Edgaras Stankevicius

Combination of Ca2+-activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

The present study investigated whether calcium‐activated K+ channels are involved in acetylcholine‐evoked nitric oxide (NO) release and relaxation.

Disruption of Na+,HCO3− Cotransporter NBCn1 (slc4a7) Inhibits NO-Mediated Vasorelaxation, Smooth Muscle Ca2+ Sensitivity, and Hypertension Development in Mice

Background— Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na+,HCO3− cotransporter NBCn1, by regulating intracellular pH (pHi), influences artery function and blood pressure regulation. Methods and Results— Knockout of NBCn1 in mice eliminated Na+,HCO3− cotransport and caused a lower steady-state pHi in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca2+ sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester–sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca2+ responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO2/HCO3−, no differences in pHi, contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. Conclusions— Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase–dependent signaling in isolated arteries and perturbs blood pressure regulation.

Novel approaches to improving endothelium-dependent nitric oxide-mediated vasodilatation

Endothelial dysfunction, which is defined by decreased endothelium-dependent vasodilatation, is associated with an increased number of cardiovascular events. Nitric oxide (NO) bioavailability is reduced by altered endothelial signal transduction or increased formation of radical oxygen species reacting with NO. Endothelial dysfunction is therapeutically reversible and physical exercise, calcium channel blockers, angiotensin converting enzyme inhibitors, and angiotensin receptor antagonists improve flow-evoked endothelium-dependent vasodilation in patients with hypertension and diabetes. We have investigated three different approaches, with the aim of correcting endothelial dysfunction in cardiovascular disease. Thus, (1) we evaluated the effect of a cell permeable superoxide dismutase mimetic, tempol, on endothelial dysfunction in small arteries exposed to high pressure, (2) investigated the endothelial signal transduction pathways involved in vasorelaxation and NO release induced by an olive oil component, oleanolic acid, and (3) investigated the role of calcium-activated K channels in the release of NO induced by receptor activation. Tempol increases endothelium-dependent vasodilatation in arteries from hypertensive animals most likely through the lowering of radical oxygen species, but other mechanisms also appear to contribute to the effect. While oleanolic acid leads to the release of NO by calcium-independent phosphorylation of endothelial NO synthase, endothelial calcium-activated K channels and an influx of calcium play an important role in G-protein coupled receptor-evoked release of NO. Thus, all three approaches increase bioavailability of NO in the vascular wall, but it remains to be addressed whether these actions have any direct benefit at a clinical level.

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SKCa or KCa2) and intermediate (IKCa or KCa3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SKCa and IKCa channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both NG,NG-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SKCa and IKCa channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IKCa channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SKCa and IKCa channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SKCa and IKCa channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Oleanolic acid induces relaxation and calcium‐independent release of endothelium‐derived nitric oxide

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO).

Physiologically Relevant Measurements of Nitric Oxide in Cardiovascular Research Using Electrochemical Microsensors

Nitric oxide (NO) plays an important role in the regulation of blood flow. Pharmacological tools and a series of other techniques have been developed for studying the NO/L-arginine pathway, but it has proved difficult to make a quantitative link between effect and tissue NO concentration. NO microsensors have been applied with success for the measurement of NO in suspensions of mitochondria and cells, such as platelets and leukocytes, and in cell cultures, which together with other interventions or measurements are particularly useful for the examination of cell signalling related to the NO/L-arginine pathway. In isolated vascular segments, studies using the NO microsensor have defined the relationship between NO concentration and relaxation and revealed residual NO release in the presence of NO synthase inhibitors. Moreover, simultaneous measurements of NO concentration and vasorelaxation in isometric preparations have shown that agonist-induced relaxation is L-arginine dependent and NO release is reduced in hypertension. By placing NO microsensors in catheters, it is possible to measure NO in the living animal and man. This approach has been applied for the measurements of NO concentration in relation to increases in flow, erection, in conditions of hypoxia, and in endotoxemia. However, further methodological development of NO microsensors is necessary to avoid the influence of changes in temperature, pH and oxygen on the measurements.

Blunted acetylcholine relaxation and nitric oxide release in arteries from renal hypertensive rats.

