Cellular microdomains for nitric oxide signaling in endothelium and red blood cells

Journal Pre-proof Cellular microdomains for nitric oxide signaling in endothelium and red blood cells Francesca Leo, Beate Hutzler, Claire A. Ruddiman, Brant E. Isakson, Miriam M. Cortese-Krott PII:

S1089-8603(19)30314-3

DOI:

https://doi.org/10.1016/j.niox.2020.01.002

Reference:

YNIOX 1957

To appear in:

Nitric Oxide

Received Date: 6 November 2019 Revised Date:

23 December 2019

Accepted Date: 2 January 2020

Please cite this article as: F. Leo, B. Hutzler, C.A. Ruddiman, B.E. Isakson, M.M. Cortese-Krott, Cellular microdomains for nitric oxide signaling in endothelium and red blood cells, Nitric Oxide (2020), doi: https://doi.org/10.1016/j.niox.2020.01.002. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Review article

Cellular microdomains for nitric oxide signaling in endothelium and red blood cells. Francesca Leo1*, Beate Hutzler1*, Claire A. Ruddiman2,3, Brant E. Isakson2,4, Miriam M. Cortese-Krott1# 1 Myocardial Infarction Research Group, Department of Cardiology, Pulmonology and Angiology Medical Faculty, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany. 2 Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia 3 Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 4 Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, Charlottesville, Virginia *Contributed equally Short title: NO signaling in microdomains #Corresponding author: Miriam M. Cortese-Krott, PhD [email protected]; Tel. +49 (0) 211 81 15115 Myocardial Infarction Research Group, Department of Cardiology, Pulmonology, and Vascular Medicine, Medical Faculty, Heinrich-Heine-University of Düsseldorf, Universitätstrasse 1, 40225 Düsseldorf, Germany. Declaration of Interest : None

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Abstract There is accumulating evidence that biological membranes are not just homogenous lipid structures, but are highly organized in microdomains, i.e. compartmentalized areas of protein and lipid complexes, which facilitate necessary interactions for various signaling pathways. Each microdomain exhibits unique composition, membrane location, and dynamics that ultimately shape their functional characteristics. In the vasculature, microdomains are crucial for organizing and compartmentalizing vasodilatory signals, which contribute to blood pressure homeostasis. In this review, we aim to describe how membrane microdomains in both the endothelium and red blood cells allow context-specific regulation of the vasodilatory signal nitric oxide (NO) and its corresponding metabolic products, and how this results in tightly controlled systemic physiological responses. We will describe (1) structural characteristics of microdomains including lipid rafts and caveolae; (2) endothelial cell caveolae and how they participate in mechanosensing and NO-dependent mechanotransduction, (3) the myoendothelial junction of resistance arterial endothelial cells and how protein-protein interactions within it have profound systemic effects on blood pressure regulation, and (4) putative/proposed NO microdomains in RBCs and how they participate in control of systemic NO bioavailability. The sum of these discussions will provide a current view of NO regulation by cellular microdomains. Keywords: endothelial nitric oxide synthase; caveolin; hemoglobin; spectrin; NO metabolites.

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Highlights • • • • •

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Microdomains are highly organized regions of proteins and lipids Caveolae and myoendothelial junctions are canonical vascular microdomains Within caveolae NO signaling is regulated by the eNOS/Cav-1 interaction Within myoendothelial junctions eNOS/Hba complexes control NO signaling and blood pressure In red blood cells putative microdomains may control NO signaling in hypoxia and normoxia

1. Introduction Compared to other radicals and reactive species, nitric oxide (NO) is stable and moderately reactive with a relatively long half-life in the cellular environment [1]. It also has a high diffusibility, which allows it to freely transverse cellular membranes and regulate numerous signaling pathways [1]. NO can bind and/or react with high affinity to molecules containing Fe2+-heme centers, including its main targets the soluble guanylate cyclase (sGC) and hemoglobin, which are of particular importance in red blood cells and endothelium [1]. NO also undergoes rapid radical-radical or oxidation reactions to produce a plethora of derivatives that are also biologically active. For instance, NO can be oxidised to form potent nitrosating agents (like N2O3 or NO2), which target thiols, can be transformed into metabolites with varying stabilities and biological activities (including NO2- and NO3-, nitrosothiols, nitrosamine, nitro fatty acids), or react with reactive species to produce peroxynitrite (ONOO-) and nitrosopersulfide (SSNO-) [1; 2]. Its conversion to these other substances multiply its possible targets and biological effects. Together these chemicophysical characteristics make NO one of the most versatile signaling molecules in biology. However, because of its high reactivity and versatility, NO signaling must be tightly controlled at multiple levels, including cellular and subcellular compartmentalization, like organelles and microdomains. Membranes are responsible for both cellular and sub-cellular compartmentalization of NO signaling. Membranes used to only be considered as homogeneous lipid barriers with randomly distributed protein components. The mosaic-fluid structure of the membranes was first recognized in electronic microscopy via the so called “freeze-etching technique”, showing that proteins were highly embedded in the membrane [3]. However, it is now clear that membranes are highly organized and compartmentalized in visible sub-structures characterised by dynamic complexes of protein and lipids, defined as microdomains [4; 5]. Generally, microdomains are crucial for facilitating directed signaling and are especially important in NO-dependent pathways [4]. The structural and functional characteristics of microdomains depend on their specific protein/lipid composition, their respective localisation on the cell surface, and the temporal dynamics of signal transduction. Microdomains allow for “disambiguation” of an otherwise common pleiotropic signal (e.g. adenosine, shear stress) by forcing a cell/condition-specific “interpretation” and response. In other words, microdomains direct an apparently “generic” signal to a specific, tightly controlled outcome. This review will describe how microdomains allow context-specific control of NO signaling and metabolism in endothelial cells (ECs) and red blood cells (RBCs), which are the critical compartments of NO production, metabolism, and signaling. Specifically, we will describe (1) structural characteristics of microdomains including lipid rafts and caveolae; (2) describe endothelial cell caveolae and how they participate in mechanosensing and NO-dependent mechanotransduction, (3) describe the myoendothelial junction (MEJ) of resistance arterial endothelial cells and how protein-protein interactions within it have profound systemic effects on blood pressure regulation, and (4) describe putative/proposed NO microdomains in RBC and how they participate in control of systemic NO bioavailability.

