Anion-modulated CoP electrode as bifunctional electrocatalyst for anion-exchange membrane hydrazine-assisted water electrolyser.

The hydrazine oxidation reaction (HzOR) is considered as a promising alternative process of the oxygen evolution reaction (OER) to realize more energy-efficient hydrogen generation. However, the lack of highly active bifunctional catalysts poses a huge challenge to this strategy. In this work, we report a novel and universal electrodeposition strategy to rationally synthesize a self-supporting electrode. The utilization of ammonium fluoride helps to modulate not only the morphology of CoP, but also the synchronous formation of an anion-modified structure, leading to an excellent bifunctional performance. The optimal F-CoP/CF exhibits small potentials of -90 mV and 41 mV at 1 A cm-2, high stability and low Tafel slopes of 28 mV dec-1 and 3.26 mV dec-1 for the HER and HzOR, respectively. The highly efficient and stable bifunctional activity of F-CoP/CF can be further confirmed in an anion-exchange membrane hydrazine-assisted water electrolyzer (0.49 V at 1 A cm-2). Utilizing the density functional theory calculations, the optimized adsorption energy of water molecules and hydrogen intermediates of the HER as well as the rate-determining step of the HzOR are demonstrated for the F-CoP.

Phosphorus-Modified Amorphous High-Entropy CoFeNiCrMn Compound as High-Performance Electrocatalyst for Hydrazine-Assisted Water Electrolysis.

Exploiting highly active and bifunctional catalysts for both hydrogen evolution reaction (HER) and hydrazine oxidation reaction (HzOR) is a prerequisite for the hydrogen acquisition. High-entropy materials have received widespread attention in catalysis, but the high-performance bifunctional electrodes are still lacking. Herein, a novel P-modified amorphous high-entropy CoFeNiCrMn compound is developed on nickel foam (NF) by one-step electrodeposition strategy. The achieved CoFeNiCrMnP/NF delivers remarkable HER and HzOR performance, where the overpotentials as low as 51 and 268 mV are realized at 100 mA cm-2 . The improved cell voltage of 91 mV is further demonstrated at 100 mA cm-2 by assessing CoFeNiCrMnP/NF in the constructed hydrazine-assisted water electrolyser, which is almost 1.54 V lower than the HER||OER system. Experimental results confirm the important role of each element in regulating the bifuncational performance of high-entropy catalysts. The main influencing elements seem to be Fe and Ni for HER, while the P-modification and Cr metal may contribute a lot for HzOR. These synergistic advantages help to lower the energy barriers and improve the reaction kinetics, resulting in the excellent bifunctional activity of the CoFeNiCrMnP/NF. The work offers a feasible strategy to develop self-supporting electrode with high-entropy materials for overall water splitting.

Coupled W-Co2P hybrid nanosheets as a robust bifunctional electrocatalyst for hydrazine-assisted hydrogen production.

Herein, we put forward a novel electrode by electrochemically integrating metal W species and Co2P nanosheets onto nickel foam (W@Co2P/NF), which delivers excellent bifunctional activity for both the HER and HzOR. The hydrazine assisted water electrolyzer provides a small cell potential of 0.18 V at 100 mA cm-2 and stability for hydrogen generation, which is superior to most of the other bifunctional materials.

Coupled MoO3-x@CoP heterostructure as a pH-universal electrode for hydrogen generation at a high current density.

Developing high-performance and low-cost self-supporting electrodes as pH-universal electrocatalysts for the hydrogen-evolution reaction (HER) and realizing high-quality hydrogen production at a high current density are highly desirable, but are hugely challenging. We created a self-supporting electrode with a coupled hierarchical heterostructure by simple electrodeposition followed by sulfurization. It comprised oxygen-deficient molybdenum oxide (MoO3-x) and cobalt phosphide (CoP) on nickel foam (NF), which represented a highly active pH-universal electrocatalyst for the HER at a high current density. Benefiting from a plethora of catalytic active sites, improved interfacial charge transfer, and strong electronic interaction, this type of MoO3-x@CoP/NF electrode delivered a superior catalytic performance. Overpotentials of only 100 mV, 135 mV, and 400 mV were needed to realize a high current density of 1 A cm-2 in alkaline, acid and neutral media, respectively, which were superior to those of most other well-developed materials based on non-noble metals. Our experimental work demonstrates the synergistic advantages of a MoO3-x@CoP heterostructure for improving the intrinsic catalytic performance but also paves a new path for the rational design of advanced electrodes for hydrogen generation in a wide range of pH conditions.

Engineering Coupled NiSx -WO2.9 Heterostructure as pH-Universal Electrocatalyst for Hydrogen Evolution Reaction.

