PGI2 Synthesis Research Articles

Studies on the effect of diadenlyated nucleotides on calcium mobilization and prostacyclin synthesis in bovine aortic endothelial cells

Extracellular adenine dinucleotides are modulators of blood vessel tone. We have previously demonstrated that Ap 2A and Ap 4A induce the synthesis of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) while Ap 3A and Ap 5A do not [FEBS Lett. 427 (1998) 320; Arch. Biochem. Biophys. 364 (1999) 280.]. In this communication we determine the effect of Ap x As ( x=2–5) on prostacyclin (PGI 2) synthesis and Ca 2+ mobilization in BAEC. Ap 2A and Ap 4A significantly enhanced the synthesis of PGI 2 while Ap 3A and Ap 5A do not. These data support the notion that Ap 2A and Ap 4A are vasodilators. All four dinucleotides significantly enhanced Ca 2+ mobilization over basal levels. Ap 5A and Ap 3A enhanced 2.0 and 1.6 times more Ca 2+ release than Ap 4A, respectively. Since neither Ap 5A nor Ap 3A enhanced the synthesis of either PGI 2 or NO but did mobilize Ca 2+, these data support the hypothesis that in BAEC Ca 2+ release is localized or compartmentalized.

Inhibition of prostacyclin by indomethacin ameliorates the splanchnic hyposensitivity to glypressin in haemorrhage-transfused common bile duct-ligated rats.

Prostacyclin (PGI2) is an important contributor to the mediation of hyporeactivity to vasoconstrictors and the development of hyperdynamic circulation in portal hypertensive states. Inhibition of PGI2 synthesis in haemorrhage-transfused partially portal vein-ligated rats could ameliorate the splanchnic hyposensitivity to glypressin, a long-acting vasopressin analogue. This study investigated whether the hyposensitivity to glypressin also exists in rats with common bile duct ligation (BDL) and whether the inhibition of PGI2 synthesis by indomethacin could potentiate the portal-hypotensive effect of glypressin in bleeding BDL rats. Two series of BDL rats were used. Series 1 investigated the haemodynamic effects of low dose glypressin (0.07 mg kg-1) in BDL rats with or without bleeding by catheterization. In series 2, haemodynamic parameters were measured in stable or bleeding BDL rats that were receiving intravenously high dose glypressin (0.2 mg kg-1) or indomethacin (5 mg kg-1) followed by high dose glypressin. In rats with a hypotensive haemorrhage, 4.5 mL of blood was withdrawn and 50% of the withdrawn blood was reinfused before the administration of glypressin or indomethacin. Splanchnic hyposensitivity to glypressin was demonstrated in haemorrhage-transfused BDL rats receiving high, but not low, doses of glypressin. Indomethacin infusion did not cause significant systemic and portal haemodynamic changes in bleeding BDL rats (P > 0.05). The addition of indomethacin significantly enhanced the portal-hypotensive effects of glypressin (P < 0.05) and potentiated the increases in mean arterial pressure induced by glypressin infusion (P < 0.001) in bleeding BDL rats. Splanchnic hyposensitivity to glypressin observed in haemorrhage-transfused BDL rats could be ameliorated by the addition of indomethacin, suggesting a role of endogenous PGI2 in its pathophysiology.

Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-δ and by mobilization of intracellular Ca2+

We reported previously that vascular endothelial growth factor (VEGF) stimulates prostacyclin (PGI2) production via activation of the extracellular signal-regulated kinase (ERK) cascade. In this paper, we examined the role of protein kinase C (PKC) in this pathway. VEGF-induced PGI2 generation and arachidonic acid release in human umbilical vein endothelial cells were inhibited by the PKC inhibitors GF109203X and calphostin C. VEGF increased PKC activity and immunoreactivity of the PKCδ, α and ε isoforms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cytosolic phospholipase A2, but had little effect on ERK activation induced by basic fibroblast growth factor. GF109203X, calphostin C and the PKCδ-selective inhibitor, rottlerin, did not inhibit activation of the KDR receptor for VEGF. Inhibition of Ca2+ fluxes using BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)] blocked VEGF-induced PGI2 production but did not inhibit ERK activation. Neither activation nor inhibition of the NO/cGMP pathway had any effect on VEGF induction of ERK activity and PGI2 synthesis. Wortmannin partially inhibited VEGF stimulation of PGI2 production, but did not inhibit VEGF-induced ERK activity. VEGF-induced ERK activation and PGI2 production were blocked by rottlerin, and VEGF increased association of PKCδ with Raf-1, the upstream activator of MEK. The PKC-selective inhibitor Go6976 did not inhibit ERK activation and had only a partial effect on PGI2 production. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade leading to PGI2 synthesis and suggest that the PKCδ isoform may be a key mediator of VEGF-induced activation of the ERK pathway via increased association with Raf-1.

Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women

Nitric oxide (NO) is a potent relaxant of smooth muscle and possibly plays a role in maintaining uterine quiescence during pregnancy. Clinical studies have shown beneficial effects of the stable NO donorglyceryl trinitrate (GTN) for the inhibition of pathological myometrial contractility that occurs in preterm labor or dysmenorrhea. Since there are contradictory results regarding the mediation of the relaxingeffect of NO ,the myometrial prostaglandin synthesis during superfusion with NO donors was studied.Human myometrial strips obtained either at term Cesarean sections before the onset of labor or afterhysterectomies in premenopausal women were studied in a superfusion system. After the manifestation of spontaneous contractions ,GTN was added in low doses comparable with in vivo levels (0.4-40nM) and the effect on myometrial activity ,intracellular cGMP and prostaglandin production was analyzed. Additionally ,the effect of sodium nitroprusside (SNP) - which releases NO spontaneously - was comparedwith that of GTN.GTN caused a significant decrease in the contraction frequency of myometrial strips from both pregnant and non-pregnant women similar to that of SNP. There was no significant changein the myometrial synthesis of PGI2 ,PGF2α and PGE2 ,whereas the intracellular cGMP content was increased. In conclusion ,GTN showed a significant inhibitory effecton human myometrium in vitro in very low doses and therefore represents an interesting therapeutic alternative for the treatment of preterm labor and dysmenorrhea. GTN in low doses did not alterthe prostaglandin synthesis of human myometrium.

Human lymphocytes stimulate prostacyclin synthesis in human umbilical vein endothelial cells. Involvement of endothelial cPLA2

Prostacyclin (PGI2) contributes to the maintenance of a nonadhesive luminal surface in blood vessels due to its anti-platelet and vasodilatory properties. Here, we sought to determine whether peripheral blood lymphocytes (PBL) may regulate the PGI2 production of human umbilical vein endothelial cells (HUVEC). Cell-cell contact between HUVEC and lymphocytes markedly enhanced PGI2 synthesis as a function of the number of lymphocytes added. This stimulated synthesis was totally suppressed when lymphocytes and HUVEC were separated by a microporous insert. It was not due to prostaglandin H synthase up-regulation. The pretreatment of lymphocytes with the PGI2 synthase inhibitor tranylcypromine partially inhibited PGI2 synthesis (47%), suggesting a transcellular metabolism of the endothelial prostaglandin endoperoxide PGH2 by the lymphocyte PGI2 synthase. Experiments using [14C]arachidonate-labeled lymphocytes coincubated with unlabeled HUVEC, and [14C]arachidonate-labeled HUVEC coincubated with unlabeled lymphocytes showed that the arachidonic acid used for PGI2 synthesis was totally of endothelial origin. Furthermore, the PGI2 synthesis was strongly inhibited by the cytosolic phospholipase A2 inhibitor, MAFP and totally suppressed by the combination of the calcium chelators, BAPTA and EGTA. Collectively, these results suggest that lymphocytes trigger an outside-in signaling in endothelial cells involving cPLA2 activation. Overall, the switch-on for PGI2 synthesis induced by lymphocytes might serve as a protection against atherothrombogenesis.

Platelet-derived growth factor causes endothelium-independent relaxation of rabbit mesenteric artery via the release of a prostanoid.

Recent evidence has indicated that the mitogen platelet-derived growth factor (PDGF) can act acutely to regulate arterial tone. In this study we demonstrate that the BB isoform of PDGF (PDGF-BB) can cause endothelium-independent relaxation of rabbit isolated mesenteric arteries. In endothelium-denuded arteries, PDGF-BB (40 pM - 8.0 nM) caused concentration-dependent relaxation of noradrenaline-induced tone. The relaxation to PDGF-BB was abolished by a cyclo-oxygenase inhibitor, indomethacin (10 microM) and by the PDGF receptor-associated tyrosine kinase inhibitor, tyrphostin AG1295 (50 microM), but not by N:(G)-monomethyl-L-arginine (L-NMMA, 200 microM), an inhibitor of nitric oxide (NO) synthase. PDGF-BB (4.0 nM) significantly increased the release of prostacyclin (PGI(2)) in endothelium-denuded arteries. Exogenously applied iloprost (1 microM), a stable analogue of PGI(2), relaxed the endothelium-denuded artery. PDGF-BB (4.0 nM) significantly increased the cyclic AMP content. In the absence of Ca(2+), PDGF-BB (4.0 nM) failed to relax the artery, and the release of PGI(2) was almost completely suppressed. In addition, the release of PGI(2) by PDGF-BB (4.0 nM) was significantly reduced in the presence of endothelium. The effect of endothelium was eliminated by L-NMMA (200 microM) and PGI(2) release increased. These data indicate that in rabbit mesenteric arteries, PDGF-BB can evoke endothelium-independent relaxation by stimulating the synthesis of PGI(2). The PDGF-BB-induced prostaglandin synthesis is dependent on both Ca(2+) and tyrosine phosphorylation of the PDGF receptor, and is attenuated by endothelium-derived NO.

Novel endothelium-derived relaxing factors: Identification of factors and cellular targets

Nitric oxide (NO), together with prostacyclin (PGI 2), mediates shear stress and endothelium-dependent vasodilator-mediated vasorelaxation. In the presence of inhibition of NO synthase (NOS) with nitroarginine analogues, such as of N w-nitro- l-arginine methyl ester ( l-NAME) and N w-nitro- l-arginine ( l-NNA), and indomethacin, to inhibit cyclooxygenase (COX) and the synthesis of PGI 2, many blood vessels still respond with an endothelium-dependent relaxation to either chemical [i.e. acetylcholine (ACh)] or mechanical (shear stress) activation. This non-NO and non-PGI 2 vasorelaxation appears to be mediated by hyperpolarization of the vascular smooth muscle cell (VSMC). Although NO can hyperpolarize VSMC, a novel mediator, the endothelium-derived hyperpolarizing factor (EDHF), which opens a VSMC K + channel(s) notably in resistance vessels, has been proposed. Little agreement exists as to the nature of this putative factor, but several candidate molecules have been proposed and evidence, notably from the microcirculation, suggests that endothelium-dependent hyperpolarization (EDH) may be mediated via low electrical resistance coupling via myoendothelial gap junctions. We describe a number of techniques that are being used to identify EDHF and present data that address the contribution of a small increase in extracellular K + as an EDHF.

Shear stress differentially regulates PGHS-1 and PGHS-2 protein levels in human endothelial cells.

The secretion of prostacyclin (PGI2) by endothelial cells is regulated by shear stress. Prostaglandin H synthase (PGHS) is considered to be a key limiting enzyme in the synthesis of PGI2 from arachidonic acid. Endothelial cells were cultured in the presence of 4, 15, or 25 dyn/cm2 shear stress using a parallel plate flow chamber to assess the effect of shear stress on both PGHS isoforms, PGHS-1 and PGHS-2. In cells exposed to 4, 15, or 25 dyn/cm2 shear stress PGHS-1 and PGHS-2 protein levels initially decreased. The decrease was followed by a sustained increase for PGHS-1 but only a transient increase for PGHS-2. The duration of the PGHS-2 increase depended on the magnitude of the shear stress. The effect of altering shear stress levels on PGHS protein levels in cells preconditioned to either 4, 15, or 25 dyn/cm2 shear stress for 48 h was also studied. Changing shear stress levels effected PGHS-2 but not PGHS-1. Increases in shear stress levels from 4 to 15 or 25 dyn/cm2 caused a decrease in PGHS-2. In contrast, decreases in shear stress levels from 15 or 25 to 4 dyn/cm2 caused PGHS-2 to increase. There was a continual decrease in PGHS-2 when the shear stress was changed from 15 to 25 or 25 to 15 dyn/cm2. In summary, the regulation of PGHS-2 by shear stress is dependent upon the magnitude of the shear stress, whereas the regulation of PGHS-1 protein levels seems to be independent of the shear stress magnitude. The regulation of PGHS-1 and PGHS-2 protein levels by shear stress indicates that these proteins play an important role in the maintenance of cardiovascular homeostasis as regulators of PGI2 production.

