NO Synthase Inhibitor Research Articles

NO-mediated anticontractile effect of the endothelium is abolished in coronary arteries of adult rats with antenatal/early postnatal hypothyroidism

IntroductionThyroid hormones are essential for proper development of many systems and organs, including circulatory system. Thyroid deficiency during pregnancy may affect the cardiovascular function in children early on and later in adulthood. However, long-term effects of early thyroid deficiency are poorly understood. We hypothesized that antenatal/early postnatal hypothyroidism will influence anticontractile effect of NO in coronary arteries of adult rats. Design and methodsTo model antenatal/early postnatal hypothyroidism dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%, w/v) from the first day of pregnancy till 2 weeks after delivery. Control females were supplied with pure water. Their male offspring was grown up till the age of 10–12 weeks. Systolic blood pressure was measured using tail cuff method. Septal coronary arteries were isolated and studied in wire myograph. Blood serum thyroid hormones concentrations (ELISA) and NO metabolites level (Griess method) were evaluated. ResultsAt the age of 10–12 weeks thyroid hormones, TSH concentrations, NO metabolites and systolic blood pressure level didn't differ between groups. Arterial responses to acetylcholine and exogenous NO-donor DEA/NO were similar in control and PTU groups. Along with that, in control rats endothelium denudation strongly potentiated basal tone of arteries and their contractile responses to thromboxane A2 receptor agonist U46619. The effects of endothelium denudation were absent in PTU rats indicating that anticontractile effect of endothelium is abolished in their arteries. Further, NO-synthase inhibitor L-NNA (100 μM) caused significant elevation of basal tone and increased U46619-induced contraction of endothelium-intact arteries only in control rats, while had no effect in PTU group. ConclusionsOur data demonstrate that NO-mediated anticontractile effect of endothelium is eliminated in coronary arteries of adult rats, which suffered from antenatal/early postnatal hypothyroidism. Therefore, maternal thyroid hormones deficiency may have detrimental consequences in adult offspring including coronary circulation pathologies, despite normal blood levels of thyroid hormones.

Dysfunction of Kidney Endothelium after Ischemia/Reperfusion and Its Prevention by Mitochondria-Targeted Antioxidant.

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen-glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.

Antinociception of petroleum ether fraction derived from crude methanol extract of Melastoma malabathricum leaves and its possible mechanisms of action in animal models.

Background Melastoma malabathricum L. (family Melastomaceae) has been traditionally used as remedies against various ailments including those related to pain. The methanol extract of M. malabathricum leaves has been proven to show antinociceptive activity. Thus, the present study aimed to determine the most effective fraction among the petroleum ether- (PEMM), ethyl acetate- (EAMM) and aqueous- (AQMM) fractions obtained through successive fractionation of crude, dried methanol extract of M. malabathricum (MEMM) and to elucidate the possible mechanisms of antinociception involved.MethodsThe effectiveness of fractions (100, 250 and 500 mg/kg; orally) were determine using the acetic acid-induced abdominal constriction test and the most effective extract was further subjected to the hot plate- or formalin-induced paw licking-test to establish its antinociceptive profile. Further elucidation of the role of opioid and vanilloid receptors, glutamatergic system, and nitric oxide/cyclic guanosine phosphate (NO/cGMP) pathway was also performed using the appropriate nociceptive models while the phytoconstituents analyses were performed using the phytochemical screening test and, HPLC-ESI and GCMS analyses.ResultsPEMM, EAMM and AQMM significantly (p < 0.05) attenuated acetic acid-induced nociception with the recorded EC50 of 119.5, 125.9 and 352.6 mg/kg. Based on the EC50 value, PEMM was further studied and also exerted significant (p < 0.05) antinociception against the hot plate- and formalin-induced paw licking-test. With regards to the mechanisms of antinociception,: i) PEMM significantly (p < 0.05) attenuated the nociceptive action in capsaicin- and glutamate-induced paw licking test.; ii) naloxone (5 mg/kg), a non-selective opioid antagonist, failed to significantly (p < 0.05) inhibit PEMM’s antinociception iii) L-arginine (a nitric oxide precursor), but not NG-nitro-L-arginine methyl esters (L-NAME; an inhibitor of NO synthase), methylene blue (MB; an inhibitor of cGMP), or their respective combination, significantly (p < 0.05) reversed the antinociception of PEMM. Phytochemical analyses revealed the presence of several antinociceptive-bearing bioactive compounds, such as triterpenes and volatile compounds like oleoamide and palmitic acid. The presence of low flavonoids, such as gallocatechin and epigallocatechin, saponins and tannins in PEMM might synergistically contribute to enhance the major compounds antinociceptive effect.ConclusionPEMM exerted a non-opioid-mediated antinociceptive activity at the central and peripheral levels via the inhibition of vanilloid receptors and glutamatergic system, and the activation of NO-mediated/cGMP-independent pathway. Triterpenes, as well as volatile oleoamide and palmitic acid, might be responsible for the observed antinociceptive activity of PEMM.

