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The roles of endothelial nitric oxide synthase (eNOS) in the ventilatory responses during and after a hypercapnic gas challenge (HCC, 5% CO2, 21% O2, 74% N2) were assessed in freely-moving female and male wild-type (WT) C57BL6 mice and eNOS knock-out (eNOS-/-) mice of C57BL6 background using whole body plethysmography. HCC elicited an array of ventilatory responses that were similar in male and female WT mice, such as increases in breathing frequency (with falls in inspiratory and expiratory times), and increases in tidal volume, minute ventilation, peak inspiratory and expiratory flows, and inspiratory and expiratory drives. eNOS-/- male mice had smaller increases in minute ventilation, peak inspiratory flow and inspiratory drive, and smaller decreases in inspiratory time than WT males. Ventilatory responses in female eNOS-/- mice were similar to those in female WT mice. The ventilatory excitatory phase upon return to room-air was similar in both male and female WT mice. However, the post-HCC increases in frequency of breathing (with decreases in inspiratory times), and increases in tidal volume, minute ventilation, inspiratory drive (i.e., tidal volume/inspiratory time) and expiratory drive (i.e., tidal volume/expiratory time), and peak inspiratory and expiratory flows in male eNOS-/- mice were smaller than in male WT mice. In contrast, the post-HCC responses in female eNOS-/- mice were equal to those of the female WT mice. These findings provide the first evidence that the loss of eNOS affects the ventilatory responses during and after HCC in male C57BL6 mice, whereas female C57BL6 mice can compensate for the loss of eNOS, at least in respect to triggering ventilatory responses to HCC.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10–HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Decreases in arterial blood oxygen stimulate increases in minute ventilation via activation of peripheral and central respiratory structures. This study evaluates the role of endothelial nitric oxide synthase (eNOS) in the expression of the ventilatory responses during and following a hypoxic gas challenge (HXC, 10% O2, 90% N2) in freely moving male and female wild-type (WT) C57BL6 and eNOS knock-out (eNOS–/–) mice. Exposure to HXC caused an array of responses (of similar magnitude and duration) in both male and female WT mice such as, rapid increases in frequency of breathing, tidal volume, minute ventilation and peak inspiratory and expiratory flows, that were subject to pronounced roll-off. The responses to HXC in male eNOS–/– mice were similar to male WT mice. In contrast, several of the ventilatory responses in female eNOS–/– mice (e.g., frequency of breathing, and expiratory drive) were greater compared to female WT mice. Upon return to room-air, male and female WT mice showed similar excitatory ventilatory responses (i.e., short-term potentiation phase). These responses were markedly reduced in male eNOS–/– mice, whereas female eNOS–/– mice displayed robust post-HXC responses that were similar to those in female WT mice. Our data demonstrates that eNOS plays important roles in (1) ventilatory responses to HXC in female compared to male C57BL6 mice; and (2) expression of post-HXC responses in male, but not female C57BL6 mice. These data support existing evidence that sex, and the functional roles of specific proteins (e.g., eNOS) have profound influences on ventilatory processes, including the responses to HXC.

As precursor cells of odontoblasts, dental papilla cells (DPCs) form the dentin-pulp complex during tooth development. Nitric oxide (NO) regulates the functions of multiple cells and organ tissues, including stem cell differentiation and bone formation. In this paper, we explored the involvement of NO in odontoblastic differentiation. We verified the expression of NO synthase (NOS) in rat odontoblasts by nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining and immunohistochemistry in vivo. The expression of all three NOS isoforms in rat DPCs was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting in vitro. The expression of neuronal NOS and endothelial NOS were upregulated during the odontoblastic differentiation of DPCs. Inhibition of NOS function by NOS inhibitor, L-NG -monomethyl arginine (L-NMMA), resulted in reduced formation of mineralized nodules and expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP1) during DPC differentiation. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.1, 1, 10, and 100 μM) promoted the viability of DPCs. Extracellular matrix mineralization and odontogenic markers expression were elevated by SNAP at low concentrations (0.1, 1, and 10 μM) and suppressed at high concentration (100 μM). Blocking the generation of cyclic guanosine monophosphate (cGMP) with 1H-(1,2,4)oxadiazolo-(4,3-a)quinoxalin-1-one (ODQ) abolished the positive influence of SNAP on the odontoblastic differentiation of DPCs. These findings demonstrate that NO regulates the odontoblastic differentiation of DPCs, and thereby influencing dentin formation and tooth development.

A characteristic aspect of the robust, systemic inflammatory state in sickle cell disease is dysfunction of endothelial nitric oxide synthase (eNOS). We identify ten aberrant endothelial cell inputs, present in the specific sickle context, that are known to have the ability to cause eNOS dysfunction. These are: endothelial arginase depletion, asymmetric dimethylarginine, complement activation, endothelial glycocalyx degradation, free fatty acids, inflammatory mediators, microparticles, oxidized low density lipoproteins, reactive oxygen species, and Toll-like receptor 4 signaling ligands. The effect of true eNOS dysfunction on clinical testing using flow-mediated dilation can be simulated by two known examples of endothelial dysfunction mimicry (hemoglobin consumption of NO; and oxidation of smooth muscle cell soluble guanylate cyclase). This lends ambiguity to interpretation of such clinical testing. The presence of these multiple perturbing factors argues that a therapeutic approach targeting only a single injurious endothelial input (or either example of mimicry) would not be sufficiently efficacious. This would seem to argue for identifying therapeutics that directly protect eNOS function or application of multiple therapeutic approaches. This article is protected by copyright. All rights reserved.

