Pannexin 1 Channels Control the Hemodynamic Response to Hypoxia by Regulating O2-Sensitive Extracellular ATP in Blood

Pannexin1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contributes to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to tissue metabolic demand. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/- erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (N=6) and WT (N=6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16±9% vs -2±8%; P<0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (N=6) vs. WT (N=6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8±6% vs -10±13%; P<0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (N=6) vs WT mice (N=6) (P<0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (N=8) was 82% lower than from WT (N=8) ( P<0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.

Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+ but not by individual K+ channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+ channels in SMCs.

Dual role of the L-arginine–ADMA–NO pathway in systemic hypoxic vasodilation and pulmonary hypoxic vasoconstriction

In healthy vascular endothelium, nitric oxide acts as a vasodilator paracrine mediator on adjacent smooth muscle cells. By activating soluble guanylyl cyclase, nitric oxide stimulates cyclic guanosine monophosphate (cGMP) which causes relaxation of vascular smooth muscle (vasodilation) and inhibition of platelet aggregation. This mechanism is active in both, the systemic and pulmonary circulation. In the systemic circulation, hypoxia results in local vasodilation, which has been shown to be brought about by stabilization of hypoxia-inducible factor-1α (HIF1α) and concomitant upregulation of endothelial nitric oxide synthase. By contrast, the physiological response to hypoxia in the pulmonary circulation is vasoconstriction. Hypoxia in the lung primarily results from hypoventilation of circumscript areas of the lung, e.g. by bronchial tree obstruction or inflammatory infiltration. Therefore, hypoxic pulmonary vasoconstriction is a mechanism preventing distribution of blood to hypoventilated areas of the lungs, thereby maintaining maximal oxygenation of blood. The exact molecular mechanism of hypoxic pulmonary vasoconstriction is less well understood than hypoxic vasodilation in the systemic circulation. While alveolar epithelial cells may be key in sensing low oxygen concentration, and pulmonary vascular smooth muscle cells obviously are the effectors of vasoconstriction, the pulmonary vascular endothelium plays a crucial role as an intermediate between these cell types. Indeed, dysfunctional endothelial nitric oxide release was observed in humans exposed to acute hypoxia, and animal studies suggest that hypoxic pulmonary vasoconstriction is enhanced by nitric oxide synthase inhibition. This may be caused, in part, by elevation of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthesis. High asymmetric dimethylarginine levels are associated with endothelial dysfunction, vascular disease, and hypertension.

Role of Nitric Oxide Carried by Hemoglobin in Cardiovascular Physiology

A continuous supply of oxygen is essential for the survival of multicellular organisms. The understanding of how this supply is regulated in the microvasculature has evolved from viewing erythrocytes (red blood cells [RBCs]) as passive carriers of oxygen to recognizing the complex interplay between Hb (hemoglobin) and oxygen, carbon dioxide, and nitric oxide—the three-gas respiratory cycle—that insures adequate oxygen and nutrient delivery to meet local metabolic demand. In this context, it is blood flow and not blood oxygen content that is the main driver of tissue oxygenation by RBCs. Herein, we review the lines of experimentation that led to this understanding of RBC function; from the foundational understanding of allosteric regulation of oxygen binding in Hb in the stereochemical model of Perutz, to blood flow autoregulation (hypoxic vasodilation governing oxygen delivery) observed by Guyton, to current understanding that centers on S-nitrosylation of Hb (ie, S-nitrosohemoglobin; SNO-Hb) as a purveyor of oxygen-dependent vasodilatory activity. Notably, hypoxic vasodilation is recapitulated by native S-nitrosothiol (SNO)–replete RBCs and by SNO-Hb itself, whereby SNO is released from Hb and RBCs during deoxygenation, in proportion to the degree of Hb deoxygenation, to regulate vessels directly. In addition, we discuss how dysregulation of this system through genetic mutation in Hb or through disease is a common factor in oxygenation pathologies resulting from microcirculatory impairment, including sickle cell disease, ischemic heart disease, and heart failure. We then conclude by identifying potential therapeutic interventions to correct deficits in RBC-mediated vasodilation to improve oxygen delivery—steps toward effective microvasculature-targeted therapies. To the extent that diseases of the heart, lungs, and blood are associated with impaired tissue oxygenation, the development of new therapies based on the three-gas respiratory system have the potential to improve the well-being of millions of patients.

