Prostaglandin synthesis in isolated glomeruli

Prostaglandins are thought to play an important role in the local regulation of glomerular blood flow and in the release of renin from the juxtaglomerular apparatus. We therefore examined prostaglandin synthesis by isolated rat glomeruli. Isolated glomeruli were either prelabeled with [14_C] arachidonic acid or were incubated with [14_C] arachidonic acid for the entire experimental incubation in Krebs buffer. Prostaglandin synthesis was determined by thin layer radiochromatography of acid extracts of the supernatant solutions. Indomethacin inhibitable synthesis of small amounts of 6-keto-PGF_1α, the metabolite of prostacyclin(PGI₂,) and larger amounts of PGF_2α, and PGE₂, and possibly thromboxane B₂ (TXB₂) by isolated glomeruli could be demonstrated with either prelabeling or direct incubation. These findings support the hypothesis that prostaglandins are produced within the glomerulus where they may affect local glomerular blood flow and function.     

Prostaglandin synthesis by human glomerular cells in culture

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable. Mesangial cells produced low amounts of PGE2, PGF2 alpha and 6 keto-PGF1 alpha and no TXB2. We also examined the effects of several agents on PG synthesis in these two types of cells scraped away from their flasks using direct RIA. Arachidonic acid produced a slight stimulation only with mesangial cells whereas angiotensin II, cyclic AMP and calcium ionophore were inactive with both cell lines. Homogenization of the cells did not enhance the stimulatory effect of arachidonic acid. Alkalinization of the incubation medium produced an increase of PG production by mesangial cells. These results suggest that two types of human glomerular cells, particularly epithelial cells, possess low cyclooxygenase activity. The low capacity of human mesangial and epithelial cells to produce PG may have consequences for the endocrine control of the glomerular microcirculation in man.     

Collagen synthesis and secretion by isolated rat renal glomeruli.

Glomeruli were isolated from rat renal cortex and incubated with radioactive lysine to study in vitro collagen synthesis in these preparations. Glomerular basement membrane was obtained by sonication, and the appearance of [-14C]lysine and hydroxylysine in medium, membrane and intracellular proteins was determined. Total glomerular incorporation of [-14C]lysine into protein linearly increased for up to 2-h period, and membrane hydroxylysine content gradually rose during this time. Hydroxy[-14C]lysine was recovered in the 105 000 times g pellet, reaching a hydroxylysine content of 22 percent in this intracellular fraction after 90 min of incubation. 60 percent of the protein secreted into the medium, and about 75 percent of newly synthesized sonicated basement membrane was acetic acid soluble. Hydroxylysine content was 33 percent in the acetic acid-insoluble fraction of sonicated membrane, suggesting that basement-membrane collagen was a significant component of total collagen synthesized by these preparation, The ability of isolated glomeruli to synthesize and secrete basement-membrane protein will be useful for studies concerning control of glomerular collagen and basement-membrane synthesis.     

Analysis of prostaglandins formed from endogenous and exogenous arachidonic acid in homogenates of human reproductive tissues

Isotope-labelled arachidonic acid has been used to study in vitro formation of prostaglandins and other products in mammalian tissue. Quantitative conclusions about cyclooxygenase activity have been drawn from such studies. However, arachidonic acid is present in all tissues, free and esterified, and therefore it can be expected that endogenous arachidonate would interfere with transformation of the radioactive exogenous substrate. (1-14C)-labelled arachidonate was, therefore, incubated with homogenates of various human tissues (amnion, chrorion, placenta and myometrium), and the two molecular forms, 12C and 14C, of arachidonic acid as well as of prostaglandin E2 and prostaglandin F2 alpha were quantitated, before and after 30 min of incubation, using gas chromatography-mass spectrometry with multiple ion detection. The results demonstrate a substantial release of arachidonic acid into the medium during incubation. There was no correlation between either the initial concentration of [12C]arachidonic acid and initial concentration of [12C]prostaglandin E2 or the percent increase of those compounds during incubation. The net formation of [12C]prostaglandin E2 and [14C]prostaglandin E2 from endogenous and exogenous precursor, respectively, were also very different. The study shows that by simply incubating (1-14C)-labelled arachidonic acid in tissue homogenates and measuring the amount of radioactivity transformed into various prostaglandins only qualitative conclusions can be drawn.     