Introduction. Nitric oxide (NO) produced by NO synthases (NOS) is involved in the regulation of vital physiological functions. At the same time, NO and NOS are involved in events associated with the tumor process: mutagenesis, proliferation, apoptosis, angiogenesis, etc., exerting a multidirectional effect on the tumor.Objectives – analyze and summarize literature data concerning the role of NO and endothelial NOS (eNOS) in the initiation and progression of tumors, as well as in the inhibition of tumor growth.Materials and methods. In preparing the review, publications of information bases of biomedical literature were used: SciVerse Scopus (538), PubMed (1327), Web of Science (905), Russian Science Citation Index (125).Results. The molecular mechanisms of the action of NO and its derivatives on the initiation and progression of carcinogenesis have been explored. Numerous factors and conditions regulating the activity of eNOS in health and tumor growth have been analyzed. The molecular signaling pathways through which the pro-tumor effects of NO and eNOS, stimulating angiogenesis, lymphangiogenesis, are realized, including through the mobilization of stem cells, are considered.Conclusion. Nitric oxide produced by activated eNOS promotes tumor progression by increasing the proliferation of tumor cells, enhancing the action of pro-angiogenic factors, stimulating angiogenesis, lymphangiogenesis, and metastasis. Selective inhibition of increased eNOS activity may be a promising therapeutic approach aimed at reducing metastasis and tumor growth.

BACKGROUND: Coronary heart disease (CHD) is the leading cause of death and start with injury to the endothelium of a coronary artery. The common feature of endothelial dysfunction is a decrease of nitric oxide (NO) bioavailability that regulated by endothelial NO synthase (eNOS) activity. AIM: The aim of our study was to study the relationship between risk factors of CHD patients with the level of eNOS. METHODS: Thirty-seven outpatients in cardiology department of the regional public hospital diagnosed as CHD were included in our study. Thirty healthy individuals were included as the control group. Risk factors of CHD were identified according to anamnesis and laboratory finding. eNOS was measured by ELISA methods. RESULTS: Endothelial NOS levels were significantly higher in the CHD when compared to the controls (p < 0.05). The most dominant risk factor for CHD is overweight, and followed by dyslipidemia, smoking, hypertension, history of CHD, and diabetes mellitus. eNOS in CHD patients who had one risk factor was 37.598 ± 0.1541 ng/ml, two risk factors 42.154 ± 22.329 ng/ml, three risk factors 25.329 ± 6.083 ng/ml, four risk factors 22.483 ± 4.022 ng/ml, and five risk factors 15.994 ± 4.774 ng/ml. There were significant differences in the average eNOS levels based on the number of risk factors (p < 0.05), and a tendency that more risk factors in CHD patients, the lower the average level of eNOS. CONCLUSION: In our study, eNOS levels showed highly significant relation with CHD and related to the number of risk factors those the CHD patients had.

Neuronal nitric oxide synthase (nNOS) is one of the three isoforms of nitric oxide synthase (NOS). The other two isoforms include inducible NOS (iNOS) and endothelial NOS (eNOS). These three isoforms of NOS are widely present in both human and other mammals and are responsible for the biosynthesis of NO. As an essential biological molecule, NO plays an essential role in neurotransmission, immune response, and vasodilation; however, the overproduction of NO can cause a series of diseases. Thus, the selective inhibition of three isoforms of NOS has been considered to be important in treating related diseases. The active sites of the three enzymes are highly conserved, causing the selective inhibition of the three enzymes to be a great challenge. (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1) has been experimentally proved to be a selective and time-dependent irreversible inhibitor of nNOS, and three pathways, including sulfide oxidation, oxidative dethiolation, and oxidative demethylation, have been suggested. In this work, we performed quantum mechanics/molecular mechanics calculations to verify the chemical conversion of inactivator 1. Although we agree with the previously suggested chemical transformation process, our calculations demonstrated that there are lower energy pathways to accomplish both oxidative dethiolation and oxidative demethylation. These three branching reactions are competitive, but only dethiolation and demethylation reactions can generate inhibitory intermediates. As a powerful time-dependent irreversible inhibitor of nNOS, the key sulfur atom and middle imine are all necessary. Our calculation results not only verified the chemical reaction of inhibitor 1 occurring in the enzymatic active site but also explained the inactivation mechanism of inhibitor 1. This is also the first verified example of the heme-enzyme-catalyzed S-demethylation mechanism.