Madagascine Induces Vasodilatation via Activation of AMPK.

Madagascine (3-isopentenyloxyemodin) can be chemically synthesized or purified from several Rhamnus species, and it is found to have more potent biological activities than the parent compound emodin. The aim of this study is to characterize the vasodilatory effect of madagascine on vasoconstriction and sphingosylphosphorylcholine induced vasospasm in ex vivo and reveal the potential mechanisms in vitro. The effects of madagascine on vasoconstriction of rat mesenteric resistance arteries (MRAs) induced by K+, methoxamine, and endothelin-1 were, respectively, studied. The cholesterol-enriched porcine coronary vascular smooth muscle (VSM) strips were used to investigate the effects of madagascine on abnormal constriction induced by sphingosylphosphorylcholine (SPC) which has a pivotal role in vasospasm. The vasodilatory effect was induced by madagascine (0.3–100 μM) in isolated rat MRAs and the vasodilatory effect was blocked by NO synthase inhibitor L-NAME and AMPK inhibitor compound C. Madagascine (10 μM) also significantly relaxed the abnormal constriction in porcine VSM induced by SPC and the effect was abolished by compound C. Madagascine significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) in endothelial cells while decreasing the phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in VSM cells. Madagascine-induced vasodilatation was abrogated using small interfering RNA knockdown of AMPK. In summary, madagascine exerted vasodilatation through activating AMPK, leading to the activation of eNOS in endothelium and inhibition of ROCK/MYPT1 in VSM. This study suggests the potential value of madagascine in amelioration of vasospasm related cardiovascular diseases.

Role of oxidative stress in oxaliplatin-induced enteric neuropathy and colonic dysmotility in mice.

Background and PurposeOxaliplatin is a platinum‐based chemotherapeutic drug used as a first‐line therapy for colorectal cancer. However, its use is associated with severe gastrointestinal side‐effects resulting in dose limitations and/or cessation of treatment. In this study, we tested whether oxidative stress, caused by chronic oxaliplatin treatment, induces enteric neuronal damage and colonic dysmotility.Experimental ApproachOxaliplatin (3 mg·kg−1 per day) was administered in vivo to Balb/c mice intraperitoneally three times a week. The distal colon was collected at day 14 of treatment. Immunohistochemistry was performed in wholemount preparations of submucosal and myenteric ganglia. Neuromuscular transmission was studied by intracellular electrophysiology. Circular muscle tone was studied by force transducers. Colon propulsive activity studied in organ bath experiments and faeces were collected to measure water content.Key ResultsChronic in vivo oxaliplatin treatment resulted in increased formation of reactive oxygen species (O2ˉ), nitration of proteins, mitochondrial membrane depolarisation resulting in the release of cytochrome c, loss of neurons, increased inducible NOS expression and apoptosis in both the submucosal and myenteric plexuses of the colon. Oxaliplatin treatment enhanced NO‐mediated inhibitory junction potentials and altered the response of circular muscles to the NO donor, sodium nitroprusside. It also reduced the frequency of colonic migrating motor complexes and decreased circular muscle tone, effects reversed by the NO synthase inhibitor, Nω‐Nitro‐L‐arginine.Conclusion and ImplicationsOur study is the first to provide evidence that oxidative stress is a key player in enteric neuropathy and colonic dysmotility leading to symptoms of chronic constipation observed in oxaliplatin‐treated mice.

