Impaired beta-adrenoceptor-induced relaxation in small mesenteric arteries from DOCA-salt hypertensive rats is due to reduced K-Ca channel activity

beta-Adrenoceptor (beta-AR)-mediated relaxation plays an important role in the regulation of vascular tone. beta-AR-mediated vascular relaxation is reduced in various disease states and aging. We hypothesized that beta-AR-mediated vasodilatation is impaired in DOCA-salt hypertension due to alterations in the cAMP pathway. beta-AR-mediated relaxation was determined in small mesenteric arteries from DOCA-salt hypertensive and control uninephrectomized (Uni) rats. To exclude nitric oxide (NO) and cyclooxygenase (COX) pathways, relaxation responses were determined in the presence of L-NNA and indomethacin, NO synthase inhibitor and COX inhibitors, respectively. Isoprenaline (ISO)-induced relaxation was reduced in arteries from DOCA-salt compared to Uni rats. Protein kinase A (PKA) inhibitors (H89 or Rp-cAMPS) or adenylyl cyclase inhibitor (SQ22536) did not abolish the difference in ISO-induced relaxation between the groups. Forskolin (adenylyl cyclase activator)-induced relaxation was similar between the groups. The inhibition of IKCa/SKCa channels (TRAM-34 plus UCL1684) or BKCa channels (iberiotoxin) reduced ISO-induced relaxation only in Uni rats and abolished the relaxation differences between the groups. The expression of SKCa channel was decreased in DOCA-salt arteries. The expression of BKCa channel a subunit was increased whereas the expression of BKCa channel p subunit was decreased in DOCA-salt arteries. The expression of receptor for activated C kinase 1 (RACK1), which is a binding protein for BKG, channel and negatively modulates its activity, was increased in DOCA-salt arteries. These results suggest that the impairment of beta-AR-mediated relaxation in DOCA-salt mesenteric arteries may be attributable to altered IKCa/SKCa and/or BKCa channels activities rather than cAMP/PKA pathway. Impaired beta-AR-stimulated BKCa channel activity may be due to the imbalance between its subunit expressions and RACK1 upregulation. (C) 2012 Elsevier Ltd. All rights reserved.

Resistance exercise acutely enhances mesenteric artery insulin-induced relaxation in healthy rats

AIMS: We evaluated the mechanisms involved in insulin-induced vasodilatation after acute resistance exercise in healthy rats. MAIN METHODS: Wistar rats were divided into 3 groups: control (CT), electrically stimulated (ES) and resistance exercise (RE). Immediately after acute RE (15 sets with 10 repetitions at 70% of maximal intensity), the animals were sacrificed and rings of mesenteric artery were mounted in an isometric system. After this, concentration-response curves to insulin were performed in control condition and in the presence of LY294002 (PI3K inhibitor), L-NAME (NOS inhibitor), L-NAME+TEA (K(+) channels inhibitor), LY294002+BQ123 (ET-A antagonist) or ouabain (Na(+)/K(+) ATPase inhibitor). KEY FINDINGS: Acute RE increased insulin-induced vasorelaxation as compared to control (CT: Rmax=7.3 ± 0.4% and RE: Rmax=15.8 ± 0.8%; p<0.001). NOS inhibition reduced (p<0.001) this vasorelaxation from both groups (CT: Rmax=2.0 ± 0.3%, and RE: Rmax=-1.2 ± 0.1%), while PI3K inhibition abolished the vasorelaxation in CT (Rmax=-0.1±0.3%, p<0.001), and caused vasoconstriction in RE (Rmax=-6.5 ± 0.6%). That insulin-induced vasoconstriction on PI3K inhibition was abolished (p<0.001) by the ET-A antagonist (Rmax=2.9 ± 0.4%). Additionally, acute RE enhanced (p<0.001) the functional activity of the ouabain-sensitive Na(+)/K(+) ATPase activity (Rmax=10.7 ± 0.4%) and of the K(+) channels (Rmax=-6.1±0.5%; p<0.001) in the insulin-induced vasorelaxation as compared to CT. SIGNIFICANCE: Such results suggest that acute RE promotes enhanced insulin-induced vasodilatation, which could act as a fine tuning to vascular tone.

Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with L-NAME (non selective NOS inhibitor, 100 mu mol/L), 7-nitroindazole (selective nNOS inhibitor, 100 mu mol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, I mu mol/L), glibenclamide (selective blocker of ATP-sensitive K+ channels, 3 mu mol/L) and 4-aminopyridine (selective blocker of voltage-dependent K+ channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O-2(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H2O2) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 mu mol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 mu mol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by L-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca2+ concentration ([Ca2+]c), O-2(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca-2]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity. (C) 2011 Elsevier Inc. All rights reserved.