Exploiting highly active and low-cost materials as pH-universal electrocatalysts for the hydrogen evolution reaction (HER) and achieving high-purity hydrogen fuel is highly desirable but remains challenging. Herein, a novel type of coupled heterostructure was designed by simple electrodeposition followed by a sulfurization treatment. This hierarchical structure was composed of nickel sulfides (NiS, NiS2 , denoted as NiSx ) and oxygen-deficient tungsten oxide (WO2.9 ), which was directly grown on nickel foam (NF) as self-supporting electrodes (NiSx -WO2.9 /NF) for HER over a wide pH range. The systematic experimental characterizations confirmed that the material had abundant catalytic active sites, fast interfacial electron transfer ability, and strong electronic interaction, resulting in the optimized reaction kinetics for HER. Consequently, the NiSx -WO2.9 /NF catalyst required low overpotentials of 96 and 117 mV to reach current densities of 50 and 100 mA cm-2 in an alkaline medium, outperforming most of the reported non-noble metal-based materials. Moreover, this self-supported electrode exhibited impressive performance over a wide pH range, only requiring 220 and 304 mV overpotential at 100 mA cm-2 in 0.5 m H2 SO4 and 1 m phosphate-buffered saline electrolytes. This work may offer a new approach to the development of advanced pH-universal electrodes for hydrogen production.

Hierarchically structured nickel/molybdenum nitride heterojunctions as superior bifunctional electrodes for overall water splitting.

Transition metal nitrides (TMNs) are considered to be some of the most promising metallic materials for electrocatalytic water splitting. However, the low density of active sites and weak reaction kinetics still limit their wide industrial application. Herein, we put forward a typical 3D hierarchical heterostructure that is composed of metallic Ni3N, Mo5N6, and Ni grown on nickel foam (denoted as Ni3N@NiMoNx/NF), presenting it as a highly-active bifunctional electrocatalyst for water splitting. This hybrid nanowire heterojunction has an abundant interface structure for more catalytically active sites, while its synergistic effects of strong electronic interaction and intrinsic high conductivity ensure fast electron transfer for rapid reaction kinetics. Remarkably, the Ni3N@NiMoNx/NF electrode delivers small overpotentials of 78 mV and 370 mV at 100 mA cm-2 for the HER and OER, respectively. By utilizing Ni3N@NiMoNx/NF as bifunctional electrodes for water splitting, an alkaline electrolyzer shows a low cell voltage of 1.68 V at 100 mA cm-2 with a superior durability of 80 h. Our work provides an experimental basis for advancing the rational design of efficient and stable bifunctional electrocatalysts for large-scale industrial water electrolysis applications.

Ni2 Se3 -CuSex Heterostructure as a Highly Efficient Bifunctional Electrocatalyst for Urea-Assisted Hydrogen Generation.

Coupling urea oxidation reaction (UOR) with hydrogen evolution reaction (HER) is an attractive alternative anode reaction for electrochemical hydrogen generation with low energy consumption. However, the development of highly efficient bifunctional electrocatalysts is still a challenge. In this work, Ni2 Se3 -CuSex heterostructure was synthesized on copper foam (Ni3 Se2 @CuSex /CF) by electrodeposition accompanied by a selenization process. Benefiting from the abundant active sites, faster reaction kinetics, and modulated electronic structure, the self-supporting Ni3 Se2 @CuSex /CF electrode exhibited superior catalytic performance. Extremely low overpotentials of 120 and 140 mV were achieved at the current density of 100 mA cm-2 for HER/UOR, respectively. Respectively, in HER||UOR coupled electrolyzer for H2 generation, the Ni3 Se2 @CuSex /CF||Ni3 Se2 @CuSex /CF delivered a low cell voltage of 1.49 V to reach a high current density of 100 mA cm-2 along with good stability, outperforming most of the other well-developed materials to date. The rational design of coupled heterostructure as bifunctional electrodes is a promising approach for energy-saving H2 production.

Heterostructure engineering of iridium species on nickel/molybdenum nitride for highly-efficient anion exchange membrane water electrolyzer.

