The binding of oxygen to heme irons in hemoglobin promotes the binding of nitric oxide (NO) to cysteine␤93, forming S-nitrosohemoglobin. Deoxygenation is accompanied by an allosteric transition in S-nitrosohemoglobin [from the R (oxygenated) to the T (deoxygenated) structure] that releases the NO group. S-nitrosohemoglobin contracts blood vessels and decreases cerebral perfusion in the R structure and relaxes vessels to improve blood flow in the T structure. By thus sensing the physiological oxygen gradient in tissues, hemoglobin exploits conformation-associated changes in the position of cysteine␤93 SNO to bring local blood flow into line with oxygen requirements.Hemoglobin (Hb) is the tetrameric protein in red blood cells (RBCs) that transports oxygen (O 2 ) from the lung to the tissues (1). As RBCs saturated in O 2 migrate through small arteries and resistance arterioles, they are exposed to an O 2 gradient (2). By the time Hb reaches the capillaries, a large fraction (ϳ50 to 65%) of the O 2 has been lost to venous exchange (a functional shunt) (2). Only about 25 to 30% of the O 2 is extracted by the tissues to meet basal metabolic requirements (1-3). Exposed to increasing oxygen tension (PO 2 ) in postcapillary venules and veins (2), Hb is ϳ75% saturated in O 2 (1, 3) upon entering the lung. Thus, on average, only one of four O 2 molecules carried by Hb is used in the respiratory cycle, even though extensive deoxygenation occurs in the flowcontrolling resistance vessels.Hemoglobin exists in two alternative structures, named R (for relaxed, high O 2 affinity) and T (for tense, low O 2 affinity) (4). Hemoglobin assumes the T structure to efficiently release O 2 (4). The allosteric transition in Hb (from R to T) controls the reactivity of two highly conserved cysteines (Cys␤93) that can react with NO or SNO (S-nitrosothiol) (5). Thiol affinity for (S)NO is high in the R structure and low in the T structure. In other words, the NO group is released from thiols of Hb in low PO 2 (5). A major function of (S)NO in the vasculature is to regulate blood flow, which is controlled by the resistance arterioles (6). We therefore proposed that partial deoxygenation of SNO-Hb in these vessels might actually promote O 2 delivery by liberating (S)NO. That is, the allosteric transition in Hb would function to release (S)NO in order to increase blood flow.Hemoglobin is mainly in the R (oxy) structure in both 95% O 2 and 21% O 2 (room air) (4). Hb and SNO-Hb both contract blood vessels in bioassays (7) at these O 2 concentrations (Fig. 1A). That is, their hemes sequester NO from the endothelium. In hypoxia [Ͻ1% O 2 (at a simulated tissue PO 2 of ϳ6 mmHg)], which promotes the T structure (4), Hb strongly contracts blood vessels, whereas SNO-Hb does not (Fig. 1B). NO group release from SNO-Hb is accelerated in RBCs by glutathione (5), which enhances SNO-Hb relaxations through formation of S-nitrosoglutathione (GSNO) (Fig. 1C). The potentiation by glutathione is inversely related to the PO 2 (Fig. 1C), because NO group transfer fr...

The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of mitochondrial biogenesis in the hypoxia-sensitive subcortex of wild-type (Wt) and neuronal NOS (nNOS) and endothelial NOS (eNOS)-deficient mice. Mice were exposed to hypobaric hypoxia for 6 h, and changes in immediate hypoxic transcriptional regulation of mitochondrial biogenesis was assessed in relation to mitochondrial DNA (mtDNA) content and mitochondrial density. There were no differences in cerebral blood flow or hippocampal PO 2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1 ␣), nuclear respiratory factor-1, and mitochondrial transcription factor A. After 24 h, new mitochondria, localized in reporter mice expressing mitochondrial green fluorescence protein, were seen primarily in hippocampal neurons. eNOS Ϫ/Ϫ mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS Ϫ/Ϫ mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS Ϫ/Ϫ mice but not in nNOS Ϫ/Ϫ mice. Hypoxia stimulated PGC-1␣ protein expression and phosphorylation of protein kinase A and cAMP response element binding (CREB) protein in Wt mice, but CREB only was activated in eNOS Ϫ/Ϫ mice and not in nNOS Ϫ/Ϫ mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1␣ and CREB.

Background and Purpose-The purpose of this study was to test the hypothesis that nitric oxide is required for preconditioning in an intact animal model of focal ischemia using neuronal and endothelial nitric oxide synthase (nNOS and eNOS) knockout mice. Methods-Cerebral blood flow was measured in wild-type, nNOS knockout, and eNOS knockout mice by hydrogen clearance (absolute) and laser Doppler flowmetry (relative). Mice were preconditioned by three 5-minute episodes of transient middle cerebral artery occlusion (MCAO) and subjected to permanent MCAO. Neurological deficit and infarct size were determined 24 hours later. Results-Although wild-type mice showed protection from ischemic preconditioning, neither eNOS nor nNOS knockout mice showed protection. Laser Doppler measurements indicated that the relative blood flow decreases in core ischemic areas were the same in all groups. Conclusions-Neither eNOS nor nNOS knockout mice show protection from rapid ischemic preconditioning, suggesting that nitric oxide may play a role in the molecular mechanisms of protection.