Excessive salt intake induces hypertension, but several gamma-aminobutyric acid (GABA) supplements have been shown to reduce blood pressure. GABA-salt, a fermented salt by L. brevis BJ20 containing GABA was prepared through the post-fermentation with refined salt and the fermented GABA extract. We evaluated the effect of GABA-salt on hypertension in a high salt, high cholesterol diet induced mouse model. We analyzed type 1 macrophage (M1) polarization, the expression of M1 related cytokines, GABA receptor expression, endothelial cell (EC) dysfunction, vascular smooth muscle cell (VSMC) proliferation, and medial thicknesses in mice model. GABA-salt attenuated diet-induced blood pressure increases, M1 polarization, and TNF-α and inducible nitric oxide synthase (NOS) levels in mouse aortas, and in salt treated macrophages in vitro. Furthermore, GABA-salt induced higher GABAB receptor and endothelial NOS (eNOS) and eNOS phosphorylation levels than those observed in salt treated ECs. In addition, GABA-salt attenuated EC dysfunction by decreasing the levels of adhesion molecules (E-selectin, Intercellular Adhesion Molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) and of von Willebrand Factor and reduced EC death. GABA-salt also reduced diet-induced reductions in the levels of eNOS, phosphorylated eNOS, VSMC proliferation and medial thickening in mouse aortic tissues, and attenuated Endothelin-1 levels in salt treated VSMCs. In summary, GABA-salt reduced high salt, high cholesterol diet induced hypertension in our mouse model by reducing M1 polarization, EC dysfunction, and VSMC proliferation.

DA-9801, a plant-based drug used for the treatment of diabetic neuropathy, is known to improve angiotensin II (Ang II)-induced vascular endothelial cell dysfunction. However, the underlying mechanism is not fully understood. We aimed to determine whether the protective effect of DA-9801 against Ang II-induced endothelial cell dysfunction was mediated via inhibition of endothelial cell inflammation and apoptosis. Ang II-induced oxidative stress was attenuated by pretreatment of human dermal microvascular endothelial cells (HDMECs) with DA-9801. This prevented the Ang II-induced upregulation of NAD(P)H oxidase (the NOX4 and p22phox subunits) and reactive oxygen species. Further, pretreatment of HDMECs with DA-9801 ameliorated Ang II-mediated nuclear factor kappa B activity via prevention of the upregulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. It also decreased the Ang II-stimulated increase in inducible nitric oxide synthase (NOS) and decreased endothelial NOS protein expression. DA-9801 decreased Ang II-induced upregulation of intercellular adhesion molecule 1, vascular adhesion molecule, and E-selectin in HDMECs. Moreover, TUNEL and annexin V-FITC fluorescence staining for apoptosis and the activities of caspases 9, 7, and 3 decreased in HDMECs pretreated with DA-9801, indicating that the drug enhanced anti-apoptotic pathways. Thus, DA-9801 modulated Ang II-induced endothelial cell dysfunction via inflammatory and apoptotic pathways.

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS–/–) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS–/– mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS–/– mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.

Endothelial dysfunction greatly contributes to microcirculation disorder. The role of prostaglandin E1 (PGE1) in cerebral microcirculation was explored in vitro. LPS (0.5 or 1 μg/ml) was added to induce injury in human brain microvascular endothelial cells (HCMEC/D3). CCK-8 was applied to check viabilities of HCMEC/D3 before and after LPS treatment. Western blot witnessed the changes in protein expressions of inflammatory cytokines, IL-6 and TNF-α. Caspase-3/7 activity was analyzed and so were the protein expressions of pro-apoptotic gene BAX and anti-apoptotic gene Bcl-2. mRNA expressions of eNOS and GTPCH1 were evaluated by RT-qPCR. After overexpressing eNOS or GTPCH1 in LPS-induced HCMEC/D3 cells, viabilities, inflammatory cytokines, caspase-3/7 activity, and apoptosis-related genes were detected. The modulation of PGE1 in eNOS and GTPCH1 production, viability, inflammation, and apoptosis was investigated. The inhibitor of eNOS or GTPCH1 was introduced to examine impacts of eNOS or GTPCH1 could have on the PGE1 function. LPS decreased cell viabilities, eNOS and GTPCH1 expression, and promoted inflammation and apoptosis in HCMEC/D3 cells. Overexpressed eNOS or GTPCH1 promoted cell viabilities and suppressed inflammation and apoptosis. PGE1 enhanced viability and decreased inflammation and apoptosis in cells treated by LPS. PGE1 activated eNOS and GTPCH1 and inhibition of eNOS or GTPCH1 led to the attenuation of the protective functions of PGE1 in LPS-induced cells. PGE1 protected HCMEC/D3 cells from injuries induced by LPS by activation of eNOS and GTPCH1, suggesting that PGE1 might be used to help maintain cerebral microcirculation in future.

Background and purpose The aim of this study is to investigate the vasorelaxant effect of exendin-4, a GLP-1 receptor agonist on retinal capillaries under normal and ischaemia-reperfusion (I/R) conditions. Experimental approach Capillary diameters in the whole-mounted retina were directly observed using infrared differential interference contrast microscopy. A model of retinal I/R was established inraats,using high perfusion pressure in an anterior chamber. To assess the effects of exendin-4, it was administered through subcutaneous injection, intravitreal injection, or eye drops. The underlying mechanism was explored by immunofluorescence, qPCR, and capillary western blots. Key results Immunofluorescence staining showed that GLP-1 receptors were expressed in endothelial cells of retinal capillaries. Exendin-4 relaxed the capillaries precontracted by noradrenaline, an effect abolished by denuding endothelium with CHAPS and inhibited by GLP-1 receptor antagonist exendin-9-39, endothelial NOS (eNOS) inhibitor l-NAME, and the guanylate cyclase blocker ODQ but not by a COX inhibitor, indomethacin. Retinal capillaries were constricted in I/R injury, an effect reversed by perfusion of exendin-4. Expression of PI3K and Akt, phosphorylation level of eNOS and NO production after I/R were lower than that in the normal control group. Administration of exendin-4 improved the changes. Conclusion and implications Exendin-4 can restore injured microvascular patency in I/R. Exendin-4 may regulate retinal capillaries through the GLP-1 receptor-PI3K/Akt-eNOS/NO-cGMP pathway. Therefore, exendin-4 may be an effective treatment for improving tissue perfusion in I/R-related conditions.

