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 [14C] arachidonic acid or were incubated with [14C] arachidonic acid for the entire experimental incubation in Krebs buffer. Prostaglandin synthesis was determined by thin layer radio-chromatography of acid extracts of the supernatant solutions. Indomethacin inhibitable synthesis of small amounts of 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2,) and larger amounts of PGF2 alpha, 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.     

In vitro prostaglandin synthesis by human glomeruli and papillae

We have investigated in vitro prostaglandin synthesis by human isolated glomeruli and papillary homogenates and compared the results with those obtained in parallel studies using rat material. Prostaglandins were measured by two methods, namely radiometric high performance liquid chromatography after incubation with ¹⁴C arachidonic acid and radioimmunoassay. The relative abundance of various prostaglandins synthesized by glomeruli was different in man (6 keto PGF_1α > TXB₂ > PGF_2α > PGE₂) and in the rat (PGE₂ TXB₂ > 6 keto PGF_1α). Unidentified peaks eluting between 6 keto PGF_1α and TXB₂ were observed only in rat glomeruli. These peaks were suppressed by indomethacin. Direct radioimmunoassay of prostaglandins in the incubation medium of human glomeruli confirmed the predominance of 6 keto PGF_1α synthesis and showed its stimulation by arachidonic acid, its progressive decrease with time and its linear relationship with glomerular protein at low concentrations. On the contrary, the profile of prostaglandin synthesis by the papilla was similar in man and in the rat, PGE₂ and PGF_2α being the major products in both species. However, related to one mg of protein, papillary synthesis of these two prostaglandins was greater in the rat. These results show that PGI₂ is the major prostaglandin synthesized in human glomeruli and suggest a role for this prostaglandin in glomerular physiology in man.     

14C-labeled arachidonic acid bioconversion in guinea pig placenta during the last third of gestation

In the presence study we investigated the arachidonic acid metabolism in guinea pig placenta during the last third of gestation. Homogenates were incubated with 14C-labeled subtrate, and eicosanoid formation was determined using rp HPLC. Arachidonic acid was substantially converted to cyclooxygenase products i.e.-keto-PGF_1α, TxB₂, PGF_2α, PGE₂, PGD₂ and 12-HHT. Lipoxygenase activity was also found but of a much lower degree and represented by the mono-hydroxy acid 12-HETE and 15-HETE. The total conversion of arachiodonic acid exhibited a progressive rise from day 50 to term, due principally to the increasing part of TxB₂, PGE₂ and 12-HHT throughout this gestational perid and in addition, near term, of 6-keto-PGF_1α and PGF_2α. The results suggest that there is an increasing concentration and/or activity of cyclooxygenase system enzymes with placenta development in guinea pig, which may contribute to the augmented intrauterine availability of prostanoids under parturition.Additional experiments were performed to compare the metabolism of exogenously added 14C-arachidonic acid and endogenously present 12C-arachidonic acid during placental homogenate incubation by means of isotopes dilution GC-MS. Although the 14C- and 12-C prostanoid patterns were comparable, the 14C/12C ratios of the prostanoids formed during incubation were significantly different. These data indicate that exogenous arachidonic acid and endogenous arachidonic acid in placental homogenate do not follow up extractly the same metabolic pathway so that assumption of biochemical identity between exogenous radio-tracer and studied endogenous substrate is not quite true.     

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI₂. The PGI₂ synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE₂ and PGF_2α were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI₂ was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI₂ are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF_1α isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE₂, PGF_2α and 6-keto PGF_1α formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI₂, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.     

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 PGF_2^α and 13,14-dihydro-15-keto-PGF_2^α (PGMF), and incorporation of [¹⁴C]-leucine into proteins were quantified and expressed per μg DNA, which gives a measure of cellular activity. Activity of trophoblastic tissue for synthesizing protein was decreased (P<.05) and for releasing PGMF was increased (P<.05) on day 20 compared to day 16 of pregnancy. Neither supercovulation nor day of pregnancy altered trophoblastic activity for releasing PGF_2^α. Supercovulation increased (P<.05) endometrial release of PGF_2^α. Endometrial release of PGF_2^α was less (P<.05) on day 20 than on day 16 of pregnancy. When arachidonic acid (0, 100, 200 or 400 μg) was added at the start of incubation, trophoblastic release of PGF_2^α changed (P<.05) quadratically with dose of arachidonic acid. When arachidonic acid was added 8 h after the start of incubation, triphoblastic release of PGF_2^α increased linearly (P<.01) with dose of arachidonic acid. Adding arachidonic acid to incubation medium did not affect trophoblastic or endometrial protein synthesis. Endometrial slices suppressed (P<.05) trophoblastic protein synthesis and release of PGF_2^α. Apparently, endometrium can modulate trophoblastic release of prostaglandins and synthesis of proteins in vitro, and trophoblastic tissue from supercovulated cattle 16 or 20 days pregnant can be used to study trophoblastic synthesis of prostaglandins and proteins.     

