Amino acid signature during sickle cell pain crisis shows significant alterations related to nitric oxide and energy metabolism.

Nitric oxide depletion secondary to arginase induced arginine deficiency has been shown to be important in the pathophysiology of vaso-occlusion in sickle cell pain crisis. Our objective of this study was to perform a comprehensive amino acid evaluation during sickle cell pain crisis. In a total of 58 subjects (29 in steady-state sickle cell disease and 29 with sickle cell pain crisis), the amino acids related to nitric oxide pathway was significantly decreased during sickle cell pain crisis compared to steady-state sickle cell disease: arginine (p = 0.001), citrulline (p = 0.012), and ornithine (p = 0.03). In addition, the amino acids related to energy metabolism was significantly decreased during a pain crisis: asparagine (p < 0.001), serine (p = 0.002), histidine (p = 0.017), alanine (p = 0.004), tyrosine (p = 0.012), methionine (p = 0.007), cystine (p = 0.016), isoleucine (p = 0.016) and lysine (p = 0.006). The amino acid related to oxidative stress were significantly higher during a sickle cell pain crisis (glutamic acid (p < 0.001). Furthermore, multivariate analysis with partial least squares-discriminant analysis (PLS-DA) showed that deficiencies of the amino acids arginine, asparagine, citrulline, methionine and alanine were the most important related to sickle cell pain crisis.

γ-Glutamylcysteine ameliorates oxidative injury in neurons and astrocytes in vitro and increases brain glutathione in vivo.

γ-Glutamylcysteine (γ-GC) is an intermediate molecule of the glutathione (GSH) synthesis pathway. In the present study, we tested the hypothesis that γ-GC pretreatment in cultured astrocytes and neurons protects against hydrogen peroxide (H(2)O(2))-induced oxidative injury. We demonstrate that pretreatment with γ-GC increases the ratio of reduced:oxidized GSH levels in both neurons and astrocytes and increases total GSH levels in neurons. In addition, γ-GC pretreatment decreases isoprostane formation both in neurons and astrocytes, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation in astrocytes in response to H(2)O(2)-induced oxidative stress. Furthermore, GSH and isoprostane levels significantly correlate with increased neuron and astrocyte viability in cells pretreated with γ-GC. Finally, we demonstrate that administration of a single intravenous injection of γ-GC to mice significantly increases GSH levels in the brain, heart, lungs, liver, and in muscle tissues in vivo. These results support a potential therapeutic role for γ-GC in the reduction of oxidant stress-induced damage in tissues including the brain.

An ethnic-specific polymorphism in the catalytic subunit of glutamate-cysteine ligase impairs the production of glutathione intermediates in vitro.

Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy-Weinberg disequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.

Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: potential therapy for postoperative pulmonary hypertension.

OBJECTIVE: Pulmonary hypertension may complicate surgical correction of congenital heart defects, resulting in increased morbidity and mortality. We have previously shown that plasma levels of the nitric oxide precursors citrulline and arginine drop precipitously after congenital cardiac surgery and that oral citrulline supplementation may be protective against the development of pulmonary hypertension. In this study, we assessed the safety and pharmacokinetic profile of intravenous citrulline as a potential therapy for postoperative pulmonary hypertension. METHODS: The initial phase of this investigation was a dose-escalation study of intravenously administered citrulline in infants and children undergoing one of five congenital cardiac surgical procedures (phase 1). The primary safety outcome was a 20% drop in mean arterial blood pressure from the baseline pressure recorded after admission to the intensive care unit. Based on our previous work, the target circulating plasma citrulline trough was 80 to 100 micromol/L. Each patient was given two separate doses of citrulline: the first in the operating room immediately after initiation of cardiopulmonary bypass and the second 4 hours later in the pediatric intensive care unit. Stepwise dose escalations included 50 mg/kg, 100 mg/kg, and 150 mg/kg. After model-dependent pharmacokinetic analysis, we enrolled an additional 9 patients (phase 2) in an optimized dosing protocol that replaced the postoperative dose with a continuous infusion of citrulline at 9 mg/(kg.h) for 48 hours postoperatively. RESULTS: The initial stepwise escalation protocol (phase 1) revealed that an intravenous citrulline dose of 150 mg/kg given after initiation of cardiopulmonary bypass yielded a trough level of in the target range of approximately 80 to 100 micromol/L 4 hours later. The postoperative dose revealed that the clearance of intravenously administered citrulline was 0.6 L/(h.kg), with a volume of distribution of 0.9 L/kg and estimated half-life of 60 minutes. Because of the short half-life, we altered the protocol to replace the postoperative dose with a continuous infusion of 9 mg/(kg.h). An additional 9 patients were studied with this continuous infusion protocol (phase 2). Mean plasma citrulline levels were maintained at approximately 125 mumol/L, with a calculated clearance of 0.52 L/(h.kg). None of the 17 patients studied had a 20% drop in mean arterial blood pressure from baseline. CONCLUSIONS: In this first report of the use of intravenous citrulline in humans, we found citrulline to be both safe and well tolerated in infants and young children undergoing congenital cardiac surgery. Because of the rapid clearance, the optimal dosing regimen was identified as an initial bolus of 150 mg/kg given at the initiation of cardiopulmonary bypass, followed 4 hours later by a postoperative infusion of 9 mg/(kg.h) continued up to 48 hours. Using this regimen, plasma arginine, citrulline, and nitric oxide metabolite levels were well maintained. Intravenous citrulline needs to be studied further as a potential therapy for postoperative pulmonary hypertension.