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2. Classification and structural characteristics of cell microdomains Microdomains are generally defined as having three characteristics: that proteins must be concentrated to a localized area within the cell, must demonstrate specific plasma membrane localization, and proteins in that area must either directly interact or regionally associate [6]. The general definition for lipid/protein complexes at the cellular membrane is lipid rafts1. Although individual lipid rafts are small in size and generally not detectable by electron microscopy, they have been shown to make up to 50% of the plasma membrane [7; 8]. They are enriched in cholesterol and glycosphingolipids, which reduces membrane fluidity compared to the surrounding plasma membrane; this high saturation allows for tight packing to facilitate microdomain formation [7]. Clustering of proteins within microdomains can facilitate signal transduction, but can also negatively regulate protein activity by sequestering proteins to prevent activating protein-protein interactions that may occur outside of the microdomain [7]. On the other hand, recruitment of a protein to a microdomain could also promote a protein-protein interaction that ultimately negatively regulates it through an inhibitory interaction [7]. An example of this is the eNOS/Caveolin-1 (Cav-1) interaction within caveolae [7]. Moreover, proteins belonging to specific signaling cascades like tyrosine kinase or G-protein coupled receptors, glycosylphosphatidylinositol-linked proteins (GPIlinked proteins), G proteins, and protein kinases may be clustered in membrane microdomains [7; 9]. Caveolae are a subset of lipid rafts and are small plasma membrane invaginations of about 50-100 nm in diameter [5], which (contrary to other lipid rafts) are visible by electron microscopy. They were initially described by G.E. Palade in 1953 from electron micrograph images of capillaries and were termed plasmalemma vesicles [10]. In 1955, E. Yamada discovered them in the gall bladder epithelium [11]. He viewed them as “little cave” structures and thus termed them caveolae intracellulares [10; 11]. These flask shaped microdomains were only defined in terms of morphology until 1992, when caveolin, the major coat protein of caveolae, was discovered [12]. Caveolin proteins distinguish caveolae from other lipid rafts, whose binding to cholesterol is thought to stabilize their invaginated structure [7]. Cells that typically express Cav-1, like EC, also express caveolin-2 [13; 14; 15; 16]; whereas vascular smooth muscle cells (VSMC) predominately express caveolin-3 [17]. The generation of global Cav-1 and -3 knockout mice revealed a reduction of the number of caveolae [18; 19; 20; 21], while caveolin-2 knockout mice retained normal occurrences [22]. Caveolin-2 therefore may act synergistically with Cav-1 in the formation of caveolae, but is not absolutely necessary [14; 23; 24; 25]. Caveolae are highly enriched in epithelial cells, adipocytes, fibroblasts, VSMC, and EC [26]. In ECs, they occur at a striking density of approximately 80 per µm2 [27]. They facilitate macromolecular endocytosis in EC, whereas in other cell types this process is largely mediated through clathrin-coated vesicles [27]. Moreover, they participate in mechanosensing and mechanotrasduction [28] (see Section 3 of this review). Another

Small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid- enriched domains that compartmentalize cellular processes. Small rafts can some- times be stabilized to form larger platforms through protein-protein and protein- lipid interactions” (Pike LJ. 2006. Rafts defined: a report on the Keystone Symposium on lipid rafts and cell function. J. Lipid Res. 47:1597–98). 1

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crucial function of caveolae is their participation in microdomain formation within the resistance arterial endothelium where they are specifically enriched within the MEJ [29] (see Section 4).

