Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Periodate-induced lymphocyte transformation : II. Character of response and comparison with phytohemagglutinin and pokeweed mitogen stimulation

Sodium metaperiodate is mitogenic for human peripheral lymphocytes. Evidence of stimulation can be detected as increased thymidine incorporation at 72 h after only 10 sec of exposure to the IO₄⁻. The degree of response varies with lymphocytes from different donors, but maximum stimulation for the healthy donors studied was obtained at concentrations of IO₄⁻ between 10⁻³ M and 4 × 10⁻³ M. Concentrations of 8 × 10⁻³ M and above are non-stimulatory and toxic. Exposures to optimum concentrations for 1 h or longer result in essentially no stimulation and inincreased cell death. However, a significant response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) remains. The kinetics of response over a 4 day culture period are similar for IO₄⁻, PHA and PWM. The morphology of the blast cells and the degree of response suggest that the IO₄⁻ responsive lymphocyte population may be more closely related to the PWM stimulated cells than the PHA responsive lymphocytes.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

The effect of arachidonic acid and prostanoids on collagen dissolution in human uterine cervix in vitro

Explants of human non-pregnant cervix produce collagenolytic enxymes which degrade collagen over a 10 day period in culture. This is significantly enhanced by the presence of very low concentrations of arachidonic acid (10⁻¹⁶−10⁻¹¹M). Prostaglandin E₂, F_2α and 6-keto-F_1α were synthesised in declining amounts over the 10 day period and synthesis was not increased by adding arachidonic acid (10−11M). Meclofenamic acid (10⁻⁶M) and indomethacin (10⁻⁵M), but not tranylcypromine (10⁻⁵) suppressed prostaglandin synthesis yet all reduced collagen dissolution. Mepacrine (phospholipase A₂ inhibitor) also suppressed collagen dissolution. Remodelling of the structure of the cervix matrix may, in part, depend upon arachidonic acid or one of its cyclo-oxygenase or lipoxygenase derived products.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

The effect of progesterone on the release of arachidonic acid from human endometrial cells stimulated by histamine

Progesterone at concentrations of 10⁻⁷M and 10⁻⁸M inhibits release of [³H]-arachidonic acid from stimulated, perfused, endometrial cells. The effect is independent of the mechanism of stimulation. Cortisol (10⁻⁵M but not 10⁻⁷M) has a similar effect in this system but estradiol (10⁻⁷M) is without effect. There was a positive correlation (p<0.05) between the magnitude of inhibition by progesterone and the day of cycle. The inhibitory action of progesterone on the release of arachidonic acid was greater in endometrial cells than in decidual cells and was apparent after fifteen minutes. The activities of commercial and endometrial cell-free preparations of phospholipase A₂ and phospholipase C were unaffected by the presence of progesterone. We conclude that progesterone modulates release of [³H]-arachidonic acid from endometrial cells by a rapid, indirect action on phospholipase activity.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (³H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-³H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

Functional incorporation of the human erythrocyte chloride exchange system into plasma membranes of Friend erythroleukemic cells by Sendai virus-induced cell fusion

Human erythrocytes (RBC) were shown to exchange Cl⁻ by an exceptionally fast mechanism ( of ³⁶Cl⁻ equilibration at 1 °C is approx. 20 sec) which is demonstrably susceptible to specific inhibitors of anion exchange such as 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS) and 4,4′-diisothyocyano-2,2′stilbene disulfonic acid (DIDS). Friend erythroleukemic cells (FELC) on the other hand, display both markedly slower Cl⁻ exchange rates ( of ³⁶Cl⁻ equilibration at 1 °C is approx. 60 min) and substantially lower susceptibilities to either DNDS or DIDS than RBC. After fusion between RBC and FELC, Cl⁻ exchange across FELC-RBC plasma membranes was noticeably enhanced compared with FELC. This enhancement was specificially abolished either by the addition of DNDS or by fusing FELC with DIDS-treated RBC.     

Biphasic effects of nonsteroidal anti-inflammatory drugs on prostaglandin production by cultured rat calvariae

We have studied the effects on bone of three structurally dissimilar non-steriodal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE₂ production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE₂ release into the medium by 80% at 10⁻⁸ M, while flurbiprofen and piroxicam produced similar inhibition at 10⁻⁶ M. However, at 10⁻¹⁰ M, treatment with all three compounds resulted in an increase in medium PGE₂ concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [³H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10⁻⁶ M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10⁻¹⁰ M, there was a substantial increase in labeled products, particularly PGE₂, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE₂ release both in control cultures and cultures which had been incubated with cortisol (10⁻⁸ M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10⁻⁵ M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.     