Abstract 12197: Increased Angiotensin II Sensitivity Contributes to Microvascular Endothelial Dysfunction in Women Who Have Had Preeclampsia

Introduction: Women with a history of preeclampsia have increased cardiovascular disease risk, however the mechanisms responsible for this association remain unclear. Microvascular damage sustained during a preeclamptic pregnancy may persist post-partum. The putative mechanisms mediating this dysfunction include a reduction in nitric oxide (NO)-dependent dilation, and an increased sensitivity to angiotensin II (ang II). In this study, we evaluated endothelium-dependent dilation, ang II sensitivity, and the therapeutic effect of ang II receptor blockade (losartan) on endothelium-dependent dilation in vivo in the microvasculature of women with a history of preeclampsia (PrEC, n=7) and control women who had a healthy pregnancy (HC, n=9). We hypothesized that PrEC would have 1) reduced endothelium-dependent dilation, 2) reduced NO-mediated dilation, and 3) increased sensitivity to ang II. We further hypothesized that losartan would increase endothelium-dependent dilation in PrEC. Methods: We assessed microvascular function by measurement of cutaneous vascular conductance responses to graded infusion of acetylcholine (ACh; 10 -10 -10 -1 M) and ang II (10 -20 -10 -4 M); and during ACh co-infusion with 15mM L-NAME (NO-synthase inhibitor) or 43μM losartan. Results: ACh-induced vasodilation was attenuated in PrEC compared to HC (PrEC -3.3±0.2 vs. HC -4.7±0.1 logEC50; p&lt;0.05). There was no difference between PrEC and HC during ACh co-infusion with L-NAME (PrEC -4.4±0.2 vs. HC -4.6±0.2 logEC50; NS). NO-mediated dilation was attenuated in PrEC compared to HC (PrEC 10±4 vs. HC 35±4%; p&lt;0.01). PrEC had a greater vasoconstriction response to ang II (PrEC -11.8±0.2 vs. HC -9.7±0.2 logEC50; p&lt;0.05. Losartan co-infusion improved the vasodilation response to ACh in PrEC (losartan -4.6±0.2 vs. ACh alone -3.3±0.2 logEC50; NS). Conclusions: These findings suggest that women who have had preeclampsia have impaired endothelium-dependent vasodilation which is mediated, in part, by reductions in NO signaling and increased sensitivity to ang II. Furthermore, losartan improved endothelium-dependent dilation, suggesting that ang II receptor blockade may have therapeutic potential to treat microvascular dysfunction in women who have had preeclampsia.

Sound Danger Signals Activate the Nitrergic System of the Medial Prefrontal Cortex

Studies on Sprague–Dawley rats using in vivo intracerebral microdialysis showed that presentation of animals with a conditioned sound signal previously combined with pain stimulation (execution of a conditioned reflex fear response) was accompanied by an increase in the extracellular citrulline (a coproduct of NO synthesis) level in the medial prefrontal cortex. Presentation of a differential sound signal (not associated with electrocutaneous stimulation) to the same animals led to no more than a small increase in this measure. The increase in the citrulline level induced by the conditioned signal was prevented by administration of the neuronal NO synthase inhibitor Nω-propyl-L-arginine (1 mM) into the medial prefrontal cortex and was not seen in rats of the control group (same procedure but no pain stimulation). These data provide the first evidence that danger-associated sound signals but not safe differential signals activate the nitrergic system of the medial prefrontal cortex.