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3. Microdomains in the endothelium: mechanotransduction and NO signaling. Mechanical stress is a fundamental physiological signal regulating vascular function and pathophysiology. Two types of mechanical forces occur naturally in the vasculature: the circumferential stress, triggered by variations of pulse pressure, and shear stress, influenced by the blood flow. Shear stress is strongly dependent on the vessel diameter and location in the vasculature (i.e., arteries, veins, or capillaries), ranging from 1 Pa in the ascending aorta to 4 Pa in capillaries and up to 5 Pa in arterioles in normal physiological conditions [30; 31]. In the venous system, it is approximately 10-fold less [30]. Endothelial caveolae play a fundamental role both for mechanosensing (i.e., allowing the cell to “sense” mechanical signals) and mechanotransduction (i.e., converting mechanical signals into biochemical signals and activating of down-stream effects), and facilitate vascular adaptation to changes in laminar flow [26; 32; 33; 34]. It was shown that mechanical forces acting on the vessel endothelium induce uniaxial stretching or deformation of the endothelial monolayer, which leads to membrane scaffolding (i.e., spatiotemporally controlled assembly of protein scaffolds at the membrane) and subsequent activation of downstream effectors [34; 35]. In ECs, eNOS is predominantly localized within caveolae forming a complex with Cav-1 [36]. The interaction of eNOS with Cav-1 occurs at the oxygenase domain of eNOS, which inhibits electron transfer and thereby NO synthesis (Figure 1) [37]. Shear stress promotes wall stretching to ultimately promote the dissociation of eNOS from Cav-1; this allows its release into the cytoplasm and its activation through binding of a Ca2+/ calmodulin (CaM) complex and/or by phosphorylation at Ser 1117 [35; 36]. Shear stress can also increase substrate availability for eNOS via increasing the velocity of the cationic amino acid transporter (CAT-1), which import the L-Arginine (L-Arg) from the extracellular space [38; 39]. (Figure 2). Under shear stress conditions addition of extracellular L-Arg increased NO production in bovine aorta EC in a dose dependent manner [39], indicating a role for shear stress mediated activation of CAT-1 in NO signaling. Interestingly, shear-stress is also responsible for both the activation of Ca2+ currents and protein kinase signaling [40; 41; 42; 43]. (recently reviewed by us in [44]). Examples of stretch activated ion channels leading to Ca2+ currents are the transient receptor cation channel V4 (TRPV4), the inwardly rectifying potassium channel, and Piezo-1 [45]. All these proteins were shown in proteomics studies to directly or indirectly (via chaperons like HSP70 and HSP90) interact with eNOS [46] and were localized at the plasma membrane of endothelial cells (see for example the proteomic study conducted by Durr et al on lung microvascular endothelial cell [47]). In addition, these studies identified direct regulatory interactions of eNOS GTP-binding (G) proteins coupled receptors like the bradykinin (BK) receptor 2 (B2) and the purinergic receptor P2Y2. GPCR are receptors located in the cell membrane with a 7-helix transmembrane structure that trigger intracellular signal cascades via G proteins in response to specific agonists; it was shown that wall stress leads to the potentiation of agonistmediated activation of GPCRs, which thereby can also act as force transducers [48]. The direct interaction with eNOS add a further regulatory level and fine tune of the combination of mechanosensing and agonist binding. A very good example of these complex regulatory mechanisms is the BK-mediated activation of eNOS. Under normal physiological conditions, the B2 receptor inhibits eNOS via binding and complex formation [49]. BK binds to its GPCR that in turn activates protein kinase C, leading to increased intracellular calcium levels. The binding of BK to the bradykinin receptor 2 activates the receptor and leads to a dissociation from eNOS [49]. Another example of a GPCR is the purinergic receptor P2Y2, which activates eNOS in response to shear stress [50] (Figure 2). After P2Y2 activation, the receptor initiates a

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signaling cascade, leading to the phosphorylation of PECAM-1, VEGRF2, Src-kinase, and Akt, all of which activate the eNOS via phosphorylation at Ser-1177 [50].Specifically, PECAM-1 forms a complex with vascular endothelial cadherin (VE-cadherin), which, following the shear stress–induced tyrosine phosphorylation of PECAM-1, recruits VEGFR2. VEGFR-2 in turn becomes tyrosine phosphorylated and activates downstream signaling events including the activation of AKT kinases, leading to eNOS activation. Other putative protein components of shear-sensitive microdomains regulating eNOS activity are proteins belonging to the cytoskeleton. The actin cytoskeleton is highly sensitive to mechanical forces and acts as a force transmitter that distributes force throughout the whole cell to sense and transmit mechanical inputs [32]. In 1976, the first electron microscopy analysis showed an connection between caveolae and stress fibers of the cytoskeleton [51]. Several cross linkers connect caveolae to actin, e.g. filamin A that connects cavin-1 (another protein associated with caveolae) with actin [52; 53]. Another example is Eps15 homology domain-containing 2 (EHD2), a membrane remodeling ATPase, that links directly the caveolae to actin [54] or even a direct bond between Cav-1 and actin [55]. Additionally, the actin cytoskeleton is also directly associated with eNOS and contributes to its regulation in response to shear stress. In pulmonary artery ECs, it was shown that there is an association of actin filaments to eNOS [56; 57], and that the polymerization state of actin plays an important role in the regulation of eNOS activity [56]. The globular single G-actin significantly increases the activity of eNOS [56] through a direct association that does not involve another protein [56]. Incubation of eNOS with G-actin resulted in a more significant increase in eNOS activity than that observed with F-actin, suggesting that depolymerization of F-actin into Gactin increases eNOS activity through a posttranslational mechanism [56]. The glycocalyx covers the luminal surface of EC and also participates in mechanosensing. Since the glycocalyx is directly connected to the cytoskeleton, flow-dependent changes in glycocalyx composition lead to a mechanotransduction. Evidence for the glycocalyx as mechanotransducer in ECs is based on the use of enzymes to selectively degrade specific components of glycocalyx (either proteoglycans, glycoproteins, and glycosaminoglycans) [58]. For example, the degradation of heparan sulfate, a type of proteoglycan, leads to an impaired shear-induced NO production in bovine aortic EC [59]. Another study showed that the removal of syndecan-1 also blocks eNOS activation [60]. Since heparan sulfate is linked to syndecans, which are in turn linked to G-protein-coupled receptors, the glycocalyx is therefore functionally linked to eNOS [43]. Taken together, canonical microdomains (e.g. caveolae) and non-canonical putative microdomains in EC (complexes at the cytoskeleton and/or at the membrane of endothelial cells) are highly specialized to regulate and fine tune eNOS activity in response to mechanical stress or specific physiological stimuli. A more specific analysis of the proteinprotein, protein-lipid, and protein-glycocalyx interactome may further contribute our understanding of NO regulation and compartmentalization.