A role of protein kinase C in the regulation of cytosolic phospholipase A2 in bradykinin-induced PGI2 synthesis by human vascular endothelial cells

A role of protein kinase C in the regulation of cytosolic phospholipase A2 in bradykinin-induced PGI2 synthesis by human vascular endothelial cells

Effect of Oral L-Arginine Administration for Three Weeks in Two Kidney-two Clip Hypertensive Rats

Nitric oxide (NO) has been identified as an effective vascular relaxant. This study analyses the contribution of the precursor L-arginine (L-arg) by oral administration in two kidney-two clip hypertension in the rat (2K-2C). Two groups were studied: sham (SH, n = 21) and hypertensive (HT, n = 15). After 4 weeks of surgery, a group of rats remained as controls (SHc and HTc, respectively), while others were supplemented with L-arg (1.25 g/L) in drinking water (SHa and HTa) for 3 weeks. Blood pressure was significantly increased in 2K-2C rats but remained unchanged after L-arg treatment. Plasma nitrite/nitrate concentrations were not different among groups. The contractile response of aorta to KCl, serotonin and the protein kinase C (PKC) stimulant, phorbol 12,13-dibutyrate (PDBu) was also evaluated. Higher contractile responses to PDBu (p < 0.001) and lower relaxation to acetylcholine (Ach 10−6M, p < 0.05 and 10−5M, p < 0.02) were observed in aortic rings of HTc vs SHc; L-arg supplementation significantly diminished tension development to all agonists (p < 0.05) but failed to modify the lower relaxation to Ach in HTa. Thromboxane (TxA2) – synthesis in rings of HTc was higher than in SHc under basal conditions (p < 0.05). In the groups with supplement of L-arg, PDBu significantly stimulated prostacyclin (PGI2) synthesis more in HTa rats than in SHa ones (p < 0.05). To conclude: 1) L-arg fails to modify hypertension development in 2K-2C rats; and 2) L-arg exerts a beneficial effect on the vascular wall, by reducing contractility in rings from HTa rats; it also improved PGI2 synthesis under PDBu stimulation. 3) greater PKC activation and TxA2 production rather than lower NO availability might result in systemic hypertension in 2K-2C rats.

A Role of Protein Kinase C in the Regulation of Cytosolic Phospholipase A2 in Bradykinin-Induced PGI2 Synthesis by Human Vascular Endothelial Cells

The purpose of this study was to elucidate the mechanism by which bradykinin (BK) enhances prostacyclin (PGI2) production in human umbilical vein endothelial cells (HUVEC). BK-induced enhancement of PGI2 synthesis was observed in a dose- and time-dependent manner, and it also increased [Ca2+]i followed by enhancement of cytosolic phospholipase A2 (cPLA2) activity. The PKC inhibitors GF109203X and H7 attenuated the BK-induced increase in [Ca2+]i and inhibited the BK-induced PGI2 synthesis. Phorbol 12-myristate 13-acetate increased cPLA2 activity and PGI2 synthesis but failed to alter [Ca2+]i. BK increased cPLA2 mRNA eightfold by 15 min, and this increase was inhibited by pretreatment with the PKC inhibitors. In response to cycloheximide pretreatment, cPLA2 mRNA was superinduced. These results suggest that BK stimulates PGI2 synthesis in HUVEC by activation of cPLA2 by dual mechanisms: an elevation of [Ca2+]i and a PKC-dependent pathway. Moreover, changes in calcium kinetics and expression of cPLA2 mRNA may underlie the BK-induced PGI2 enhancement in these cells.

Urinary prostacyclin metabolites in patients with IgA nephropathy

Background. Urinary 6-keto-prostaglandin F1α (6kPGF) is an index of renal prostacyclin (PGI2) synthesis, while urinary 2,3-dinor-6-keto-prostaglandin F1α (23d6kPGF) reflects extrarenal PGI2 synthesis. We therefore examined the relationship between the urinary excretion of PGI2 metabolites and the deterioration of renal function and histopathological findings.