The effect of pre-eclampsia-like syndrome induced by L-NAME on learning and memory and hippocampal glucocorticoid receptor expression: A rat model

ABSTRACTObjective: We aimed to study the impacts of pre-eclampsia on the cognitive and learning capabilities of adolescent rat offspring and to explore the possible underlying mechanisms at the molecular level. Methods: Pregnant rats were subcutaneously injected with saline solution (control) (n = 16) or NG-nitro-L-arginine methyl ester (L-NAME) (n = 16) from the 13th day of gestation until parturition. The brain tissues from fetal rats delivered by cesarean section were examined in both groups with hematoxylin and eosin (H&E) staining. Rats born vaginally in both groups were subjected to the Morris water maze test when 8-week-old and their hippocampi were analyzed for glucocorticoid receptor (GR) expression. Results: A pre-eclampsia-like model was successfully built in pregnant rats by infusion of the NO synthase inhibitor L-NAME, including phenotypes as maternal hypertension and proteinuria, high stillbirth rate, and fetal growth retardation. Neuroepithelial cell proliferation was found in the hippocampus of fetal rats in the L-NAME group. Grown to 8-week-old, the L-NAME group showed significantly longer escape latency than the control group in the beginning as well as in the end of navigation trials. At the same time, the swimming distance achieved by the L-NAME group was significantly longer than that of the control group. Such differences in cognitive and learning capabilities between the two groups were not gender dependent. Besides, the 8-week-old rats in the L-NAME group had increased GR expression in the hippocampus than the control group. Conclusion: Pre-eclampsia would impair cognitive and learning capabilities in adolescent offspring, and the upregulated expression of hippocampal GR may be involved in the underlying mechanisms.

Nitrergic and opioidergic systems affect radiographic density ‎and histomorphometric indices in bile-duct-ligated cirrhotic rats.

Metabolic bone disease is a major issue in chronic liver disease. Increased production of nitric oxide (NO) and elevation of endogenous opioids have been suggested to occur during cholestasis/cirrhosis. We aimed to investigate the involvement of nitrergic and opioidergic systems in bone loss after bile-duct-ligation (BDL) in rats using optical density (OD) evaluation and histomorphometric analysis. BDL- and sham-operated (SO) rats received injections of 3 mg/kg Nω-Nitro-L-arginine methyl-ester-hydrochloride (L-NAME) as an NO-synthase inhibitor, 10 mg/kg naltrexone (NTX) as an opioid-receptors antagonist or saline once daily for 28 days. Lateral cephalometric radiographs were taken on days 0 and 28 and histomorphometric and biochemical indices were measured. Plasma levels of total bilirubin and alkaline-phosphate were markedly increased in BDL compared with SO rats (p≤0.05). Among the studied variables, osteoclast number/mm trabecular surface showed significant increase in BDL animals compared to controls, which was significantly reduced following NO-synthase inhibition (p≤0.05). Similarly, cortical area slightly decreased in BDL animals in comparison to controls, whereas both L-NAME and NTX significantly increased this variable. Following BDL, optical density increased in the skulls of cirrhotic animals and showed a significant decrease after blocking opioid-receptors (p≤0.05). Inhibition of NO-synthase and/or opioid receptors caused significant changes in OD and histomorphometric parameters in BDL rats, both in favor of reducing bone loss. If confirmed by further studies, it seems that manipulation of these systems might be able to improve bone problems in subjects with cholestasis/cirrhosis.

MCRT, a chimeric peptide based on morphiceptin and PFRTic-NH2, regulates the depressor effects induced by endokinin A/B