A new NO donor failed to release NO and to induce relaxation in the rat basilar artery

Nitric oxide (NO)-donors are pharmacologically active substances that in vivo or in vitro release NO. Their most common side effect is headache caused by cerebral vasodilatation. We previously demonstrated that the new NO-donor Ru(terpy)(bdq)NO](3+) (Terpy), synthesized in our laboratory, induces relaxation of rat aorta. This study aimed to verify the effect of Terpy and sodium nitroprusside (SNP) in basilar artery. We conducted vascular reactivity experiments on endothelium-denuded basilar rings. The concentrations of iron (Fe) and ruthenium (Ru) complex were analyzed in basilar artery lysates after incubation with NO donors by mass spectrometry. We also evaluated the NO released from SNP and Terpy by using confocal microscopy. Interestingly, Terpy did not induce relaxation of the basilar artery. SNP induced relaxation in a concentration-dependent way. NO donors cross the membrane of vascular smooth muscle and entered the cell. In spite of its permeability, Terpy did not release NO in the basilar artery. Otherwise, SNP released NO in the basilar artery cells cytoplasm. Taken together, our results demonstrate that the new NO donor (Terpy) failed to release NO and to induce relaxation in the basilar artery. The NO donor SNP induces vascular relaxation due to NO release in the vascular smooth muscle cells. (C) 2011 Elsevier B.V. All rights reserved.

Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

The protective effect of cilostazol on isolated rabbit femoral arteries under conditions of ischemia and reperfusion: the role of the nitric oxide pathway

OBJECTIVES: The clinical significance of ischemia/reperfusion of the lower extremities demands further investigation to enable the development of more effective therapeutic alternatives. This study investigated the changes in the vascular reactivity of the rabbit femoral artery and nitric oxide metabolites under partial ischemia/reperfusion conditions following cilostazol administration. METHODS: Ischemia was induced using infrarenal aortic clamping. The animals were randomly divided into seven groups: Control 90 minutes, Ischemia/Reperfusion 90/60 minutes, Control 120 minutes, Ischemia/Reperfusion 120/90 minutes, Cilostazol, Cilostazol before Ischemia/Reperfusion 120/90 minutes, and Ischemia 120 minutes/Cilostazol/Reperfusion 90 minutes. Dose-response curves for sodium nitroprusside, acetylcholine, and the calcium ionophore A23187 were obtained in isolated femoral arteries. The levels of nitrites and nitrates in the plasma and skeletal muscle were determined using chemiluminescence. RESULTS: Acetylcholine- and A23187-induced relaxation was reduced in the Ischemia/Reperfusion 120/90 group, and treatment with cilostazol partially prevented this ischemia/reperfusion-induced endothelium impairment. Only cilostazol treatment increased plasma levels of nitrites and nitrates. An elevation in the levels of nitrites and nitrates was observed in muscle tissues in the Ischemia/Reperfusion 120/90, Cilostazol/Ischemia/Reperfusion, and Ischemia/Cilostazol/Reperfusion groups. CONCLUSION: Hind limb ischemia/reperfusion yielded an impaired endothelium-dependent relaxation of the femoral artery. Furthermore, cilostazol administration prior to ischemia exerted a protective effect on endothelium-dependent vascular reactivity under ischemia/reperfusion conditions.

Hypotensive and vasorelaxing effects of the new NO-donor [Ru(terpy)(bdq)NO+](3+) in spontaneously hypertensive rats

Drugs that release nitric oxide (NO) usually have limitations due to their harmful effects. Sodium nitroprusside (SNP) induces a rapid hypotension that leads to reflex tachycardia, which could be an undesirable effect in patients with heart disease, a common feature of hypertension. The nitrosyl ruthenium complex [Ru(terpy)(bdq)NO+](3+) (TERPY) is a NO donor that is less potent than SNP in denuded aortic rings. This study evaluated the hypotension and vasorelaxation induced by this NO donor in Wistar (W) and spontaneously hypertensive rats (SHR) and compared to the results obtained with SNP. Differently from the hypotension induced by SNP, the action of TERPY was slow, long lasting and it did not lead to reflex tachycardia in both groups. The hypotension induced by the NO-donors was more potent in SHR than in W. TERPY induced relaxation with similar efficacy to SNP, although its potency is lower in both strains. The relaxation induced by TERPY is similar in W and SHR, but SNP is more potent and efficient in SHR. The relaxation induced by TERPY is partially dependent on guanylate cyclase in SHR aorta. The NO released from the NO donors measured with DAF-2 DA by confocal microscopy shows that TERPY releases similar amounts of NO in W and SHR, while SNP releases more NO in SHR aortic rings. (c) 2012 Elsevier Inc. All rights reserved.