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4. NO signaling in the MEJ - control of vascular tone and blood pressure The arterial wall is composed of three main layers. One layer is the endothelium that lines the lumen and is oriented to flow direction. Ad second layer is constituted by VSMCs, which are aligned perpendicularly to EC and mediate vasoconstriction. Between these two layers is an extracellular matrix layer termed the internal elastic lamina, which has a high density of holes. In order to communicate with the VSMCs, ECs sends projections through these internal elastic lamina holes and contacts the VSMC. These projections are called MEJs and are one of the most well recognized signaling microdomains in the vascular wall. At the MEJ heterocellular communication can occur either through the close juxtaposition of membranes or through transfer of cytosolic signaling molecules, such as electrical signals, Ca2+ and inositol trisphosphate (IP3) via gap junctions [61; 62; 63]. Gap junctions located at the inter-endothelial junctions are also associated with specific phospholipids in the membrane [64; 65; 66; 67; 68; 69; 70].These microdomains are an anatomical hallmark of the resistance arterioles, which are the main regulators of total peripheral resistance, and are not characteristic of larger conduit vessels [71]. The MEJ, also referred to as the myoendothelial gap junction or the myoendothelial projection, was first identified and described by Moore and Ruska in 1957 using electron microscopy on isolated arterioles from dog heart [72]. The focus of their morphological studies was on pinocytotic vesicles in capillary and small artery ECs; the description of endothelial projections through windows in the internal elastic lamina was an additional observation that was not elaborated on until 1967 by Rhodin, who used electron microscopy on rabbit kidney arterioles [73]. It was Rhodin who drove the idea forward that these cellular projections contributed to vascular physiology. Indeed, research since this initial description has demonstrated the MEJ as a unique signaling microdomain that facilitates heterocellular communication. The MEJ is most commonly studied for its crucial role in vasodilatory signaling. One major pathway is endothelial derived hyperpolarization (EDH) of the VSMC. For EDH-mediated vasodilation at the MEJ in resistance arteries, Ca2+ influx into the EC activates intermediateand small-conductance potassium channels (IK and SK, respectively) that are enriched within the MEJ [74], leading to K+ efflux and VSMC membrane hyperpolarization [75; 76; 77]. This hyperpolarization leads to deactivation of voltage dependent calcium channels in the smooth muscle and decreases the cytosolic calcium, which ultimately reduces myosin light chain (MLC)- mediated contraction. There has also been extensive work on TRPV4 localization to MEJ, which also facilitates Ca2+ influx into the EC and can activate EDHbased vasodilatory signals. EDH was initially termed EDH factor (EDHF) due to the assumption that this hyperpolarization occurred downstream of a single factor. Since then, several factors have been identified that hyperpolarize the VSMC, like EETs and prostacyclins, and thus this pathway is now more commonly referred to as EDH. For or an extensive review on EDH-based signaling please see the following review ([78]). Binding of catecholamines to their respective receptors on VSMC membranes activates GqPCR mediated pathways that lead to increases in cytosolic Ca2+ and IP3, which can diffuse into EC through MEJ [62] (composed of connexons), and activate vasodilatory pathways in a process called myoendothelial feedback [75]. Specifically, the IP3 receptor 1 (IP3R1) is localized on the endoplasmic reticulum within this projection and is the target of IP3 described in the myoendothelial feedback loop [63]. Studying these microdomains in vivo is difficult and a lot of the mechanistic details that we know about these cellular projections came from work done on an in vitro vascular co-culture technique (VCCC). ECs and VSMCs are plated on opposite sides of a Transwell and endothelial projections form through holes in the Transwell, representing the cellular projections that occur in vivo within the vascular wall. The MEJ fraction can then be isolated by scraping each side of the trans well of the two cell types and digesting the protein still left on the trans well. This technique was imperative for