The interactions of the chimeric peptide MCRT (YPFPFRTic-NH2), based on morphiceptin and neuropeptide FF derivative PFRTic-NH2, on the effects of endokinin A/B (EKA/B) on mean arterial blood pressure of the urethane-anaesthetized rat have been investigated in the absence and presence of tachykinin receptor antagonists, naloxone and NO synthase inhibitors. While MCRT produced dose dependent decreases in mean arterial pressure, in its presence only a small but statistically insignificant decreases in the magnitude and the time course of the depressor effect of EKA/B (10nmol/kg) were observed. MCRT had little influence on the depressor effect of EKA/B (1 nmol/kg), but strongly potentiated that of EKA/B (100nmol/kg). The tachykinin NK1 receptor antagonist SR140333B (1mg/kg) and the NK3 antagonist SR142891 (2.79mg/kg) both reduced the hypotensive effects of EKA/B and MCRT alone and blocked those of the two peptides in combination. The NK2 antagonist GR159897 (4mg/kg) partially blocked the depressor effects of EKA/B and MCRT alone. Naloxone (2mg/kg) completely blocked the depressor effect of MCTR, but partially blocked that of EKA/B. The NO synthase inhibitor l-NAME (50mg/kg) partially blocked the depressor effects of EKA/B, MCRT, and EKA/B + MCRT. These results could help to better understand the role of tachykinin receptors, opioid receptors and neuropeptide FF receptors in cardiovascular system.

Activity of the Nitrergic System of the Medial Prefrontal Cortex in Rats with High and Low Levels of Generalization of a Conditioned Reflex Fear Reaction

In vivo intracerebral microdialysis/HPLC studies on Sprague–Dawley rats showed that acquisition of a conditioned reflex fear reaction (combined conditioned sound stimulus and unavoidable electrocutaneous shock) was accompanied by an increase in the level of extracellular citrulline (a coproduct of NO synthesis) in the medial area of the prefrontal cortex. This increase was almost completely blocked by administration of the neuronal NO synthase inhibitor Nω-propyl-L-arginine (1 mM) into this area of the cortex and was not seen in animals of the control group. These increases were large in animals subsequently showing low levels of freezing (a measure of fear) in response to the differential signal not previously combined with the pain stimulus and low in rats with high levels of freezing in response to the differential signal, though they did not correlate with the level of freezing in response to the conditioned signal. These data provide the first evidence that the formation of a conditioned reflex fear reaction is accompanied by activation of the nitrergic system of the medial prefrontal cortex, reflected in the extent of subsequent generalization but not expression of this conditioned reflex reaction.

Adenosine A2A receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies.

We aim to investigate whether A2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A2A agonist, 24h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC50 10-9.2M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (∼1.5-fold) and pre-eclamptic pregnancies (∼1.2-fold), an effect blocked by the A2A antagonist, ZM-241385 (10-5M) and the inhibitor of NO synthase, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A2A. CGS-21680 also increased (∼1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A2A decrease (∼30%) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.

CB1 receptor activation in the rat paraventricular nucleus induces bi-directional cardiovascular effects via modification of glutamatergic and GABAergic neurotransmission

We have shown previously that the cannabinoid receptor agonist CP55940 microinjected into the paraventricular nucleus of the hypothalamus (PVN) of urethane-anaesthetized rats induces depressor and pressor cardiovascular effects in the absence and presence of the CB1 antagonist AM251, respectively. The aim of our study was to examine whether the hypotension and/or hypertension induced by CP55940 given into the PVN results from its influence on glutamatergic and GABAergic neurotransmission. CP55940 was microinjected into the PVN of urethane-anaesthetized rats twice (S1 and S2, 20 min apart). Antagonists of the following receptors, NMDA (MK801), β2-adrenergic (ICI118551), thromboxane A2–TP (SQ29548), angiotensin II–AT1 (losartan) or GABAA (bicuculline), or the NO synthase inhibitor L-NAME were administered intravenously 5 min before S2 alone or together with AM251. The CP55940-induced hypotension was reversed into a pressor response by AM251, bicuculline and L-NAME, but not by the other antagonists. The CP55940-induced pressor effect examined in the presence of AM251 was completely reversed by losartan, reduced by about 50–60 % by MK801, ICI118551 and SQ29548, prevented by bilateral adrenalectomy but not modified by bicuculline and L-NAME. Parallel, but smaller, changes in heart rate accompanied the changes in blood pressure. The bi-directional CB1 receptor-mediated cardiovascular effects of cannabinoids microinjected into the PVN of anaesthetized rats depend on stimulatory glutamatergic and inhibitory GABAergic inputs to the sympathetic tone; the glutamatergic input is related to AT1, TP and β2-adrenergic receptors and catecholamine release from the adrenal medulla whereas the GABAergic input is reinforced by NO.