Metabolism of [C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

The pericardium as a source of prostacyclin in the dog, ox and rat

Cyclo-oxygenase products of arachidonic acid metabolism formed by the pericardium and epicardial surface of dog heart were identified and quantitated by radioimmunoassay after separation by high-pressure liquid chromatography. Pieces of pariental pericardium, of dog, ox and rat, when incubated produced mainly 6-keto-PGF_1α, with lesser amounts of PGE₂, PGF_2α and thromboxane B₂. Biosynthesis of all prostanoids increased during incubation of the pariental pericardium of each species with arachidonic acid, but 6-keto-PGF_1α was still the major metabolite. When slices of dog heart were incubated with arachidonic acid (1 μg/ml) the rates of 6-keto-PGF_1α formation by the pariental pericardium was much greater than that of the myocardium and endocardium. Epicardial slices appeared to be intermediate in 6-keto-PGF_1α formation. The hearts of anesthetized dogs were also irrigated with Krebs' solution, and during the first 5 min of epicardial irrigation the pericardial fluid leaving the heart again contained high levels of 6-keto-PGF_1α, with lesser amounts of the other prostanoids. Addition of arachidonic acid (3 μg/ml) to the irrigating fluid caused an increase in all measured prostanoid levels, although 6-keto-PGF_1α remained the predominant metabolite. In contrast, intravenous infusion of isoproterenol selectively increased the release of 6-keto-PGF_1α from the irrigated heart. It is concluded that the pericardium and epicardium continuously release prostacyclin into the pericardial fluid, and that the increased release of this substance observed when cardiac workload increases derives mainly from these membranous sources. This raises the interesting possibility that pericardial prostacyclin might influence coronary vascular tone and chemoreflexes which arise from the epicardium during myocardial ischemia.     

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.     

Prostaglandin production from homogenates of separated glandular epithelium and stroma from human endometrium

The biosynthesis of prostaglandins by isolated epithelial glandular and stromal cells was studied after collagenase digestion of endometrium collected from women at various stages of the menstrual cycle. Homogenates of the separate cell types were incubated for 60 minutes with 2.08 μg 1¹⁴C arachidonic acid and the products separated by thin layer chromatography. Both glandular and stromal homogenates synthesised PGF_2α. More PGF_2α was synthesised by glandular epithelium separated from both proliferative and secretory endometrium than by stroma. The ratio of PGF_2α/PGE₂ was greater in glands and stroma isolated from secretory than proliferative endometrium. Small but significant amounts of 6-keto-PGF₁α were produced by all cell types. These results suggest that the increased synthesis of PGF₂α from secretory endometrium is due, at least in part, to increased activity of cyclo-oxygenase enzyme in the glandular epithelium.     

Effects of 17 β estradiol on the metabolism of labelled arachidonic acid in uteri isolated from intact and ovariectomized rats. Influence of a restricted diet

The effects of restricted diet (50% of the normal intake during 25 days) on the metabolism of ¹⁴U arachidonic acid, were explored in uterine horn strips isolated from intact and ovariectomized rats, treated by 17 β-estradiol or controls. The metabolism of arachidonic acid into different eicosanoids, PGE₂, PGF_2α, 6-keto PGF_1α and TXB₂, showed that the restricted diet diminished PGE₂ and PGF_2α, in intact rats, significantly. In contrast, this kind of feeding did not produce any change in castrated rats.Tissue preparations from previously estrogenized intact and castrated normal-fed rats showed that the production of different metabolites decreased. A similar result was obtained in intact rats subjected to a restricted diet. Nevertheless, in castrated underfed rats, estrogens did not produce any effect on the various eicosanoids analysed.These results showed that in isolated uteri, the effects of 17 β-estradiol, on metabolite production from labelled arachidonic acid, are different from controls in ovariectomized diet-restricted rats.     

