The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.

Mechanisms that protect against asthma remain poorly understood. S-nitrosoglutathione (GSNO), an endogenous bronchodilator, is depleted from asthmatic airways, suggesting a protective role. We report that, following allergen challenge, wild-type mice exhibiting airway hyperresponsivity have increased airway levels of the enzyme GSNO reductase (GSNOR) and are depleted of lung S-nitrosothiols (SNOs). In contrast, mice with genetic deletion of GSNOR exhibit increases in lung SNOs and are protected from airway hyperresponsivity. Our results indicate that endogenous SNOs, governed by GSNOR, are critical regulators of airway responsivity and may provide new therapeutic approaches to asthma.

SUMMARYHomologous chromosome segregation errors during meiosis I are common and generate aneuploid embryos. Here, we provide a reason for this susceptibility to mis-segregation by live cell imaging of mouse oocytes. Our results show that stable kinetochoremicrotubule attachments form in mid-prometaphase, 3-4 hours before anaphase. This coincided with the loss of Mad2 from kinetochores and with the start of anaphase-promoting complex/cyclosome (APC/C)-mediated cyclin B1 destruction. Therefore, the spindle assembly checkpoint (SAC) ceased to inhibit the APC/C from mid-prometaphase. This timing did not coincide with bivalent congression in one-third of all oocytes examined. Non-aligned bivalents were weakly positive for Mad2, under less tension than congressed bivalents and, by live-cell imaging, appeared to be in the process of establishing correct bi-orientation. The time from when the APC/C became active until anaphase onset was affected by the rate of loss of CDK1 activity, rather than by these non-aligned bivalents, which occasionally persisted until anaphase, resulting in homolog non-disjunction. We conclude that, in oocytes, a few erroneous attachments of bivalent kinetochores to microtubules do not generate a sufficient SAC 'wait anaphase' signal to inhibit the APC/C.

The mechanism by which hypoxia [low partial pressure of O2 (pO2)] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO 2-governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). hemoglobin ͉ red blood cell vasodilation ͉ S-nitrosylation I n the systemic microcirculation, blood flow is regulated by physiological O 2 gradients that couple the O 2 content of blood to regulated vasodilation and vasoconstriction (1-3). Blood flow is thereby matched to tissue O 2 demand. An analogous mechanism operates in the lungs, where O 2 uptake (ventilation) is optimized through regulated vasodilation and vasoconstriction (perfusion). Blood flow is thereby matched to alveolar ventilation (2). Because it is Hb O 2 saturation, not the partial pressure of O 2 (pO 2 ), that is coupled to blood flow in vivo (1, 3) it has been deduced that RBCs may serve as O 2 sensors within the integrated vascular system. In support of this idea, it has been shown recently that RBCs can act as O 2 -responsive transducers of vasodilator and vasoconstrictor activity (4-10), at least partly by modulating the availability of [6][7][8]10,11). According to these studies, RBCs release NO bioactivity under hypoxia and sequester it at hyperoxia. The release of NO bioactivity would facilitate hypoxic vasodilation in peripheral tissues and oppose hypoxic pulmonary vasoconstriction (HPV) in the lungs. S-nitrosothiol (SNO)-deficientThe mechanism by which NO bioactivity escapes from RBCs is incompletely understood. It is generally accepted that the rapid reaction of NO with the hemes of Hb produces a heme-iron nitrosyl adduct (Hb [FeNO]) that exhibits no vasodilator activity (4,7,12). Hb also sustains S-nitrosylation at two cysteine residues conserved in all mammals and birds. Biochemical and mutational analyses (␤93Cys3Ala) indicate that S-nitrosohemoglobin (SNO-Hb) is formed upon oxygenation of Hb [FeNO] by means of heme-to-Cys NO transfer (13-15) and by transnitrosylative transfer from low-mass S-nitrosothiols (SNOs) (16,17). SNO-Hb is very stable in the oxygenated (or R) structure and thus cannot effectively dilate blood vessels (5, 10, 18). However, upon deoxygenation [or with change in the spin state of the hemes (3)], the vasodilator potency of SNO-Hb is markedly potentiated (5,16,18). Crystal structures and molecular models show that the ␤-Cys NO gains solvent access in the deoxygenated (or T) state (3, 19). Solvent-exposed NO can exchange with acceptor thiols within the N-terminal cytoplasmic domain of the RBC membrane anion exchange protein (AE1; band 3) (4, 15). Transnitrosylation of AE1 by SNO-Hb involves a direct protein-protein interaction. The st...