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 ...

Bone marrow-derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients ( = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecF neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecF neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.