Respiratory syncytial virus infection reduces beta2-adrenergic responses in human airway smooth muscle.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract illness in infants, the effect of RSV on human airway smooth muscle (HASM) has not been studied. We hypothesized that RSV has direct effects on cAMP formation and beta(2)-adrenergic receptor (ADRB2) density and that ADRB2 haplotype influences this response. A recombinant green-fluorescent protein (rg) expressing RSV was used to determine whether RSV could infect cultured HASM. Influence of RSV infection on beta(2)-adrenergic responsiveness was determined by measuring differences in isoproterenol (ISO)-induced cyclic AMP (cAMP) formation, ADRB2 density, and G(i) expression in HASM cells challenged with RSV, with ultraviolet-inactivated RSV, and with mock infection. The rgRSV efficiently infected cultured HASM cells. ISO-induced cAMP formation was significantly reduced in cells infected with RSV, compared with mock-infected and ultraviolet-inactivated RSV, in a time- and concentration-dependent manner. Forskolin-induced cAMP formation and Gi expression were not altered in cells infected with RSV, suggesting that the influence of RSV on beta(2)-adrenergic relaxation was upstream of cAMP formation. ADRB2 density was reduced in cells infected with RSV, compared with mock infection, and the Arg16Gln27 ADRB2 haplotype was associated with decreased ISO-induced cAMP formation (P < 0.05) and with decreased ADRB2 density at baseline (P < 0.05). The implications of these results are that limitations of beta(2)-agonists in the treatment of any airway obstruction associated with RSV infection may be related to direct effects of RSV on HASM, and ADRB2 genotype may predict beta(2)-adrenergic responses.

Environmentally determined genetic expression: clinical correlates with molecular variants of carbamyl phosphate synthetase I.

Carbamyl phosphate synthetase I (CPSI) determines the rate-limiting entry of free ammonia into the urea cycle. Disruption of CPSI affects the liver's ability to remove waste nitrogen and produce arginine, citrulline, and urea. Arginine is the necessary precursor for the critical biomolecule, nitric oxide (NO). We have studied the classic model of CPSI deficiency, which results in severe hyperammonemia, and identified a large number of molecular defects. A number of CPSI polymorphisms have been found that appear to result in functional consequences. We have examined the association of these polymorphisms with various environmental stress conditions and found that certain CPSI alleles are associated with clinical outcome. We refer to these associations as environmentally determined genetic expression (EDGE) affects. In addition to studies of classic CPSI deficiency, we have developed data for the EDGE concept in post-cardiac surgery-related pulmonary hypertension, hepatic veno-occlusive disease after bone marrow transplantation, and persistent pulmonary hypertension of the newborn. We have linked these outcomes and genotypes to the availability of the urea cycle intermediates, citrulline and arginine, and their role in NO synthesis. We hypothesize that these polymorphisms affect the functional efficiency of CPSI and thus the entire urea cycle and as such, the availability of the NO substrates. By piecing together the various functional aspects of the urea cycle changes we have seen, we can better understand the clinical vulnerabilities of patients in environmentally stressful situations. This knowledge should allow us to design intervention strategies to either predict or modify the associated adverse outcomes.

HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium.

This study examines the notion that heat shock protein (HSP) 90 binding to nitric oxide (NO), endothelial NO synthase (eNOS), and PI3K-Akt regulate angiopoietin (Ang)-1-induced angiogenesis in porcine coronary artery endothelial cells (PCAEC). Exposure to Ang-1 (250 ng/ml) for periods up to 2 h resulted in a time-dependent increase in eNOS phosphorylation at Ser 1177 that occurred by 5 min and peaked at 60 min. This was accompanied by a gradual increase in NO release. Ang-1 also led to stimulation of HSP90 binding to eNOS and a significant increase in Akt phosphorylation. Thirty minutes of pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of HSP90) or 500 nM wortmannin [a specific phosphatidylinositol 3 (PI3)-kinase (PI3K) inhibitor] significantly attenuated Ang-1-stimulated eNOS phosphorylation and NO production. Exposure to Ang-1 caused an increase in endothelial cell migration, tube formation, and sprouting from PCAEC spheroids, and pharmacological blockage of HSP90 function or inhibition of PI3K-Akt pathway completely abolished these effects. Inhibition of nitric oxide synthase by NG-nitro-l-arginine methyl ester (2.5 mM) also resulted in a significant decrease in Ang-1-induced angiogenesis. We conclude that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to Ang-1-induced eNOS phosphorylation, NO production, and angiogenesis in PCAEC.

Conopy architecture of Larrea tridentata (DC.) Cov., a desert shrub: foliage orientation and direct beam radiation interception.

At sites in the United States, creosote bushes (Larrea tridentata (DC.) Cov.) orient foliage clusters predominantly toward the southeast. Foliage of bushes at the southernmost distribution extreme in Mexico shows no predominant orientation. Clusters at all sites are inclined between 33° and 71° from the horizontal. Inclinations are steeper in the drier and hotter Mojave Desert than in the Chihuahuan Desert. Individual leaflets, though not measured, appear more randomly oriented than foliage clusters. In several populations studied, branches were shorter in the southeastern sectors of the crown, reducing self-shading early in the morning. Measurements of direct beam radiation interception by detached branches, using digital image processing, indicated that foliage clusters oriented toward the southeast exhibited less self-shading during spring mornings than clusters oriented northeast. This effect was not apparent at the summer solstice. This type of canopy architecture may tend to minimize self-shading during the morning hours when conditions are more favorable for photosynthesis, resulting in an improved daily water use efficiency.