Retinal Vessel Diameter Responses to Central Electrical Stimulation in the Rat: Effect of Nitric Oxide Synthase Inhibition.

Recent histological data suggest autonomic innervation of the central retinal artery. In the present study, we investigated the effect of electrical brain stem stimulation at the superior salivatory nucleus (SSN) on the retinal vessel diameter in rats and whether nitric oxide mediates a possible effect. Sprague-Dawley rats (n = 12) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN. Stimulations were performed at 20 Hz, 9 μA, 1 ms pulse duration and 200 pulses. Retinal vessel diameters were measured continuously with the Imedos DVA-R, a noncontact fundus camera for rats with image analysis software. After control measurements, L-NAME, a nonspecific inhibitor of NO synthase, was applied intravenously (10 mg/kg), and the SSN stimulations were repeated. Stimulation at the SSN coordinates increased the retinal arterial diameter by 6.41% ± 1.65% and the venous diameter by 3.48% ± 1.93% (both P < 0.05). Application of L-NAME reduced the arterial response significantly to 2.93% ± 0.91%, but did not change the venous response. Mean arterial pressure, carotid blood flow, and heart rate remained unaltered (by the stimulation). The present study demonstrates that the retinal circulation reacts to electric stimulation at the SSN coordinates in rats. Nitric oxide is involved in the response, but it is not the sole neurotransmitter.

ISH ADA-01 Critical role of telomerase in regulating cerebral vascular function and redox environment

Flow mediated dilation (FMD) is the most physiological relevant form of endothelial-mediated vasodilation. Our laboratory has previously shown that telomerase, a ribo-nucleoprotein that counteracts telomere shortening, has a protective effect on endothelial function under conditions of oxidative stress in the human microcirculation. In the presence of coronary artery disease, decreased telomerase activity contributes to a shift in the mediator of FMD from atheroprotective nitric oxide (NO) to pro-inflammatory and atherogenic hydrogen peroxide (H2O2). Endothelial dysfunction in the cerebral vasculature has been directly linked to cerebral microbleeds and cognitive decline in models of stroke and dementia, thus we hypothesized that TERT plays a critical role in maintaining normal NO-mediated vasodilation in the cerebral vasculature. Using Crisp/Cas9 we generated a novel deletion model for TERT (the catalytic subunit of telomerase) in rats on the WKY background. Middle cerebral arteries (MCA) were isolated from wildtype (WT) and TERT−/− rats, prepared for videomicroscopy, and FMD was measured. The mediator of FMD was determined using a pharmacological inhibitor of NO-synthase (L-NAME) and a scavenger of H2O2 (PEG-catalase). No changes in the magnitude of FMD were observed in MCA from TERT−/− compared to WT rats (max dilation: TERT−/− 71.97 ± 14.7% vs. WT 80.9 ± 10.6%; n≥3; p > 0.05 two way ANOVA RM). In WT animals, pre-incubation with L-NAME abolished FMD while PEG-catalase had no effect on FMD (max dilation: L-NAME -5.8 ± 7%; Peg-catalase 76.6 ± 8.6%; n≥3; p < 0.05). Conversely, in TERT−/− animals FMD was not affected in the presence of L-NAME but abolished in presence of Peg-catalase (max dilation: L-NAME 57.8.0 ± 16.5%; Peg-catalase −4.3 ± 2.4%; n≥3; p < 0.05). Telomerase deficiency causes a switch in the vasoactive mediator of FMD from NO to H2O2, creating a pro-oxidative environment in the cerebral vasculature. Understanding the regulatory role of telomerase in mediating this mechanistic switch may provide a novel therapeutic strategy for the treatment or prevention of cerebrovascular dysfunction.

