TET2 confers a mechanistic link of microRNA-210 and mtROS in hypoxia-suppressed spontaneous transient outward currents in uterine arteries of pregnant sheep.

Hypoxia during pregnancy impairs uterine vascular adaptation via microRNA-210 (miR-210)-mediated mitochondrial dysfunction and mitochondrial reactive oxygen species (mtROS) generation. TET methylcytosine dioxygenase 2 (TET2) participates in regulating inflammation and oxidative stress and its deficiency contributes to the pathogenesis of multiple cardiovascular diseases. Thus, we hypothesize a role of TET2 in hypoxia/miR-210-mediated mtROS suppressing spontaneous transient outward currents (STOCs) in uterine arteries. We found that gestational hypoxia downregulated TET2 in uterine arteries of pregnant sheep and TET2 was a target of miR-210. Knockdown of TET2 with small interfering RNAs suppressed mitochondrial respiration, increased mtROS, inhibited STOCs and elevated myogenic tone. By contrast, overexpression of TET2 negated hypoxia- and miR-210-induced mtROS. The effects of TET2 knockdown in uterine arteries on mtROS, STOCs and myogenic contractions were blocked by the mitochondria-targeted antioxidant MitoQ. In addition, the recovery effects of inhibiting endogenous miR-210 with miR-210-LNA on hypoxia-induced suppression of STOCs and augmentation of myogenic tone were reversed by TET2 knockdown in uterine arteries. Together, our study reveals a novel mechanistic link between the miR-210-TET2-mtROS pathway and inhibition of STOCs and provides new insights into the understanding of uterine vascular maladaptation in pregnancy complications associated with gestational hypoxia. KEY POINTS: Gestational hypoxia downregulates TET methylcytosine dioxygenase 2 (TET2) in uterine arteries of pregnant sheep. TET2 is a downstream target of microRNA-210 (miR-210) and miR-210 mediates hypoxia-induced TET2 downregulation. Knockdown of TET2 in uterine arteries recapitulates the effect of hypoxia and miR-210 and impairs mitochondrial bioenergetics and increases mitochondrial reactive oxygen species (mtROS) . Overexpression of TET2 negates the effect of hypoxia and miR-210 on increasing mtROS. TET2 knockdown reiterates the effect of hypoxia and miR-210 and suppresses spontaneous transient outward currents (STOCs) and elevates myogenic tone, and these effects are blocked by MitoQ. Knockdown of TET2 reverses the miR-210-LNA-induced reversal of the effects of hypoxia on STOCs and myogenic tone in uterine arteries.

Iron nitrosyl complexes are formed from nitrite in the human placenta.

Placental nitric oxide (NO) is critical for maintaining perfusion in the maternal-fetal-placental circulation during normal pregnancy. NO and its many metabolites are also increased in pregnancies complicated by maternal inflammation such as preeclampsia, fetal growth restriction, gestational diabetes, and bacterial infection. However, it is unclear how increased levels of NO or its metabolites affect placental function or how the placenta deals with excessive levels of NO or its metabolites. Since there is uncertainty over the direction of change in plasma levels of NO metabolites in preeclampsia, we measured the levels of these metabolites at the placental tissue level. We found that NO metabolites are increased in placentas from patients with preeclampsia compared to healthy controls. We also discovered by ozone-based chemiluminescence and electron paramagnetic resonance that nitrite is efficiently converted into iron nitrosyl complexes (FeNOs) within the human placenta and also observed the existence of endogenous FeNOs within placentas from sheep and rats. We show these nitrite-derived FeNOs are relatively short-lived, predominantly protein-bound, heme-FeNOs. The efficient formation of FeNOs from nitrite in the human placenta hints toward the importance of both nitrite and FeNOs in placental physiology or pathology. As iron nitrosylation is an important posttranslational modification that affects the activity of multiple iron-containing proteins such as those in the electron transport chain, or those involved in epigenetic regulation, we conclude that FeNOs merit increased study in pregnancy complications.

