Nitric oxide (NO) is a pivotal signaling molecule that regulates blood flow and hemorheology, stimulating vasoactivity, inhibiting smooth muscle cell proliferation, inhibiting platelet adhesion, and regulating cell adhesion, vascular permeability, and erythrocyte deformability [1
]. NO can be enzymatically synthesized in a reaction catalyzed by nitric oxide synthase (NOS). Three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). Since the discovery of NO as an important signaling molecule more than 30 years ago, the NO molecules synthesized by endothelial NOS present in the vascular endothelium have been considered the dominant source in the vasculature [34
NOS3 is localized in the endothelial lining of the vascular wall, adjacent to the smooth muscle of the vessel. This enzyme catalyzes the degradation of L-arginine to L-citrulline, with the release of NO molecules [1
]. Once formed, NO diffuses out of the endothelial cells and enters the extravascular and intraluminal spaces [43
]. In the vascular wall, NO reacts with soluble guanylate cyclase (sGC), which catalyzes the formation of 3’,5’-cyclic guanosine monophosphate (cGMP) to induce vasodilation. In the extravascular region, NO can inhibit mitochondrial respiration by binding to the heme group of cytochrome c oxidase, further regulating oxygen delivery to tissue [19
]. Inside the lumen, NO can reach its cellular targets and regulates certain rheologic factors, such as erythrocyte deformability and platelet-platelet adhesion [9
] at least partially through the transient interaction with the heme group of guanylate cyclase expressed on erythrocytes and platelets [4
]. Other proposed regulation mechanisms may involve the interaction between NO and ion transport channels on erythrocytes and platelets [21
]. Thus, in the vasculature, NO can be consumed as a result of its reactivity with a variety of species in the vasculature, including O2, reactive oxygen species, reactive nitrogen species, and cytochrome c oxidase, while carrying out its signaling role.
The life-time of NO in the extravascular tissue is relatively longer, on the scale of sub-seconds to a few seconds [43
]. However, inside the lumen of the blood vessel, the NO that is formed is rapidly scavenged by hemoglobin contained in erythrocytes and converted into relatively inert nitrite and nitrate [59
], and its lifetime can be as short as a few milliseconds [36
]. The surviving NO in the lumen has the capacity to regulate hemorheological properties. However, for many years the blood was viewed solely as a sink for NO molecules. Targets of NO signaling in the lumen were thought to be regulated by locally produced NO from the endothelium. One interesting possibility is that NO could be “carried” through the circulation by sources in the blood itself, with the NO bioactivity being released when needed and thereby providing an endocrine regulation of hemorheology. Also, if a significant amount of NO from these sources could eventually reach the smooth muscle and react with sGC, vascular tone could be regulated by the blood-borne NO. However, in vitro experiments and theoretical modeling suggested that most of the free NO in the bloodstream is rapidly converted to relatively inert nitrite and nitrate [28
] because of the fast reactivity of NO with hemoglobin contained in erythrocytes and the abundance of such hemoglobin in the blood. These findings appeared to close the door on the possibility that NO bioactivity could be stored in the blood and that blood-borne NO could regulate vascular tone and other biological functions.
The NO concentration around blood vessels has been measured, and the values reported have been in the range of 200–1000 nM [54
], with the measured NO generally considered to be endothelial in origin. However, the microelectrodes used in these experiments measured the local NO level (discussed in Ref. [14
]) that is derived from all possible NO sources in the vasculature. Furthermore, theoretical biochemical pathway analyses [15
] have shown that NOS3-derived NO cannot account for the large concentration of NO measured in the perivascular region, suggesting a discrepancy between the predicted and measured NO concentrations. This discrepancy, coupled with real-time NO concentration mapping evidence, indicated that a significant amount of NO might be produced from the intraluminal sources and might reach the vascular wall [14
]. Indeed, several hypotheses have been proposed since the 1990s to account for the possible release of NO from the blood (reviewed below).
Despite the theoretical prediction that NO cannot escape the scavenging of erythrocyte-contained hemoglobin, experiments have provided strong evidence of NO bioactivity after its release from erythrocytes. For example, inhalation of NO gas can reduce pulmonary hyptertension through NO-related mechanisms [76
]. In the 1990s, a novel endocrine signaling pathway was proposed [63
] in which NO bioactivity was hypothesized to be preserved, rather than destroyed, by blood proteins, with the NO molecules being released under certain conditions, particularly hypoxia. Furthermore, nitrite, which in the past has been considered a stable species, has also been proposed to be reduced to NO radicals in low-pH environment or by hemoglobin in erythrocytes (and possibly by other heme-containing globins in other cells beyond the intraluminal region) [33
]. Recent studies have also shown evidence of NO release from other sources, such as iron-nitrosyl-hemoglobin (HbNO) and via the activity of NOS3 in erythrocytes. The likely pathways that lead to NO release are summarized in . In addition to in vitro and ex vivo experiments that have focused on the specific pathways for the release of NO, in vivo measurements in animals have been conducted to detect NO in the blood. Kashiwagi et al. [38
] observed strong fluorescent signals from intraluminal sources from their in vivo experiments using 4,5-diaminofluorescein diacetate in rat mesentery, suggesting the existence of intraluminal NO within the mesenteric vessels.
Fig. 1 Major intraluminal pathways of NO release. Intraerythrocytic NO release can potentially come from S-nitrosylated blood proteins, nitrite reduction by heme-containing proteins, and iron-nitrosyl-hemoglobin, and NOS3 expressed on erythrocytes. EC, endothelial (more ...)
The potential release of intraluminal NO poses several conceptual challenges: First, it is not clear how the NO molecules that are formed can escape being scavenged by hemoglobin. The large quantity of erythrocyte-contained hemoglobin acts as a natural sink for the highly reactive NO molecules. Second, we still lack direct and specific measurements of the amount of NO derived from each source, especially from the intraluminal region. Finally, we have only an incomplete understanding of the quantity of NO released from the blood and how this blood-derived NO compares to endothelial NO in terms of physiological activity. The NO synthesized by NOS3 in endothelial cells diffuses to other parts of the vascular wall and to the extravascular region, and its periarteriolar level is reported as several hundred nanomolar [77
]. How much NO can reach that region from the blood and there regulate a variety of physiological functions?
In the next section, we will review the current hypotheses regarding the release of bioactive NO from the blood and discuss the likely mechanisms by which NO molecules might survive the scavenging activity of hemoglobin.