A dynamic cycle exists in which haemoglobin is S-nitrosylated in the lung when red blood cells are oxygenated, and the NO group is released during arterial-venous transit. The vasoactivity of S-nitrosohaemoglobin is promoted by the erythrocytic export of S-nitrosothiols. These findings highlight newly discovered allosteric and electronic properties of haemoglobin that appear to be involved in the control of blood pressure and which may facilitate efficient delivery of oxygen to tissues. The role of S-nitrosohaemoglobin in the transduction of NO-related activities may have therapeutic applications.

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...

In vitro drug metabolism studies, which are inexpensive and readily carried out, serve as an adequate screening mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further in vivo testing. This publication is a sequel to part I in a series and aims at providing a general framework to guide designs and protocols of the in vitro drug metabolism studies considered good practice in an efficient manner such that it would help researchers avoid common pitfalls and misleading results. The in vitro models include hepatic and non-hepatic microsomes, cDNA-expressed recombinant human CYPs expressed in insect cells or human B lymphoblastoid, chemical P450 inhibitors, S9 fraction, hepatocytes and liver slices. Important conditions for conducting the in vitro drug metabolism studies using these models are stated, including relevant concentrations of enzymes, co-factors, inhibitors and test drugs; time of incubation and sampling in order to establish kinetics of reactions; appropriate control settings, buffer selection and method validation. Separate in vitro data should be logically integrated to explain results from animal and human studies and to provide insights into the nature and consequences of in vivo drug metabolism. This article offers technical information and data and addresses scientific rationales and practical skills related to in vitro evaluation of drug metabolism to meet regulatory requirements for drug development.