Alterations in relaxation to lactate and H(2)O(2) in human placental vessels from gestational diabetic pregnancies.

We determined whether alterations in the mechanism of relaxation to H(2)O(2) potentially contribute to the enhanced prostaglandin-mediated contractile response to H(2)O(2) and posthypoxic reoxygenation seen in human placental vessels of pregnancies with gestational diabetes mellitus (GDM). Isolated placental arteries and veins from GDM and uncomplicated full-term pregnancies were precontracted with prostaglandin F(2alpha) (PO(2) 35-38 Torr) and then exposed to lactate (1-10 mM), arachidonic acid (0.01-10 microM), nitroglycerin (1 nM-1 microM), forskolin (0.01-10 microM), or H(2)O(2) (1 microM-1 mM + 10 microM indomethacin). The rates of tissue H(2)O(2) metabolism by catalase and nitrite production were measured. The relaxation to lactate was reduced in GDM placental arteries and veins by 54-85 and 66-80%, and the relaxation to H(2)O(2) was inhibited by 80-94% in GDM placental veins compared with vessels from uncomplicated full-term pregnancies. H(2)O(2) caused only minimal relaxation of placental arteries. Responses to other relaxing agents were not altered in the GDM placental vessels. Diabetic vessels showed rates of nitrite production that were increased by 113-195% and rates of H(2)O(2) metabolism by catalase that were decreased by 44-61%. The loss of relaxation to H(2)O(2) and lactate (mediated via H(2)O(2)), perhaps as a result of the inhibition of catalase by nitric oxide, may explain the previously reported enhancement of prostaglandin-mediated contractile responses to H(2)O(2) and posthypoxic reoxygenation seen in GDM placental vessels.

Roles for NAD(P)H oxidases and reactive oxygen species in vascular oxygen sensing mechanisms.

Observations that physiological levels of O2 control the rates of production of reactive O2 species by systems including NAD(P)H oxidases and that certain of these species have signalling mechanisms that regulate vascular tone has resulted in consideration of these systems in processes that mediate the sensing of changes in P(O2). Evidence exists for the participation of hydrogen peroxide-dependent regulation of prostaglandin production and soluble guanylate cyclase activity, resulting from the metabolism of peroxide by cyclooxygenase and catalase, respectively, in P(O2)-elicited signalling mechanisms that regulate vascular force generation. A microsomal NADH oxidase whose activity is controlled by the redox status of cytosolic NAD(H) appears to function as a P(O2) sensor in bovine pulmonary and coronary arteries where changes in O2 levels control the production of superoxide anion-derived hydrogen peroxide and a cGMP-mediated relaxation response. Interactions with nitric oxide and superoxide anion, and the activity of glutathione peroxidase appear to influence the function of these O2 sensing systems, and some of these interactions, along with the activation of other oxidases, may contribute to alterations in P(O2) sensing mechanisms under pathophysiological conditions that affect vascular function.

Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD.

We have previously reported that inhibition of Cu/Zn superoxide dismutase (SOD) in endothelium-removed bovine pulmonary arteries (BPA) attenuates nitrovasodilator-elicited relaxation and that a NADH oxidase linked to the redox status of cytosolic NADH is the major detectable source of superoxide (O-2) production in this tissue. In the present study, we investigated whether NADH oxidase-derived O-2 participated in inhibition of nitrovasodilator-elicited relaxation and soluble guanylate cyclase (sGC) stimulation. Lactate (10 mM) and pyruvate (10 mM) were employed to increase and decrease, respectively, NADH-dependent O-2 production in the BPA presumably by modulating cytosolic NAD(H) through the lactate dehydrogenase reaction. A 30-min pretreatment with 10 mM diethyldithiocarbamate (DETCA) was used to inhibit Cu/Zn SOD, and S-nitroso-N-acetylpenicillamine (SNAP) was employed as a source of nitric oxide (NO). Lactate or pyruvate did not alter relaxation to NO. However, when the response to NO was inhibited by DETCA, lactate potentiated and pyruvate reduced the inhibitory effects of DETCA. SOD attenuated the inhibitory effects of DETCA plus lactate. In the presence of 10 microM SNAP, the activity of sGC in a BPA homogenate preparation (which was reconcentrated to approximate tissue conditions) was not altered by SOD. However, NADH (0.1 mM) decreased sGC activity by 70%, and this effect of NADH was attenuated in the presence of SOD. Thus cytosolic NADH redox and Cu/Zn SOD activity have important roles in controlling the inhibitory effects of O-2 derived from NADH oxidase on sGC activity and cGMP-mediated relaxation to nitrovasodilators in BPA.

Potential role of a membrane-bound NADH oxidoreductase in nitric oxide release and arterial relaxation to nitroprusside.

