Prostaglandin release from the human umbilical artery in vitro

Release of prostaglandins from human umbilical artery preparations into the surrounding bathing fluid was studied by radioimmunoassay using PGF_2α antibodies. A significant release of prostaglandins was found under conditions where a spontaneous tone of the artery could be maintained. Indometacin reduced the prostaglandin release and the spontaneous tone of the artery. Intramural synthesis of prostaglandins in the human umbilical arteries is postulated.     

The effect of prostacyclin on the human umbilical artery

Prostacyclin was tested on human umbilical artery obtained after spontaneous delivery or by Cesarean section. Isometric and isotonic responses were measured on spiral preparations in Krebs-bicarbonate buffer at 37°C equilibrated with 95% O₂ and 5% CO₂. Spiral artery strips, whether superfused or mounted in organ baths isometrically or isotonically, responded in a dose-dependent manner to both prostacyclin and serotonin; the PGI₂ response was biphasic in that low doses (2.5 × 10^-8 M - 1.0 × 10^-6 M) elicited a dose-dependent relaxation which changed with higher concentrations (1.0 × 10^-6 M - 2.53 × 10⁵ M) to a contractile response. The maximum tension exerte was 50% less than that elicited by serotonin. The data indicate that the human umbilical artery is responsive to prostacyclin and may be involved in the regulation of fetal placenta blood flow.     

The effect of prostacyclin on the human umbilical artery.

Prostacyclin was tested on human umbilical artery obtained after spontaneous delivery or by Cesarean section. Isometric and isotonic responses were measured on spiral preparations in Krebs-bicarbonate buffer at 37 degrees C equilibrated with 95% O2 and 5% CO2. Spiral artery strips, whether superfused or mounted in organ baths isometrically or isotonically, responded in a dose-dependent manner to both prostacyclin and serotonin; the PGI2 response was biphasic in that low doses (2.5 x 10(-8) M -1.0 x 10(-6) M) elicited a dose-dependent relaxation which changed with higher concentrations (1.0 x 10(-6) M -2.53 X 10(-5) M) to a contractile response. The maximum tension exerted was 50% less than that elicited by serotonin. The data indicate that the human umbilical artery is responsive to prostacyclin and may be involved in the regulation of fetal placenta blood flow.     

Human recombinant lipocortin 1 inhibits prostacyclin production by human umbilical artery

Submicromolar concentrations of human recombinant Lipocortin 1 inhibit the release of prostacycin from human umbilical artery rings in a dose-dependent fashion. This is the first demonstration that the recombinant protein is effective in human cells.     

Human recombinant lipocortin 1 inhibits prostacyclin production by human umbilical artery in vitro

Submicromolar concentrations of human recombinant Lipocortin 1 inhibit the release of prostacyclin from human umbilical artery rings in a dose-dependent fashion. This is the first demonstration that the recombinant protein is effective in human cells.     

17 alpha-ethinylestradiol decreases production and release of prostacyclin in cultured human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC) were incubated with the estrogen 17 alpha-Ethinylestradiol (30 ng/ml) in order to examine its regulating influence on synthesis and release of prostacyclin (PGI2). ATP (1 mg/ml) was used to stimulate PGI2-production through the purine receptors. We demonstrated that this level of estrogen decreases PGI2-synthesis by 11% and PGI2-release by 32% within 300 sec. Longer incubation times (48 hrs) resulted in the same inhibitory effect. Intracellular ATP content and methyl-3H-thymidine uptake demonstrated that the decrease of prostacyclin-concentration is not caused by reduced viability of the cells but by a direct inhibitory effect on prostacyclin synthesis and release.     