Spectral Induced Polarization (SIP) signatures of clayey soils containing toluene

We performed laboratory experiments to investigate the sensitivity of the Spectral Induced Polarization (SIP) method to toluene contamination in clayey soils. We used mixtures of quartzitic sand and montmorillonite as soil samples, artificially contaminated with varying amounts of toluene. Care was taken to quantify the experimental uncertainty resulting from packing since such effects must be quantified if variations in SIP signatures between samples are to be reliably interpreted in terms of the effects of hydrocarbon concentration. The SIP response of all samples following addition of toluene was monitored for a period of 40 days following sample preparation. Stepwise regression was used to examine the statistical significance of correlations between (i) clay content and (ii) toluene concentration and SIP parameters. Both single-frequency real and imaginary conductivity measurements, along with the integral chargeability, normalized chargeability, DC conductivity and time constant obtained from a Debye decomposition fitting, were examined in this regression analysis. The SIP measurements show a clear time dependence following sample preparation, indicating that samples containing toluene may take significant time to reach an equilibrium electrical response. SIP measurements are significantly related to toluene content shortly after sample preparation, when the expected dependence of SIP on clay concentration is apparently suppressed. However, for the state of electrical equilibrium after 40 days (interpreted to indicate surface chemistry at equilibrium) there is no significant relation between SIP measurements and toluene content; instead SIP measurements are then significantly correlated with clay concentration. The total chargeability, normalized chargeability and relaxation time obtained from the Debye decomposition show no correlation with toluene content, indicating that this procedure, which likely integrates over multiple mechanisms, may not be suitable for understanding relationships between SIP and hydrocarbon contamination. We find only small low-frequency polarization signals observed in relation to toluene concentration (2 mrad at 0.01 Hz), which initially decreases the interfacial polarization. Unlike earlier works, our results do not support the use of the SIP method as a tool for monitoring toluene contamination in clay soils.

Endothelial dysfunction in the pulmonary artery induced by concentrated fine particulate matter exposure is associated with local but not systemic inflammation

Clinical evidence has identified the pulmonary circulation as an important target of air pollution. It was previously demonstrated that in vitro exposure to fine particulate matter (aerodynamic diameter <= 2.5 mu m, PM2.5) induces endothelial dysfunction in isolated pulmonary arteries. We aimed to investigate the effects of in vivo exposure to urban concentrated PM2.5 on rat pulmonary artery reactivity and the mechanisms involved. For this, adult Wistar rats were exposed to 2 weeks of concentrated Sao Paulo city air PM2.5 at an accumulated daily dose of approximately 600 mu g/m(3). Pulmonary arteries isolated from PM2.5-exposed animals exhibited impaired endothelium-dependent relaxation to acetylcholine without significant changes in nitric oxide donor response compared to control rats. PM2.5 caused vascular oxidative stress and enhanced protein expression of Cu/Zn- and Mn-superoxide dismutase in the pulmonary artery. Protein expression of endothelial nitric oxide synthase (eNOS) was reduced, while tumor necrosis factor (TNF)-alpha was enhanced by PM2.5 inhalation in pulmonary artery. There was a significant positive correlation between eNOS expression and maximal relaxation response (E-max) to acetylcholine. A negative correlation was found between vascular TNF-alpha expression and E-max to acetylcholine. Plasma cytokine levels, blood cells count and coagulation parameters were similar between control and PM2.5-exposed rats. The present findings showed that in vivo daily exposure to concentrated urban PM2.5 could decrease endothelium-dependent relaxation and eNOS expression on pulmonary arteries associated with local high TNF-alpha level but not systemic pro-inflammatory factors. Taken together, the present results elucidate the mechanisms underlying the trigger of cardiopulmonary diseases induced by urban ambient levels of PM2.5. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Long-lasting Hypotensive Effect in Renal Hypertensive Rats Induced by Nitric Oxide Released From a Ruthenium Complex