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defining heterocellular communication via gap junctions in the regulation of vasodilation. Specifically, this model was used to identify that IP3R1, and not IP3R2 or IP3R3, localize to the endoplasmic reticulum that is present in the cellular projection [63]. The in vitro vascular co-culture technique was also used to identify that the eNOS localized to the MEJ is functionally distinct from eNOS elsewhere in the EC [79]. This could be due to the enrichment of diacylglycerol and phosphatidylserine in the MEJ plasma membrane; thus, further demonstraing the importance of signaling microdomains within the vascular wall. In addition, since NO has a short biological half-life of approximately five seconds, its localization to endothelial MEJs facilitates its function as a vasodilatory signal, since its close proximity to the VSMC allows for it to quickly diffuse and induce vasodilation prior to its degradation. Once in the VSMC, NO can activate sGC, which results in an increase in cyclic GMP leading to a reduction of cytosolic calcium in the smooth muscle and increased myosin light chain phosphatase activity. Ultimately, these events actively reduce the contraction within the smooth muscle layer (Figure 1). Signaling microdomains in the endothelium are important regulators of vascular function and modulation of proteins within these domains represent potential therapeutic targets for treatment of hypertension. A prime example of this is eNOS, which has several known modulators of activity, including its inhibitory interaction with hemoglobin alpha (Hbα). Hemoglobin is canonically known for its role in gaseous transport within the RBC. Hemoglobin tetramers in RBC are composed of two alpha and two beta subunits that exhibit binding properties for various gases, most notably oxygen and carbon dioxide, and are well recognized for their role in regulating gas exchange in RBC [80]. Recently, a new role for Hbα, but not beta, has been described within the MEJs of the resistance artery. A proteomic analysis of the MEJ fraction isolated from the VCCC in vitro model system revealed an enrichment for Hbα within the MEJ [29]. Additionally, a proximity ligation assay on resistance arteries demonstrated that Hbα associates with eNOS within the endothelium and specifically at MEJs [29]. Although other globin proteins are also expressed within the vascular wall, the discovery of Hbα in the resistance artery endothelium, and specifically at the MEJ, makes its localization to the vascular wall unique compared to other globins endothelium (Figure 2) [81; 82]. In order to study the systemic effects of the Hbα-eNOS interaction on blood pressure regulation, Straub et al. created a peptide that mimics Hbα’s binding site on eNOS. This peptide, termed HbαX, has since been used to demonstrate the physiological impact of disrupting this protein-protein interaction [83]. Arterioles treated with this HbαX demonstrated a blunted vasoconstriction in response to phenylephrine that could be restored in the presence of the NOS inhibitor L-NAME [29]. In addition, mice injected with this mimetic peptide demonstrated markedly reduced blood pressure after only two days of treatment [84]. The peptide did not demonstrate any toxicity or affect oxygen binding to Hbα, indicating the function of Hbα within RBC is maintained in the presence of this peptide [84]. Taken together, this suggests that Hbα’s interaction with eNOS at MEJs normally functions as a sink for newly produced NO in order to prevent its diffusion to and vasoactive properties within the smooth muscle layer. It therefore represents a potential therapeutic target for lowering blood pressure. HbαX peptide is a way to pharmacologically intervene with the eNOS-Hbα interaction. However, there are several other known mediators of eNOS activity in vivo, including Cav-1, which, like Hbα, is a negative regulator of eNOS activity and is also enriched within the MEJ signaling microdomain. Upon an increase in cytosolic calcium, the Cav-1-eNOS interaction is disrupted and eNOS is able to catalyse the conversion of L-Arg into L-Citrullin to produce NO. Conversely, a number of activating interactions with eNOS have also been described, such as with heat shock protein (HSP)-90 [85; 86; 87] and NOS interacting protein [88; 89]. In addition to protein-protein interactions, eNOS is also regulated by different phosphorylation states. For an extensive review on eNOS regulation, see [90].

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5. Microdomains for NO signaling in RBC Although RBC were first identified in 1668 by Dutch biologist and microscopist Jan Swammerdam [91], their membrane structure was not described until 1925 by Gorter and Grendel [92]. Through advances in biochemical and molecular biology methods since then, we now know that the RBC membrane shows peculiar structural characteristics and a biconcave “donut-like” shape, both of which are essential to their physiological function as carriers of respiratory gases. Unlike other cells, RBC lack nuclei, organelles, and generally any type of intracellular compartmentalization [93]. Thus, microdomains within their plasma membrane could be particularly important for regulating signaling pathways in RBCs. The RBC surface is arranged as in any other cell in three layers: the glycocalyx, the lipid bilayer containing transmembrane proteins, and the cytoskeleton connected to the lipid bilayer. However, molecular composition of these layers is peculiar for RBCs compared to other cells [91; 94]. The cytoskeleton is mainly composed of spectrin forming an elastic network of hexagonal units shaping the cell in its characteristic donut-like form. The glycocalyx is composed of membrane-bound glycolipids and glycoproteins [95].The glycocalyx has a large impact on adhesion forces (due to ionic and hydrogen bonds) between cells and it may play an important role in control RBC interactions in health and disease [96] . The lipid bilayer in RBCs is approximately made up of equal parts phospholipids (including phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine) and cholesterol [97; 98]. Additionally, in contrast to other cell types, the lipids are asymmetrically distributed across the bilayer plane, in a trans asymmetry organization [99]. Interestingly, disruption of this asymmetrical distribution can lead to cell lysis [100; 101]; thus, its homeostasis is generated and maintained by several different types of energy-dependent and energy-independent phospholipid transport proteins, which are not specific to, but especially important in RBC biology: (1) the “flippases” are Mg2+ dependent enzymes and P-glycoprotein ATPases family and are responsible for phospholipids translocation from the outer to the inner monolayer, while (2) the “floppases” are members of the multidrug resistance protein 1 (MRP1) family and catalyse the translocation of from the inner to the outer leaflet of the bilayer against a concentration gradient in an energy-dependent manner[102] [102]. In contrast, (3) the “scramblases” move phospholipids bi-directionally down their concentration gradients in an energy-independent manner. This specific composition and distribution of lipids in the membrane of RBCs a may be related to their imperative role in maintaining erythrocyte curvature and regulating their reshaping processes [103]. For example, cholesterol is highly associated with high curvature areas and upon stretching is further recruited to these areas [103]. Although a depletion of cholesterol-rich domains also reduced the deformability of RBCs [103], increasing the cholesterol content within a RBC plasma membrane can also contribute to membrane stiffness and reduced fluidity[104] [104]. Fatty acid composition in mammalian erythrocytes has also an enormous influence on cellular aggregation [105]. These findings clearly show a necessary homoeostasis for this lipid rafts to properly facilitate gas exchange within the microcirculation. While the lipid distribution in red cells is well described the protein composition of RBC lipid rafts and the presence of canonical microdomains is still largely unknown. There are doubts regarding the presence of caveolae on the membrane of normal RBCs; the presence of Cav1 in RBCs was found in detergent-resistant membrane rafts by ELISA [106], but its functional significance is still unknown. It was proposed that detergent-resistant membrane rafts may play a fundamental role in regulating and/or facilitating parasite infections, such Plasmodium falciparum (malarial parasite) [107; 108]; in fact, depletion of lipids from the rafts inhibits infection and was proposed as possible pharmacological target for targeting 11