The effects of somatostatin on the arachidonate cascade of platelets

Somatostatin (10⁻⁹ M) significantly elevated the synthesis of thromboxane B₂ in rat platelets. The transformation of arachidonic acid to active lipoxygenase metabolites was suppressed by somatostatin (10⁻⁹ and 10⁻⁸ M). The ratio of the lipoxygenase/cyclooxygenase products was significantly reduced by the polypeptide (10⁻⁹ and 10⁻⁸ M) in rat platelets. Higher concentrations (10⁻⁷, 10⁻⁶ and 10⁻⁵ M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor — proaggregatory cyclooxygenase products was stimulated by the polypeptide (10⁻⁹ and 10⁻⁸ M), while the formation of vasodilatator - antiaggregatory cyclooxygenase metabolites was induced by higher concentrations of somatostatin (10⁻⁷ and 10⁻⁶ M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose.     

Stimulation of DNA synthesis and mitosis of hepatocytes in primary cultures of neonatal rat liver by arachidonic acid and prostaglandins

At low concentrations (i.e. 10⁻¹²–10⁻⁹ mol/l) arachidonic acid intensely stimulated both DNA synthetic and mitotic activities of hepatocytes in 4-day-old primary cultures of neonatal rat liver. This effect of arachidonate was completely suppressed by the simultaneous administration to the cultures of a high dose (i.e. 10⁻⁴ mol/l) of indomethacin. A similar, but much weaker proliferogenic activity was exerted on neonatal hepatocytes by quite low concentrations of some of the main products of arachidonic acid metabolism, namely prostaglandins A₁, E₁, and E₂. Although these data support the possibility that arachidonate and prostaglandins are involved in the regulation of hepatocytic proliferative activation, the exact role of prostaglandins remains to be ascertained, because such agents might as well have acted by inducing intracellular surges of known mitogenic compounds, such as cAMP and cGMP.     

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca²⁺]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca²⁺]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca²⁺ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca²⁺]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca²⁺]i, with NDGA exerting a greater stimulation of [Ca²⁺i than indomethacin. Prostaglandin F_2α (PGF_2α) as well as prostaglandin E₂ (PGE₂) increased [Ca²⁺ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca²⁺]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF_2α and PGE₂) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca²⁺]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

The mechanism of enhancement by fatty acid hydroperoxides of anaphylactic mediator release

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml⁻¹). Both 15-HPAA (1–20 μg ml⁻¹ min⁻¹) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml⁻¹ min⁻¹) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml⁻¹ min⁻¹) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml⁻¹ min⁻¹). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml⁻¹ min⁻¹) but was inhibited by PGE₂ (5 and 10 μg ml⁻¹ min⁻¹). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pig

Pulmonary responses to intravenous leukotrienes C₄, D₄ and E₄ administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD₄, LTC₄ and LTE₄ (respective ED₅₀ of 0.21 ± .1, 0.64 ± .2 and 2.0 ± .1 μg kg⁻¹) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-Rössler). Bronchoconstriction was antagonized by FPL-55712 (50–200 μg kg⁻¹), and indomethacin (50–200 μg kg⁻¹) but was not significantly altered by mepyramine (1.0 mg kg⁻¹), methysergide (0.1 mg kg⁻¹), intal (10 mg kg⁻¹) mepacrine (5 mg kg⁻¹) or dexamethasone (10 mg kg⁻¹). The beta adrenoceptor blocker, timolol (5 μg kg⁻¹) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE₄ but not LTD₄ or LTC₄ were partially antagonized by atropine (100 μg kg⁻¹) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD₄ and LTE₄ (2.8–3.2 μg kg⁻¹ min⁻¹) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE₄ and LTD₄ was partly reversed by intravenous FPL-55712 (1.0 mg kg⁻¹) and atropine (100 μg kg⁻¹) but was almost completely reversed by FPL-55712 (3 – 10 mg kg⁻¹). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE₄ as well as a small part of the initial response to continuous infusion of LTD₄ and LTE₄. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

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

The effect of various anti-inflammatory drugs on the production of prostaglandins E₂ and F₂α, 6 keto PGF₁α and thromboxane B₂ 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 E₂ and F₂, and to a lesser extent, 6 keto PGF₁α, but T×B₂ 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×B₂ and stimulating PGE₂ and 6 keto PGF₁α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A₂ 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 ³⁵SO₄ labeling methodology. The results showed that the concentrations tested (10⁻⁵M to 10⁻⁷M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited ³⁵SO₄ 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.