The expression levels of microRNA (miR)‑340‑5p are reportedly decreased in the peripheral blood during acute ischemic stroke; however, the direct effect and mechanism of action of miR‑340‑5p in ischemic stroke remains largely unknown. The present study aimed to investigate the effects of miR‑340‑5p, and its mechanism of action, on PC12 cells following oxygen‑glucose deprivation/reperfusion (OGD/R) induction. OGD/R‑induced PC12 cells served as the cellular model and subsequently, mRNA expression levels of miR‑340‑5p and neuronal differentiation 4 (Neurod4) were analyzed using reverse transcription‑quantitative PCR. Tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6 expression levels were detected using ELISA kits, and flow cytometry was used to determine the rate of cellular apoptosis. In addition, a nitric oxide (NO) synthase activity assay kit was used to detect NO levels and a NADPH assay kit was used to measure NADPH levels. Western blotting was also performed to analyze protein expression levels of bax, bcl‑2, cleaved caspase 3 and phosphorylated endothelial NOS (eNOS), and the target gene of miR‑340‑5p was predicted using TargetScan software and verified using a dual‑luciferase reporter assay. The expression levels of miR‑340‑5p were decreased in PC12 cells following OGD/R induction and Neurod4 was identified as a target gene of miR‑340‑5p. In addition, miR‑340‑5p overexpression reduced inflammation, apoptotic rate, NO production and NADPH levels, in addition to increasing eNOS expression in PC12 cells following OGD/R induction. Notably, the overexpression of Neurod4 reversed the aforementioned effects of miR‑340‑5p on PC12 cells following OGD/R induction. In conclusion, the findings of the present study suggested that miR‑340‑5p may protect PC12 cells against OGD/R through targeting Neurod4, which could provide important implications for the treatment of ischemia‑reperfusion injury based on miR‑340‑5p expression levels in vivo.

To explore the role of calpain and its signaling pathway in lipopolysaccharide (LPS)-induced acute kidney injury (AKI), animal models of endotoxemia were established by administration of LPS to mice with endothelial-specific Capn4 knockout (TEK/Capn4−/−), mice with calpastatin (an endogenous calpain inhibitor) overexpression (Tg-CAST) and mice with myeloid-specific Capn4 knockout (LYZ/Capn4−/−). Mouse pulmonary microvascular endothelial cells (PMECs) were used as a model of the microvascular endothelium and were stimulated with LPS. Renal function, renal inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expression, cellular apoptosis, plasma and renal levels of NO and reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinase (MAPK) family members (p38, ERK1/2, and JNK1/2) were examined. Moreover, a calpain inhibitor, calpastatin overexpression adenoviruses and MAPK inhibitors were used. Significant renal dysfunction was induced by LPS stimulation, and recovery was observed in TEK/Capn4−/− and Tg-CAST mice but not in LYZ/Capn4−/− mice. Endothelial Capn4 knockout also abrogated the LPS-induced increases in renal iNOS expression, caspase-3 activity and apoptosis and plasma and renal NO and ROS levels but did not obviously affect renal eNOS expression. Moreover, LPS increased both calpain and caspase-3 activity, and only the expression of iNOS in PMECs was accompanied by increased phosphorylation of p38 and JNK. Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression, NO/ROS production, and cellular apoptosis induced by LPS. These results suggest that endothelial calpain plays a protective role in LPS-induced AKI by inhibiting p38 phosphorylation, thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis., Acute kidney injury: enzyme block could combat failure Therapies that inhibit the enzyme calpain could alleviate the effects of acute kidney injury according to researchers in China and Canada. Acute kidney injury is induced by endotoxemia, in which changes in the permeability of the intestine allow lipopolysaccharides (LPS) to pass from gut bacteria into the bloodstream. Calpain is known to be active during this process. Zhifeng Liu at the General Hospital of Guangzhou Military Command and co-workers induced endotoxemia in various mouse models by injecting them with LPS. The LPS induced significant kidney dysfunction and cell death, but these were alleviated in mice that were genetically modified to block calpain activity in the blood vessel lining, and in mice that overexpressed calpastatin, a calpain inhibitor. Blocking calpain reduces the expression of nitric oxide synthases that damage endothelial cells.

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd3+, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the KV channel and inhibiting the L-type Ca2+ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

Nitric oxide (NO) is a free radical acting as a cellular signaling molecule in many different biochemical processes. NO is synthesized from l-arginine through the action of the nitric oxide synthase (NOS) family of enzymes, which includes three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). iNOS-derived NO has been associated with the pathogenesis and progression of several diseases, including liver diseases, insulin resistance, obesity and diseases of the cardiovascular system. However, transient NO production can modulate metabolism to survive and cope with stress conditions. Accumulating evidence strongly imply that iNOS-derived NO plays a central role in the regulation of several biochemical pathways and energy metabolism including glucose and lipid metabolism during inflammatory conditions. This review summarizes current evidence for the regulation of glucose and lipid metabolism by iNOS during inflammation, and argues for the role of iNOS as a metabolic enzyme in immune and non-immune cells.

L-arginine is a conditionally essential amino acid, which is the cellular regulator of many vital functions of the body. He is involved in the regulation of the tone of the smooth muscle component of the blood vessels wall, bronchi, and intestines. L-arginine is a substrate for the nitric oxide synthase (NOS), which produces nitric oxide (NO). NO, produced in the vascular endothelium of endothelial NOS, is responsible for the relaxation of smooth muscles and is necessaryto lower blood pressure. L-arginine has a high functional priority in the production of NO and, therefore, in the physiology of the cardiovascular and cerebrovascular systems. L-arginine can reduce obesity, lower blood pressure, suppress oxidative processes and normalize endothelial dysfunction, contributing to remission in type 2 diabetes. Larginine is also used by cells of the immune system, can reduce the level of infection, especially in case of impaired immune function. L-arginine slows down aging, inhibits platelet aggregation, and regulates multiple metabolic pathways associated with the metabolism of fatty acids, glucose, amino acids and proteins. Therefore, one should take into account the great therapeutic potential of L-arginine and continue to study the possibilities of its use as a promising drug against the progression of vascular dysfunction associated with aging, diabetes and cardiovascular diseases.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and G(q)/G(11)-mediated signaling, as well as by Ca(2+) and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ- and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity.

Mechanisms that regulate nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions and is activated by calmodulin (CaM) binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two NOS electron transfer domains in an FRET dye-labeled endothelial NOS reductase domain (eNOSr) and to understand how CaM affects the dynamics to regulate catalysis by shaping the spatial and temporal conformational behaviors of eNOSr. In addition, we developed and applied a new imaging approach capable of recording three-dimensional FRET efficiency versus time images to characterize the impact on dynamic conformal states of the eNOSr enzyme by the binding of CaM, which identifies clearly that CaM binding generates an extra new open state of eNOSr, resolving more detailed NOS conformational states and their fluctuation dynamics. We identified a new output state that has an extra open conformation that is only populated in the CaM-bound eNOSr. This may reveal the critical role of CaM in triggering NOS activity as it gives conformational flexibility for eNOSr to assume the electron transfer output FMN-heme state. Our results provide a dynamic link to recently reported EM static structure analyses and demonstrate a capable approach in probing and simultaneously analyzing all of the conformational states, their fluctuations, and the fluctuation dynamics for understanding the mechanism of NOS electron transfer, involving electron transfer among FAD, FMN, and heme domains, during nitric oxide synthesis.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. The majority of HGPS cases are caused by a de novo point mutation in the LMNA gene (c.1824C>T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes normal angiogenic function in HGPS. Here, we use human iPSC‐derived endothelial cell (iPSC‐EC) models to study angiogenesis in HGPS. We cultured normal and HGPS iPSC‐ECs under both static and fluidic culture conditions. HGPS iPSC‐ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared with normal controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary‐like microvascular network formation. Furthermore, the expression of matrix metalloproteinase 9 (MMP‐9) was reduced in HGPS iPSC‐ECs, while the expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) was upregulated relative to healthy controls. Finally, we used an adenine base editor (ABE7.10max‐VRQR) to correct the pathogenic c.1824C>T allele in HGPS iPSC‐ECs. Remarkably, ABE7.10max‐VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized the misregulated TIMPs, and restored angiogenic competence in HGPS iPSC‐ECs. Together, these results provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE could be a promising therapeutic approach for correcting HGPS‐related cardiovascular phenotypes., Progerin antagonizes the normal angiogenic function via inhibiting endothelial nitric oxide synthase (eNOS) expression and activity and concomitant decrease in intracellular nitric oxide (NO) level.

Background: Capsaicin, a pungent component of chili pepper, has been reported to decrease blood pressure (BP) and to cause vasorelaxation via nitric oxide (NO) production. However, it is still unclear how dietary capsaicin effects on renovascular hypertension. To examine this, we observed the effects of dietary capsaicin on BP in 2-kidney, 1-clip renovascular hypertension (2K1C) rats, and investigated the participation of NO in the mechanism.Methods: Rats with 2K1C or sham-operated rats (SHAM) were treated with 0.006% capsaicin diet (CAP) or control diet (CTL) for 6 weeks. Systolic BP (SBP) was measured by tail-cuff method once a week. In the end, mean arterial BP (MAP) was measured in the rats under anesthesia. These observations were performed also in the rats taking a NO synthase (NOS) inhibitor (LN). After rats were euthanized, thoracic aortas were collected and used for western blot analyses to evaluate the phosphorylated ratio of endothelial NOS (eNOS), protein kinase A (PKA) and B (Akt), in order to explore a mechanism of the effects on BP by dietary capsaicin.Results: SBP and MAP in 2K1C rats were significantly higher than in SHAM rats when fed CTL, but not when fed CAP. Those in 2K1C-CAP rats were significantly lower than in 2K1C-CTL rats. LN suppressed the effect of dietary capsaicin. The ratios of phosphorylated (p-) eNOS/eNOS and p-Akt/Akt, but not p-PKA/PKA, were significantly increased in rats fed CAP compared with rats fed CTL.Conclusion: Dietary capsaicin may alleviate 2K1C renovascular hypertension, probably via enhancing phosphorylation of Akt and eNOS.Abbreviations: 2K1C: 2-kidney, 1-clip hypertension model; Akt: protein kinase B; Ang II: angiotensin II; ANOVA: measures analysis of variance; BP: blood pressure; EC: endothelial cell; eNOS: endothelial nitric oxide synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L-NAME, LN: Nω-Nitro-L-arginine methyl ester hydrochloride; MA: mesenteric arteries; MAP: mean arterial blood pressure; NO: nitric oxide; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SBP: Systolic blood pressure; SHR: spontaneously hypertensive rats; SN: sympathetic nervous; TRPV1: transient receptor potential vanilloid type 1; WKY: Wistar Kyoto rats.