malaria [107; 108]. Additionally, non-canonical signaling microdomains may be present in RBCs. In fact, several studies pointed out that there are adrenergic GPCRs leading to activation of cAMP-dependent pathways [109], as well as a (probably) highly compartmentalized NO metabolic activity in hypoxia and normoxia [110]. Biochemical and physiological studies suggest that during hypoxia, hemoglobin possesses an allosterically regulated nitrite reductase activity that reduces nitrite to NO, with a maximal effect at about 50% oxygen saturation [111; 112]. It was proposed that nitrite is taken up by RBCs through the anion-exchange protein (AE-1/ band 3) or through the membrane as nitrous acid in a pH-dependent process that accelerates nitrite uptake during tissue hypoxia [113; 114]. There is strong evidence of NO formation from RBCs under hypoxic conditions that is released from the cell, as demonstrated by measuring NO gas formation or NO bioactivity in an NO-specific bioassay (using vessels or platelets after nitrite addition to deoxygenated or partially deoxygenated RBCs) [111; 115; 116; 117]. Instead, under normoxic conditions RBC are a major sink of NO because of the high amount of heme they contain. In fact, NO reacts with oxyHb with a near-diffusion-limited reaction rate [111; 118]. Therefore, a major challenge for a full explanation of these highly reproducible observations is understanding how the NO that is produced by deoxyhemogobin (via nitrite reduction) can escape NO scavenging and be released from RBCs. Gladwin et al [119] proposed the presence of a “nitrite reductase metabolon” complex, composed of anion exchange proteins (AE-1 / band 3), CA, deoxyHb, aquaporin, and Rh channels, and all localized within a RBC lipid raft or in a caveola homologue. This metabolic microdomain would concentrate the chemical reactants nitrite, proton, and deoxyheme with highly hydrophobic channels at the membrane complex and allow channelling nitrite and H+ to deoxyHb for catalytic NO generation by RBCs. Such microdomain could explain the protonation of nitrite and facilitate the export of NO or its metabolites. However, electron microscopy evidence of a nitrite reductase microdomain in RBCs is still lacking (Figure 3). Another putative microdomain for NO synthesis and signaling in RBCs was proposed by Cortese-Krott et al.[120] to explain how intracellular eNOS-derived NO may play a significant role in RBCs. It is now well established that RBCs carry a full Arginase-1/ eNOS/ soluble guanylate cyclase (sGC) dependent pathway [121; 122; 123; 124] and that an eNOSdependent nitrosation (of diamino-fluorescein) occurs in RBCs under normoxic conditions as assessed by quantitative analytical techniques [122]. In RBCs, eNOS is localized to the internal side of the membrane as shown via electron microscopy [123]. It was proposed [120] that local formation of metHb may protect NO from degradation by oxyHb through allowing NO to interact with its target proteins in the immediate vicinity of eNOS, which promotes their signaling function and leads to the activation of NO signaling pathways. One possible and potentially ideal target for NO signaling in RBCs may be the recently described red cell sGC [125]. However, it is important to note that evidence showing co-localisation or a functional link between eNOS and sGC is still lacking (Figure 3). Similar to the vasculature, shear stress appears to stimulate mechanosensitive channels in the RBC plasma membrane that facilitate calcium influx into the cell, transforming mechanical stimulation into biochemical signal transduction. The shear-stress-mediated deformation of the RBCs causes the release of ATP [126] and the ATP released by RBCs may induce NO signaling in the vasculature [6; 127] Sprague et al. [128] proposed that of cAMP signaling and the CFTR receptor may participate in deformation-induced release of ATP from RBCs, there by inducing flow-induced NO synthesis in particular in the pulmonary circulation. However, we could not reproduce the effects of cAMP-mediated ATP release from RBCs, at least under static conditions [109]. More studies focused on adenine nucleotide signaling in RBCs are needed to clarify how ATP release from RBCs is controlled. It is tempting to speculate that eNOS is part of mechanosensing microdomains in RBCs and may be activated by calcium influx and/or mechanical stimuli similar to EC; thus participating in physiological adaptation to shear stress within the circulation [129]. Interestingly there are

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several putative mechanosensors / mechanosening systems on the surface of RBCs, which are known to play a fundamental role in shear stress-induced activation of eNOS in endothelial cells (Figure 2). The glycocalyx forms a negatively charged “cushion” on the surface of both EC and RBCs preventing RBCs to adhere to the endothelial surface via electrostatic repulsive forces [130] [131]. The thickness of the glycocalyx of RBCs progresslively decrease with aging of the cells likely through mechanical damage in zone of turbulent flow or the microcirculation [131]. Whether the glycocalyx in RBCs may also participate to mechanosensing and transduction in RBCs (e.g. via activation of specific GPCR receptors) is unknown. Potentially mechanosensing GPCRs receptors including P2Y2 [132; 133] and β adrenergic receptors were also found on the surface of RBCs and were proposed to regulate cell signaling, but their role in mechanosensing were not investigated in detail. A furthermechanosensing candidate potentially leading to eNOS activation is the Ca2+ channel Piezo-1 [134], which was also shown to participate to shear stress-mediated ATP release [135]. Whether shear-stress induced calcium influx may also activate eNOS via the calcium/CaM-complex pathway is unknown. L-Arg the essential substrate for eNOS to generate NO is transported through the cationic amino acid transporter CAT-1 into the cell lumen [136; 137]. Shear stress leads to an increased transport velocity of L-Arg and in turn to an enhanced NO production in endothelial cells. Whether the same mechanisms are also found in RBCs is still not clear. There is some experimental evidence of shear stress-induced increase in intracellular NO levels, determined as nitrosation events by using DAF and fluorescence microscopy [138; 139; 140] or by showing phosphorylation of eNOS at Ser-1177 as a “marker” for eNOS activation [141; 142]. It is important to point out that as discussed by the authors of these papers, there are limitations to DAF usage in microscopy or fluorimetry (instead of quantitative analytical techniques like high pressure liquid chromatography) [143]. There are also limitations with using eNOS phosphorylation as an index of eNOS activity via immunohistochemistry due to the resistance of inhibiting phosphorylation sites [144], and background signal from nonspecific antibody binding in RBCs (discussed in [120]). Therefore, a role of eNOS in RBCs for mechanotransduction is still not fully established and needs to be investigated further with more specific and/or independent techniques. As pointed out at the beginning of this section, the very peculiar characteristics of the membrane of RBCs allow them to significantly change their shape in response to mechanical forces, such as shear stress or changes in flow velocity, and altering their maximum diameter to pass through the capillaries. These properties are mainly controlled by the interactions of the structural flexibility of the spectrin cytoskeleton [94]. Initially identified in a pioneering paper by the Baskurt group [145], NO, and specifically eNOS-derived NO, may participate in regulating RBC deformability as assessed by ekacytometry [142; 146]. However, in addition to two other independent laboratories, we were unable to reproduce the effects of NO donors on RBC deformability [147; 148; 149]. Moreover, we found that RBCs from global eNOS KO mice showed no difference in RBC deformability as compared to wildtype mice [149]; thus excluding a direct effect of eNOS-derived NO on RBC deformability. However, we observed that NO donors or nitrosothiols rescued RBCs from oxidant-induced loss of deformability, which supports an antioxidant role for NO in RBCs [149]. Thus, the source of NO is important for determining the effects on RBC deformability, and further studies on NO microdomains in RBCs may reveal how sub-compartmentalisation of NO contributes to this regulation.

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6. Summary and Perspective The biophysical characteristics and complex chemical biology of NO and its metabolites allow it to exert multiple pleiotropic physiological effects, as well as potential toxic effects. The action of NO is controlled at multiple levels including rates of production and breakdown, and the compartmentalization of signaling machinery in cellular and subcellular compartments. A particularly important compartmentalization system is signaling microdomains within the cellular membrane. While the cellular membrane was originally considered a homogenous “sea of lipids” with transmembrane proteins distributed randomly, it is now clear that membrane lipids and proteins are highly organised in unique dynamic structures or “lipid rafts”, of a specific lipid composition, which contain structural proteins, such as caveolinCav-1, and functional/signaling proteins (like receptors, channels, transporters) that are either connected to the cytoskeletal network or glycocalyx. These structures allow context-specific signaling of different potentially pleiotropic signals. The last characterized microdomains regulating NO signaling in the vasculature includes caveolin-coated caveolae where Cav-1 binds to eNOS and limits its activity in the vascular wall, and the MEJ where Hbα interacts with eNOS to fine tune NO bioavailability. Both of these microdomains, ultimately contribute to the regulation of vascular tone and blood pressure. Other putative regulators of eNOS activity, which may further contribute to localization of NO signaling, HSP90, and HSP70 and protein kinases (like PUK2)/phosphatases. Moreover, microdomains are hypothesized to be within RBCs to allow NO signaling in a cell lacking organelles and carrying 10 mM of NO-scavenging heme. Microdomain regulation has a clear potential for clinical translation as demonstrated by peptides targeting the binding side of Hbα on eNOS is effective in regulating blood pressure in vivo [84]. Ongoing research on NO signaling microdomains, the involvement of lipids and binding partners on enzymatic-mediated production and half-life will continue to reveal how local regulation contributes to its systemic effects and pathophysiology. The regulation of specific pathways by localization of signaling proteins in microdomains is likely to play an important role in cardiovascular diseases, where dysregulation of signaling pathway may be linked to dysfunctional microdomains.

