ABSTRACT
BACKGROUND: Airway acidification plays a role in disorders of the pulmonary tract. We hypothesized that the inhalation of alkalinized glycine buffer would measurably alkalinize the airways without compromising lung function or causing adverse events. We evaluated the safety of an inhaled alkaline glycine buffer in both healthy subjects and in subjects with stable obstructive airway disease. METHODS: This work includes 2 open-label safety studies. The healthy controls were part of a phase 1 safety study of multiple inhalations of low-dose alkaline glycine buffer; nebulized saline was used as a comparator in 8 of the healthy controls. Subsequently, a phase 2 study in subjects with stable obstructive airway disease was completed using a single nebulized higher-dose strategy of the alkaline inhalation. We studied 20 non-smoking adults (10 healthy controls and 10 subjects with obstructive airway disease), both at baseline and after inhalation of alkaline buffer. We used spirometry and vital signs as markers of clinical safety. We used changes in fraction of exhaled nitric oxide (NO) and exhaled breath condensate (EBC) pH as surrogate markers of airway pH modification. RESULTS: Alkaline glycine inhalation was tolerated by all subjects in both studies, with no adverse effects on spirometric parameters or vital signs. Airway alkalinization was confirmed by a median increase in EBC pH of 0.235 pH units (IQR 0.56-0.03, P = .03) in subjects after inhalation of the higher-dose alkaline buffer (2.5 mL of 100 mmol/L glycine). CONCLUSIONS: Alkalinization of airway lining fluid is accomplished with inhalation of alkaline glycine buffer and causes no adverse effects on pulmonary function or vital signs.
Subject(s)
Glycine , Hydrogen-Ion Concentration , Lung Diseases, Obstructive/drug therapy , Administration, Inhalation , Adult , Biomarkers, Pharmacological/analysis , Breath Tests/methods , Buffers , Dose-Response Relationship, Drug , Drug Monitoring , Exhalation , Female , Glycine/administration & dosage , Glycine/adverse effects , Glycine Agents/administration & dosage , Glycine Agents/adverse effects , Humans , Lung Diseases, Obstructive/diagnosis , Lung Diseases, Obstructive/metabolism , Male , Middle Aged , Nebulizers and Vaporizers , Nitric Oxide/analysis , Respiratory Function Tests/methods , Statistics, Nonparametric , Treatment OutcomeABSTRACT
Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.
Subject(s)
Endothelial Cells/metabolism , Gene Expression Regulation , Hemoglobins/metabolism , Nitric Oxide/metabolism , Peptide Fragments/metabolism , Signal Transduction , Adrenergic alpha-1 Receptor Agonists/pharmacology , Animals , Cells, Cultured , Diffusion , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Gene Expression Profiling , Gene Expression Regulation/drug effects , Hemoglobins/genetics , Humans , Iron/chemistry , Mice , Nitric Oxide Synthase/metabolism , Oxidation-Reduction , Peptide Fragments/genetics , Phenylephrine/pharmacologyABSTRACT
OBJECTIVE: To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. METHODS AND RESULTS: Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NO(x) concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. CONCLUSIONS: This study provides evidence for compartmentalized S-nitrosylation/denitrosylation in the regulation of smooth muscle cell to endothelial cell communication.