Developing highly active electrocatalysts is a pivotal issue for anion-exchange membrane water electrolyzers (AEMWE). However, realizing the continuous hydrogen generation at a large current density remains challenging. Herein, a novel kind of hybrid electrode is successfully developed by introducing trace iridium (Ir) species onto a hierarchical Ni/Mo5N6 heterostructure on Ni foam (Ir-Ni/Mo5N6/NF). The synergistic advantages of high conductivity, abundant active sites, and strong electronic interaction endow superior reaction kinetics, presenting a highly-active bifunctional electrocatalyst. Remarkably, the Ir-Ni/Mo5N6/NF exhibit extremely low overpotentials of 52 mV and 250 mV at 100 mA cm-2 for hydrogen evolution reaction (HER) and oxygen evolution reaction (OER). By exploiting the Ir-Ni/Mo5N6 as both anode/cathode, the constructed AEMWE device delivers superior performance. The current density reaches 2.1 A cm-2 at a voltage of 2.0 V and 250 mA cm-2 at 1.8 V in alkaline/neutral media. This work put forward a facile and effective strategy to synthesize advanced bifunctional electrocatalysts for water electrolysis.

Fluorine-anion engineering endows superior bifunctional activity of nickel sulfide/phosphide heterostructure for overall water splitting.

Designing advanced transition metal-based materials for electrocatalytic water splitting is of significance, but their wide application is still limited due to the lack of an effective regulation strategy. Herein, a synergistic regulation strategy of surface/interface is developed to optimize the catalytic activity of nickel sulfide (Ni3S2). The construction of nickel phosphide with Ni3S2 heterostructure by using fluorine (F)-anion modification is successfully developed on nickel foam (F-NiPx/Ni3S2-NF) via a simple fluorination and phosphating treatment. This new kind of electrocatalyst contains plenty of active sites and strong electronic interactions, presenting superior bifunctional activity for both hydrogen evolution reaction (HER) and oxygen evolution reaction (OER). The overpotentials only need 182 mV and 370 mV to reach the current density of 100 mA cm-2 for HER and OER, respectively. In addition, the F-NiPx/Ni3S2-NF-based electrolyzer delivers promising performance for overall water splitting. A low potential of 1.55 V and 1.7 V can be achieved at the current density of 10 mA cm-2 and 50 mA cm-2. This work provides a new surface/interface regulation strategy for high-efficient bifunctional electrocatalysts.

Electronic regulation of platinum species on metal nitrides realizes superior mass activity for hydrogen production.

Developing high-active electrocatalyst to improve the efficiency of hydrogen evolution reaction (HER) is critical to achieve clean hydrogen. However, the low mass activity and high cost of this technology still limits its wide commercial application. Herein, a new kind of hybrid material is designed by introducing trace Pt species onto a mixed metal nitride matrixs (denoted as NiWNx), presenting as an excellent electrocatalyst for HER. The prepared Pt-NiWNx hybrid possesses abundant heterointerfaces, high conductivity and strong electron interactions, facilitating the reaction kinetics for hydrogen production. As a result, the Pt-NiWNx only needs a small overpotential of 61 mV to reach the geometric current density of 100 mA cm-2 in alkaline electrolyte. Notably, this kind of catalyst delivers a superior mass activity of 32.8 A mgPt-1 at -0.1 V and high durability, exhibiting the promising prospects for industrial application. This work offers a novel design strategy for high-efficient hybrid materials for scaled hydrogen generation.

Hierarchical Metal Sulfides Heterostructure as Superior Bifunctional Electrode for Overall Water Splitting.

The development of highly active bifunctional electrocatalysts for overall water splitting is of significant importance, but huge challenges remain. The key element depends on engineering the electronic structure and surface properties of material to achieve improved catalytic activity. Herein, a hierarchical nanowire array of metal sulfides heterostructure on nickel foam (FeCoNiSx /NF) was designed as a novel type of hybrid electrocatalyst for overall water splitting. The hybrid structure endowed plenty of catalytic active sites, strong electronic interactions, and high interfacial charge transferability, leading to superior bifunctional performance. As a result, the FeCoNiSx /NF catalyst delivered low overpotentials of 97 and 260 mV at the current density of 50 mA cm-2 for hydrogen and oxygen evolution reactions, respectively. Moreover, the FeCoNiSx /NF-based water electrolyzer exhibited a small potential of 1.57 V for a high current density of 50 mA cm-2 . These results indicate the promising application potential of FeCoNiSx /NF electrode for hydrogen generation. This work provides a new approach to develop robust hybrid materials as the highly active electrode for electrocatalytic water splitting.

Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors.

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.

Ethanol is a fast channel inhibitor of P2X4 receptors.

P2X receptors (P2XRs) are ion channels gated by synaptically released ATP. The P2X4 is the most abundant P2XR subtype expressed in the central nervous system and to date is the most ethanol-sensitive. In addition, genomic findings suggest that P2X4Rs may play a role in alcohol intake/preference. However, little is known regarding how ethanol causes the inhibition of ATP-gated currents in P2X4Rs. We begin to address this issue by investigating the effects of ethanol in wild-type and mutant D331A and M336A P2X4Rs expressed in human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp methods. The results suggest that residues D331 and M336 play a role in P2X4R gating and ethanol inhibits channel functioning via a mechanism different from that in other P2XRs. Key findings from the study include: 1) ethanol inhibits ATP-gated currents in a rapid manner; 2) ethanol inhibition of ATP-gated currents does not depend on voltage and ATP concentration; 3) residues 331 and 336 slow P2X4 current deactivation and regulate the inhibitory effects of ethanol; and 4) ethanol effects are similar in HEK293 cells transfected with P2X4Rs and cultured rat hippocampal neurons transduced with P2X4Rs using a recombinant lentiviral system. Overall, these findings provide key information regarding the mechanism of ethanol action on ATP-gated currents in P2X4Rs and provide new insights into the biophysical properties of P2X4Rs.

A point mutation in the ectodomain-transmembrane 2 interface eliminates the inhibitory effects of ethanol in P2X4 receptors.

ATP-gated P2X4 receptors (P2X4R) are abundantly expressed in the CNS. However, little is known about the molecular targets for ethanol action in P2X4Rs. The current investigation tested the hypothesis that the ectodomain-transmembrane (TM) interface contains residues that are important for the action of ethanol in P2X4Rs. Wild type (WT) and mutant P2X4R were expressed in Xenopus oocytes. ATP concentration-response curves and ethanol (10-200 mM)-induced changes in ATP EC(10)-gated currents were determined using two-electrode voltage clamp (-70 mV). Alanine substitution at the ectodomain-TM1 interface (positions 50-61) resulted in minimal changes in ethanol response. On the other hand, alanine substitution at the ectodomain-TM2 interface (positions 321-337) identified two key residues (D331 and M336) that significantly reduced ethanol inhibition of ATP-gated currents without causing marked changes in ATP I(max), EC(50), or Hill's slope. Other amino acid substitutions at positions 331 and 336 significantly altered or eliminated the modulatory effects of ethanol. Linear regression analyses revealed a significant relationship between hydropathy and polarity, but not molecular volume/molecular weight of the residues at these two positions. The results support the proposed hypothesis and represent an important step toward developing ethanol-insensitive receptors for investigating the role of P2X4Rs in mediating behavioral effects of ethanol.

Purinergic type 2 receptors at GABAergic synapses on ventral tegmental area dopamine neurons are targets for ethanol action.

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1-200 microM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, alpha,beta-methylene ATP, a selective P2X(1) and P2X(3) receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[beta-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X(1) and/or P2X(3) receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.

Ethanol inhibits functional activity of the human intestinal dipeptide transporter hPepT1 expressed in Xenopus oocytes.

BACKGROUND: The pathological effects of high alcohol (ethanol) consumption on gastrointestinal and hepatic systems are well recognized. However, the effects of ethanol intake on gastric and intestinal absorption and transport systems remain unclear. The present study investigates the effects of ethanol on the human peptide transporter 1 (hPepT1) which mediates the transport of di-and tripeptides as well as several orally administered peptidomimetic drugs such as beta-lactam antibiotics (e.g., penicillin), angiotensin-converting enzyme inhibitors, the anti-neoplastic agent bestatin, and prodrugs of acyclovir. METHODS: Xenopus oocytes were injected with hPepT1 cRNA and incubated for 3 to 10 days. Currents induced by glycyl-sarcosine (Gly-Sar), Ala-Ala (dipeptides), penicillin and enalapril measured in the presence or absence of ethanol were determined using an 8-channel 2-electrode voltage clamp system, with a membrane potential of -70 mV and 11 voltage steps of 100 milliseconds (from +50 mV to -150 mV in -20 mV increments). RESULTS: Ethanol (200 mM) inhibited Gly-Sar and Ala-Ala currents by 42 and 30%, respectively, with IC(50)s of 184 and 371 mM, respectively. Ethanol reduced maximal transport capacity (I(max)) of hPepT1 for Gly-Sar without affecting Gly-Sar binding affinity (K(0.5) and Hill coefficient). Penicillin- and enalapril-induced currents were significantly less than those induced by dipeptides and were not inhibited by ethanol. CONCLUSION: Ethanol significantly reduced transport of dipeptides via a reduction in transport capacity, rather than competing for binding sites in hPepT1. Ethanol inhibition or alteration of transport function may be a primary causative factor contributing to both the nutritional deficits as well as the immunological deficiencies that many alcoholics experience including alcohol liver disease and brain damage.

Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of alpha1 glycine receptors.