Cyclooxygenase Inhibition Converts the Effect of Nitric Oxide Synthase Inhibition from Infarct Size Reduction to Expansion in Isolated Rabbit Hearts

Nitric oxide (NO) and prostacyclin (PGI2) are putative cardioprotective agents. Evidence indicates that there may be a reciprocal relationship involved in the synthesis of NO and PGI2, so that inhibiting the release of one mediator may promote the synthesis of the other. Therefore, we investigated the effects of concomitantly inhibiting NO and PGI2synthesis, using NG-nitro- l -arginine (l -NOARG) or indomethacin, respectively, on infarct size. Langendorff-perfused rabbit hearts were assigned randomly to one of five treatment groups of n=6: control l -NOARG 100 μ mol/l; indomethacin 3 μ mol/l l -NOARG 100 μ mol/l + indomethacin 3 μ mol/l; or l -NOARG 100 μ mol/l +l -arginine 1 mmol/l. After 30 min regional ischaemia and 120 min reperfusion, infarct size was assessed by tetrazolium staining. Infarct size was reduced significantly in hearts treated with l -NOARG (20.8±1.3%) compared to control hearts (34.7±0.4%). This reduction in infarct size was abolished by co-perfusing with a 10-fold excess of l -arginine (30.7±1.7%). While indomethacin alone had no effect (33.4±2.3%), perfusion with bothl -NOARG and indomethacin resulted in a significant increase in infarct size (44.0±1.9%) compared to controls. Treatment with l -NOARG alone increased 6-keto PGF1αin coronary effluent prior to ischaemia (30.5±1.2 vs 16.6±1.3 pg/min/g in controls, P<0.05). This effect was reversed by co-perfusion with either l -arginine or indomethacin. These results indicate that the reduction in infarct size byl -NOARG may be due to increased PGI2release. Concomitant administration of indomethacin negated this effect and revealed an adverse effect of NO synthase inhibition on infarct size.

Effect of Propofol on Arachidonate Cascade by Vasopressin in Aortic Smooth Muscle Cells

The mechanisms underlying the vascular effects of propofol are not fully understood. Vasopressin, a potent vasoactive peptide, stimulates the arachidonate cascade and the synthesis of prostacyclin (PGI2; the main metabolite of the cascade in vascular smooth muscle cells). Arachidonic acid (AA) release by phospholipases is the rate-limiting step in the cascade. We investigated the mechanisms underlying vasopressin-induced AA release and the effect of propofol on PGI2 synthesis in a rat aortic smooth muscle cell line: A10 cells. In cultured A10 cells pretreated with propofol, the stimulation by vasopressin of AA release and PGI2 synthesis was evaluated by measuring [3H]AA and 6-keto PGF1alpha, respectively, in the culture medium. The effects of propofol on vasopressin-induced activation of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D were evaluated by measuring inositol phosphate formation and choline formation, respectively. A phospholipase C inhibitor and a phosphatidic acid phosphohydrolase inhibitor both attenuated vasopressin-induced AA release and PGI2 synthesis, as did a phospholipase A2 inhibitor. Propofol inhibited vasopressin-induced activation of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D, but this effect of propofol was significant only at supraclinical concentration (0.1 mM). Propofol reduced vasopressin-induced PGI2 synthesis. The inhibitory effect was observed at concentrations (10 microM-0.1 mM) higher than those used clinically. Propofol suppresses the arachidonate cascade caused by vasopressin at least partly by inhibiting phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D, resulting in the inhibition of PGI2 synthesis. Propofol-mediated inhibition of vasopressin-stimulated synthesis of PGI2 may reduce the vasorelaxation by propofol.

Interleukin 1beta decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation.

We have reported that peroxynitrite (PON) selectively inactivated prostacyclin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe-Seyler 378, 707-713]. We have now extended our studies on rat mesangial cells (RMC) and show that nitration can occur under the influence of cytokines. Pretreatment of RMC with interleukin 1beta (IL-1beta), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conversion of [14C]prostaglandin H2 (PGH2) into the stable prostacyclin (PGI2) metabolite 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha). The presence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhibitor Nomega-monomethyl-l-arginine (100 microM) as well as cycloheximide (10 microM) plus actinomycin (10 microM) abolished IL-1beta-mediated down-regulation of 6-oxo-PGF1alpha from PGH2. At the same time, 6-oxo-PGF1alpha production from arachidonate (AA) increased at the expense of prostaglandin E2 (PGE2). Neither NO alone generated from different NO donors nor superoxide from xanthine/xanthine oxidase (1-100 m-units/ml) inhibited PGI2 synthesis, either from PGH2 or from AA. Bolus additions of chemically synthesized PON or the PON generator 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF1alpha release from both PGH2 and AA. In addition, immunoprecipitation of nitrotyrosine-containing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1beta-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly suggest that IL-1beta pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.

Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial cells.

Several thrombogenic abnormalities are associated with diabetes. To investigate the underlying molecular mechanisms, we examined the effects of advanced glycation endproducts (AGE), non-enzymatically glycated protein derivatives, on the production of prostacyclin (PGI2), an anti-thrombogenic prostanoid, and of plasminogen activator inhibitor-1 (PAI-1), a fast-acting serine protease inhibitor of fibrinolysis, in human microvascular endothelial cells (EC). Firstly, AGE-bovine serum albumin (BSA) but not non-glycated BSA, was found to considerably decrease the production of PGI2 to about two-thirds of the control value. Secondly, quantitative reverse transcription-polymerase chain reaction showed that AGE-BSA increased the EC levels of mRNA coding for PAI-1, this being associated with a concomitant increase in the immunoreactive PAI-1 contents and the anti-fibrinolytic activity. Thirdly, the effects of AGE on PGI2 and PAI-1 syntheses in EC were found to be mediated by a receptor for AGE (RAGE) because antisense DNA against RAGE mRNA could reverse the AGE effects. Further, it was found that AGE decreased the intracellular cyclic AMP concentrations in EC and that cyclic AMP agonists such as dibutyryl cyclic AMP, forskolin and PGI2 analogue reduced the AGE-stimulated PAI-1 production, suggesting the involvement of cyclic AMP in the AGE-signalling pathway. The results thus suggest that AGE have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis and thereby contributing to the development and progression of diabetic vascular complications.

Estrogen upregulates cyclooxygenase-1 gene expression in ovine fetal pulmonary artery endothelium.

Prostacyclin (PGI2) is a key mediator of pulmonary vasodilation in the perinatal period and its synthesis in the pulmonary vasculature increases markedly during late gestation due to enhanced expression of the rate-limiting enzyme cyclooxygenase-1 (COX-1). The hormone estrogen may play a role in COX-1 upregulation since fetal estrogen levels rise dramatically during late gestation and estrogen enhances PGI2 synthesis in nonpulmonary vascular cells. We therefore studied the direct effects of estrogen on COX-1 expression in ovine fetal pulmonary artery endothelial cells (PAEC). Exposure to estradiol-17beta (E2beta, 10(-)10 to 10(-)6 M) caused a dose-related increase in COX-1 mRNA expression that was evident after 48 h and maximal at 10(-)8 M (fourfold increase). COX-1 mRNA stability was unchanged, suggesting that the upregulation is mediated at the level of transcription. E2beta treatment (10(-)8 M for 48 h) also caused a threefold increase in COX-1 protein expression and a threefold increase in PGI2 synthesis stimulated by bradykinin, the calcium ionophore A23187, or arachidonic acid. The estrogen receptor (ER) antagonist ICI 182,780 fully reversed the effects of the hormone on COX-1 protein expression and on arachidonic acid-stimulated PGI2 synthesis, and ER expression was evident in the PAEC by immunoblot analysis. These findings indicate that physiologic levels of estrogen cause upregulation of COX-1 expression and PGI2 synthesis in fetal PAEC via activation of PAEC ER. This process may play a critical role in optimizing the capacity for PGI2-mediated pulmonary vasodilation at birth, and it may also be involved in estrogen responsiveness in other vascular beds.

5,6-Epoxyeicosatrienoic acid reduces increases in pulmonary vascular resistance in the dog.

We recently reported that canine pulmonary microsomes metabolize arachidonic acid to all four regioisomeric epoxyeicosatrienoic acids (EET). 5,6-EET dilates blood vessels in several nonpulmonary vascular beds, often in a cyclooxygenase-dependent manner. The present study was designed to determine whether 5,6-EET can decrease pulmonary vascular resistance (PVR) in the intact pulmonary circulation. In isolated canine lungs perfused with physiological salt solution, a constant infusion of U-46619 (3.28 +/- 0.99 nmol/min) increased PVR 62.1 +/- 4.5%. Administration of 5,6-EET (10(-5) M) into the perfusate reduced the U-46619-mediated increase in PVR by 23.6 +/- 6.1%. These effects of U-46619 and 5,6-EET were limited to changes in resistance solely in the pulmonary venous segment. In contrast, venous as well as arterial segmental resistances were increased in 5-hydroxytryptamine (5-HT)-treated lungs. However, in the latter instance, 5,6-EET reduced arterial but not venous segmental resistance. 5,6-EET increased pulmonary PGI2 synthesis from 70.5 +/- 18.4 to 675.9 +/- 125.4 ng/min. In the presence of indomethacin (10(-4) M), 5,6-EET did not increase PGI2 synthesis nor did it decrease U-46619- or 5-HT-mediated increases in PVR. In canine intrapulmonary vessels, 5,6-EET decreased active tension in veins contracted with U-46619. 5,6-EET decreased active tension in arteries but not veins contracted with 5-HT, consistent with results in the perfused lungs. These results demonstrate that 5, 6-EET is a vasodilator in the intact pulmonary circulation. Its dilator activity depends on the constrictor agent present, the segmental resistance, and cyclooxygenase activity.

Dietary docosahexaenoic acid reduces the thromboxane/prostacyclin synthetic ratio in humans

The effect of fish oils, rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on eicosanoid production in vivo has been extensively studied, but data on the effects of dietary DHA alone on the synthesis of thromboxane (TXA 2) and prostacyclin (PGI 2) in humans are lacking. We quantified the effect of an isocaloric shift in DHA intake from trace (low-DHA diet) to about 6% of total fatty acids (high-DHA diet), ca. 2 en%, on the excretion of 11-dehydrothromboxane B 2 (11-DTXB 2) and 2, 3-dinor-6-oxo-prostaglandin F 1α (PGI 2-M). In a longitudinal study, seven healthy men, living in a metabolic unit, were fed a 30% fat low-DHA diet for 30 days, then a high-DHA diet containing 6g/day of DHA for 90 days. A control group of four subjects remained on the low-DHA diet for the duration of the study (120 days). Three-day urine pools were collected at the end of each dietary period (around day 30 and day 120) and analyzed for eicosanoids by gas chromatography-electron capture negative ion-tandem mass spectrometry. Mean excretion of 11-DTXB 2 was 590 ± 256 ng/d (SD; n = 7) with the low-DHA diet, and 385 ± 148 ng/d ( n = 7) with the high-DHA diet, a 35% reduction ( p = 0.013, n = 11 including the control group, when log transformed data were used). Production of 11-DTXB 2 in the control group was unchanged. Mean excretion of PGI 2-M was 229 ± 73 ng/day (SD; n = 7) and 210 ± 102 ng/day with the low-DHA and the high DHA diet, respectively (a nonsignificant reduction). This study confirms that the synthesis of TXA 2 is more open to diet-induced modulation than the synthesis of PGI 2. The observed reduction of 11-DTXB 2 excretion may be associated with measurable effects on several physiologically significant cellular functions, such as platelet aggregation in vivo and inflammation in response to immune challenges.

Effect of high glucose concentrations on prostacyclin-stimulating factor mRNA expression in cultured aortic smooth muscle cells.

Prostacyclin (PGI2) is a potent vasoactive prostanoid regulating vascular tone. We recently purified and cloned a PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular endothelial cells (ECs). Previous study demonstrated that PSF is predominantly located in vascular smooth muscle cells (SMCs) and present in serum. PSF may act on vascular ECs to regulate PGI2 synthesis for maintaining vessel wall homeostasis. Decreased PSF production in the vessel wall may result in an imbalance of prostanoid synthesis, leading to the development of vascular lesions such as diabetic angiopathy. In the present study, to investigate the regulatory mechanisms of PSF gene expression, we examined the effect of high glucose concentrations on PSF mRNA expression in cultured bovine aortic SMCs. Expression of PSF mRNA was significantly decreased to 66+/-6% of control value (p < 0.01), when the glucose level was raised from 5.5 to 27.8 mmol/l. We also examined the effect of osmolarity on PSF mRNA expression by addition of an appropriate dose of mannitol to the culture medium. We confirmed that high glucose concentration itself reduced the expression of PSF mRNA and glucose had much more effect than the osmolarity control. The expression of PSF mRNA was significantly decreased to 72+/-5% of control value (p < 0.05) by a protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA). The decreased expression of PSF mRNA in the presence of high glucose or PMA was restored by co-incubation with a PKC-specific inhibitor (GF109203X). These results suggest that PSF gene expression in vascular SMCs may be decreased via a specific effect of high glucose concentrations. High glucose-induced activation of PKC is suggested to participate partly in the regulation of PSF gene expression.