The site of metabolism in vascular smooth muscle responsible for the release of nitric oxide (NO) from nitroprusside is not well established. In this study we observed that a membrane-bound NADH oxidoreductase in the pulmonary artery activates nitroprusside to release NO, and we examined whether this process could potentially participate in relaxation to nitroprusside. Relaxation to nitroprusside in bovine calf pulmonary artery is inhibited by a scavenger of NO and by an antagonist of NO stimulation of guanylate cyclase. A flavoprotein probe that inhibits pulmonary artery NADH oxidoreductase (1 micromol/L diphenyliodonium) and electron acceptors for NADH oxidoreductase (0.3 mmol/L nitroblue tetrazolium and 0.1 mmol/L ferricyanide) inhibited pulmonary artery relaxation to nitroprusside, but not to nitroglycerin. Pulmonary arteries were observed to promote the release of NO from nitroprusside in vitro, and NO release was inhibited by the presence of nitroblue tetrazolium, ferricyanide, and diphenyliodonium. In homogenates of pulmonary arteries, NADH (0.1 mmol/L) increased the release of NO from nitroprusside by approximately 6-fold, whereas NADPH, mitochondrial substrates, and other redox cofactors had minimal effects on NO release, and the action of NADH on nitroprusside was inhibited by nitroblue tetrazolium, ferricyanide, and diphenyliodonium. A membrane fraction enriched in NADH oxidoreductase activity showed a NADH-dependent release of NO from nitroprusside; nitroprusside caused NADH consumption, and it also inhibited the NADH-dependent reduction of nitroblue tetrazolium. Thus, a membrane-bound NADH oxidoreductase appears to contribute to the release of NO from nitroprusside, but not nitroglycerin, in calf pulmonary artery.

Influence of glutathione peroxidase on coronary artery responses to alterations in PO2 and H2O2.

Our previous work suggests that relaxation of endothelium-removed bovine coronary arteries (BCA) to posthypoxic reoxygenation is mediated by NADH oxidase-dependent superoxide anion-derived H2O2 and cGMP. The purpose of this study was to investigate if altering BCA GSH peroxidase activity by enhancing its activity with a GSH peroxidase-mimetic (0.1 mM Ebselen) or by inhibiting its activity with an inhibitor of GSH peroxidase [10 mM mercaptosuccinic acid (MS)] causes a selective modulation of responses to exogenously (1 microM-1 mM H2O2) and endogenously generated (reoxygenation and 1-10 mM lactate) H2O2. Ebselen inhibited and MS enhanced all of the responses that are thought to be mediated by H2O2, without having significant effects on relaxation to hypoxia or a nitric oxide donor [1 nM-10 microM S-nitroso-N-acetylpenicillamine (SNAP)]. Thus enhancement of BCA GSH peroxidase activity with Ebselen inhibits relaxation to reoxygenation, lactate, and H2O2, whereas inhibition of GSH peroxidase with MS causes potentiation of responses thought to be mediated by H2O2 in BCA. Inactivation of catalase by pretreatment of BCA with 3-amino-1,2,4-triazole (50 mM, 30 min) inhibited relaxation to H2O2 and the potentiation by MS. Whereas the actions of these probes are not consistent with a role for oxidation of GSH in the relaxation to H2O2, their effects are potentially a result of modulating the metabolism of H2O2 by endogenous catalase, which is thought to mediate the stimulation of the cytosolic or soluble form of guanylate cyclase.

Oxidant--nitric oxide signalling mechanisms in vascular tissue.

Nitric oxide has several signalling mechanisms that can potentially control force generation by vascular smooth muscle. Some of these mechanisms include the stimulation of cGMP production by the soluble heme-containing form of guanylate cyclase (sGC), inhibition of mitochondrial respiration, and the modulation of vasoactive mediator release by the endothelium. Reactive O2 species (ROS) can also regulate force generation by vascular smooth muscle through mechanisms including the stimulation of production of vasoactive prostaglandins, the stimulation of sGC by catalase-mediated metabolism of H2O2 and inhibition of sGC activation by superoxide, the activation of protein kinase C, and the modulation of mediator release from the endothelium. Interactions between NO and ROS signalling mechanisms result in additional processes which modulate vascular force generation. For example, NO-elicited stimulation of sGC can be attenuated by superoxide, and this results in the formation of peroxynitrite (ONOO-). However, high levels of NO result in a ONOO- and thiol dependent formation of a species which regenerates NO in a time-dependent manner. It appears that NO inhibits catalase through an O2 and superoxide dependent process which results in inhibition of relaxation mediated by H2O2-elicited stimulation of sGC. Furthermore, evidence exists suggesting additional signalling mechanisms resulting from interactions between regulatory systems involving NO and ROS which appear to be important in control of vascular force generation in pathophysiological states.

Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: potential role of NADH oxidase.

