Endogenous Prostacyclin Production Research Articles

Nitric oxide and prostacyclin play a role in the regulation of microvascular protein and hydraulic permeability in cat skeletal muscle.

To evaluate a possible role of nitric oxide (NO) and prostacyclin in the regulation of basal microvascular protein and hydraulic permeability. The study was performed on the autoperfused cat calf muscle. Changes in the osmotic reflection coefficient for albumin, calculated from the extended Starling equation, were used as a measure of altered protein permeability, whereas changes in capillary filtration coefficient (CFC) were used as a measure of altered hydraulic permeability. Inhibition of the endogenous NO production with L-nitro-arginine methyl ester given intra-arterially to the muscle decreased the reflection coefficient to 70% of the control (p < 0.05) and increased the CFC by 17% (p < 0.05). The addition of a simultaneous intra-arterial infusion of the NO precursor L-arginine restored both the reflection coefficient and the CFC back to control level. Blockade of the endogenous prostacyclin production with tranylcypromine given intra-arterially decreased the reflection coefficient to 72% of the control (p < 0.05) and increased the CFC by 24% (p < 0.05), and they were both restored to control levels by a simultaneous intravenous infusion of a nonvasodilating dose of prostacyclin (1.0 ng/kg per minute). The results support the view that endogenous NO and prostacyclin decrease both protein and hydraulic permeability. Assuming a dynamic endogenous release of these substances, they may act as bidirectional regulators of protein and hydraulic permeability.

Prostaglandins and the antiarrhythmic effect of preconditioning in the isolated rat heart.

Our study evaluated the relationship between the endogenous production of prostacyclin and the antiarrhythmic effect of ischemic preconditioning against ischemic and reperfusion-induced tachyarrhythmia. Langendorff perfused rat hearts underwent 30 min regional ischemia with reperfusion. Preconditioning was induced by a single episode of 5 min ischemia and 15 min reperfusion. Prostaglandin 6-keto F1 alpha (a stable metabolite of prostacyclin) was determined in the coronary effluent. In the control group the incidence of tachyarrhythmia was 31% during ischemia and 67% during reperfusion. Preconditioning did not affect ischemic arrhythmias but attenuated arrhythmias a reperfusion (8%, p < 0.01) and was associated with increased release of prostacyclin prior to reperfusion. Aspirin abolished the antiarrhythmic effect of preconditioning against reperfusion tachyarrhythmias. However, no relationship was found between suppression of prostacyclin production and the occurrence of arrhythmia in individual hearts. Thus, our findings suggest that metabolites of arachidonic acid via the cyclooxygenase pathway are involved in the protective effect of ischemic preconditioning against reperfusion-induced tachyarrhythmias.

Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury.

Simplified assay for the quantification of 2,3-dinor-6-ketoprostaglandin F 1α by gas chromatography—mass spectrometry

Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F 1α (2,3-dinor-6-keto-PGF 1α). Gas chromatographic—mass spectrometric (GC—MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF 1α employing GC—MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is ± 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 ± 54 pg/mg creatinine (mean ± S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF 1α is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.

Experimental evaluation of myocardial protective effect of prostacyclin analog (OP-41483) as an adjunct to cardioplegic solution

A stable prostacyclin analog (OP-41483) was evaluated for myocardial protective effect against global ischemia with the use of cardioplegia. Isolated canine hearts (n = 25) were exposed to 60 minutes of warm (37 degrees C) global ischemia after the arrest by crystalloid cardioplegia. Prostaglandin analog was given in three different ways: preadministration (700 ng/kg body weight per minute) before ischemia for 30 minutes (group I, n = 5), given as a component of cardioplegic solution (600 ng/ml, group II, n, = 6), and post-administration (25 ng/kg body weight per minute) during reperfusion for 30 minutes (group III, n = 7). During reperfusion, coronary sinus blood flow, 6-keto-prostaglandin F1 alpha in coronary sinus blood, and myocardial oxygen consumption were measured during reperfusion. As a result, groups II and III showed significantly better global left ventricular function (developed pressure, maximum dP/dt, and diastolic compliance) than the control group (without prostaglandin analog, n = 7) and group I. Myocardial oxygen consumption at reperfusion (1 minute) was significantly larger in group II than in the control group. 6-keto-prostaglandin F1 alpha flux was significantly larger in group II than in the other three groups during reperfusion. The results indicated that prostaglandin analog has a beneficial effect on myocardial protection under global ischemia with cardioplegia, particularly when used as a component of cardioplegic solution and also during reperfusion. The mechanism may relate to the cytoprotective effect (including protection of endothelium with enhanced endogenous prostacyclin production at reperfusion and also to the modulation of reperfusion per se.

The influence of extra-umbilical serotonin, PGE2and PGF2αon prostanoid production in human umbilical arteries

The influence of extra-umbilical applications of serotonin and prostaglandins PGE2 and PGF2 alpha on the endogenous production of prostacyclin and thromboxane in human umbilical arteries was registered during in vitro perfusion by use of specific radio-immunoassays. PGE2 and PGF2 alpha caused significant dose-dependent increases in the production of prostacyclin, but no specific alterations in that of thromboxane. Serotonin did not lead to any changes in the prostanoid formation. The results provide indirect support for the assumption that prostanoids synthesized in the vessel wall, predominantly in the endothelial cells, serve as mediators in the action of autacoids on vascular smooth muscle tension.

A clinical trial of nafazatrom (Bay g 6575) in advanced cancer.

Nafazatrom (Bay g 6575) has been shown to be a potent inhibitor of tumor metastasis in preclinical models. It is believed to work by stimulating endogenous prostacyclin production. The drug has also been shown to inhibit the growth of certain experimental tumors, to be cytostatic for certain cell lines in tissue culture, and to induce differentiation in HL-60, neuroblastoma, and Friend erythroleukemia cell lines. Furthermore, no toxicity has been seen in animals or human volunteers. We report here a clinical trial of oral nafazatrom at five dose levels in patients with advanced cancer. Thirty patients with a wide variety of advanced malignancies were treated for 26-638+ days (median 82 days). No tumor responses were seen. Toxicity included two cases with mild skin rashes, one case with nausea and vomiting, and one case with diarrhea. Nafazatrom is a safe and well-tolerated agent. Maximum activity would be predicted to occur in the adjuvant treatment of cancer and we feel that further efforts should proceed to identify the appropriate dose for such a trial.

15-Hydroxy-5,8,11,13-eicosatetraenoic acid inhibits human vascular cyclooxygenase. Potential role in diabetic vascular disease.

Human umbilical arteries converted arachidonic acid to three hydroxyeicosatetraenoic acids (HETEs) as well as prostaglandins. The mono-HETEs have been identified by reverse-phase high pressure liquid chromatography and gas chromatography-mass spectroscopy as 15-HETE and 11-HETE. 15-HETE in arterial segments appears to be derived mainly via the 15-lipoxygenase pathway, whereas 11-HETE, and the presumed di-HETE(s) were products of cyclooxygenase. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, stimulated prostanoid production with a concomitant inhibition of 15-HETE formation. These results suggested that 15-HETE may function as an endogenous regulator of prostacyclin. In human umbilical arterial microsomes, 15-HETE was found to inhibit 6-keto-prostaglandin F1 alpha and total prostanoid production in a concentration-dependent manner (median inhibition constant [IC50] of 52 +/- 3 and 63 +/- 4 microM respectively). The relative distribution of prostaglandins, however, remained unaffected, indicating that the site of action was cyclooxygenase. Kinetic analysis revealed that 15-HETE was a competitive inhibitor of the enzyme. Although no changes in maximum velocity occurred, the apparent Km was significantly different (9.3 +/- 6.9 microM [1 SD] for control vs. 37.6 +/- 17.7 microM for the 15-HETE-treated enzyme). Furthermore, the inhibitory effect of 15-HETE on prostacyclin production was confirmed using cultured bovine endothelial cells. In this cell system, not only did 15-HETE inhibit endogenous prostacyclin production, but also the conversion of exogenous [1-14C]arachidonic acid to prostacyclin (IC50 of 40 +/- 17 microM). No effect on arachidonic acid release was noted. To investigate whether our in vitro finding that 15-HETE inhibited prostacyclin production could be relevant to the in vivo situation, our final studies were performed on vasculature obtained from the diabetic milieu. We found that the production of 15-HETE was significantly increased in vasculature obtained from the infant of the diabetic mother (1.14 +/- 0.26 pmol/mg) when compared to control neonates (0.77 +/- 0.22; P less than 0.01). A concomitant decrease in prostacyclin production was seen (51.6 +/- 12.6 pmol/mg in infants of diabetic mothers vs. 71 +/- 22.3 in controls). Moreover, an inverse correlation between these two eicosanoids was also noted. Our results suggest a potential in vivo regulatory role for 15-HETE on prostacyclin production.

Functional injury of vein graft endothelium. Role of hypothermia and distention.

The cause of endothelial injury during vein harvesting and preservation is complex. Hypothermia is thought necessary to preserve cell viability but has been implicated in morphologic injury to the endothelium. This study explored the effect of temperature on preserving endothelial function using prostacyclin production as a metabolic marker. Canine veins were atraumatically excised and matched segments were stored at three temperatures using either nutrient medium or heparinized saline. After storage, endogenous production of prostacyclin by the luminal surface of each vein was collected in a closed perfusion system at 37 degrees C and assayed by radioimmunoassay. Optimal prostacyclin production was observed in veins stored in tissue culture medium at normothermia. Preservation of normal endothelial function may require revision of traditional vein graft-harvesting techniques.

