Thromboxane synthase inhibition: implications for prostaglandin endoperoxide metabolism. II. Testing the 'redirection hypothesis' in an acute intravenous challenge model

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.     

Testing the "redirection hypothesis" of prostaglandin metabolism in the kidney

Furosemide increases the synthesis of two major renal eicosanoids, prostacyclin (PGI2) and thromboxane A2 (TXA2), by stimulating the release of arachidonic acid which in turn is metabolized to PGG2/PGH2, then to PGI2 and TXA2. PGI2 may mediate, in part, the early increment in plasma renin activity (PRA) after furosemide. We hypothesized that thromboxane synthetase inhibition should direct prostaglandin endoperoxide metabolism toward PGI2, thereby enhancing the effects of furosemide on renin release. Furosemide (2.0 mg . kg-1 i.v.) was injected into Sprague-Dawley rats pretreated either with vehicle or with U-63,557A (a thromboxane synthetase inhibitor, 2 mg/kg-1 followed by 2 mg/kg-1 X hr-1). Urinary 6ketoPGF1 alpha and thromboxane B2 (TXB2), reflecting renal synthesis of PGI2 and TXA2, as well as PRA and serum TXB2, were measured. Serum TXB2 was reduced by 96% after U-63,557A. U-63,557A did not affect the basal PRA. Furosemide increased PRA in both vehicle and U63,557A treated rats. However, the PRA-increment at 10, 20 and 40 min following furosemide administration was greater in U-63,557A-treated rats than in vehicle-treated rats and urine 6ketoPGF1 alpha excretion rates were increased. These effects of thromboxane synthesis inhibition are consistent with a redirection of renal PG synthesis toward PGI2 and further suggest that such redirection can be physiologically relevant.     

Thromboxane synthase inhibition: Implications for prostaglandin endoperoxide metabolism: I Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH₂ metabolism in favour of PGI₂, a potent vasodilator and anti-platelet agent. While redirection has been observed there are conflicting reports of its occurrence . We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH₂ for the study of PGH₂ metabolism. Following challenge, plasma concentrations of TXB₂, 6-oxo-PGF_1α, alleged metabolites of PGI₂ (PGI₂m) and PGE₂ were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB₂ while AA and PGH₂, in addition, elevated 6-oxo-PGF_1α and PGI₂m. Injection of PGH₂ elevated 6-oxo-PGF_1α, PGI₂m, TXB₂ and PGE₂ levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg⁻¹), collagen (100 kg⁻¹), AA (1mg kg⁻¹) and PGH₂ (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge (5μg kg⁻¹) and measurement of eicosanoids 0.5min following challenge were optimal for detection of redirection of PGH₂ metabolism . The identity of immunoreactive TXB₂ and 6-oxo-PGF_1α was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [³H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect biosynthesis.     

Metabolism of prostaglandin endoperoxide by microsomes from cat lung

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.     

Metabolism of prostaglandin endoperoxide by microsomes from human lung parenchyma and comparison with metabolites produced by pig, bovine, rat, mouse and guinea-pig

The metabolism of PGH2 by human lung parenchymal microsomes was characterized by radiometric high performance liquid chromatography and compared with metabolism by pig, bovine, rat, mouse, and guinea pig lung microsomes. Microsomes from human lung synthesized 0.74 nmoles/mg protein and 0.72 nmoles/mg protein, PGI2 (6-Keto-PGF1 alpha) and TxA2 (TxB2) respectively, upon incubation with 4.0 nmoles of PGH2. Pig, bovine, rat, mouse, and guinea pig microsomes respectively synthesized 1.0, 1.0, 0.9, 0.4, and 0.1 nmoles of PGI2/mg protein, and 0.9, 1.0, 0.7, 0.3, 1.8 nmoles of TxA2/mg protein, and preparations formed some PGE2, PGF2 alpha, and PGD2. Mouse lung microsomes were unique in synthesizing PGE2 as the major prostaglandin. The thromboxane synthetase inhibitor 1-benzylimidazole was a specific inhibitor in these six species.     

