Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

The role of globins in cardiovascular physiology.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

Vascular Expression of Hemoglobin Alpha in Antarctic Icefish Supports Iron Limitation as Novel Evolutionary Driver.

Frigid temperatures of the Southern Ocean are known to be an evolutionary driver in Antarctic fish. For example, many fish have reduced red blood cell (RBC) concentration to minimize vascular resistance. Via the oxygen-carrying protein hemoglobin, RBCs contain the vast majority of the body's iron, which is known to be a limiting nutrient in marine ecosystems. Since lower RBC levels also lead to reduced iron requirements, we hypothesize that low iron availability was an additional evolutionary driver of Antarctic fish speciation. Antarctic Icefish of the family Channichthyidae are known to have an extreme alteration of iron metabolism due to loss of RBCs and two iron-binding proteins, hemoglobin and myoglobin. Loss of hemoglobin is considered a maladaptive trait allowed by relaxation of predator selection since extreme adaptations are required to compensate for the loss of oxygen-carrying capacity. However, iron dependency minimization may have driven hemoglobin loss instead of a random evolutionary event. Given the variety of functions that hemoglobin serves in the endothelium, we suspected the protein corresponding to the 3' truncated Hbα fragment (Hbα-3'f) that was not genetically excluded by icefish may still be expressed as a protein. Using whole mount confocal microscopy, we show that Hbα-3'f is expressed in the vascular endothelium of icefish retina, suggesting this Hbα fragment may still serve an important role in the endothelium. These observations support a novel hypothesis that iron minimization could have influenced icefish speciation with the loss of the iron-binding portion of Hbα in Hbα-3'f, as well as hemoglobin ß and myoglobin.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Heterocellular Contact Can Dictate Arterial Function.

RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Replacement Therapies in Metabolic Disease.

BACKGROUND: Replacement therapies have revolutionized treatment paradigms in metabolic diseases by restoring defective enzymes and supplementing missing downstream metabolites. Through most of the 20th century, no targeted therapies existed for these conditions, the only treatment options available focusing on symptoms rather than the underlying disorders. Improved understanding of the molecular pathways underlying metabolic disease has allowed not only supplementation of missing metabolites and reduction of upstream substrates, but replacement of defective or missing enzymes. OBJECTIVE: Modern genetic technologies have facilitated steady progress in recombinant enzyme innovation, providing treatments that replicate not only endogenous enzymes, but also their posttranslational modifications to optimize their delivery and function. The advent of the gene therapy revolution brings a possibility of new therapeutic opportunities in which the enzymes at the core of metabolic diseases may not only be added back, but genetically replaced. CONCLUSION: With the next generation of treatments approaching, this review examines the recent decades of replacement therapy innovation in metabolic disease and discusses the challenges and opportunities for the next generation of treatments.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

The Evolving Role of Diverse Gaseous Transmitters Mediating Heterocellular Communication Within the Vasculature.

Heterocellular communication between different cell types of the vasculature, both within the blood vessel wall and cells interacting with the blood vessel wall, is absolutely vital and must be tightly regulated. In this Forum, the role of four different gaseous transmitters [nitric oxide [NO], carbon monoxide (CO), hydrogen sulfide (H2S), and superoxide (O2•-)] is examined by four different research groups in detail, with two original articles and two reviews of the literature. In this editorial, we discuss how each of them may contribute their own component to heterocellular signaling in the vasculature. Antioxid. Redox Signal. 26, 881-885.

Endothelial nitric oxide synthase in the microcirculation.

Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

Emerging concepts regarding pannexin 1 in the vasculature.

Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.

Flame Retardant Applications in Camping Tents and Potential Exposure.

Concern has mounted over health effects caused by exposure to flame retardant additives used in consumer products. Significant research efforts have focused particularly on exposure to polybrominated diphenyl ethers (PBDEs) used in furniture and electronic applications. However, little attention has focused on applications in textiles, particularly textiles meeting a flammability standard known as CPAI-84. In this study, we investigated flame retardant applications in camping tents that met CPAI-84 standards by analyzing 11 samples of tent fabrics for chemical flame retardant additives. Furthermore, we investigated potential exposure by collecting paired samples of tent wipes and hand wipes from 27 individuals after tent setup. Of the 11 fabric samples analyzed, 10 contained flame retardant additives, which included tris(1,3-dichloroisopropyl) phosphate (TDCPP), decabromodiphenyl ether (BDE-209), triphenyl phosphate, and tetrabromobisphenol-A. Flame retardant concentrations were discovered to be as high as 37.5 mg/g (3.8% by weight) in the tent fabric samples, and TDCPP and BDE-209 were the most frequently detected in these samples. We also observed a significant association between TDCPP levels in tent wipes and those in paired hand wipes, suggesting that human contact with the tent fabric material leads to the transfer of the flame retardant to the skin surface and human exposure. These results suggest that direct contact with flame retardant-treated textiles may be a source of exposure. Future studies will be needed to better characterize exposure, including via inhalation and dermal sorption from air.