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7. Acknowledgements This work was supported by the by the German Research Council (DFG) (CO 1305/2-1, SPP1710 CO 1305/3-1 to MCK and IRTG1902, Project P9 to MCK, Malte Kelm, BEI;), the Forschungskommission of the Medical faculty of the Heinrich-Heine University (to MCK). FL is a scholar of the IRTG1902. BH is a scholar of the SPP1710. BEI was supported by NIH HL088554 and CAR was supported by NHLBI 1F31HL149228-01.

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8. Figures

Figure 1: NO signaling microdomains in endothelium. NO signaling microdomains can be found either on the luminal side of the EC cell membrane, or between cells as junctional complexes and MEJs. Mechanical forces or receptor activating substrates lead to the activation of NO signaling at those signaling microdomains. As demonstrated in this figure, eNOS is activated downstream of extracellular signals ,channel activation, or shear stress, which all ultimately either increase intracellular Ca2+, alter eNOS phosphorylation, or provide more substrate . On the luminal surface of EC membranes, shear stress activated ion channels, the glycocalyx, GPCR and Cav-1 can lead to an influx of calcium, activating eNOS by the Ca2+/CAM-pathway. The direct association of Cav-1 to eNOS in contrast inhibits the enzyme activity. Heat shock proteins (HSP) are binding partners of eNOS, increasing the activity (HSP90) or downregulating activity (HSP70) via CHIP and trafficking eNOS into the Golgi complex. Integrins bind to collagen and laminin, activating eNOS via the PI3-aktkinase pathway. Moreover, influences the polymerization state of actin filaments the eNOS activity. Between EC are junctional complexes, regulating eNOS via PYK-2 or PI3-akt-kinase pathway. In resistance vessels, MEJ are a heterocellular communication microdomain between EC and SMC. Hemoglobin α, enriched in MEJ, forms a complex with eNOS and the reductase CytB5R3. The diffusion of eNOS generated NO is tightly dependent of the redox state of Hbα. When Hbα resides in the Fe3+ state, NO is able to diffuse to the smooth muscle cell layer; however, if it is reduced to Fe2+ by CytB5R3, NO will be scavenged by Hbα. CAT1 is sensitive to shear stress, increasing the transport velocity of L-Arginine, the substrate for eNOS. GPCR: G-protein coupled receptor, PKA: protein kinase a, PKB: protein kinase B, CaM:Calmodulin, PYK2: proline-rich tyrosine kinase 2, HSP: heat shock protein, CHIP: Cterminal Hsp70-interacting protein, CAT-1: cationic amino acid transporter, CytB5R3: cytochrome b 5 reductase 3, MEJ: MEJs, NO: nitric oxide.

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Figure 2

Figure 2: Putative mechanosensing microdomains in RBCs. In panel (I-IV) are depicted example of putative RBCs mechanosensors on RBCs. The glycocalyx, the GPCR receptors like P2Y2 and β1 adrenergic receptors, the Ca2+ channel piezo-1 and the arginine trasporter CAT-1 were all show to play a crucial role in endothelial mechanosensing and induce eNOSdependent downstream signalling in endothelial cells; all these were also demonstrated to be present on the surface of RBCs. Whether they participate to erythrocytic mechanosensing and activation of eNOS needs to be investigated. .

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Fig 3. Putative NO signaling microdomains in RBCs. RBCs are highly rich in hemoglobin (10 mM of heme), a tetrameric molecule composed of two alpha and two beta subunits, well recognized for their role in regulating gas exchange in RBCs. Under normoxic conditions NO reacts with oxyHb with a near-diffusion-limited reaction rate. It is speculated a nitrite reductase metabolon complex at the lipid bilayer of erythrocyte membrane facilitating the export of NO or its metabolites from RBCs. Such complex of anion-exchange proteins, CA, deoxyHb, aquaporin, and Rh channels may be localized specifically in RBC lipid rafts and it could serve for catalytic NO generation by the erythrocyte (picture adapted from Gladwin et al, Am J Physiol Heart Circ Physiol, 2006). Another hypothesised microdomain for NO synthesis and signaling in RBC may contain eNOS and related proteins allowing NOmediated downstream signaling in RBC. This microdomain could comprise cationic aminoacid transporters y+/CAT and/or y+L, which allow L-arginine entering into the cell via the. L-Arg may be used by arginase 1, which is highly abundant in RBC or alternatively by eNOS. The mechanisms regulating L-Arg flux to Arg1 or eNOS are unknown. After the reaction of NO with OxyHb, metHb may act as a shield protecting NO from further oxidation. If eNOS and sGC were colocalized eNOS-derived NO may be channelled to its target/receptor and be protected by degradation by oxyHb, promoting its signaling function. Evidence showing co-localisation of the proteins or a functional link between eNOS and sGC activites is still lacking.

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Graphical Abstract

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