Predominance of locally-produced prostaglandin E2 over prostacyclin in skin incisions in man

Blood was collected from skin incisions made by the 'Simplate' technique in 8 healthy men. Prostaglandin (PG) E2, but not 6-oxo-PGF1 alpha, the stable hydrolysis product of prostacyclin (PGI2), was tentatively identified using capillary column gas chromatography/electron capture mass spectrometry. It was not possible to quantify PGE2 because of the small volumes of blood generated by this method. Larger blood samples were then collected from 22 skin incisions in 13 patients undergoing cardiothoracic surgery. Concentrations of PGE2 were substantially greater than 6-oxo-PGF1 alpha in every sample. 13,14-Dihydro-15-oxo-PGF2 alpha, a pulmonary metabolite of PGE2, was not elevated, indicating that the PGE2 was synthesised locally at the site of incision. In 6 further patients undergoing cardiac surgery, blood sampled from an antecubital vein before and during operation contained little or no PGE2. We conclude that a substantial proportion of the PGE2 in blood emerging from skin incisions may be formed locally by the traumatised microvessels, consistent with the hypothesis that PGE2 is the principle prostaglandin synthesised by human cutaneous microvessels in vivo.     

Binding of prostacyclin by plasma glycoproteins

The binding of prostacyclin (PGI2) to plasma proteins and the resulting increase in PGI2 stability was investigated. Using gel filtration to separate bound and free PGI2, we have found that Cohn Fraction VI can bind PGI2, and retard its hydrolysis to 6-keto-PGF1 alpha (6KPGF1 alpha). The biological activity of the bound PGI2 correlated well with the quantity of bound PGI2, measured as 6KPGF1 alpha by RIA. Fraction VI bound a greater percentage of PGI2 than the other eicosanoids tested (i.e., PGI2 greater than TXB2 greater than LTB4 greater than PGE1 greater than PGF2 alpha). The PGI2 binding activity of Fraction VI was lost after neuraminidase treatment. Our data suggest that Fraction VI glycoproteins may play an important role in the binding and stabilization of PGI2 by plasma proteins.     

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys.

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE2, PGF2 alpha and the stable metabolites of PGI2 (6-keto-PGF1 alpha) and TXA2 (TXB2). PGI2 was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI2 by about 50 % in both organs. TXA2 formation was similarily decreased in lungs. In kidneys, the decrease in PGI2 formation was accompanied by an increase in PGE2 formation.     

Inhibition of GSH-dependent PGH2 isomerase in mammary adenocarcinoma cells by allicin

We have reported that allicin, a constituent of garlic oil, has no effect on the activities of platelet cyclooxygenase or thromboxane synthase, or vascular PGI2 synthase. The effect of allicin on glutathione (GSH) dependent PGH2 to PGE2 isomerase is unknown. We therefore studied the effect of allicin on PGE2 biosynthesis in a murine mammary adenocarcinoma cell line (No 4526). Intact or sonicated cells were incubated with either 14C-arachidonic acid (AA) or 14C-PGH2, respectively. Following metabolism, products were extracted, separated by TLC and analyzed by radiochromatographic scan. PGE2 was predominantly formed with minimal amounts of PGF2 alpha and PGD2. Formation of 6-keto-PGF1 alpha or TXB2 was not detected indicating the absence of TXA2 and PGI2 synthase activity. Indomethacin and ibuprofen inhibited the PGE2 formation (p less than 0.05). The enzymatic PGE2 formation in sonicates was blocked by depletion of the cellular non-protein thiols by buthionine sulfoximine and was shown to be dependent on GSH. Allicin, over the range of 10-1000 microM, inhibited the formation of PGE2 in cells exposed to 2.0 microM 14C-AA for 20 min. and in sonicated cells incubated with 20.0 microM 14C-PGH2 for 2 min (p less than 0.05). Allicin did not alter cyclooxygenase-mediated oxygen utilization in ram seminal vessicle microsomes, suggesting that allicin selectively inhibits the GSH-dependent PGH2 to PGE2 isomerase in this adenocarcinoma cell line.     

The major product of PGG2 metabolism by aortic microsomes is 6, 15-dioxo-PGF1 alpha.

Incubations of PGG2 with aortic microsomes yielded two products which were not formed in boiled enzyme control, one of which was 6-oxo-PGF1 alpha. The major metabolite was identified by gas-liquid chromatography-mass spectrometry as 6,15-dioxo-PGF1 alpha. Thus, unlike PGH2, PGG2 is probably converted to 15-hydroperoxy PGI2 which subsequently decomposes to 6, 15-dioxo-PGF1 alpha.     

