Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were also shown to stimulate PGE₂ and 6-keto-PGF_1α (the hydration product of PGI₂) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE₂ and PGI₂ (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occuring at approximately 10⁻⁷ M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments which have indicated that the compounds stimulated PGI₂ production. Finally, since the osteoblasts is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early time EGF stimulated PGE₂ release, however, the predominant effect of the growth factor was an inhibition of both PGE₂ and PGI₂ production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the esteoblast and that any increase in PG production by these cells may be in response to vascular agents     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

Influence of angiotensins (I,II, & III), bradykinin and arachidonic acid on renomedullary PGE production in vitro

Renomedullary tissue from rabbit or rat was incubated with angiotensin I, II, III, arachidonic acid, bradykinin, indomethacin and meclofenamate to study their effect on PGE₂ production.Arachidonic acid and bradykinin enhanced PGE₂ production significantly. Indomethacin and meclofenamate inhibited PGE₂ production by more than 70%. Angiotensin I, II and III did not influence PGE₂ production. These results suggest that bradykinin and arachidonic acid stimulate PGE₂ production by a direct cellular action whereas the angiotensins do not.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

Effect of various prostaglandins on the release of arachidonic acid from cultured fibroblasts

Effect of various prostaglandins on the release of arachidonic acid from [¹⁴C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F_2α was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF_2α(200.1%)>PGF_1α (141.8%)>PGD₂ (137.1%)>thromboxane B₂ (113.7%)>PGE₂ (109.4%). On the other hand, PGI₂ showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF_1α, an end metabolite of PGI₂, had no effect.     

Prostaglandin production in cultured gastric mucosal cells: Role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE₂, PGI₂ and thromboxaneA₂ (TXA₂) in amounts of 316±18, 100±7 and 30±5 pg per 10⁵ cells in 10 min, respectively, in response to 10μM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1–5mM) did not alter the amount of subsequent AA-induced PGE₂ production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE₂ synthesis, while it increased intracellular cAMP accumulation. Our studies suggest (1) AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, (2) AA-induced PG production is limited in these cells, and (3) it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Renal arachidonic acid metabolism and cellular changes in the rabbit renal vein constricted kidney: Inflammation as a common process in renal injury models

The process of renal inflammation was examined using the partial renal vein constricted rabbit kidney (RVC) as a model. Forty eight hours of partial renal vein constriction in the rabbit was associated with an increase in prostaglandin (PG) and thromboxane (Tx) production. The perfused RVC kidney showed an enhanced time-dependent increase in PG and Tx production in response to bradykinin stimulation when compared with the unlatered contralateral (CLK) or normal kidney. At 6 hrs of perfusion bradykinin stimulation lateral (CLK) or normal kidney. At 6 hrs of perfusion bradykinin stimulation released 2950±350 ng PGE₂, 61±15 ng TxB₂ from the RVC, and 225±85 ng PGE₂ and undetectable TxB₂ from the CLK. Histological examination of the RVC cortex showed an increase in fibroblast-like cells, a modest increase in the interstitial space and an appearance of macrophages and lymphocytes not seen in the normal of CLK. Endotoxin has been reported to stimulate macrophages in culture to produce PGE₂ and TxB₂. Endotoxin (100 ng)_stimulation of the perfused RVC kidney caused an immediate, followed by a chronically increasing, release of PGs and Tx. Two hours after endotoxin injection 50 ml of effluent fromt the RVC contained 1450±107 ng PGE₂ and 15.0±4.5 ng TxB₂. Other models of renal inflammation (e.g., the hydronephrotic kidney, chronic glomerulonephritis) also show the histological appearance of macrophages. In addition, hydronephrotic kidneys undergo fibroblast proliferation and changes in arachidonic acid metabolism similar to what we observed in the RVC. This work suggests that the inflammatory process (mononuclear cell infiltration), fibroblast-like cell proliferation, and accompanying changes in arachidonate metabolism) is common among different forms of renal injury.     

Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin

Because infiltration of mononuclear cells and fibroblast proliferation are associated in chronic inflammatory lesions, we tested the hypothesis that leukotrienes (LT), a product of activated mononuclear cells, may modulate fibroblast growth. Proliferation of cultured human skin fibroblasts was estimated by [3H]thymidine incorporation and cell count at increasing concentrations (0.1 nM to 0.1 microM) of LTC4 or LTD4. LTC4 and LTD4 stimulated cell growth in a dose-dependent manner only in the presence of 50 microM indomethacin. Under similar conditions, LTE4 but not LTB4 (0.1 microM) was active. Both asynchronous, growing cells and synchronous, quiescent cells were sensitive to LT when prostaglandin (PG) synthesis was suppressed by indomethacin. Other blockers of cyclooxygenase such as ibuprofen and aspirin exhibited identical permissive activity, and the effect of indomethacin was totally abolished by addition of PGE2. LTC4 modified neither [3H]arachidonic acid release from prelabeled fibroblasts nor PGE2 production by fibroblasts. These results demonstrate that the sulfidopeptide LT stimulate fibroblast proliferation only when the endogenous synthesis of PG is blocked, but they do not enhance the synthesis of PG in their target cells showing no evidence for a negative feed-back loop. Nevertheless, it seems likely that the initiation and development of the fibrotic process in the different tissues depends in part on the local balance between PG and LT productions.     

Production of arachidonic acid metabolites by operationally defined macrophage subsets

The antitumor activity and arachidonic acid metabolism of operationally defined macrophage populations was examined. Macrophages from mice injected with (strain BCG) or with pyran-copolymer were cytotoxic for tumor cells. The major arachidonic acid metabolite of these cells was PGE₂. Neither resident nor elicited macrophages were cytotoxic. However, elicited macrophages as well as macrophages from BCG injected mice inhibited tumor cell growth. The production of arachidonic acid metabolites by elicited cells, while low initially, was followed by a rapid increase in PGE₂. The major metabolites of resident cells were PGE₂ and prostacyclin. The cAMP:cGMP ratio correlated with the metabolic activity of the cells.     

Prostaglandin synthesis by cells comprising the calf pulmonary artery

Prostaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PGI2) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGI2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGI2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10(-12) M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10(-12) M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a "quiescent" nongrowth state.     

Prostaglandin controls Neuromuscular Transmission in Guinea-pig Vas Deferens

PROSTAGLANDINS of the E type (PGE₁, PGE₂) inhibit sympathetic neurotransmission in several tissues and species^1–4. On the basis of their natural occurrence and availability for release, as well as observations on the pharmacological actions of the PGs, endogenous PGE₁ and PGE₂ are postulated to operate on sympathetic neurotransmission by a feedback mechanism and thereby modulate the effector responses to nerve activity^{1, 5}. Inhibition by 5,8,11,14-eicosatetraynoic acid (ETA) of PG synthesis in the cat spleen and in the rabbit heart increases the release of noradrenaline (NA) in response to nerve stimulation, thus strongly supporting the hypothesis^{6, 7}. We report here that guinea-pig vas deferens releases PG in response to nerve stimulation and that the neuromuscular transmission is facilitated after inhibition of PG synthesis. PG synthesis was irreversibly inhibited using ETA⁸.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.     

Differences in types of prostaglandins produced by two MDCK canine kidney cell sublines

The pattern of prostaglandins produced from arachidonic acid by two sublines of MDCK canine kidney epithelia cells was different. In one subline designated MDCK₁, the most prevalent prostaglandin product was PGE₂, whereas the most prevalent product in the subline designated MDCK₂ was PGF_2α. This difference was observed when cells previously labeled with [1^?14C]arachidonic acid were stimulated with either bradykinin or the calcium ionophore A23187, or when prostaglandins were produced from labeled arachidonic acid added directly to the assay medium. In the latter case, the difference was maintained over a 38-fold range of extracellular arachidoante concentrations. These findings indicate the there is a persistent difference in the distribution of prostaglandins produced by the two commonly used sublines of MDCK cells.     