The role of inwardly rectifying potassium channels in the relaxation of rat hind-limb arteries

An increase in the extracellular K+ concentration, which causes relaxation of arteries due to the activation of inwardly rectifying potassium channels, can occur in some organs under intensive metabolism, as well as endothelium-dependent hyperpolarization. The aim of this work was a comparison of the contribution of these channels in the regulation of the tone of arteries that supply skeletal muscles and the skin. The reactions of skin-region arteries (a subcutaneous artery and its branch) and gastrocnemius muscle arteries were recorded in the isometric mode. During the contraction caused by α1-adrenoceptor agonist, the relaxation reactions upon an increase in extracellular K+ concentration and on acetylcholine in the presence of inhibitors of NO-synthase and cyclooxygenase were recorded (to detect the effects of endothelium-dependent hyperpolarization). The muscle arteries at both effects showed a pronounced relaxation, which was strongly suppressed by Ba2+ ions (blockers of inwardly rectifying potassium channels); both reactions did not exceed 20% in the skin arteries. Thus, the regulatory effect of inwardly rectifying potassium channels in the muscle arteries is much higher than in the skin arteries which is consistent with the idea about the functioning of these arteries in the organism.

Spatial Memory in the Progeny of Rats Subjected to Different Types of Experimental Preeclampsia.

Spatial memory was studied in 2-month-old offspring of rats subjected to different types of experimental preeclampsia (replacement of drinking water with 1.8% NaCl from day 1 to 21 of gestation or intraperitoneal administration of non-selective NO-synthase inhibitor L-NAME to pregnant rats in a daily dose of 25 mg/kg for 7 days on gestation days 14-20). Spatial memory was evaluated in an elevated 8-arm radial maze. Both types of experimental preeclampsia impaired spatial (long-term and short-term) memory and can be used in the development of drugs correcting negative effects of this pregnancy complication on memory.

OP.2D.02] LYCOPENE INCREASES NITRIC OXIDE BIOAVAILABILITY AND INHIBITS ENDOTHELIAL CELLS MIGRATION

Objective: Epidemiologic and intervention studies suggest that dietary vegetables containing lycopene may associate with a reduction in cardiovascular risk. Lycopene has antioxidant activity, being the most efficient carotenoid singlet oxygen quencher. Aim of the present study was to explore the effects of lycopene on the synthesis and release of nitric oxide (NO) in endothelial cells and endothelial cells migration, a key step in angiogenesis. Design and method: Cultured human umbilical endothelial cells (HUVECs) were incubated for 30 minutes in the presence or absence of lycopene (2 micromol/L), vascular endothelial growth factor-A (VEGF-A, 10 ng/mL) and the NO-synthase inhibitor L-NAME (300 micromol/L). Confocal microscopy and Diaminofluorescein-FM diacetate (DAF-FM-DA) as NO indicator were used to analyze NO generation. Nitrite/nitrate concentration was measured in the cell culture medium using 2,3-diaminonaphthalene fluorimetric assay after 30 minutes of exposure to agonists and inhibitors. HUVECs migration was quantified by counting the number of migrated cells (10 fields per well) up to 20 hours after the addition of VEGF using the scratch wound healing assay. Results: An increase in DAF-FM-DA fluorescence was observed in HUVECs with a perinuclear pattern after exposure to lycopene (486.5 ± 142.0 Arbitrary Units, M ± SD, n = 7, p < 0.01), VEGF (472.7 ± 103.4 AU, p < 0.01) or the combination of the two agents with no synergistic effect (460.4 ± 183.2 AU, p < 0.05) compared to controls (293.5 ± 110.9 AU). Nitrite/nitrate concentration in HUVECs culture medium was higher in the presence of lycopene (35.9 ± 25.0 nmol/L, n = 4, P < 0.05), compared to controls (10.4 ± 1.0 nmol/L) and VEGF-A which had no effects. Nitrite/nitrate concentration was reduced by L-NAME (8.6 ± 2.6 nmol/L), demonstrating their generation via NO-synthase. HUVECs migration was stimulated by VEGF-A (967 ± 235 Cells/Field, control: 652.9 ± 118.5, n = 8, p < 0.05) and inhibited by the addition of lycopene (676.1 ± 204.8 C/F, p < 0.01 vs VEGF alone). Conclusions: The present data demonstrate that lycopene increases NO generation and release, as shown by the increase in NO and nitrite/nitrate concentrations, by preventing peroxynitrite formation. Lycopene has biological activity, blunting the increase in cell migration induced by VEGF-A, possibly through NO-depended modulation of VEGF signaling system. The potential of lycopene bioactivity should be further investigated in the setting of vascular inflammation and pathological angiogenesis.