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.     

Transformation of steroids by Bacillus strains isolated from the foregut of water beetles (Coleoptera:Dytiscidae): I. Metabolism of androst-4-en-3,17-dione (AD)

Two Bacillus strains were isolated from the foregut of the water beetle Agabus affinis (Payk.) and tested for their steroid transforming ability. After incubation with androst-4-en-3,17-dione (AD), 13 different transformation products were detected. AD was hydroxylated at C₆, C₇, C₁₁ and C₁₄, resulting in formation of 6β-, 7α-, 11α- and 14α-hydroxy-AD. One strain also produced small amounts of 6β,14α-dihydroxy-AD. Partly, the 6β-hydroxy group was further oxidized to the corresponding 6-oxo steroids. In addition, a specific reduction of the Δ⁴-double bond was observed, leading to the formation of 5α-androstane derivatives. In minor yields the carbonyl functions at C₃ and C₁₇ were reduced leading to the formation of 3ξ-OH or 17β-OH steroids. EI mass spectra of the trimethylsilyl and O-methyloxime trimethylsilyl ether derivatives of some transformation products are presented for the first time.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Synthesis of prostaglandins by the ductus arteriosus of the bovine fetus

Previous studies demonstrated that prostaglandins are local or tissue hormones which can be released from blood vessel walls. In the present study, we investigated the capacity of bovine ductus arteriosus to synthetize prostaglandins . After incubation of slices of ductus arteriousus in Krebs' solution with (1-¹⁴C) arachidonic acid for 3 hours, more than 40% of the radiolabeled material recovered from the incubating medium were metabolites of arachidonic acid. The major product was indistinguisable from 6 keto-PGF_1α as determined by its chromatographic mobility and resistance to alkaline conversion to PGB.The PGI₂ synthetic capacity of the ductus arteriosus, as revealed by the predominance of its major metabolite 6 keto-PGF_1α, suggests that this metabolic pathway of arachidonic acid may contribute to the hemodynamic changes occurring during fetal life and at birth.     

Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions

The effects of gibberellic acid (GA₃) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA₃ or Ca²⁺ show qualitative and quantitative changes in hydrolase production following incubation in either GA₃ or Ca²⁺ or both. Incubation in H₂O or Ca²⁺ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA₃ to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca²⁺ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA₃ and Ca²⁺ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA₃ or GA₃ plus Ca²⁺. The synthesis of α-amylases 3 and 4 requires the presence of both GA₃ and Ca²⁺ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA₃ and Ca²⁺, while only one group of antigens is found in media of layers incubated in GA₃ alone. Strontium ions can be substituted for Ca²⁺ in increasing hydrolase production, although higher concentrations of Sr²⁺ are required for maximal response. We conclude that GA₃ is required for the production of α-amylase 1 and that both GA₃ and either Ca²⁺ or Sr²⁺ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.     

Altered PGE2 production by glomeruli and papilla of rats with hereditary diabetes insipidus

PGE₂ synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of Brattleboro heterozygous control rats. Incubations were carried out in isotonic buffer at 37°C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE₂ production was measured in the supernatant by specific radioimmunoassay. Results were compared by analysis of variance. PGE₂ production was significantly decreased in the papilla (p < 0.01) and increased in the glomeruli (p < 0.01) of DI rats compared to controls. Stimulation by arachidonic acid was similar in both groups. Chronic ADH deficiency thus modifies the ability of the kidney to produce PGE₂ in vitro. The opposite effects observed in glomeruli and papilla suggest a different hormonal control of PGE₂ synthesis in both tissues.     

Synthesis of thromboxane B2 and 6-keto-prostaglandin F1α by bovine gastric mucosal and muscle microsomes

Bovine gastric mucosal and muscle microsomes synthesize prostaglandins and thromboxane B₂ (TXB₂) from arachidonic acid (AA). TXB₂ and 6-keto-prostaglandin F₁α (6-keto-PGF₁α) were the major products synthesized by pylorus, body, and cardiac region of the gastric mucosa. Gastric muscle mainly synthesized 6-keto-PGF₁α. TXB₂ and 6-keto-PGF₁α synthesis occurs at an appreciable rate from endogenous precursors but more rapidly with added arachidonate. Prostaglandins E₂, F₂α and D₂ were synthesized in smaller amounts under the conditions studied.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.