Sites of lipase activation and prostaglandin synthesis in isolated, perfused rabbit hearts and hydronephrotic kidneys

The tissue lipids of isolated, perfused rabbit hearts and hydronephrotic kidneys were labelled with [¹⁴C]-arachidonic acid by two different techniques: direct infusion of [¹⁴C]-arachidonic acid in a protein free media into the perfused organ (method A), and recirculation of [¹⁴C]-arachidonic acid in a solution containing albumin (method B). Autoradiography of the labelled organs demonstrated that method A resulted in selective labelling of arteries and arterioles in both perfused organs as well as glomeruli in the kidney. Labelling with method B resulted in a non-specific radioisotope incorporation in both the vasculature and myocardial cells in the heart; and of the vasculature and renal tubules in the perfused kidneys. Analysis of the tissue lipids shows similar patterns of incorporation of radioactivity between methods A and B.Peptide hormone stimulation (bradykinin) and non-specific noxious stimulation (with transient ischemia) were employed to elicit lipase activation (i.e., release of [¹⁴C]-arachidonate) and prostaglandin (PG) synthesis. It was found that in both hearts and hydronephrotic kidneys, the radioactive PG release in response to bradykinin and ischemia was much higher with method A (vascular labelling) than with method B (diffuse labelling) despite the appearance of comparable amounts of bioassayable PG release, thus indicating the sites of PG synthesis in these organs is predominantly localized in the vascular tissue. Furthermore, the radioactive arachidonic acid release in response to bradykinin stimulation in the hydronephrotic kidneys was 3 times higher with method A than with method B, suggesting the predominant sites of hormone specific lipase activation in the renal cortex is also in the vasculature. However, the radioactive arachidonic acid release in response to ischemia was much higher with method B than with method A in both hearts and hydronephrotic kidneys, indicating the sites of non-specific lipase activation in these organs are more diffusely distributed, and present also in the myocardial cells and renal tubules.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Dissociation of aldosterone and prostaglandin biosynthesis in the rat adrenal glomerulosa

The relationship between aldosterone production and prostaglandin E₂ synthesis was evaluated using the responses of isolated rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. Simultaneous PGE₂ and aldosterone measurements were made during timed incubations with these stimuli, and in incubations with arachidonic acid, meclofenamate, indomethacin, and aminoglutethamide. PGE₂ and aldosterone production were assessed by radioimmunoassay. We were not able to demonstrate stimulation of PGE₂ by angiotensin II, ACTH, or potassium despite significant increments in aldosterone production with these stimuli. Arachidonic acid enhanced PGE₂ synthesis, but had no effect on aldosterone release. Indomethacin and meclofenamate inhibited aldosterone secretion. Aminoglutethimide depressed aldosterone production, but had little effect on PGE₂ levels in the media.These studies demonstrate that dienoic prostaglandins play no direct role in aldosterone production stimulated by angiotensin II, ACTH, or potassium in rat adrenal glomerulosa cells. Since inhibitors of cyclo-oxygenase decreased aldosterone synthesis, it is possible that fatty acids other than arachidonic acid may be cyclo-oxygenated to products which regulate aldosterone production.     

Modification of upper gastrointestinal prostaglandin synthesis by dietary fatty acids

The effects of dietary iols on gastric, duodenal mucosa and liver were investigated ina rat model. Unsaturated fatty acid profles and in vitro prostaglandin (PG) synthesis (PGE₂, PGF_2α, 6-oxo-PGF_1α and thromboxane B₂). were measured after 14 days of dietary oil supplements.There were no significant differences in prostanoid synthesis between rats fed coconut oil (high saturated fat content) and standard diet. After fish oil supplement, tissue eicosapentaenoic acid and docosahexaenoic acid levels were higher, arachidonic acid levels were lower, and prostanoid synthesis was reduced in both stomach and duodenum. After corn oil and evening primrose oil, linoleic acid levels were variaby increased, bt there were no significant differences in arachidonic acid or prostanoid synthesis. Dihomogamma-linolenic acid levels were slightly increased after evening primrose oil.Dietary incorporation of fatty acids into gastroduodenal tissue is not uniform. When incorporated, fatty acids can modify prostaglandin synthesis.     

Effect of essential polyunsaturated fatty acid modifications on prostaglandins production by MDCK canine kidney cells

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE₂ release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9-times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglycerie fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE₂ release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE₂ accounting for 55 to 68% of the total output from [1-¹⁴C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE₂ formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. it is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substrate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids bocome highly enriched in arachidonate.