Recent insights into nitrite signaling processes in blood

Nitrite was once thought to be inert in human physiology. However, research over the past few decades has established a link between nitrite and the production of nitric oxide (NO) that is potentiated under hypoxic and acidic conditions. Under this new role nitrite acts as a storage pool for bioavailable NO. The NO so produced is likely to play important roles in decreasing platelet activation, contributing to hypoxic vasodilation and minimizing blood-cell adhesion to endothelial cells. Researchers have proposed multiple mechanisms for nitrite reduction in the blood. However, NO production in blood must somehow overcome rapid scavenging by hemoglobin in order to be effective. Here we review the role of red blood cell hemoglobin in the reduction of nitrite and present recent research into mechanisms that may allow nitric oxide and other reactive nitrogen signaling species to escape the red blood cell.

Skeletal muscle vasodilation during systemic hypoxia in humans

In humans, the net effect of acute systemic hypoxia in quiescent skeletal muscle is vasodilation despite significant reflex increases in muscle sympathetic vasoconstrictor nerve activity. This vasodilation increases tissue perfusion and oxygen delivery to maintain tissue oxygen consumption. Although several mechanisms may be involved, we recently tested the roles of two endothelial-derived substances during conditions of sympathoadrenal blockade to isolate local vascular control mechanisms: nitric oxide (NO) and prostaglandins (PGs). Our findings indicate that 1) NO normally plays a role in regulating vascular tone during hypoxia independent of the PG pathway; 2) PGs do not normally contribute to vascular tone during hypoxia, however, they do affect vascular tone when NO is inhibited; 3) NO and PGs are not independently obligatory to observe hypoxic vasodilation when assessed as a response from rest to steady-state hypoxia; and 4) combined NO and PG inhibition abolishes hypoxic vasodilation in human skeletal muscle. When the stimulus is exacerbated via combined submaximal rhythmic exercise and systemic hypoxia to cause further red blood cell (RBC) deoxygenation, skeletal muscle blood flow is augmented compared with normoxic exercise via local dilator mechanisms to maintain oxygen delivery to active tissue. Data obtained in a follow-up study indicate that combined NO and PG inhibition during hypoxic exercise blunts augmented vasodilation and hyperemia compared with control (normoxic) conditions by ∼50%; however, in contrast to hypoxia alone, the response is not abolished, suggesting that other local substances are involved. Factors associated with greater RBC deoxygenation such as ATP release, or nitrite reduction to NO, or both likely play a role in regulating this response.

Contribution of epoxyeicosatrienoic acids to the cerebral blood flow response to hypoxemia

Adenosine A2A receptors and ATP-activated K+ (KATP) channels contribute to part of the cerebral vasodilatory response to systemic hypoxia, but other mediators are likely involved. Epoxyeicosatrienoic acids (EETs) are cerebral vasodilators and are released from astrocytes exposed to hypoxia. Moreover, stimulation of metabotropic glutamate receptors (mGluR) produces vasodilation by an EET-dependent mechanism. Here, we tested the hypothesis that EET signaling and mGluR activation contribute to hypoxic vasodilation. Laser-Doppler flow was measured over cerebral cortex of anesthetized rats subjected to stepwise reductions in arterial oxygen saturation to 50-70%. Hypoxic reactivity was calculated as the slope of the change in laser-Doppler flow vs. the reciprocal of arterial oxygen content. Hypoxic reactivity significantly decreased from 9.2 ± 1.9 (±95% confidence interval) in controls with vehicle treatment to 2.6 ± 1.4 with the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid, to 3.0 ± 1.5 with the EET synthesis inhibitor MS-PPOH, to 1.9 ± 2.3 with the combined mGluR subtype 1 and 5 antagonists 2-methyl-6-(phenylethynyl)pyridine and LY367385 , to 5.6 ± 1.2 with the KATP channel inhibitor glibenclamide, and to 5.8 ± 2.3 with the A2A receptor antagonist SCH58261. However, reactivity was not significantly altered by the A2B receptor antagonist MRS1754 (6.7 ± 1.8; P = 0.28 Dunnett's test) or by the 20-hydroxyeicosatetraenoic acid synthesis inhibitor HET0016 (7.5 ± 2.3; P = 0.6). These data indicate that, in addition to the known contributions of A2A receptors and KATP channels to the increase in cerebral blood flow during hypoxia, EETs and mGluRs make a major contribution, possibly by mGluR stimulation and hypoxia-induced release of EETs. In contrast, A2B receptors do not make a major contribution, and 20-hydroxyeicosatetraenoic acid does not significantly limit hypoxic vasodilation.