Objective Investigation of the effect of hypertension on endothelium-dependent relaxation and release of nitric oxide (NO) in normotensive and renal hypertensive rats. Design and methods Sprague-Dawley rats were randomly allocated into two groups: uninephrectomized controls and one-kidney one-clip (Goldblatt hypertension) hypertensive rats, a non-renin dependent model of hypertension. After 10 weeks and in the presence of the cyclooxygenase inhibitor indomethacin, simultaneous measurements of the NO concentration, measured with a NO-specific microelectrode and endothelium-dependent relaxation were performed in isolated rat superior mesenteric arteries. Results Addition of the NO scavenger, oxyhaemoglobin, showed that basal NO concentration was unaltered in arterial segments from hypertensive rats. In norepinephrine-contracted arteries, acetylcholine increased the NO concentration and caused relaxations, and both parameters were significantly reduced in renal hypertensive arteries. Relaxations induced by the NO donor, S-nitroso-N-acetylpenicillamine were reduced. The superoxide scavenger, superoxide dismutase, and the NO synthase substrate, l-arginine, did not change the increase in NO concentration or acetylcholine relaxation in arteries from normotensive or renal hypertensive animals. In contrast, the NO synthase inhibitor, asymmetric dimethyl l-arginine, reduced the NO concentration and acetylcholine relaxation, while these responses were abolished in the presence of oxyhaemoglobin. Conclusions This study provides direct evidence that reduced endothelium-dependent relaxations in the superior mesenteric artery from renal hypertensive rats is due, at least in part, to diminished NO release. The reduced NO release and relaxation persist in the presence of excess of substrate for NO synthase.

Hyperoxia reduces basal release of nitric oxide and contracts porcine coronary arteries

Aim:  The purpose of the present study was to investigate whether changes in nitric oxide (NO) concentration is involved in hyperoxia‐induced vasoconstriction in porcine conduit coronary arteries.

NS11021, a novel opener of large-conductance Ca2+-activated K+ channels, enhances erectile responses in rats

Background and purpose:  Large‐conductance Ca2+‐activated K+ channels (BKCa), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1‐(3,5‐Bis‐trifluoromethyl‐phenyl)‐3‐[4‐bromo‐2‐(1H‐tetrazol‐5‐yl)‐phenyl]‐thiourea), a novel opener of BKCa channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5.

Antiatherogenic effects of oleanolic acid in apolipoprotein E knockout mice

Oleanolic acid (OA) is a plant triterpenoid steroid with potentially antiatherogenic properties. We investigated whether OA affected atherosclerosis development and vascular function in apolipoprotein E knockout (ApoE(-/-)) mice. ApoE(-/-) mice were fed a high cholesterol Western-type diet in combination with OA (100 mg/kg/day), fluvastatin (5 mg/kg/day) or vehicle, with wild type (WT) mice serving as controls. After 8 weeks of treatment atherosclerotic plaque areas in the aortic arch and plasma lipid concentrations were determined. Vasoconstriction and relaxation of the proximal part of aorta were investigated in vitro. Inducible nitric oxide synthase (iNOS) was visualized using immunoblotting. As opposed to WT and fluvastatin- and vehicle-treated mice, OA-fed ApoE(-/-) mice gained no weight during the treatment period. Plasma concentrations of total-cholesterol and triglyceride were not significantly reduced by OA- or fluvastatin treatment. Plaque area of vehicle-treated mice was 25%, but only 14% in OA- and 19% in fluvastatin-treated mice. As compared to WT, vasoconstriction to phenylephrine was attenuated in ApoE(-/-) mice. The NOS inhibitor asymmetric dimethylarginine (ADMA) enhanced phenylephrine constriction, but significantly more so in vehicle- and fluvastatin-treated than in OA-treated and WT mice. Relaxation to acetylcholine was only slightly attenuated in ApoE(-/-) mice and not affected by OA or fluvastatin treatment. ADMA abolished acetylcholine relaxation almost completely. In ApoE(-/-) mice iNOS expression was reduced by OA treatment. In conclusion OA exerts potent antiatherogenic effects independent of plasma lipid levels and without major changes in eNOS-mediated acetylcholine relaxation. However, OA reduced iNOS expression possibly altering vascular reactivity to phenylephrine.