Cocaine use and its effect on umbilical artery prostacyclin production

Cocaine's association with adverse perinatal outcome has been attributed to its inhibition of norepinephrine uptake. This study examined the effect of cocaine on umbilical artery prostacyclin (PGI2) production. Umbilical arteries from pregnant cocaine users and controls were incubated in vitro and PGI2 levels in the media determined by measuring its stable metabolite, 6-keto-PGF1 alpha, by RIA. Cocaine users showed a significant decrease (p less than .05) in PGI2 production from their umbilical arteries when compared to controls. This appears to be through a direct effect of cocaine, as it decreases PGI2 production when added in vitro to umbilical arteries from controls. In addition, in vitro phospholipase A2 activity is inhibited by cocaine in a dose-dependent manner. These results suggest that the adverse perinatal outcome associated with cocaine use may be due in part to reduced vascular PGI2 production in the fetus.     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Histamine-induced phosphoinositide metabolism in cultured human umbilical vein endothelial cells. Association with thromboxane and prostacyclin release

Histamine stimulation of cultured human umbilical vein endothelial cells induced dose- and time-dependent increases in glycerophosphoinositol (GroPIns), inositol-1-phosphate (InsP), inositolbisphosphate (InsP2) and inositoltrisphosphate (InsP3) in addition to release of thromboxane A2 and prostacyclin. Increases in InsP2 and InsP3 were immediate while increases in GroPIns and InsP occurred only after 1 min. Thromboxane A2 and prostacyclin release paralleled GroPIns and InsP production. The data indicate that, in endothelial cells, histamine evokes early hydrolysis of polyphosphoinositides, and that subsequent mobilization of arachidonic acid for thromboxane and prostacyclin synthesis involves both deacylation and phosphodiesteratic cleavage of phosphatidylinositol.     

Prostanoid synthesis by human umbilical artery

A discrepancy between published values of PGI₂ production by human umbilical artery measured by platelet bioassay, compared with values of 6-oxo-PGF_1α by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI₂ determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF_1α in hydrolysed and non-hydrolysed samples. 6-oxo-PGF_1a, PGF_2α and TXB₂ were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI₂ concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p < 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI₂ production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59–1.53 ng/mg/h) differed from previously reported values (range 70–325 ng/mg/h). 6-oxo-PGF_1α concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI₂ production by this tissue overestimated it by approximately 100 times.     

Prostanoid synthesis by human umbilical artery

A discrepancy between published values of PGI2 production by human umbilical artery in vitro measured by platelet bioassay, compared with values of 6-oxo-PGF1 alpha by radioimmunoassay, raised the possibility that another anti-aggregatory prostanoid was produced by this tissue. To test this hypothesis, umbilical artery rings were incubated in buffer and PGI2 determined by platelet bioassay and by a more specific radioimmunoassay based on comparison of 6-oxo-PGF1 alpha in hydrolysed and non-hydrolysed samples. 6-oxo-PGF1 alpha, PGF2 alpha and TXB2 were also measured by gas chromatography negative ion chemical ionisation mass spectrometry. PGI2 concentrations by radioimmunoassay and bioassay were significantly correlated (r = 0.92, p less than 0.01). There was no difference between them, disproving the presence of an additional antiaggregatory substance. PGI2 production determined by bioassay (mean 1.21 ng/mg wet weight/h, range 0.59-1.53 ng/mg/h) differed from previously reported values (range 70-325 ng/mg/h). 6-oxo-PGF1 alpha concentrations were confirmed by gas chromatography negative ion chemical ionisation mass spectrometry. Previous determinations of PGI2 production by this tissue overestimated it by approximately 100 times.     

Thromboxane and prostacyclin generation by human umbilical veins after thrombin.

Prostacyclin (PGI2) and Thromboxane B2 (TxB2) production induced by thrombin in human umbilical veins (HUV) was studied. Successive stimulations of HUV segments were performed with and without restoration of arachidonic acid (AA). Thrombin consistently stimulated the production of both substances. The magnitude of the increment declined with progressive stimuli. The addition of exogenous AA could restore the production of TXB2 but not that of PGI2. These results suggest that sustained stimulation of AA release may lead to an imbalance in the TXA2/PGI2 ratio perhaps through an effect of unknown products of AA oxidation on PGI2 synthase.     