Arachidonic acid metabolites mediate the radiation-induced increase in glomerular albumin permeability

Radiation-induced renal injury is characterized by proteinuria, hypertension, and progressive decline in renal function. We have previously shown that in vivo or in vitro irradiation of glomeruli with a single dose of radiation (9.5 Gy) increases glomerular albumin permeability (P(alb)) within 1 hr. The current studies tested the hypothesis that this early radiation-induced increase in P(alb) is caused by the release of arachidonic acid and by the generation of specific arachidonic acid metabolites. Glomeruli obtained from WAG/Rij/MCW rats and cultured rat glomerular epithelial and mesangial cells were studied after irradiation (9.5 Gy, single dose). Arachidonic acid release and eicosanoid synthesis by glomeruli or cultured glomerular cells were measured after irradiation, and the effect of inhibitors of phospholipase A2 (PLA2) and cyclooxygenase (COX) on the irradiation-induced increase in P(alb) was assessed. Arachidonic acid release was demonstrated within 10 mins of irradiation of isolated glomeruli and monolayer cultures of glomerular epithelial and mesangial cells. Prostaglandin F(2alpha) (PGF(2alpha)) and PGE2 release was increased after irradiation of isolated glomeruli. Blocking arachidonic acid release or COX activity before irradiation completely prevented the increase in P(alb). COX inhibition immediately after irradiation also diminished the radiation-induced increase in P(alb). We conclude that arachidonic acid and its COX metabolites play an essential role in the early cellular changes that lead to the radiation-induced increase in P(alb). Understanding of the early epigenetic effects of irradiation may lead to new intervention strategies against radiation-induced injury of normal tissues.     

Atrial natriuretic factor does not stimulate prostaglandin synthesis in isolated rat glomeruli

The effect of alpha-human atrial natriuretic polypeptide (alpha-hANP) on the synthesis of prostaglandins was studied in isolated rat glomeruli. Glomeruli were isolated by a passive sieving technique according to the method of Misra and were incubated at 37 degrees C for 60 min in the presence (Group II: 10(-6) M, Group III: 10(-5) M) or absence (Group I: control) of alpha-hANP. Furthermore, glomeruli were incubated with arachidonic acid (10(-5), 10(-4), and 10(-3) M) and at the end of the incubation period trypan blue was added to the glomerular suspension. The presence of alpha-hANP (10(-6) and 10(-5) M) caused no significant difference in prostaglandin synthesis as compared with the control. On the other hand, arachidonic acid stimulated prostaglandin synthesis and the glomerular preparation was not stained by trypan blue, indicating that they remained viable. These results suggest that alpha-hANP does not directly affect the prostaglandin synthesis in isolated rat glomeruli.     

Hydroperoxy fatty acid formation in selenium deficient rat platelets: coupling of glutathione peroxidase to the lipoxygenase pathway

After incubation with [1-14C]-arachidonic acid, washed platelets from selenium deficient rats produced a sevenfold greater amount of 12-hydroperoxytetraenoic acid than platelets from control animals. When stimulated with either arachidonic acid or t-butyl-hydroperoxide, antimycin-A1 treated platelets from the deficient rats also converted markedly lower amounts of [1-14C]-glucose to [14C]-CO2 than platelets from control rats. These results indicate a significant role for platelet selenium-dependent glutathione peroxidase in the enzymatic reduction of platelet-produced hydroperoxides.     

Effect of dexamethasone on prostaglandin synthesis and on lipocortin status in human endothelial cells. Inhibition of prostaglandin I2 synthesis occurring without alteration of arachidonic acid liberation and of lipocortin synthesis

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.     