Does third generation left ventricular assist device alter heart failure-related microvascular dysfunction?

Background Left ventricular assist device (LVAD) is a promising new therapy in patients with advanced heart failure. Previous studies suggested that continuous blood flow impairs endothelial function. Whether the third-generation LVAD (HeartWareR, HeartWare Inc., Framingham, MA, USA) system affects endothelial regulation of microvascular flow, endothelial nitric oxide (NO) bioavailability and endothelial production of vWF is not known.Methods Fifteen LVAD-supported heart failure patients and 13 age/body mass index matched heart failure patients (HF) were included. The microvascular endothelial function of skin vessels was assessed with laser Doppler imager (LDI) using 3 different hyperaemic challenges: acetylcholine (Ach) iontophoresis, sodium nitroprusside (SNP) iontophoresis and local heating to 44°C. NO-mediated vasodilation was further evaluated by comparing heating hyperaemic response in skin area pretreated either by a saline solution (control) or a specific NO-synthase inhibitor (L-N-arginine-methyl-ester, L-NAME). vWF antigen was also measured in LVAD and HF patients.Results SNP-induced vasodilation did not differ between LVAD and HF patients, and we observed a trend towards an increased vasodilator response to Ach in LVAD patients (P =0.06). Compared to HF patients, skin thermal hyperaemia was increased in LVAD patients in both control and L-NAME pretreated skin (all P < 0.001). The hyperaemic reaction attributable to NO-mediated vasodilation correlated negatively with HF duration (r = –0.50, P < 0.01, n = 28), but did not differ between LVAD and HF patients. Both groups disclosed also similar vWF antigen serum levels.Conclusion This case-control study indicates that third-generation LVAD therapy does not alter skin microvascular endothelial function and vWF production of patients with HF

Long-term Treatment with Hesperidin Improves Endothelium-dependent Vasodilation in Femoral Artery of Spontaneously Hypertensive Rats: The Involvement of NO-synthase and Kv Channels.

Hesperidin is the most common flavonoid found in citrus fruits and is expected to exert vasodilation action relevant to its health benefits. The present study aimed to explore the effect of hesperidin on the vascular responses in normotensive and hypertensive rats and the involvement of NO-synthase and Kv channels. The 15-week-old Wistar and spontaneously hypertensive rats (SHR) were randomized to orally receive either hesperidin (50 mg/kg/day) or a corresponding volume of the water for 4 weeks. Vascular responses of isolated femoral arteries were studied with myograph in control conditions and during inhibition of NO-synthase with l-NNA and Kv channels with 4-AP. Hesperidin had no effect on blood pressure. Endothelium-dependent vasodilation in Wistar and SHR was significantly improved by the treatment with hesperidin. The contraction responses after l-NNA were increased in all groups of rats to similar extent, but relaxatory responses were significantly attenuated only in SHR. The inhibition of Kv channels significantly reduced endothelium-dependent vasodilatory responses in only in SHR administered with hesperidin. The results of our experiment indicate that hesperidin might improve the endothelium-dependent vasodilation during hypertension, possibly through the enhancement of Kv channels function. Copyright © 2016 John Wiley & Sons, Ltd.