Influence of interleukin-1alpha and COX-2 over the metabolism of arachidonic acid and glucose in isolated uterus of restricted diet rats

Isolated uteri from rats fed with a normal diet convert [14C]arachidonate into eicosanoids: PGE(2), PGF(2alpha), TXB(2) and 6-keto-F(1alpha). Restricted diet (50% of the normal diet, during 25 days) diminishes the levels of PGE(2), PGF(2alpha) and TXB(2). The addition of Interleukin-1alpha to the Krebs-Ringer bicarbonate medium increases sharply the production of eicosanoids. Inhibitors of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester or aminoguanidine, do not prevent eicosanoids increase. Conversely, NS-398 (a selective inhibitor of COX-2) blocks the increase of eicosanoids while PGE(2) blocks eicosanoids production mediated by IL-1alpha. Other experiments with uteri of underfed rats confirm that interleukin-1alpha produces an increase in the glucose metabolism. The addition of Nomega-nitro-L-arginine methyl ester, aminoguadinine or NS-398 blocked such stimulation. It is concluded that Interleukin-1alpha produces an increase of glucose metabolism in uteri isolated from underfed rats by two different mechanisms, both involving COX-2: (1) nitric oxide independent and (2) nitric oxide dependent.     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Actions of prostacyclin (PGI2) and its product, 6-oxo-PGF1alpha on the rat gastric mucosa in vivo and in vitro.

The effects of prostacyclin (PGI2) and its breakdown product 6-oxo-PGF1alpha on various aspects of gastric function were investigated in the rat. PGI2 increased mucosal blood flow when infused intravenously. PGI2 was a more potent inhibitor of gastric acid secretion in vivo than PGE2. Like PGE2, PGI2 inhibited acid secretion from the rat stomach in vitro. PGI2 had comparable activity to PGE2 in inhibiting indomethacin-induced gastric erosions. Thus prostacyclin shares several of the activities of PGE2, and may be involved in the regulation of gastric mucosal function.     

Urinary levels of 2,3-dinor-6-oxo-PGF1 alpha: a reliable index of the production of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M), a major metabolite of PGI2, are determined by the balance between the amount of PGI2 synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI2-M can be used as a reliable index of the in vivo production of PGI2 in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI2-M during paired intravenous infusions of 6-oxo-PGF1 alpha (the stable product of the spontaneous hydrolysis of PGI2) in conscious, unrestrained SHR and WKY rats aged 12-15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI2 was infused instead of 6-oxo-PGF1 alpha. The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF1 alpha and PGI2 into PGI2-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF1 alpha infused and the amount of PGI2-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha as an index of PGI2 production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI2 than WKY rats in vivo, contrary to the situation prevailing in vitro.     

Arachidonic acid metabolism in isolated rat aorta. Dependence of prostacyclin biosynthesis on extracellular potassium concentration

Slices of rat aorta were incubated in Krebs-Ringer bicarbonate buffer for measurements of immunoreactive 6-ketoprostaglandin F1 alpha, thromboxane (TX) B2, prostaglandin (PG)E2, and PGF2 alpha, and in Tris buffer (pH 9.3) for determination of prostacyclin (PGI2)-like activity. No significant generation of TXB2, PGE2, or PGF2 alpha by rat aortic tissue could be detected. The time-dependent release of 6-keto-PGF1 alpha Krebs-Ringer bicarbonate buffer closely correlated with PGI2 generation in alkaline Tris buffer. During a 30-min incubation period, 6-keto-PGF1 alpha, release was 79.8 +/- 3.3 pmol/mg at a buffer potassium concentration of 3.9 mmol/liter and significantly increased by 23% to 98.3 +/- 8.5 pmol/mg (P less than 0.025) in the absence of potassium in the incubation medium. A smaller decrease in buffer potassium concentration to 2.1 mmol/liter and an increase to 8.8 mmol/liter did not significantly alter aortic 6-keto-PGF1 alpha release. Changes in the incubation buffer sodium concentration from 144 mmol/liter to either 138 or 150 mmol/liter at a constant potassium concentration of 3.9 mmol/liter did not alter the recovery of 6-keto-PGF1 alpha. Our results support the concept that PGI2 is the predominant product of arachidonic acid metabolism in rat aorta. They further show that PGI2 can be recovered quantitatively as 6-keto-PGF1 alpha under the present in vitro conditions. In addition, this in vitro study points to the potassium ion as a modulator of vascular PGI2 synthesis with a stimulation at low potassium concentrations.     