Effects of superovulation, arachidonic acid or endometrium on release of prostaglandins and synthesis of proteins by bovine trophoblast

This study was conducted in vitro to examine factors that may regulate prostaglandin release by bovine trophoblast and endometrial slices. Trophoblastic tissues and endometrial slices were recovered from superovulating and normally-ovulating cattle on day 16 or 20 of pregnancy and incubated for 24 h. Release of PGF2 alpha and 13,14-dihydro-15-keto-PGF2 alpha (PGFM), and incorporation of [14C]-leucine into proteins were quantified and expressed per microgram DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P less than .05) and for releasing PGFM was increased (P less than .05) on day 20 compared to day 16 of pregnancy. Neither superovulation nor day of pregnancy altered trophoblastic activity for releasing PGF2 alpha. Superovulation increased (P less than .05) endometrial release of PGF2 alpha. Endometrial release of PGF2 alpha was less (P less than .05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 micrograms) was added at the start of incubation, trophoblastic release of PGF2 alpha changed (P less than .05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, trophoblastic release of PGF2 alpha increased linearly (P less than .01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P less than .05) trophoblastic protein synthesis and release of PGF2 alpha. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from superovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Hormonal stimulation of arachidonate release from isolated perfused organs. Relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labelled with [14C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [14C]prostaglandins; little or no release of the precursor [14C]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [14C]arachidonic acid was detected following hormonal stimulation. The release of [14C]arachidonic acid was dose-dependent and greater than 3 fold that of [14C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [14C]prostaglandins and only slightly inhibited release of [14C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.     

Inhibition of microcystin-induced release of cyclooxygenase products from rat hepatocytes by anti-inflammatory steroids

We showed previously that exposure to microcystin causes eicosanoid release. That study was extended further to test the effect of glucocorticoids on microcystin-induced release of [14C]arachidonic acid and its metabolites from rat hepatocytes previously treated with [14C]arachidonic acid. Release of total radioactivity was 4-fold greater from hepatocytes after 2-hr incubation with 1 microM microcystin than after incubation with control medium. Fluocinolone pretreatment decreased the microcystin-induced synthesis and release of prostacyclin by 24 +/- 2.6% (P less than 0.05) and thromboxane B2 by 39 +/- 3% (P less than 0.025). Treatment of hepatocyte cultures with either microcystin (1 microM) or steroids had no effect on cell viability or total cell protein. Total radioactivity released into the incubation medium was not affected by glucocorticoid alone. Under these conditions, the quantities of both prostaglandin F2 alpha and prostaglandin E2 released were not significantly different when control and microcystin-treated cultures were compared. The half-maximal inhibition (IC50) values obtained from the dose-response data for the inhibition of arachidonic acid release by steroids were comparable with normal cortisol levels in humans. Dose-response curves gave the following rank order of inhibitory potency: fluocinolone greater than dexamethasone greater than hydrocortisone. These results suggest that glucocorticoid therapy might be beneficial in microcystin toxicosis.     

Renal cyclo-oxygenase and lipoxygenase products in health and disease

Renal glomeruli have cyclo-oxygenase and lipoxygenase enzymes which convert arachidonic acid to prostaglandins, thromboxane and 12-hydroxyeicosatetraenoic acid. Glomerular epithelial and mesangial cells, in culture, also synthesize these arachidonate products. Angiotensin and vasopressin contract mesangial cells and stimulate mesangial synthesis of PGE2. PGE2, in the glomerulus, antagonizes the actions of angiotensin on the mesangium and hence reduces angiotensin-mediated glomerular contraction. Glomerular immune injury (nephrotoxic serum nephritis) augments glomerular production of prostaglandins and thromboxane. Thromboxane reduces glomerular function and inhibition of thromboxane synthesis preserves glomerular filtration rate and renal plasma flow in this disease model. Spontaneously hypertensive rats also have enhanced glomerular prostaglandin and thromboxane synthesis. Although acute inhibition of thromboxane synthesis will vasodilate the hypertensive rat kidney, chronic inhibition does not reduce blood pressure or increase renal blood flow.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Protein synthesis in isolated rabbit forelimb muscles. The possible role of metabolites of arachidonic acid in the response to intermittent stretching.