Nitric Oxide Affects Heme Oxygenase-1, Hepcidin, and Transferrin Receptor Expression in the Placenta.

Nitric oxide (NO) is a gasotransmitter that avidly binds both free and heme-bound iron, forming relatively stable iron nitrosyl compounds (FeNOs). We have previously demonstrated that FeNOs are present in the human placenta and are elevated in preeclampsia and intrauterine growth restriction. The ability of NO to sequester iron raises the possibility of the NO-mediated disruption of iron homeostasis in the placenta. In this work, we tested whether exposure of placental syncytiotrophoblasts or villous tissue explants to sub-cytotoxic concentrations of NO would elicit the formation of FeNOs. Furthermore, we measured changes in the mRNA and protein expression levels of key iron regulatory genes in response to NO exposure. Ozone-based chemiluminescence was used to measure concentrations of NO and its metabolites. Our results showed a significant increase in FeNO levels in placental cells and explants treated with NO (p < 0.0001). The mRNA and protein levels of HO-1 were significantly increased in both cultured syncytiotrophoblasts and villous tissue explants (p < 0.01), and the mRNA levels of hepcidin and transferrin receptor were significantly increased in culture syncytiotrophoblasts and villous tissue explants, respectively, (p < 0.01), while no changes were seen in the expression levels of divalent metal transporter-1 or ferroportin. These results suggest a potential role for NO in iron homeostasis in the human placenta and could be relevant for disorders of pregnancy such as fetal growth restriction and preeclampsia.

Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca2+ Imaging Datasets.

Intracellular Ca2+ signals are key for the regulation of cellular processes ranging from myocyte contraction, hormonal secretion, neural transmission, cellular metabolism, transcriptional regulation, and cell proliferation. Measurement of cellular Ca2+ is routinely performed using fluorescence microscopy with biological indicators. Analysis of deterministic signals is reasonably straightforward as relevant data can be discriminated based on the timing of cellular responses. However, analysis of stochastic, slower oscillatory events, as well as rapid subcellular Ca2+ responses, takes considerable time and effort which often includes visual analysis by trained investigators, especially when studying signals arising from cells embedded in complex tissues. The purpose of the current study was to determine if full-frame time-series and line-scan image analysis workflow of Fluo-4 generated Ca2+ fluorescence data from vascular myocytes could be automated without introducing errors. This evaluation was addressed by re-analyzing a published "gold standard" full-frame time-series dataset through visual analysis of Ca2+ signals from recordings made in pulmonary arterial myocytes of en face arterial preparations. We applied a combination of data driven and statistical approaches with comparisons to our published data to assess the fidelity of the various approaches. Regions of interest with Ca2+ oscillations were detected automatically post hoc using the LCPro plug-in for ImageJ. Oscillatory signals were separated based on event durations between 4 and 40 s. These data were filtered based on cutoffs obtained from multiple methods and compared to the published manually curated "gold standard" dataset. Subcellular focal and rapid Ca2+ "spark" events from line-scan recordings were examined using SparkLab 5.8, which is a custom automated detection and analysis program. After filtering, the number of true positives, false positives, and false negatives were calculated through comparisons to visually derived "gold standard" datasets. Positive predictive value, sensitivity, and false discovery rates were calculated. There were very few significant differences between the automated and manually curated results with respect to quality of the oscillatory and Ca2+ spark events, and there were no systematic biases in the data curation or filtering techniques. The lack of statistical difference in event quality between manual data curation and statistically derived critical cutoff techniques leads us to believe that automated analysis techniques can be reliably used to analyze spatial and temporal aspects to Ca2+ imaging data, which will improve experiment workflow.

Nitric oxide metabolism in the human placenta during aberrant maternal inflammation.

KEY POINTS: Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre-eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre-eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. ABSTRACT: Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone-based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre-eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non-villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre-eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre-eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre-eclampsia and fetal growth restriction.

Evidence for placental-derived iron-nitrosyls in the circulation of the fetal lamb and against a role for nitrite in mediating the cardiovascular transition at birth.

