calcimycin and sepiapterin

To investigate whether decidual endothelial cells (DEC) contribute to the pathogenesis of preeclampsia through abnormal nitric oxide production. Decidual endothelial cells from normal (NDEC) and preeclamptic (PEDEC) pregnancies, and also human umbilical vein endothelial cells (HUVEC), were examined.. HUVEC, NDEC, and PEDEC were incubated for 45 min in serum-free media with the addition of potential stimulators [calcium ionophore (A23187), sepiapterin, and a combination of cytokines (TNF-alpha, gamma-IFN and LPS)], and the competitive inhibitor, NG-monomethyl-L-arginine (L-NMMA). These were added alone or in combination. Supernatants were measured for nitrate/nitrite (NOx) levels and the cells acid-extracted for measurement of cyclic guanosine monophosphate (cGMP). The effect of 30 min of shear stress (approximately 20 dynes/cm2) on NO and cGMP production by NDEC and PEDEC and on production of prostacyclin and thromboxane A2, was assessed.. PEDEC and HUVEC both produced more NO than NDEC under all conditions examined. Cell-associated cGMP levels, however, were not different among the cell groups but were increased by A23187 and inhibited by L-NMMA. In control conditions, shear stress stimulated cGMP levels 5-fold (p<0.01) in both NDEC and PEDEC, and PGI2 production 2-fold (p<0.05).. DEC from preeclamptic women do not have reduced NO production and respond normally to shear stress by increasing cGMP and PGI2 production. Our results are consistent with other reports of equal or higher NO levels in preeclampsia and indicate that reduced NO production by endothelial cells is not the explanation for the vasoconstriction of uterine vessels.

Topics: Adult; Calcimycin; Case-Control Studies; Cells, Cultured; Cyclic GMP; Cytokines; Decidua; Endothelium, Vascular; Epoprostenol; Female; Humans; Nitric Oxide; omega-N-Methylarginine; Pre-Eclampsia; Pregnancy; Pteridines; Pterins; Thromboxane A2; Umbilical Veins

Tetrahydrobiopterin (BH4) is of fundamental importance for the normal function of endothelial NO synthase. The purpose of this study was to investigate the effects of hyperlipidemia on vascular BH4 levels and the effect of supplementation with sepiapterin in the presence and absence of N-acetylcysteine (NAC).. New Zealand White rabbits were fed normal chow (normocholesterolemic [NC] group) or hyperlipidemic chow (hyperlipidemic [HL] group) for 8 to 10 weeks. Mean cholesterol levels were 1465+/-333 and 53+/-17 mg/dL in the HL and NC group, respectively. Markedly diminished BH4 levels were found in the HL group compared with the NC group, but these levels could be restored after 6 hours of incubation with sepiapterin. Peak relaxations to acetylcholine and A23187 were impaired in the HL group. Supplementation with sepiapterin resulted in a further diminution of relaxation in the HL but not NC group. Incubation with NAC for 6 hours failed to raise BH4 levels, whereas NAC in conjunction with sepiapterin raised BH4 levels approximately 221-fold. However, this increase did not improve relaxations to A23187 and acetylcholine.. Prolonged exposure to sepiapterin impairs vasorelaxation in hyperlipidemia despite repletion of endogenous BH4. Antioxidant thiols do not correct this impairment. These studies have implications for the use of sepiapterin in the correction of vasomotor tone in atherosclerosis.

Topics: Acetylcholine; Acetylcysteine; Animals; Antioxidants; Aorta, Thoracic; Arteriosclerosis; Biopterins; Calcimycin; Cholesterol; Diet; Endothelium, Vascular; Free Radical Scavengers; Hyperlipidemias; Ionophores; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxygen; Pteridines; Pterins; Rabbits; Sulfhydryl Compounds; Vasodilator Agents

Altered endothelial cell nitric oxide (NO(*)) production in atherosclerosis may be due to a reduction of intracellular tetrahydrobiopterin, which is a critical cofactor for NO synthase (NOS). In addition, previous literature suggests that inactivation of NO(*) by increased vascular production superoxide (O(2)(*-)) also reduces NO(*) bioactivity in several disease states. We sought to determine whether these 2 seemingly disparate mechanisms were related.. Endothelium-dependent vasodilation was abnormal in aortas of apoE-deficient (apoE(-/-)) mice, whereas vascular superoxide production (assessed by 5 micromol/L lucigenin) was markedly increased. Treatment with either liposome-entrapped superoxide dismutase or sepiapterin, a precursor to tetrahydrobiopterin, improved endothelium-dependent vasodilation in aortas from apoE(-/-) mice. Hydrogen peroxide had no effect on the decay of tetrahydrobiopterin, as monitored spectrophotometrically. In contrast, superoxide modestly and peroxynitrite strikingly increased the decay of tetrahydrobiopterin over 500 seconds. Luminol chemiluminescence, inhibitable by the peroxynitrite scavengers ebselen and uric acid, was markedly increased in apoE(-/-) aortic rings. In vessels from apoE(-/-) mice, uric acid improved endothelium-dependent relaxation while having no effect in vessels from control mice. Treatment of normal aortas with exogenous peroxynitrite dramatically increased vascular O(2)(*-) production, seemingly from eNOS, because this effect was absent in vessels lacking endothelium, was blocked by NOS inhibition, and did not occur in vessels from mice lacking eNOS.. Reactive oxygen species may alter endothelium-dependent vascular relaxation not only by the interaction of O(2)(*-) with NO(*) but also through interactions between peroxynitrite and tetrahydrobiopterin. Peroxynitrite oxidation of tetrahydrobiopterin may represent a pathogenic cause of "uncoupling" of NO synthase.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Apolipoproteins E; Biopterins; Calcimycin; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; In Vitro Techniques; Ionophores; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroglycerin; Pteridines; Pterins; Superoxides; Vasodilation; Vasodilator Agents

Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose-dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO(2)(-) + NO(3)(-)) production and l-arginine to l-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by approximately 50% without altering the K(m) for l-arginine. The effect of ascorbate was tetrahydrobiopterin (BH(4))-dependent, because ascorbate was ineffective with BH(4) concentrations >10 microm or in PAECs treated with sepiapterin to increase intracellular BH(4). The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH(4). These data suggest that ascorbic acid enhances NO bioactivity in a BH(4)-dependent manner by increasing intracellular BH(4) content.

Topics: Animals; Aorta; Arginine; Ascorbic Acid; Atrial Natriuretic Factor; Biopterins; Calcimycin; Cattle; Cell Line; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Kinetics; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Pteridines; Pterins; Recombinant Proteins; Spodoptera; Swine; Transfection

The 4-amino analogue of tetrahydrobiopterin (4-ABH(4)) is a potent pterin-site inhibitor of nitric oxide synthases (NOS). Although 4-ABH(4) does not exhibit selectivity between purified NOS isoforms, a pronounced selectivity of the drug towards inducible NOS (iNOS) is apparent in intact cells. This work was carried out to investigate the potential iNOS selectivity of 4-ABH(4) in isolated pig pulmonary and coronary arteries. Endothelium-dependent relaxations of pig pulmonary and coronary artery strips to bradykinin or calcium ionophore A23187 were inhibited by 4-ABH(4) in a concentration-dependent manner. Half-maximal inhibition was observed at 60 - 65 microM (pulmonary artery) and 200 - 250 microM 4-ABH(4) (coronary artery). Pig coronary artery strips precontracted with 0.1 microM 9, 11-dideoxy-9, 11-methanoepoxy-prosta-glandin F(2alpha) (U46619) showed a time-dependent relaxation (monitored for up to 18 h) upon incubation with 1 microg ml(-1) lipopolysaccharide (LPS). Addition of 10 microM 4-ABH(4) 1 h after LPS led to a pronounced inhibition of the LPS-triggered relaxation, whereas the pterin antagonist had no effect when given> or =4 h after LPS. Incubation of pulmonary and coronary artery strips with 1 microg ml(-1) LPS attenuated contractile responses to norepinephrine (1 microM) and U46619 (0.1 microM). This hyporeactivity of the blood vessels to vasoconstrictor agents was inhibited by 4-ABH(4) in a concentration-dependent manner [IC(50)=17.5+/-5.9 microM (pulmonary artery) and 20.7+/-3 microM (coronary artery)]. The effect of 0.1 mM 4-ABH(4) was antagonized by coincubation with 0.1 mM sepiapterin, which is known to supply intracellular BH(4) via a salvage pathway. These results demonstrate that 4-ABH(4) is a fairly selective inhibitor of iNOS in an in vitro model of endotoxaemia, suggesting that this drug and/or related pterin-site NOS inhibitors may be useful to increase blood pressure in severe infections associated with a loss of vascular responsiveness to constrictor agents caused by endotoxin-triggered iNOS induction in the vasculature.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biopterins; Bradykinin; Calcimycin; Coronary Vessels; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxins; Enzyme Inhibitors; Hydrazines; In Vitro Techniques; Lipopolysaccharides; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Nitrogen Oxides; Norepinephrine; Pteridines; Pterins; Pulmonary Artery; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilation