It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-␤ 93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased Ϸ20-fold in human septic shock (P ‫؍‬ 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.sepsis ͉ nitric oxide ͉ vascular physiology E vidence has accumulated for an S-nitrosothiol (SNO)-based vascular signaling system in which hemoglobin (Hb) reactions with nitric oxide (NO) transduce redox gradients into bioactivities (1-6). There is agreement that human Hb undergoes Snitrosylation at Cys-␤ 93 (3,(7)(8)(9)(10)(11). Erythrocytes are proposed to couple O 2 tension to the distribution of NO activities (such as control of blood flow) by linking the allosteric transition of Hb (12, 13) to conformation-dependent changes in the redox activity of this Cys-␤ 93 (13-18) and the stereochemistry of this SNO bond at Cys-␤ 93 (6, 7). Indeed, Cys-␤ 93 SNO in human Hb (SNOHb) can be crystallized only with the Hb tetramer in the relaxed (R, oxygenated) conformation; the SNO bond is unstable with Hb in the tense (T, deoxygenated) conformation (7). These observations support a paradigm in which NO binding to Cys-␤ 93 is favored in the R state and NO binding to Fe(II) (and͞or transnitrosation to an alternate thiol) is favored in the T state (19-21). Thus, the change in stability of Cys-␤ 93 SNO during Hb transition between R and T states may serve to couple regional O 2 gradients to the deployment or quenching of NO bioactivities in the microcirculation (2, 6, 22).However, assaying SNOHb has been problematic. First, detection of the SNO bond has required dilution and͞or pretreatment of Hb to (i) control for artifactual identification of nitrite and Fenitrosyl species and (ii) prevent autocapture of NO on Fe during analysis (8,19,(23)(24)(25). As a result, attempts to quantify Cys-␤ 93 SNO density can be biased ...

The endogenous bronchodilator, S-nitrosoglutathione (GSNO), increases expression, maturation, and function of both the wild-type and the ⌬F508 mutant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR). Though transcriptional mechanisms of action have been identified, GSNO seems also to have post-transcriptional effects on CFTR maturation. Here, we report that 1) GSNO is only one of a class of S-nitrosylating agents that, at low micromolar concentrations, increase ⌬F508 and wild-type CFTR expression and maturation; 2) NO itself (at these concentrations) and 8-bromocyclic GMP are minimally active on CFTR; 3) a novel agent, S-nitrosoglutathione diethyl ester, bypasses the need for GSNO bioactivation by ␥-glutamyl transpeptidase to increase CFTR maturation; 4) surprisingly, expression-but not S-nitrosylation-of cysteine string proteins (Csp) 1 and 2 is increased by GSNO; 5) the effect of GSNO to increase full maturation of wild-type CFTR is inhibited by Csp silencing (si)RNA; 6) proteins relevant to CFTR trafficking are SNO-modified, and SNO proteins traffic through the endoplasmic reticulum (ER) and Golgi after GSNO exposure; and 7) GSNO alters the interactions of ⌬F508 CFTR with Csp and Hsc70 in the ER and Golgi. These data suggest that GSNO is one of a class of S-nitrosylating agents that act independently of the classic NO radical/cyclic GMP pathway to increase CFTR expression and maturation. They also suggest that the effect of GSNO is dependent on Csp and on intracellular SNO trafficking. We speculate that these data will be of relevance to the development of NO donor-based therapies for CF.Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene encoding the CF transmembrane conductance regulatory (CFTR) protein (Riordan, 1999). CFTR has several functions but is typically regarded as an apical membrane Cl Ϫ channel in epithelial cells. Its posttranslational processing involves a complex and incompletely defined series of interactions with a variety of chaperones and cochaperones that fold and glycosylate the protein and screen it for defects. The most common mutation associated with CF, ⌬F508, results in a single amino acid deletion (Drumm et al., 1991;Riordan, 1999;Gibson et al., 2003). The majority of wild-type (wt) CFTR-and virtually all ⌬F508 CFTR-is degraded before reaching the cell surface (Drumm et al., 1991). Certain agents and conditions increase expression, maturation, and (in the presence of cyclic AMP-stimulating agonists) function of ⌬F508 CFTR. Therefore, there is an interest in identifying compounds with a favorable pharmacological profile that could have this effect in vivo, reversing the molecular defect, and preventing disease progression (Denning et al., 1992;Ward and Kopito, 1994;Zeitlin et al., 2002). High-throughput screening has been used to identify 1435at ASPET Journals on May 11, 2018 molpharm.aspetjournals.org Downloaded fromcompounds that, by augmenting ⌬F508 CFTR trafficking, increase its functional expression on the surf...

. S-nitrosothiols regulate cellsurface pH buffering by airway epithelial cells during the human immune response to rhinovirus. Am J Physiol Lung Cell Mol Physiol 290: L827-L832, 2006; doi:10.1152/ajplung.00406.2005.-Human rhinovirus infection is a common trigger for asthma exacerbations. Asthma exacerbations and rhinovirus infections are both associated with markedly decreased pH and ammonium levels in exhaled breath condensates. This observation is thought to be related, in part, to decreased activity of airway epithelial glutaminase. We studied whether direct rhinovirus infection and/or the host immune response to the infection decreased airway epithelial cell surface pH in vitro. Interferon-␥ and tumor necrosis factor-␣, but not direct rhinovirus infection, decreased pH, an effect partly associated with decreased ammonium concentrations. This effect was 1) prevented by nitric oxide synthase inhibition; 2) independent of cyclic GMP; 3) associated with an increase in endogenous airway epithelial cell S-nitrosothiol concentration; 4) mimicked by the exogenous S-nitrosothiol, S-nitroso-N-acetyl cysteine; and 5) independent of glutaminase expression and activity. We then confirmed that decreased epithelial pH inhibits human rhinovirus replication in airway epithelial cells. These data suggest that a nitric oxide synthase-dependent host response to viral infection mediated by S-nitrosothiols, rather than direct infection itself, plays a role in decreased airway surface pH during human rhinovirus infection. This host immune response may serve to protect the lower airways from direct infection in the normal host. In patients with asthma, however, this fall in pH could be associated with the increased mucus production, augmented inflammatory cell degranulation, bronchoconstriction, and cough characteristic of an asthma exacerbation.asthma; S-nitrosothiol; airway epithelium; glutaminase ACUTE ASTHMA EXACERBATIONS are often triggered by viral upper respiratory tract infections (4,36,42). In adults and in children Ͼ2 yr old, the virus most commonly associated with asthma attacks is human rhinovirus (HRV) (12,27,40). Patients with acute asthma exacerbations have low pH (25) in their exhaled breath condensate (EBC). Notably, there is also a reduction in EBC pH during experimental upper airway infection with HRV (35). Indeed, the decrease in airway pH caused by HRV infection has been proposed to have a role in the pathophysiology of acute asthma attacks; exposure of airways to exogenous acid causes increased mucus production, impaired ciliary motility, cough, and bronchoconstriction (25,26).During an experimental HRV upper respiratory infection, HRV mRNA can be detected in the intrathoracic airways (16), but there are also modifications of lower airway reactivity and of bronchial inflammation that may be independent of direct lower airway infection (12,13,19). For example, cytokines produced by T helper (Th) 1 lymphocytes in response to viral infection can affect the lower airways (24), and it has been proposed that upper a...