Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O2. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs. Under this model, the delivery of SNO-based NO bioactivity by Hb redefines the respiratory cycle as a triune system (NO/O2/CO2). However, the physiological ramifications of RBC-mediated vasodilation are unknown, and the apparently essential nature of βCys93 remains unclear. Here we report that mice with a βCys93Ala mutation are deficient in hypoxic vasodilation that governs blood flow autoregulation, the classic physiological mechanism that controls tissue oxygenation but whose molecular basis has been a longstanding mystery. Peripheral blood flow and tissue oxygenation are decreased at baseline in mutant animals and decline excessively during hypoxia. In addition, βCys93Ala mutation results in myocardial ischemia under basal normoxic conditions and in acute cardiac decompensation and enhanced mortality during transient hypoxia. Fetal viability is diminished also. Thus, βCys93-derived SNO bioactivity is essential for tissue oxygenation by RBCs within the respiratory cycle that is required for both normal cardiovascular function and circulatory adaptation to hypoxia.

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO ϳ20 -40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser 239 induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.As a small sized gaseous free radical, nitric oxide (NO) 2 presents unique challenges toward understanding the nature of its signaling in biological systems. Numerous levels of regulation at the level of nitric oxide synthase catalysis influence the rate of NO generation. Spatial localization of the biological signal is secondarily determined by a combination of the distinct physical and chemical properties of NO within the context of the microenvironment in which it was formed. The lifetime of NO molecules is governed chiefly by their relative abundance in relation to other radicals (1, 2), transition metal centers (3, 4), and oxygen (O 2 ) (5, 6). In addition to its very small size, the diffusional path of NO from the point of origin is affected by its preferentially hydrophobic solubility (7,8), resulting in an enrichment of NO in biological membranes relative to the aqueous milieu. In the present work, the hypothesis that cells may utilize plasma membrane cholesterol to further orchestrate spatial heterogeneity in NO biological signaling activity was tested.Cholesterol, a major lipid component of the plasma membrane in eukaryotic cells, plays an essential role in maintaining membrane fluidity and architecture (9 -11). Cells tightly control the ratio ...

Background: Excitation-contraction coupling in striated muscle requires intracellular Ca 2ϩ release through ryanodine receptor/Ca 2ϩ -release channels (RyRs). Results: S-Palmitoylation is a previously unidentified post-translational modification of skeletal muscle RyR1. Diminishing S-palmitoylation significantly diminishes RyR1 activity including stimulus-dependent Ca 2ϩ release. Conclusion: S-Palmitoylation provides a previously unidentified mechanism to regulate Ca 2ϩ flux in skeletal muscle. Significance: S-Palmitoylation is likely to regulate Ca 2ϩ flux in many cell types.

This study was conducted to determine the relationship between dysglycemia and the coronary artery vasa vasorum density. RESEARCH DESIGN AND METHODSThe left anterior descending coronary artery was removed from 57 deceased individuals during autopsy, and the capillaries in the vessel wall were identified using fluorescent immunohistochemical staining. HbA 1c was determined in postmortem whole blood for each individual. The density of the vasa vasorum in the intima-media and the adventitia was manually quantified and recorded by readers unaware of the individual's other characteristics. RESULTSThe individuals with diabetes had a lower density of the coronary vasa vasorum than those without diabetes. The higher the HbA 1c , the lower the density of these vessels in the adventitia and entire vessel wall. CONCLUSIONSDysglycemia-induced damage to the vasa vasorum may promote ischemic heart disease in people with diabetes.Diabetes promotes retinal, renal, neurologic, vascular, cardiac, and other organ damage. Although many reasons for this have been suggested, dysglycemia-mediated vasculopenia due to capillary damage in various tissues would provide an overarching explanation. In the walls of medium to large arteries, these damaged capillary beds would be the vasa vasorum (1), a possibility supported by animal models (2) and in vivo measurements in humans (3,4).If the above hypothesis is true, the density of the vasa vasorum should be reduced in the coronary arteries of people with diabetes. This cross-sectional autopsy study was therefore conducted to compare the coronary artery vasa vasorum in individuals with normal versus high HbA 1c at the time of death and to assess the relationship between HbA 1c and vasa vasorum density. RESEARCH DESIGN AND METHODSDeceased individuals undergoing forensic autopsy whose families consented to a full or limited postmortem examination were included if they were aged 50 or older, their coronary arteries could be sectioned and stained, a postmortem blood sample was available, and the autopsy occurred during the pathologist's (V.N.) working hours. Age, sex, history of diabetes, and cause of death were collected, and a postmortem whole-blood sample was sent for HbA 1c . The study was approved by the Research Ethics Board at Hamilton Health Sciences and McMaster University.