Aggregated immunoglobulin G (AggIgG) caused a concentration-dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Generation of the 5-lipoxygenase product of arachidonic acid (AA) metabolism, 5(S), 12(R)-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid [leukotriene B 4 (LTB 4)], by neutrophils exposed to AggIgG occurred in the presence but not absence of exogenous AA. U-60,257B (piriprost potassium), an inhibitor of leukotriene synthesis, caused a dose-related suppression of LTB 4 production and granule exocytosis by AggIgG-treated cells. These data suggest that a lipoxygenase product of AA metabolism may mediate AggIgG-induced phagocytic release of granule constituents from neutrophils.

Characterization of effect of N-formyl-methionyl-leucyl-phenylalanine on leukotriene synthesis in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNLs) were exposed to N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) in the presence or absence of exogenous arachidonic acid. Analysis of incubation mixtures by high-performance liquid chromatography showed that f-Met-Leu-Phe stimulated the synthesis of 5-hydroxy-eicosatetraenoic acid (HETE) and of leukotriene B4 (LTB4) which was rapidly metabolized into 20-OH-LTB4 and 20-COOH-LTB4. The stimulatory effect of f-Met-Leu-Phe was dose and time dependent. The tripeptide showed maximum stimulatory activity at the concentration of 1 microM and after 20-30 min of incubation. Addition of arachidonic acid to the f-Met-Leu-Phe-stimulated PMNLs resulted in an increase in the synthesis of LTB4 and 5-HETE. Pretreatment of the PMNLs with cytochalasin B strongly potentiated (up to six-fold) the stimulatory effect of f-Met-Leu-Phe on 5-lipoxygenase product synthesis, whereas cytochalasin B alone or with arachidonic acid had no significant effect. The tripeptide did not increase the synthesis of platelet-derived 12-HETE, and 12-hydroxyheptadecatrienoic acid, or of PMNL-derived 15-HETE, suggesting that its action was selective for PMNL 5-lipoxygenase. The present data indicate that f-Met-Leu-Phe causes the release of arachidonic acid from cellular lipids and activates the 5-lipoxygenase.

Inflammation and hypersensitivity.

The leukotrienes are a family of potent mediators of hypersensitivity and inflammation. The application of sensitive and specific radioimmunoassays for leukotrienes has revealed the generation of significant quantities of several leukotrienes by leukocytes exposed to natural stimuli in the absence of exogenous arachidonic acid. The C6 peptide leukotrienes, LTC4 and LTD4, are potent vasoactive and smooth muscle contractile factors. LTB4 and other di-HETEs stimulate polymorphonuclear leukocyte function and suppress T-lymphocyte activities. The effects of LTB4 on T lymphocytes are subset-specific and include the activation of suppressor and cytotoxic T cells. Receptors for LTB4 on polymorphonuclear leukocytes are specific but exhibit considerable heterogeneity of binding affinity and may mediate different functional effects. The complexity of the pathways of generation and metabolism of leukotrienes, and of the leukotriene receptors, suggest that carefully defined systems will be needed to test the effects of pharmacological inhibitors and antagonists.

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E 2 and F 2α, 6 keto PGF 1α and thromboxane B 2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10 −7M – 10 −3M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E 2 and F 2, and to a lesser extent, 6 keto PGF 1α, but T×B 2 production was only slightly inhibited by the drug in the absenced of arachidonic acid and markedly increased in its presence. Colchicine (10 −7M – 10 −3M) had the opposite effect, causing an inhibition of T×B 2 and stimulating PGE 2 and 6 keto PGF 1α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A 2 and cyclo-oxygenase, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO 4 labeling methodology. The results showed that the concentrations tested (10 −5M to 10 −7M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO 4 incorporation into newly synthesized proteoglycan molecules both in the presence (10 −6M) and absence of exogenous arachidonic acid. In the same concentration range choroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.

Arachidonic acid metabolism by neonatal lungs perfused with krebs bicarbonate buffer

We characterized and quantified products of exogenous arachidonic acid metabolism by neonatal lamb lungs perfused with Krebs solution and determined effects of alveolar hypoxia upon this metabolism. The predominant metabolite was PGI 2 (62.4 ± 5.8 (SEM)% of the total prostanoids (PGs) produced). An additional 23.1 ± 6.7% was metabolized PGI 2. PGE 2 (1.7 ± .3.0X) was produced also. Little thromboxane B 2 (TXB 2) (3.3 ± 1.7%) was synthesized by these platelet-free lungs. Although the pulmonary vasoconstrictor response to arachidonic acid infusion reached a plateau in 71 ± 19 seconds, the PG efflux was greater 8–15 minutes after beginning arachidonic acid infusion (3066 ± 1353 ng/min) than 1–8 minutes into the infusion (899 ± 343 ng/min). Reduction of F I O2 from 20% to 3% 02 which produces vasoconstriction in the absence of exogenous arachidonic acid, did not alter the PG profile nor quantity produced. With respect to the neonatal lamb pulmonary vasculature perfused with Krebs solution, we conclude; 1) exogenous arachidonic acid ultimately is metabolized predominantly to PGI 2, 2) arachidonic acid-induced pulmonary vasoconstriction is not caused by TXA 2, 3) short term hypoxia does not alter the conversion of excess arachidonic acid to PGs, and 4) a lag between intrapulmonary PG synthesis and pulmonary venous efflux may occur.