We investigated the mechanisms by which propiverine hydrochloride influenced bladder activity in rats with pelvic venous congestion (PC) and urinary frequency. To create PC rats, female rats were anesthetized with isoflurane and the bilateral common iliac veins and bilateral uterine veins were ligated. At 4 weeks after ligation, we assessed voiding behaviour, locomotor activity, and urinary 8-hydroxydeoxyguanosine (8-OHdG) and nitric oxide metabolites (NOx). We also performed cystometry and measured mRNAs for nitric oxide synthase (NOS) and several receptors in the bladder wall. PC rats showed a decrease in locomotor activity and an increased frequency of urination. There was a decrease in endothelial NOS (eNOS), M3, and TRPV1 mRNA expression in the bladder wall, as well as an increase in inducible NOS (iNOS) mRNA. Administration of propiverine to PC rats increased locomotor activity to the level in sham rats, improved bladder function, decreased urinary 8-OHdG excretion, and increased urinary NOx excretion. In addition, propiverine increased neuronal NOS (nNOS) mRNA expression, and decreased expression of iNOS, M3 and TRPV1 mRNA in the bladder wall. Therefore, propiverine not only improved bladder dysfunction through its previously reported actions (anti-muscarinic effect, Ca antagonist effect, and inhibition of noradrenaline re-uptake), but also by reducing inflammation.

This study aimed at the participation of lncRNA H19, endothelial NADPH oxidases (NOX4) and miR-148b-3p in hypoxia stress in human hepatic sinusoidal endothelial cells (HHSEC), and clarifying the relationship among them. The expression of lnc H19, NOX4 and miR-148b-3p in cirrhotic patients and hypoxic HHSEC were measured by RT-PCR. The nitric oxide and hydrogen peroxide content in HHSEC were determined using ultraviolet chromatometry. The protein levels of NOX4, endothelial NOS (eNOS) and phosphorylated eNOS (p-eNOS) were measured via western blotting. The interaction between NOX4 promoter and lnc H19/miR-148b-3p was measured by dual-luciferase reporter gene detection system. The present results indicated that the expressions of NOX4 mRNA and lnc H19 were increased but miR-148b-3p was decreased in both cirrhotic patients and hypoxic HHSEC. Further, hypoxia induced the up-regulation of hydrogen peroxide and the down-regulation of eNOS/NO signaling in HHSEC. And these symptoms were ameliorated by lnc H19 shRNA and miR-148b-3p mimics. But the beneficial effects of lnc H19 shRNA and miR-148b-3p mimics were further abolished by miR-148b-3p inhibitor and NOX4 over-expression, respectively. In addition, NOX4 was a direct, negatively regulated target of miR-148b-3p, and miR-148b-3p was negatively regulated by lnc H19. Collectively, lnc H19 is a negatively regulator of microRNA-148b-3p, and participate in hypoxia stress in HHSEC via positively regulating NOX4 and negatively regulating eNOS/NO signaling.

This study aimed to evaluate the effects of combined use of tetrahydrobiopterin (BH4) and nebivolol on cardiac diastolic dysfunction in spontaneously hypertensive rats (SHRs). Twelve-week-old male SHRs were treated with BH4, nebivolol, or a combination of both. Left ventricle function was evaluated, and reactive oxygen species (ROS) production (including dihydroethidium (DHE) and 3-nitrotyrosine (3-NT)), nitric oxide synthase (NOS) activity and the level of NO in myocardial tissue were determined. The expression levels of endothelial NOS (eNOS), phospholamban (PLN), sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), β3-adrenoceptor, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) were assayed. Treatment with BH4, nebivolol, or both reversed the noninvasive indexes of diastolic function, including E/E' and E'/A', and the invasive indexes, including time constant of isovolumic left ventricle (LV) relaxation (tau), -dP/dtmin, -dP/dtmin/LV systolic pressure (LVSP), and LV end-diastolic pressure (LVEDP) in SHRs. mRNA and protein expression levels of eNOS dimer, phosphorylated PLN, SERCA2a, cGMP, and PKG in the myocardium of treated SHRs were significantly up-regulated compared with those in control rats (p < 0.05 or p < 0.01). The expression levels of 3-NT and DHE were reduced in all treated groups (p < 0.05 or p < 0.01). Notably, combined use of BH4 and nebivolol had better cardioprotective effects than monotherapies. BH4 or nebivolol has a protective effect on diastolic dysfunction in SHRs, and BH4 combined with nebivolol may exert a synergistically cardioprotective effect through activation of β3-adrenoceptor and the NO/cGMP/PKG signaling pathway.