In this study, we investigated the effect of the ruthenium complex [Ru(terpy)(bdq)NO+](3+) (TERPY) on the arterial pressure from renal hypertensive 2 kidney-1 clip (2K-1C) rats, which was compared with sodium nitroprusside (SNP). The most interesting finding was that the intravenous bolus injection of TERPY (2.5, 5.0, 7 mg/kg) had a dose-dependent hypotensive effect only in 2K-1C rats. On the other hand, SNP (35 and 70 mu g/kg) presented a similar hypotensive effect in both normotensive (2K) and 2K-1C although the effect of 70 mu g/kg was >35 mu g/kg. The injection of the nonselective NO-synthase inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME) increased the arterial pressure in 2K and 2K-1C rats with a similar magnitude. After infusion of L-NAME, the hypotensive effect induced by TERPY and SNP was potentiated in both 2K and in 2K-1C rats. The administration of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl increased the hypotensive effect induced by TERPY or SNP in both 2K and 2K-1C rats. The hypotensive effect induced by TERPY was longer than that produced by SNP. Taken together, our results show that the TERPY has a long-lasting hypotensive effect, which has a dose dependence and higher magnitude in 2K-1C compared with in 2K rats. In comparison with SNP, TERPY is less potent in inducing arterial pressure fall, but it presents a much longer hypotensive effect.

A simple analysis of the influence of the solvent-induced electronic polarization on the N-15 magnetic shielding of pyridine in water

Electronic polarization induced by the interaction of a reference molecule with a liquid environment is expected to affect the magnetic shielding constants. Understanding this effect using realistic theoretical models is important for proper use of nuclear magnetic resonance in molecular characterization. In this work, we consider the pyridine molecule in water as a model system to briefly investigate this aspect. Thus, Monte Carlo simulations and quantum mechanics calculations based on the B3LYP/6-311++G (d,p) are used to analyze different aspects of the solvent effects on the N-15 magnetic shielding constant of pyridine in water. This includes in special the geometry relaxation and the electronic polarization of the solute by the solvent. The polarization effect is found to be very important, but, as expected for pyridine, the geometry relaxation contribution is essentially negligible. Using an average electrostatic model of the solvent, the magnetic shielding constant is calculated as -58.7 ppm, in good agreement with the experimental value of -56.3 ppm. The explicit inclusion of hydrogen-bonded water molecules embedded in the electrostatic field of the remaining solvent molecules gives the value of -61.8 ppm.

Low-dose Enalapril Reduces Angiotensin II and Attenuates Diabetic-induced Cardiac and Autonomic Dysfunctions

Activation of renin-angiotensin system has been linked to cardiovascular and autonomic dysfunctions in diabetes. Experiments were performed to investigate the effects of angiotensin-converting enzyme inhibitor (ACEI), enalapril, on cardiac and autonomic functions in diabetic rats. Diabetes was induced by streptozotocin (50 mg/kg), and rats were treated with enalapril (1 mg.kg(-1).d(-1)). After 30 days, evaluations were performed in control, diabetic, and enalapril-treated groups. Cardiac function was evaluated by echocardiography and through cannulation of the left ventricle (at baseline and in response to volume overload). Heart rate and systolic blood pressure variabilities were evaluated in the time and frequency domains. Streptozotocin rats had left ventricular systolic and diastolic dysfunctions, expressed by reduced ejection fraction and increased isovolumic relaxation time. The ACEI prevented these changes, improved diastolic cardiac responses to volume overload and total power of heart rate variability, reduced the ACE1 activity and protein expression and cardiac angiotensin (Ang) II levels, and increased angiotensin-converting enzyme 2 activity, despite unchanged blood pressure. Correlations were obtained between Ang II content with systolic and diastolic functions and heart rate variability. These findings provide evidence that the low-dose ACEI prevents autonomic and cardiac dysfunctions induced by diabetes without changing blood pressure and associated with reduced cardiac Ang II and increased angiotensin-converting enzyme 2 activity.

Atomically resolved study of the morphology change of InAs/GaAs quantum dot layers induced by rapid thermal annealing

The optoelectronic properties of InAs/GaAs quantum dots can be tuned by rapid thermal annealing. In this study, the morphology change of InAs/GaAs quantum dots layers induced by rapid thermal annealing was investigated at the atomic-scale by cross-sectional scanning tunneling microscopy. Finite elements calculations that model the outward relaxation of the cleaved surface were used to determine the indium composition profile of the wetting layer and the quantum dots prior and post rapid thermal annealing. The results show that the wetting layer is broadened upon annealing. This broadening could be modeled by assuming a random walk of indium atoms. Furthermore, we show that the stronger strain gradient at the location of the quantum dots enhances the intermixing. Photoluminescence measurements show a blueshift and narrowing of the photoluminescence peak. Temperature dependent photoluminescence measurements show a lower activation energy for the annealed sample. These results are in agreement with the observed change in morphology. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4770371]