Circulating metabolites of nitric oxide, such as nitrite, iron nitrosyls (FeNO), and nitrosothiols, have vasodilatory bioactivity. In both human and sheep neonates, plasma concentrations of these NO metabolite (NOx) concentrations fall >50% within minutes after birth, raising the possibility that circulating NOx plays a role in maintaining low fetal vascular resistance and in the cardiovascular transition at birth. To test whether the fall in plasma NOx concentrations at birth is due to either ligation of the umbilical cord or oxygenation of the fetus to newborn levels, plasma NOx concentrations were measured during stepwise delivery of near-term fetal lambs. When fetal lambs were intubated and mechanically ventilated with 100% O2 to oxygenate the arterial blood while still in utero with the umbilical circulation still intact, there was no change in plasma NOx levels. In contrast, when the umbilical cord was ligated while fetal lambs were mechanically ventilated with O2 levels that maintained fetal arterial blood gases, plasma NOx levels decreased by nearly 50%. Characterization of the individual NOx species in plasma revealed that the overall fall in NOx at birth was attributable mainly to FeNO compounds. Finally, when the typical fall in NOx after birth was prevented by intravenous nitrite infusion, birth-related changes in blood pressure, heart rate, and carotid flow changes were little affected, suggesting the cardiovascular transition at birth is not dependent on a fall in plasma NOx. In conclusion, this study shows FeNO is released from the placenta and that its decline accounts for most of the measured fall in plasma NOx at birth.

The role of gasotransmitters in neonatal physiology.

The gasotransmitters, nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO), are endogenously-produced volatile molecules that perform signaling functions throughout the body. In biological tissues, these small, lipid-permeable molecules exist in free gaseous form for only seconds or less, and thus they are ideal for paracrine signaling that can be controlled rapidly by changes in their rates of production or consumption. In addition, tissue concentrations of the gasotransmitters are influenced by fluctuations in the level of O2 and reactive oxygen species (ROS). The normal transition from fetus to newborn involves a several-fold increase in tissue O2 tensions and ROS, and requires rapid morphological and functional adaptations to the extrauterine environment. This review summarizes the role of gasotransmitters as it pertains to newborn physiology. Particular focus is given to the vasculature, ventilatory, and gastrointestinal systems, each of which uniquely illustrate the function of gasotransmitters in the birth transition and newborn periods. Moreover, given the relative lack of studies on the role that gasotransmitters play in the newborn, particularly that of H2S and CO, important gaps in knowledge are highlighted throughout the review.

Cerebral and Renal Oxygenation in Infants Undergoing Laparoscopic Gastrostomy Tube Placement.

AIM: The aim of this study was to evaluate the effects of a carbon dioxide pneumoperitoneum on cerebral and renal oxygenation and oxygen extraction, in a cohort of infants from the neonatal intensive care unit, undergoing laparoscopic gastrostomy. METHODS: After institutional review board approval, between February 2018 and June 2019, infants 0-3 mo corrected age, undergoing laparoscopic gastrostomy tube placement, were included. Strict exclusion criteria created a homogeneous cohort. Cerebral and renal tissue oxygen saturation (rSO2) by near-infrared spectroscopy, skin surface oxygen saturation (SpO2), by pulse oximetry, and amplitude-integrated electroencephalography were measured. Monitoring was divided into preoperative, intraoperative and postoperative time periods. Cerebral and renal fractional tissue oxygen extraction was calculated using arterial (SpO2) and tissue oxygen saturation (rSO2): (SpO2-rSO2SpO2)X100. Data were averaged into one-minute epochs and significant changes from baseline during the intraoperative and postoperative periods were detected using one-way analysis of variance with repeated measures. RESULTS: This pilot study examined sixteen infants, born at a median gestational age of 34.2 wk (range: 23.0-40.6) with a median corrected age of 42.9 wk (range: 40.0-46.3) at operation. None had seizure activity or altered sleep-wake cycles. No statistically significant variations in cerebral and renal tissue oxygenation and extraction were observed. Pulse oximetry did demonstrate significant variation from baseline on analysis of variance, but post hoc analysis did not identify any one specific time point at which this difference was significant. CONCLUSIONS: During a short infant laparoscopic procedure, no significant alteration in cerebral or renal oxygenation or oxygen extraction was observed. No seizure activity or changes in infant sleep-wake cycles occurred.

Human milk oligosaccharide composition predicts risk of necrotising enterocolitis in preterm infants.

OBJECTIVE: Necrotising enterocolitis (NEC) is one of the most common and often fatal intestinal disorders in preterm infants. Markers to identify at-risk infants as well as therapies to prevent and treat NEC are limited and urgently needed. NEC incidence is significantly lower in breast-fed compared with formula-fed infants. Infant formula lacks human milk oligosaccharides (HMO), such as disialyllacto-N-tetraose (DSLNT), which prevents NEC in neonatal rats. However, it is unknown if DSLNT also protects human preterm infants. DESIGN: We conducted a multicentre clinical cohort study and recruited 200 mothers and their very low birthweight infants that were predominantly human milk-fed. We analysed HMO composition in breast milk fed to infants over the first 28 days post partum, matched each NEC case with five controls and used logistic regression and generalised estimating equation to test the hypothesis that infants who develop NEC receive milk with less DSLNT than infants who do not develop NEC. RESULTS: Eight infants in the cohort developed NEC (Bell stage 2 or 3). DSLNT concentrations were significantly lower in almost all milk samples in NEC cases compared with controls, and its abundance could identify NEC cases prior to onset. Aggregate assessment of DSLNT over multiple days enhanced the separation of NEC cases and control subjects. CONCLUSIONS: DSLNT content in breast milk is a potential non-invasive marker to identify infants at risk of developing NEC, and screen high-risk donor milk. In addition, DSLNT could serve as a natural template to develop novel therapeutics against this devastating disorder.

Hemodynamic Effects of Glutathione-Liganded Binuclear Dinitrosyl Iron Complex: Evidence for Nitroxyl Generation and Modulation by Plasma Albumin.

Glutathione-liganded binuclear dinitrosyl iron complex (glut-BDNIC) has been proposed to be a donor of nitric oxide (NO). This study was undertaken to investigate the mechanisms of vasoactivity, systemic hemodynamic effects, and pharmacokinetics of glut-BDNIC. To test the hypothesis that glut-BDNICs vasodilate by releasing NO in its reduced [nitroxyl (HNO)] state, a bioassay method of isolated, preconstricted ovine mesenteric arterial rings was used in the presence of selective scavengers of HNO or NO free radical (NO•); the vasodilatory effects of glut-BDNIC were found to have characteristics similar to those of an HNO donor and markedly different than an NO• donor. In addition, products of the reaction of glut-BDNIC with CPTIO [2-(4-carboxyphenyl)-4,4,5-tetramethyl imidazoline-1-oxyl-3-oxide] were found to have electron paramagnetic characteristics similar to those of an HNO donor compared with an NO• donor. In contrast to S-nitroso-glutathione, which was vasodilative both in vitro and in vivo, the potency of glut-BDNIC-mediated vasodilation was markedly diminished in both rats and sheep. Wire myography showed that plasma albumin contributed to this loss of hypotensive effects, an effect abolished by modification of the cysteine-thiol residue of albumin. High doses of glut-BDNIC caused long-lasting hypotension in rats that can be at least partially attributed to its long circulating half-life of ∼44 minutes. This study suggests that glut-BDNIC is an HNO donor, and that its vasoactive effects are modulated by binding to the cysteine residue of plasma proteins, such as albumin.

A novel rodent model of pregnancy complications associated with genetically determined angiotensin-converting enzyme (ACE) activity.

Brown Norway (BN) and Lewis (LW) inbred rat strains harbor different angiotensin-converting enzyme ( Ace) polymorphisms that result in higher ACE activity in BN than LW rats. Thus we hypothesized that pregnant BN rats would show pregnancy complications linked to angiotensin II (AII) activity. We performed longitudinal and cross-sectional studies in pregnant LW and BN rats. We found that BN rats have significantly higher ACE activity and AII levels at prepregnancy and throughout pregnancy compared with LW rats, except at midgestation. BN placentas and maternal kidneys had significantly higher expression of AII receptor 1 (AGTR1) and lower expression of AGTR2 than the respective LW placentas and maternal kidneys. Renin-angiotensin system activation in BN rats correlated with hypertension and proteinuria at gestational days 17-21, which were resolved after delivery. In addition, BN rat pregnancies were characterized by significant fetal loss, restricted growth in surviving fetuses, decreased uteroplacental blood flows, and decreased trophoblast remodeling of uterine arteries compared with LW pregnancies. Short-term losartan treatment significantly increased uteroplacental blood flow and fetal weight and decreased maternal blood pressure (BP) and proteinuria in BN pregnancies. In contrast, losartan treatment significantly decreased uteroplacental blood flow and fetal weight but had no significant effect on maternal BP in LW pregnancies. We conclude that Ace polymorphisms play an important role in the reproductive phenotype of BN and LW rats and that BN rats are a novel model of pregnancy complications in association with genetically controlled, increased ACE activity.

Long-term hypoxia uncouples Ca2+ and eNOS in bradykinin-mediated pulmonary arterial relaxation.

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca2+ with confocal microscopy. eNOS was inhibited with 100 µM NG-nitro-l-arginine methyl ester (l-NAME), PGI2 production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the ß1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols.

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 µM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

Detection of dinitrosyl iron complexes by ozone-based chemiluminescence.

Dinitrosyl iron complexes (DNICs) are important intermediates in the metabolism of nitric oxide (NO). They have been considered to be NO storage adducts able to release NO, scavengers of excess NO during inflammatory hypotensive shock, and mediators of apoptosis in cancer cells, among many other functions. Currently, all studies of DNICs in biological matrices use electron paramagnetic resonance (EPR) for both detection and quantification. EPR is limited, however, by its ability to detect only paramagnetic mononuclear DNICs even though EPR-silent binuclear are likely to be prevalent. Furthermore, physiological concentrations of mononuclear DNICs are usually lower than the EPR detection limit (1 µM). We have thus developed a chemiluminescence-based method for the selective detection of both DNIC forms at physiological, pathophysiological, and pharmacologic conditions. We have also demonstrated the use of the new method in detecting DNIC formation in the presence of nitrite and nitrosothiols within biological fluids and tissue. This new method, which can be used alone or in tandem with EPR, has the potential to offer insight about the involvement of DNICs in many NO-dependent pathways.

Use of Esophageal Hemoximetry to Assess the Effect of Packed Red Blood Cell Transfusion on Gastrointestinal Oxygenation in Newborn Infants.

Objectives There are no widely accepted methods of continuously monitoring gut oxygenation in the newborn during packed red blood cell transfusion. We investigated the use of an orally inserted light spectroscopy probe to measure lower esophageal oxyhemoglobin saturations (eStO2) before, during, and after transfusion and made comparisons with abdominal near-infrared spectroscopy (NIRS) and superior mesenteric artery (SMA) flow. Study Design Thirteen neonates with corrected gestational ages ranging from 22 weeks, 0 day to 37 weeks, 5 days were enrolled. eStO2 and NIRS measurements were recorded continuously for a 25-hour period starting 1 hour prior to starting the 4-hour transfusion. Transabdominal ultrasound was used to measure SMA flow prior to, upon completion, and 20 hours after the transfusion. Results Twelve infants completed the study. eStO2 was well-tolerated and was weakly (r = 0.06) correlated (p < 0.001) with NIRS. Compared with NIRS, eStO2 demonstrated a markedly greater variation in oxyhemoglobin values. NIRS and SMA flow measurements did not change, while eStO2 increased from 48 ± 5% and 45 ± 5% in the pre- and intratransfusion periods to 57 ± 4% in the posttransfusion period (p = 0.03). Conclusion Measurement of eStO2 is feasible in neonates and may provide a continuous and sensitive index of rapid changes in mesenteric oxygenation in this patient population.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth.

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca(2+)-activated K(+) (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca(2+). Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca(2+) signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼ 140 days gestation) and newborn, 10- to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca(2+) responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 µM nitro-L-arginine methyl ester or 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel α1-subunit expression but increased ß1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes.

Postprandial lipids accelerate and redirect nitric oxide consumption in plasma.

Nitric oxide (NO) and O2 are both three-to four-fold more soluble in biological lipids than in aqueous solutions. Their higher concentration within plasma lipids accelerates NO autoxidation to an extent that may be of importance to overall NO bioactivity. This study was undertaken to test the hypothesis that increased plasma lipids after a high-fat meal appreciably accelerate NO metabolism and alter the byproducts formed. We found that plasma collected from subjects after consumption of a single high-fat meal had a higher capacity for NO consumption and consumed NO more rapidly compared to fasting plasma. This increased NO consumption showed a direct correlation with plasma triglyceride concentrations (p = 0.006). The accelerated NO consumption in postprandial plasma was reversed by removal of the lipids from the plasma, was mimicked by the addition of hydrophobic micelles to aqueous buffer, and could not be explained by the presence of either free hemoglobin or ceruloplasmin. The products of NO consumption were shifted in postprandial plasma, with 55% more nitrite (n = 12, p = 0.002) but 50% less SNO (n = 12, p = 0.03) production compared to matched fasted plasma. Modeling calculations indicated that NO autoxidation was accelerated by about 48-fold in the presence of plasma lipids. We conclude that postprandial triglyceride-rich lipoproteins exert a significant influence on NO metabolism in plasma.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Changes in plasma and urinary nitrite after birth in premature infants at risk for necrotizing enterocolitis.

BACKGROUND: Plasma nitrite serves as a reservoir of nitric oxide (NO) bioactivity. Because nitrite ingestion is markedly lower in newborns than adults, we hypothesized plasma nitrite levels would be lower in newborns than in adults, and that infants diagnosed with necrotizing enterocolitis (NEC), a disease characterized by ischemia and bacterial invasion of intestinal walls, would have lower levels of circulating nitrite in the days prior to diagnosis. METHODS: Single blood and urine samples were collected from 9 term infants and 12 adults, 72 preterm infants every 5 d for 3 wk, and from 13 lambs before and after cord occlusion. RESULTS: Nitrite fell 50% relative to cord levels in the first day after birth; and within 15 min after cord occlusion in lambs. Urinary nitrite was higher in infants than adults. Plasma and urinary nitrite levels in infants who developed NEC were similar to those of preterm control infants on days 1 and 5, but significantly elevated at 15 and 20 d after birth. CONCLUSION: Plasma nitrite falls dramatically at birth while newborn urinary nitrite levels are significantly greater than adults. Acute NEC is associated with elevated plasma and urinary nitrite levels.

A novel, noninvasive, predictive epilepsy biomarker with clinical potential.

A significant proportion of temporal lobe epilepsy (TLE), a common, intractable brain disorder, arises in children with febrile status epilepticus (FSE). Preventative therapy development is hampered by our inability to identify early the FSE individuals who will develop TLE. In a naturalistic rat model of FSE, we used high-magnetic-field MRI and long-term video EEG to seek clinically relevant noninvasive markers of epileptogenesis and found that reduced amygdala T2 relaxation times in high-magnetic-field MRI hours after FSE predicted experimental TLE. Reduced T2 values likely represented paramagnetic susceptibility effects derived from increased unsaturated venous hemoglobin, suggesting augmented oxygen utilization after FSE termination. Indeed, T2 correlated with energy-demanding intracellular translocation of the injury-sensor high-mobility group box 1 (HMGB1), a trigger of inflammatory cascades implicated in epileptogenesis. Use of deoxyhemoglobin-sensitive MRI sequences enabled visualization of the predictive changes on lower-field, clinically relevant scanners. This novel MRI signature delineates the onset and suggests mechanisms of epileptogenesis that follow experimental FSE.