Increased prostacyclin biosynthesis in patients with severe atherosclerosis and platelet activation.

Prostacyclin is a potent vasodilator and platelet inhibitor produced by vascular endothelium. Endogenous production of prostacyclin under physiologic conditions is extremely low, far below the capacity of vascular tissue to generate this substance in response to stimulation in vitro. This may reflect a low frequency or intensity of stimulation of prostacyclin production. We postulated that if prostacyclin does act as an endogenous platelet-inhibitory agent, it should be produced in greater amounts in a clinical setting in which platelet-vascular interactions are likely to be increased. To test this hypothesis, we examined prostacyclin biosynthesis in patients with severe atherosclerosis and evidence of platelet activation in vivo. Excretion of 2,3-dinor-6-keto-prostaglandin F1 alpha, a major urinary prostacyclin metabolite, was significantly higher in 9 patients with severe atherosclerosis and evidence of platelet activation (251 to 1859 pg per milligram of creatinine) than in 54 healthy volunteers (45 to 219 pg per milligram of creatinine; P less than 0.001). This difference represented an alteration in biosynthesis rather than in metabolism, since the fractional conversion of infused prostacyclin to the dinor metabolite was identical in both groups. Prostacyclin production may be low in healthy persons because there is almost no stimulus for its production but enhanced in patients with severe atherosclerosis as a consequence of platelet interactions with endothelium or other vascular insults. These observations are compatible with a role for prostacyclin as a local regulator of platelet-vascular interactions.

ChemInform Abstract: PROSTACYCLIN: A POTENT ANTIMETASTATIC AGENT

Metastasis is the principal cause of failures to cure human cancers. Prostacyclin is a powerful antimetastatic agent against B16 amelanotic melanoma cells. This effect, which may result from the platelet antiaggregatory action of prostacyclin, is potentiated by a phosphodiesterase inhibitor. Inhibitors of prostacyclin synthesis increase metastasis. Prostacyclin and agents that may increase endogenous prostacyclin production or prolong its activity are suggested as new antimetastatic agents.

Prostacyclin: a potent antimetastatic agent.

Metastasis is the principal cause of failures to cure human cancers. Prostacyclin is a powerful antimetastatic agent against B16 amelanotic melanoma cells. This effect, which may result from the platelet antiaggregatory action of prostacyclin, is potentiated by a phosphodiesterase inhibitor. Inhibitors of prostacyclin synthesis increase metastasis. Prostacyclin and agents that may increase endogenous prostacyclin production or prolong its activity are suggested as new antimetastatic agents.

Production of prostacyclin by vascular endothelial cells.

One of the earliest events in thrombus formation is the adherence of blood platelets to the exposed subendothelium. Under normal conditions no platelet adherence to the vascular endothelium occurs. To study a possible interaction between endothelial cells and blood platelets, endothelial cells were isolated from the umbilical cord vein. These isolated cells could be identified as endothelial cells by the presence of the so-called Weibel Palade bodies and the F&lt;sub&gt;VIII&lt;/sub&gt;:AG. After incubation of monolayers of endothelial cells with blood platelets an unstable substance which inhibits platelet aggregation is released from the endothelial cell. This substance could be identified as prostacyclin according to the following criteria: (1) Its formation could be blocked by pretreatment of the endothelial monolayers with indomethacin or tranylcypromin. (2) After preincubation of endothelial cells with [&lt;sup&gt;14&lt;/sup&gt;C]-endoperoxide, chemically detectable amounts of [&lt;sup&gt;14&lt;/sup&gt;C]-6-keto PGF&lt;sub&gt;1α&lt;/sub&gt;, the stable end product of prostacyclin could be detected. (3) In incubation supernatants the presence of 6-keto PGF&lt;sub&gt;1α&lt;/sub&gt; could be shown by means of gas chromatography. Monolayers of endothelial cells only synthesize prostacyclin when the cells are stimulated for example by mechanical stress or after addition of substances like arachidonic acid or thrombin. It has been suggested that apart from the aforementioned endogenous production of prostacyclin the endothelial cell may also synthesize prostacyclin using endoperoxides provided by platelets. In these studies the production of prostacyclin was measured as the inhibition of platelet aggregation. Comparison of the amount produced in the presence of buffer alone, with the amount formed in the presence of platelets, led to the suggestion that endoperoxides liberated by platelets were used by endothelial cells to synthesize prostacyclin. If, however, the retainment of the biological activity in these two systems is compared, then it appears that in the presence of plasma or platelets, prostacyclin is stabilized to a