Protein synthesis was measured in isolated intact rabbit muscles by the incorporation of [3H]phenylalanine added at a high concentration (2.5 mM) to the incubation medium. Intermittent mechanical stretching substantially increased the rate of protein synthesis relative to that in control muscles incubated under a constant tension. Indomethacin and meclofenamic acid, inhibitors of the enzyme cyclo-oxygenase, which converts free arachidonic acid into the prostaglandins, prostacyclins and thromboxanes, decreased the rate of protein synthesis in intermittently stretched muscles, but had no effect on synthesis rates in the unstimulated controls. Arachidonic acid at concentrations of 0.2 and 1.0 microM gave a highly significant increase in the rate of protein synthesis in muscles incubated under a constant tension. The ability of arachidonic acid to increase protein-synthesis rates was abolished by the addition of indomethacin. Activation of protein synthesis by intermittent stretching persisted for 10-20 min after the stretch stimulation had ceased. Indomethacin, added either during the initial incubation with intermittent stretching or during the subsequent period when protein synthesis was measured after stimulation had ceased, decreased protein-synthesis rates. This decrease was similar whether indomethacin was present during the initial, final or entire incubation period. In experiments analogous with those in (4) above, when Ca2+ was withheld and EGTA added for the entire incubation, rates of protein synthesis were again decreased. The rates of protein synthesis observed when Ca2+ was present during either an initial stimulation phase or a final, unstimulated, measurement phase were similar, and were intermediate between control rates and those in muscles incubated without Ca2+ for the whole experiment. Two prostaglandins, F2 alpha (2.8 microM) and A1 (28 microM), increased rates of protein synthesis in unstimulated muscles, but prostaglandins E2 and D2 and the leukotrienes C4 and D4 failed to do so. It is concluded that the stretch-stimulated increase in protein synthesis may be caused by activation of membrane phospholipases, release of arachidonic acid and a consequent increase in prostaglandin synthesis.     

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

Hormonal stimulation of arachidonic release from isolated perfused organs relationship to prostaglandin biosynthesis

The lipids of isolated Krebs perfused rabbit kidneys and hearts were labeled with [¹⁴C]arachidonic acid. Subsequent hormonal stimulation (e.g. bradykinin, ATP) of the pre-labelled tissue resulted in dose-dependent release of [¹⁴C]prostaglandins; little or no release of the precursor [¹⁴]arachidonic acid was observed. When fatty acid-free bovine serum albumin was added to the perfusion medium as a trap for fatty acids substantial release of [¹⁴C]arachidonic acid was detected following hormonal stimulation. The release of [¹⁴C]arachidonic acid was dose-dependent and >;3 fold that of [¹⁴C]prostaglandin release. Indomethacin by inhibiting the cyclo-oxygenase, completely inhibited release of [¹⁴C]prostaglandins and only slightly inhibited release of [¹⁴C]arachidonic acid. These results demonstrate that in both rabbit kidney and heart much more substrate is released by hormonal stimulation than is converted to prostaglandins. This suggests that either the deacylation reaction is not tightly coupled to the prostaglandin synthetase system or that there are two deacrylation mechanisms, one which is coupled to prostaglandin synthesis while the other is non-specific. It has previously been shown that prostaglandin release due to hormones such as bradykinin is transient despite continued presence of the hormone (tachyphylaxis). By utilizing albumin to trap released fatty acid, it was found that hormone-stimulated release of arachidonic acid is also transient. This directly demonstrates that tachyphylaxis occurs at a step prior to the cyclo-oxygenase.     

Arachidonic acid metabolic pathways in the rabbit pericardium

Minced rabbit pericardium actively converts [1-14C]arachidonic acid into the known prostaglandins (6-[1-14C]ketoprostaglandin F1 alpha, [1-14C]prostaglandin E2 and [1-14C]prostaglandin F2 alpha) and into several unidentified metabolites. The major metabolite was separated by C18 reverse-phase high-pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) to be 6,15-[1-14C]diketo-13,14-dihydroprostaglandin F1 alpha. The other nonpolar metabolites were 15-[1-14C]hydroxy-5,8,11,13-eicosa-tetraenoic acid (15-HETE), 11-[1-14C]hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) and 12-[1-14C]hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Arachidonic acid metabolites actively produced by the pericardium could influence the tone of surface blood vessels on the myocardium.