BACKGROUND: Lactate increases lucigenin chemiluminescence (CL)-detectable superoxide anion (O2.-) generation in bovine vascular smooth muscle and endothelium, and a microsomal flavoprotein-containing NADH oxidase whose activity is regulated by PO2 and cytosolic NAD(H) redox appears to be the detected source of O2.- production. Little is known about the importance of this O2.(-)-producing system in cardiac myocytes. METHODS AND RESULTS: In isolated bovine cardiac myocytes, lactate (10 mmol/L) increased lucigenin-detectable O2.- levels to approximately 1.8 times baseline, whereas pyruvate (10 mmol/L) and mitochondrial probes did not increase the detection of O2.-. A nonmitochondrial NADH oxidase activity, found in microsomes containing a cytochrome b558, was a major source of O2.- production in the homogenate of myocytes, because NADH (0.1 mmol/L) increased basal lucigenin CL >100-fold. NADPH oxidases, mitochondria, and xanthine oxidase were minor sources of detectable O2.- production. However, mitochondria released H2O2 in the presence of 5 mmol/L succinate and 30 micromol/L antimycin, based on its detection as catalase-inhibitable luminol (+horseradish peroxidase)-elicited CL. Diphenyliodonium (DPI), an inhibitor of flavoprotein-containing oxidases, significantly attenuated basal, lactate, and NADH-elicited lucigenin CL. Hypoxia eliminated myocyte lucigenin CL, and posthypoxic reoxygenation caused an 8.6-fold increase in the detection of O2.- that was potentiated by lactate and inhibited by DPI. CONCLUSIONS: NADH oxidase activity linked to cytosolic NAD(H) redox appears to be a key source of O2.- production in cardiac myocytes that could contribute to oxidant signaling mechanisms and injury upon exposure to changes in PO2 and metabolites produced under hypoxia, such as lactate. These processes could contribute to the previously observed potentiation of injury caused by lactate in cardiac ischemia/reperfusion.

Nitric oxide inhibits pulmonary artery catalase and H2O2-associated relaxation.

Our previous studies on the mechanism of relaxation of calf pulmonary arteries to H2O2 detected a role for increased formation of guanosine-3',5'-cyclic monophosphate as a result of a catalase-elicited activation of soluble guanylate cyclase. We have also shown that lactate elicits relaxation through increasing H2O2 produced from NADH oxidase-derived superoxide anion (O2-.). Because nitric oxide (NO) is a potential inhibitor of catalase, we examined the effects of exposure of endothelium-denuded bovine calf pulmonary arteries to an elevated physiological level of NO on relaxation to H2O2 and lactate. Treatment of pulmonary arteries with approximately 50 nM of NO gas for 2 min caused a subsequent inhibition of relaxation to H2O2 (10(-6) to 10(-3)M) and lactate (1-10 mM), without markedly altering relaxation responses to S-nitroso-N-acetylpenicillamine (10(-9) to 10(-6) M) or isoproterenol (10(-9) to 10(-6) M). This NO exposure caused a 63 and 70% inhibition of the metabolism by smooth muscle catalase of both endogenously produced and exogenous (100 microM) H2O2, respectively, as measured by the H2O2-dependent cooxidation of methanol to formaldehyde. A similar treatment of purified catalase with NO caused subsequent inhibition of its ability to metabolize H2O2, associated with changes in the spectra of catalase (increases in the absorbance at 535 and 570 nm) to a species that resembled compound II, an inactive form of catalase. The exposure of pulmonary arteries to NO also resulted in the detection of H2O2 release (by catalase-inhibitable luminol/ peroxidase-chemiluminescence). Thus exposure of pulmonary arteries to increased physiological levels of NO may promote altered vasoactive responses involving H2O2 as a result of the inhibition of catalase.

Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide.

Our previous studies in isolated endothelium-removed calf pulmonary arteries suggest that PO2-elicited responses are primarily mediated through modulation of guanosine 3',5'-cyclic monophosphate via changes in the generation of H2O2 originating from superoxide anion (O2-.) produced by NADH oxidase activity. In the present study we examined the importance of this mechanism in PO2-elicited responses of endothelium-removed calf coronary arteries. NADH oxidase activity was found to be the major source of O2-. in the homogenate of endothelium-removed calf coronary arteries detected by lucigenin-elicited chemiluminescence. Precontracted endothelium-removed calf coronary arteries show a relaxation to hypoxia, and reoxygenation causes a transient additional relaxation before the recovery of normoxic levels of force. Under these conditions the detection of O2-. was decreased by hypoxia and a transient overproduction was observed during reoxygenation. The relaxation to reoxygenation, but not to hypoxia, was significantly inhibited by a scavenger of O2-. that prevents the formation of H2O2 (nitro blue tetrazolium), an inhibitor of NAD(P)H oxidases and other O2(-.)-generating flavoproteins (diphenyliodonium), and inhibition of the stimulation of soluble guanylate cyclase (LY-83583). A scavenger of O2-. that promotes H2O2 formation (Tiron) did not inhibit the PO2-elicited responses examined. Hypoxia and diphenyliodonium (but not Tiron) decreased the metabolism of endogenous H2O2 by catalase (as measured by the H2O2-dependent co-oxidation of methanol to formaldehyde by catalase), and reoxygenation caused a stimulation of H2O2 metabolism by catalase. The presence of endothelium resulted in minor modifications of the PO2 responses, which were partially mediated via prostaglandins and nitric oxide on the basis of the effects of indomethacin and nitro-L-arginine, respectively. These results suggest that in calf coronary arteries the stimulation of guanylate cyclase via H2O2 originating from NADH-derived O2-(.) production contributes to the transient relaxation to posthypoxic reoxygenation, but not the response to hypoxia.