Histamine stimulates prostacyclin synthesis in cultured human umbilical vein endothelial cells

Human venous endothelial cells synthesize prostacyclin (PGI₂) in response to treatment with histamine. The amount of PGI₂ produced is proportional to the histamine concentration over the range of 10^?7 to 10^?5 M, with a maximal response at 2–5 × 10^?6 M. PGI₂ synthesis occurs as a burst lasting less than 3 minutes after histamine addition. The H1 histamine receptor antagonist pyrilamine causes an 87% inhibition of PGI₂ synthesis, whereas the H2 antagonist cimetidine gives no significant inhibition, suggesting that PGI₂ synthesis in response to histamine is mediated by an H1 receptor.     

Effect of ethanol on thromboxane and prostacyclin synthesis by fetal platelets and umbilical artery

The effect of ethanol (10-500 mmol/l) on platelet thromboxane production and on vascular thromboxane and prostacyclin was studied in human fetal tissues. The release of thromboxane B2 (a metabolite of thromboxane A2) during thrombin-induced spontaneous aggregation of fetal platelets was inhibited by ethanol concentrations of 50 mmol/l or higher. Ethanol at concentration from 100 mmol/l also inhibited umbilical artery production of thromboxane B2 and that of 6-keto-prostaglandin F1 alpha (a metabolite of prostacyclin). However, it stimulated the conversion of exogenous arachidonic acid to thromboxane B2 in fetal platelets and to 6-keto-prostaglandin F1 alpha in the umbilical artery. This suggests that ethanol inhibits phospholipase A2, but stimulates the enzymes distal from phospholipase A2 in the prostaglandin-synthesizing enzyme cascade.     

Element content of human umbilical artery and vein in umbilical cord

To elucidate the element content of newborn blood vessels, umbilical arteries and veins in human umbilical cords, which had the advantage of easy sampling, were examined by ICP-AES. Umbilical cords were removed after birth. Mothers’ ages ranged from 26 to 35 yr. It was found that the content of sulfur was the highest in both umbilical arteries and veins, being higher than the content of calcium and phosphorus. With respect of the content of sulfur, calcium, and magnesium, there were significant differences between the arteries and veins.     

Shear stress enhances prostacyclin release from endocardial endothelial cells

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.     

Angiogenin activates human umbilical artery smooth muscle cells

Angiogenin stimulates proliferation of human umbilical artery smooth muscle cells. This activity of angiogenin depends on the cell density and requires nuclear translocation of the ligand as well as activation of SAPK/JNK MAP kinase. Angiogenin binds to a 170-kDa putative receptor on the cell surface and induces phosphorylation of SAPK/JNK. It also undergoes nuclear translocation in a time and concentration dependent manner. Neomycin inhibits nuclear translocation of angiogenin and abolishes angiogenin-induced cell proliferation but does not inhibit SAPK/JNK phosphorylation. The data demonstrate that smooth muscle cells are targets for angiogenin and that both SAPK/JNK phosphorylation and nuclear translocation of the ligand are required for angiogenin to activate smooth muscle cells.     

Effects of arsenate and ergot alkaloid compounds on prostacyclin synthesis in human umbilical endothelium

Cultured human umbilical endothelium was incubated with various concentrations of o-arsenilic acid and ergotamine tartrate respectively for 72 hr at 37 degrees C, 5% CO2/95% air in 100% humidity. At the end of incubation, medium was removed for the determination of 6-keto-PGF1 alpha concentration by RIA method. Compared to the normal controls, arsenate (0.1 to 10.0uM) showed inhibition (17 to 24%) on the PGI2 production. Ergotamine tartrate gave an activatory effect (20 to 12%) in low concentration (0.1 to 1.0uM) incubation, but had inhibitory effect (25%) on the PGI2 production in higher concentration incubation (10.0uM). These results indicate arsenate in low and high concentrations does not play an important role on prostacyclin synthesis in human umbilical endothelium. However, ergotamine tartrate at higher concentration might be a main factor for the lower prostacyclin synthesis in Blackfoot disease, an endemic disease of the peripheral vascular system found in the southwest coast of Taiwan.