Inhibition of prostaglandin biosynthesis by SQ 28,852, a 7-oxabicyclo[2.2.1]heptane analog

7-Oxabicyclo[2.2.1]heptane analogs of prostaglandin (PG) H2 can act as thromboxane (Tx) A2 receptor antagonists or agonists, PGI2 and/or PGD2 receptor agonists, or exhibit a mixture of the above activities. SQ 28,852, a new analog with a hexyloxymethyl omega side chain, is a potent inhibitor of PG synthesis. SQ 28,852 inhibited collagen and arachidonic acid (AA)-induced platelet aggregation and TxB2 and PGE2 formation, but did not block platelet aggregation induced by ADP or the TxA2 mimics, 9,11-azo PGH2, SQ 26,655, and U-46,619. It also blocked conversion of AA to TxB2, PGE2, and 6-keto PGF1 alpha by microsomal preparations of human platelets, bovine seminal vesicles, and bovine aortas, respectively, but did not inhibit the conversion of PGH2 to TxA2 by the platelet microsomal preparation. SQ 28,852 (p.o.) protected mice against the lethal effects of AA (75 mg/kg, i.v.). The I50 values for SQ 28,852, indomethacin and aspirin were 0.025, 0.05 and 15 mg/kg, respectively. Neither SQ 28,852 nor indomethacin protected mice from death caused by 9,11-azo PGH2. SQ 28,852 (0.01 to 1 mg/kg, i.v.) inhibited AA-induced bronchoconstriction in anesthetized guinea pigs for at least 60 min. As an inhibitor of AA-induced bronchoconstriction, SQ 28,852 was 16- and 45-times more potent than indomethacin at 3 and 60 min after i.v. administration, respectively. SQ 28,852 did not inhibit bronchoconstriction induced by histamine or 9,11-azo PGH2, indicating its specificity of action in vivo. SQ 28,852 is the first example of a new class of cyclooxygenase inhibitors whose structure is similar to that of the naturally occurring endoperoxide, PGH2.     

Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon

Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2 alpha and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2 alpha greater than TXB2 greater than 6-keto-PGF1 alpha, the stable degradation product of PGI2 = PGD2 = PGE2 = 13,14-dihydro-15-keto-PGF2 alpha). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 much greater than PGD2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGF2 alpha = TXB2 = 6-keto-PGF1 alpha greater than 13,14-dihydro-15-keto-PGF2 alpha). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF2 alpha. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.     

Evidence for a bidirectional prostaglandin endoperoxide shunt between platelets and the bovine coronary artery

While platelets have been shown to be capable of supplying prostaglandin (PG) H2 to endothelial cells in culture for PGI2 synthesis, endothelial cells have been shown unable to supply PGH2 to platelets for thromboxane (TX) A2 synthesis. We incubated rings of the bovine coronary artery (BCAR) with human platelets treated with aspirin (to inhibit cyclooxygenase) or CGS 13080 (to inhibit TXA2 synthase) in the presence of 20 microM arachidonic acid. BCAR, with damaged endothelium, produced significantly less PGI2 than that with intact endothelium. However, co-incubation with CGS 13080-treated platelets resulted in an increase in PGI2 independent of endothelium, demonstrating a shunt of PGH2 from platelets to BCAR. Co-incubation of BCAR with aspirin-treated platelets resulted in a net increase in TXA2 demonstrating a shunt of PGH2 from BCAR to platelets. Employing [14C]PGH2 as substrate, BCAR with and without intact endothelium produced similar amounts of 6-keto-[14C]PGF1 alpha. Likewise, homogenates (50 micrograms protein) of intimal and subintimal regions of BCAR and BCAR converted similar amounts of PGH2 to 6-keto-PGF1 alpha. These data suggest that vascular production of PGH2 is more dependent on an intact endothelium than is the conversion of PGH2 to PGI2. These data also suggest a potential for a bidirectional exchange of PGH2 between platelets and vascular wall during platelet-vascular wall interactions.     

Effects of experimental diabetes on spontaneous contractions, on the output of prostaglandins and on the metabolism of labelled arachidonic acid, in uteri isolated from ovariectomized rats. Influences of estradiol

Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (Eo) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic Eo injected animals (0.5 + 1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from Eo injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F2 alpha, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of Eo to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1 alpha (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with Eo formed less 6-keto-PGF1 alpha and PGE2 and similar amounts of PGF2 alpha or of TXB2 from AA, than Eo injected controls, whereas uteri from castrated diabetic animals injected with Eo, formed a similar % of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of Eo is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulat