The postnatal changes in red blood cell NO levels.

AIM: To determine the levels of RBC HbSNO and HbFe(II)NO using chemiluminescence in very low birth weight infants breathing room air, during the first 2 days of life. METHOD: RBC NO values were compared to the levels obtained in cord blood at birth from infants of similar gestational age. Five infants ranging from 25 to 27 weeks of gestation were sampled between 12 and 24 h after birth. These infants were considered as the postnatal group and had normal blood gases at room air. RESULTS: The HbSNO levels were increased in the postnatal group from 49.0 +/- 17.4 nm to 152.3 +/- 54.3 nm (p = 0.0006). There was no difference in HbFe(II)NO levels between the two groups (mean of 267.6 +/- 186.5 nm in cord blood and 180.3 +/- 89.2 nm in the postnatal sample. CONCLUSION: The increase in HbSNO postnatally could be an important mechanism for the neonatal pulmonary adaptation to extra-uterine life.

Nitric oxide signaling via nuclearized endothelial nitric-oxide synthase modulates expression of the immediate early genes iNOS and mPGES-1.

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.

The effect of adult hemoglobin on red blood cell nitric oxide levels during fetal development.

OBJECTIVE: To compare the levels of S-nitrosohemoglobin (HbSNO) at different gestational ages in newborn infants and correlate the levels of HbSNO with HbA and HbF. METHOD: Cord blood samples of 22 newborn infants of different gestational ages (25-41 weeks) were analyzed. The levels of HbF and HbA were determined by HPLC and of HbSNO by chemiluminescence. RESULTS: The level of HbSNO varied from 28.1 to 145.3 nM. There was a significant correlation with gestational age (r(2) = 0.5469, p < 0.0001) and with the relative amount of HbA (r(2) = 0.8144, p < 0.0001). CONCLUSION: The increases in HbSNO in fetal red cells is directly related to the relative amount of HbA.

Developmental changes in NO bioavailability in fetal erythrocytes.

S-nitrosohemoglobin (HbSNO), where hemoglobin (Hb) is nitrosated at Cysbeta93, presumably controls delivery of the vasorelaxant nitric oxide (NO) to hypoxic tissues in an oxygen-sensitive manner. Little is known about how Hb regulates NO bioavailability during fetal development. A study was planned to determine the levels of HbSNO and HbFe(II)NO (NO bound to FeII of heme) in the cord blood of newborn infants of different gestational ages and establish their relationship with the levels of fetal Hb (HbF). Blood samples were collected from umbilical cord obtained from normal newborns between 24 and 41 weeks of gestation. Determinations of HbSNO and HbFe(II)NO were performed using chemiluminescence. The proportion of HbF was determined by HPLC. There were 11 preterm (24-34 weeks of gestation) and 11 term infants (37-41 weeks of gestation). The levels of HbSNO varied from 0.37 to 1.72 x 10(-5) mol/mol heme. There was a significant correlation with gestational age (r2 = 0.46, P = 0.0005) due to the effect of the decrease in the amount of HbF (r2 = 0.81, P < 0.0001). The relationship of HbFe(II)NO was not affected by gestational age or the level of HbF (mean 1.68+/-1.15 x 10(-5) mol/mol heme). Under physiological in utero conditions, fetal erythrocytes have lower levels of HbSNO, which increase in the later stage of fetal development. The levels of HbSNO in the fetal red cell are dependent on the level of adult Hb (HbA). The low HbSNO levels at physiological fetal O2 saturations during early development could protect the fetal circulation from an excess release of NO and O2.

Fetal hemoglobin synthesis determined by gamma-mRNA/gamma-mRNA + beta-mRNA quantitation in infants at risk for sudden infant death syndrome being monitored at home for apnea.

OBJECTIVE: Fetal hemoglobin (HbF) levels in the hemolysates obtained from infants who died from sudden infant death syndrome (SIDS) are reported to be markedly increased compared with controls. This finding could have been explained by increased HbF synthesis caused by episodes of hypoxemia in the SIDS infants. A prospective study in a group of infants being monitored at home after an apparent life-threatening event (ALTE) and considered at increased risk for SIDS was conducted with an improved ribonuclease protection assay. The ribonuclease protection assay allowed for the quantitation of [gamma/(gamma+beta)]-globin mRNAs, which has a highly significant correlation with the levels of HbF synthesis. METHODS: Thirty-five infants who were admitted for an ALTE were included in the study. All infants were at home under surveillance with a cardiorespiratory monitor and followed in an apnea clinic with monthly appointments. Seventy-three blood samples were obtained between 38 and 61 weeks of postconceptional age. For control purposes, a similar group of 37 normal infants (99 samples) whose HbF synthesis was previously determined were included. RESULTS: Mean [gamma/(gamma+beta)]-globin mRNAs were increased in the ALTE group at 42 to 45 and 46 to 49 weeks of postconceptional age (mean: 55.2 +/- 17.4% and 33.9 +/- 14%) in comparison with HbF synthesis in controls (mean: 42.6 +/- 13.7% and 23.6 +/- 9.8%). CONCLUSIONS: The data obtained in this report from infants who were considered at risk for SIDS show that HbF synthesis is increased between 42 and 49 weeks of postconceptional age. Determining HbF synthesis as described in this study may have value as a marker for episodes of hypoxemia for certain infants who are at risk for SIDS.

The effect of blood transfusion on the hemoglobin oxygen dissociation curve of very early preterm infants during the first week of life.

A study was conducted during the first week of life to determine the changes in P50 (PO2 required to achieve a saturation of 50% at pH 7.4 and 37 degrees C) and the proportions of fetal hemoglobin (HbF) and adult hemoglobin (HbA) prior to and after transfusion in very early preterm infants. Eleven infants with a gestational age < or = 27 weeks have been included in study. The hemoglobin dissociation curve and the P50 was determined by Hemox-analyser. Liquid chromatography was also performed to determine the proportions of HbF and HbA. The mean gestational age of the 11 infants was 25.1 weeks (+/- 1 weeks) and their mean birth weight was 736 g (+/- 125 g). They received 26.9 mL/kg of packed red cells. The mean P50 prior and after transfusion was 18.5 +/- 0.8 and 21.0 +/- 1 mm Hg (P = .0003) while the mean percentage of HbF was 92.9 +/- 1.1 and 42.6 +/- 5.7%, respectively. The data of this study show a decrease of hemoglobin oxygen affinity as a result of blood transfusion in very early preterm infants prone to O2 toxicity. The shift in HbO2 curve after transfusion should be taken into consideration when oxygen therapy is being regulated for these infants.