A considerable number of human diseases have an inflammatory component, and a key mediator of immune activation and inflammation is inducible nitric oxide synthase (iNOS), which produces nitric oxide (NO) from l-arginine. Overexpressed or dysregulated iNOS has been implicated in numerous pathologies including sepsis, cancer, neurodegeneration, and various types of pain. Extensive knowledge has been accumulated about the roles iNOS plays in different tissues and organs. Additionally, X-ray crystal and cryogenic electron microscopy structures have shed new insights on the structure and regulation of this enzyme. Many potent iNOS inhibitors with high selectivity over related NOS isoforms, neuronal NOS, and endothelial NOS, have been discovered, and these drugs have shown promise in animal models of endotoxemia, inflammatory and neuropathic pain, arthritis, and other disorders. A major issue in iNOS inhibitor development is that promising results in animal studies have not translated to humans; there are no iNOS inhibitors approved for human use. In addition to assay limitations, both the dual modalities of iNOS and NO in disease states (ie, protective vs harmful effects) and the different roles and localizations of NOS isoforms create challenges for therapeutic intervention. This review summarizes the structure, function, and regulation of iNOS, with focus on the development of iNOS inhibitors (historical and recent). A better understanding of iNOS' complex functions is necessary before specific drug candidates can be identified for classical indications such as sepsis, heart failure, and pain; however, newer promising indications for iNOS inhibition, such as depression, neurodegenerative disorders, and epilepsy, have been discovered.

The fertility of female mammals depends on the quality of oocytes, which decreases in the process of biological aging. We found an inhibitory effect of two related pituitary hormones (prolactin (PRL) and growth hormone (GH)) on destructive modifications of metaphase chromosomes in the eggs of domestic cow (Bos taurus taurus), aging in vitro. The involvement of different NO synthase (NOS) isoforms in realization of the effect of PRL and GH on age-related changes of MII chromosomes with prolonged cultivation of in vitro matured cow oocytes was studied in the present work. In the absence of hormones NPLA, neuronal NOS (nNOS) inhibitor had no effect on the frequency of chromosomal abnormalities in aging oocytes. At the same time, 1400 W (inducible NOS (iNOS) inhibitor) and L-NAME (efficient endothelial NOS (eNOS and nNOS ) inhibitor) decreased this frequency, while the blocking of all three NO synthase isoforms led to the opposite effect. The inhibitory effect of PRL on destructive chromosome modifications in aging eggs increased in the presence of L-NAME, but was not expressed with the inhibition of nNOS, iNOS, or all NO synthase isoforms simultaneously. Neither NPLA nor L-NAME modulated the inhibitory effect of GH on abnormal chromosome changes in oocytes. On the contrary, the blocking of iNOS, as well as all three NO synthase isoforms, led to an increase in the portion of oocytes with destructive changes of MII chromosomes. At the same time, the level of total activity of NO synthase in oocytes did not depend on the presence of the studied hormones or NOS inhibitors during the prolonged cultivation of oocyte–cumulus complexes. Data obtained suggest that the inhibitory effect of PRL and GH on abnormal changes of metaphase chromosomes in aging cow eggs is associated with a decrease in the activity of endothelial NO synthase, as well as (in the case of PRL) with an increase in the activity of neuronal NO synthase in the cumulus cells surrounding the oocytes.

Rationale: Passive heat therapy improves vascular endothelial function, likely via enhanced nitric oxide (NO) bioavailability, although the mechanistic stimuli driving these changes are unknown. Objective: To determine the isolated effects of circulating (serum) factors on endothelial cell function, particularly angiogenesis, and NO bioavailability. Methods and Results: Cultured human umbilical vein endothelial cells (HUVECs) were exposed to serum collected from 20 healthy young (22 ± 1 years) adults before (0 wk), after one session of water immersion (Acute HT), and after 8 wk of either heat therapy (N = 10; 36 sessions of hot water immersion; session 1 peak rectal temperature: 39.0 ± 0.03°C) or sham (N = 10; 36 sessions of thermoneutral water immersion). Serum collected following acute heat exposure and heat therapy improved endothelial cell angiogenesis (Matrigel bioassay total tubule length per frame, 0 wk: 69.3 ± 1.9 mm vs. Acute HT: 72.8 ± 1.4 mm, p = 0.04; vs. 8 wk: 73.0 ± 1.4 mm, p = 0.03), with no effects of sham serum. Enhanced angiogenesis was NO-mediated, as addition of the NO synthase (NOS) inhibitor L-NNA to the culture media abolished differences in tubule formation across conditions (0 wk: 71.3 ± 1.8 mm, Acute HT: 71.6 ± 1.9 mm, 8 wk: 70.5 ± 1.6 mm, p = 0.69). In separate experiments, we found that abundance of endothelial NOS (eNOS) was unaffected by Acute HT serum (p = 0.71), but increased by 8 wk heat therapy serum (1.4 ± 0.1-fold from 0 wk, p < 0.01). Furthermore, increases in eNOS were related to improvements in endothelial tubule formation (r(2) = 0.61, p < 0.01). Conclusions: Passive heat therapy beneficially alters circulating factors that promote NO-mediated angiogenesis in endothelial cells and increase eNOS abundance. These changes may contribute to improvements in vascular function with heat therapy observed in vivo. Abbreviations: Ang-1: angiopoietin-1; ANOVA: analysis of variance; bFGF: basic fibroblast growth factor; CV: cardiovascular; CVD: cardiovascular diseases; eNOS: endothelial nitric oxide synthase; HSPs: heat shock proteins; HT: heat therapy; HUVECs: human umbilical endothelial cells; L-NNA: Nω-nitro-L-arginine; MnSOD: manganese superoxide dismutase; NO: nitric oxide; NOS: nitric oxide synthase; PBMCs: peripheral blood mononuclear cells; RM: repeated measures; sFlt-1: soluble VEGF receptor; SOD: superoxide dismutase; TGF-β: transforming growth factor- β; VEGF: vascular endothelial growth factor