Considerable evidence indicates that ethanol acts on specific residues in the transmembrane domains of glycine receptors (GlyRs). In this study, we tested the hypothesis that the extracellular domain is also a target for ethanol action by investigating the effect of cysteine substitutions at positions 52 (extracellular domain) and 267 (transmembrane domain) on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs expressed in Xenopus oocytes. In support of the hypothesis: (i) The A52C mutation changed ethanol sensitivity compared to WT GlyRs; (ii) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs; and (iii) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one site in order to determine its effect at other site(s). In these situations, ethanol caused negative modulation when acting at position 52 and positive modulation when acting at position 267. Collectively, these findings parallel the evidence that established the TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the same alcohol pocket and indicate that the net effect of ethanol on GlyR function reflects the summation of its positive and negative modulatory effects on different targets.

Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes.

While Ca2+ influx is essential for activation of the cell cycle machinery, the processes that regulate Ca2+ influx in this context have not been fully elucidated. Electrophysiological and molecular studies have identified multiple Ca2+ channel genes expressed in mammalian cells. Ca(v)3.x gene family members, encoding low voltage-activated (LVA) or T-type channels, were first identified in the central nervous system and subsequently in non-neuronal tissue. Reports of a potential role for T-type Ca2+ channels in controlling cell proliferation conflict. The present study tested the hypothesis that T-type Ca2+ channels, encoded by Ca(v)3.x genes, control pulmonary artery smooth muscle cell proliferation and cell cycle progression. Using quantitative RT/PCR, immunocytochemistry, and immunohistochemistry we found that Ca(v)3.1 was the predominant Ca(v)3.x channel expressed in early passage human pulmonary artery smooth muscle cells in vitro and in the media of human pulmonary arteries, in vivo. Selective blockade of Ca(v)3.1 expression with small interfering RNA (siRNA) and pharmacological blockade of T-type channels completely inhibited proliferation in response to 5% serum and prevented cell cycle entry. These studies establish that T-type voltage-operated Ca2+ channels are required for cell cycle progression and proliferation of human PA SMC.

Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels.

Cytosolic reducing cofactors, such as NADPH and NADH, are thought to regulate vascular smooth muscle ion channel activity and vascular tone. In this study, the effects of pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN), epiandrosterone (EPI), and dehydroepiandrosterone (DHEA), on vascular tone were studied in isolated perfused lungs and pulmonary artery (PA) and aortic rings from rats. In addition, effects of 6-AN on voltage-gated K(+) (K(v)) current in PA smooth muscle cells (SMCs) were also examined. Pretreatment of lungs with 6-AN and EPI reduced the pressor response to acute hypoxia and decreased tissue NADPH levels. 6-AN, EPI, and DHEA relaxed isolated PA and aortic rings precontracted with 30 mM KCl in a dose-dependent manner. The PPP inhibitor-induced PA relaxations were reduced in PA rings precontracted with 80 mM KCl but not by pretreatment with nitro-L-arginine or endothelial removal. Pretreatment of PA rings with tetraethylammonium chloride or 4-aminopyridine caused rightward shifts of concentration-relaxation curves for 6-AN, EPI, and DHEA. In contrast, glybenclamide, charybdotoxin, or apamin did not inhibit the relaxant effects of 6-AN, EPI, and DHEA. 6-AN caused an increase in K(v) current in PASMC. These results indicate that reduction of NADPH by the PPP inhibitors causes vasodilation at least partly through opening of K(v) channels.

Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme.

The subcellular localization of endothelial nitric-oxide synthase (eNOS) is critical for optimal coupling of extracellular stimulation to nitric oxide production. Because eNOS is activated by Akt-dependent phosphorylation to produce nitric oxide (NO), we determined the subcellular distribution of eNOS phosphorylated on serine 1179 using a variety of methodologies. Based on sucrose gradient fractionation, phosphorylated-eNOS (P-eNOS) was found in both caveolin-1-enriched membranes and intracellular domains. Co-transfection of eNOS with Akt and stimulation of endothelial cells with vascular endothelial growth factor (VEGF) increased the ratio of P-eNOS to total eNOS but did not change the relative intracellular distribution between these domains. The proper localization of eNOS to intracellular membranes was required for agonist-dependent phosphorylation on serine 1179, since VEGF did not increase eNOS phosphorylation in cells transfected with a non-acylated, mistargeted form of eNOS. Confocal imaging of P-eNOS and total eNOS pools demonstrated co-localization in the Golgi region and plasmalemma of transfected cells and native endothelial cells. Finally, VEGF stimulated a large increase in NO localized in both the perinuclear region and the plasma membrane of endothelial cells. Thus, activated, phosphorylated eNOS resides in two cellular compartments and both pools are VEGF-regulated to produce NO.