We investigated the protective effects of swimming exercise on high-fat diet-induced heart and aorta damage by evaluating oxidative stress and the endothelial nitric oxide (NO) system. Sprague Dawley rats were fed either standard chow (STD, 6% fat) or high-fat diet (HFD; 45% fat) for 18 weeks, with half of the animals trained by daily swimming sessions (EXC; 1 h per day for 5 days/week) for the last 6 weeks of the experimental period and half kept sedentary (SED). Heart and aorta tissues were prepared for routine light and electron microscopy evaluation. Endothelial NOS (eNOS) and inducible NOS (iNOS) distribution in the tissue samples were examined by immunohistochemistry. Biochemical examinations, including blood serum lipid profiles, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and tissue NO levels were measured. Deteriorated heart and aorta morphology, increased MDA levels and iNOS-immunoreactivity (iNOS-ir), as well as decreased GSH, NO, SOD, and eNOS-ir parameters were observed in the HFD + SED group. These morphological and biochemical parameters were ameliorated in the HFD + EXC group. Our study revealed that obesity-induced iNOS activation and increased oxidative stress in cardiac and aorta tissues. Exercise protected the obesity-induced cardiac and aortic tissue damage by modulating oxidant/antioxidant balance via involvement of the NO system.

Systemic infusion of nitric oxide synthase (NOS) inhibitors increases peripheral vascular resistance due to inhibition of endothelial NOS leading to the activation of the arterial baroreceptor mechanisms and inhibition of central sympathetic outflow. In the current study, we explored that systemic NOS blockage activates protein kinase A (PKA)-mediated signaling pathway through maintained cGMP-dependent protein kinase (PKG) activation. Rats were treated with 3 different concentrations of N(ω)-nitro-l-arginine methyl ester (L-NAME) for 14 days. Systemic L-NAME treatment induced a dose-dependent increase in blood pressure and increased mRNA levels of atrial natriuretic peptide (ANP) and phosphorylation levels of p44/42 MAPK without any change in cardiac mass. The cardiac cGMP levels and PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) (Ser239) did not alter in any group. At the highest dose of treatment (100 mg/kg per day), PKA-mediated phosphorylations of VASP (Ser157) and troponin I (TnI) (Ser23/24) were enhanced significantly indicating the increase in PKA activation in response to chronic NOS blockage. Alterations in both phosphorylated phospholamban (Ser16/Thr17) and sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA2) levels can increase cytosolic Ca2+ load and impair Ca2+ handling. Our data suggest that the increased PKA activation in response to chronic NOS blockage appears to be responsible for cardiac abnormalities that occur due to prolonged L-NAME treatment.

© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. Background and Purpose: Apelin is an endogenous vasodilatory and inotropic peptide that is down-regulated in human pulmonary arterial hypertension, although the density of the apelin receptor is not significantly attenuated. We hypothesised that a G protein-biased apelin analogue MM07, which is more stable than the endogenous apelin peptide, may be beneficial in this condition with the advantage of reduced β-arrestin-mediated receptor internalisation with chronic use. Experimental Approach: Male Sprague–Dawley rats received either monocrotaline to induce pulmonary arterial hypertension or saline and then daily i.p. injections of either MM07 or saline for 21 days. The extent of disease was assessed by right ventricular catheterisation, cardiac MRI, and histological analysis of the pulmonary vasculature. The effect of MM07 on signalling, proliferation, and apoptosis of human pulmonary artery endothelial cells was investigated. Key Results: MM07 significantly reduced the elevation of right ventricular systolic pressure and hypertrophy induced by monocrotaline. Monocrotaline-induced changes in cardiac structure and function, including right ventricular end-systolic and end-diastolic volumes, ejection fraction, and left ventricular end-diastolic volume, were attenuated by MM07. MM07 also significantly reduced monocrotaline-induced muscularisation of small pulmonary blood vessels. MM07 stimulated endothelial NOS phosphorylation and expression, promoted proliferation, and attenuated apoptosis of human pulmonary arterial endothelial cells in vitro. Conclusion and Implications: Our findings suggest that chronic treatment with MM07 is beneficial in this animal model of pulmonary arterial hypertension by addressing disease aetiology. These data support the development of G protein-biased apelin receptor agonists with improved pharmacokinetic profiles for use in human disease.