Sona Cacanyiova

The reaction products of sulfide and S-nitrosoglutathione are potent vasorelaxants.

The chemical interaction of sodium sulfide (Na2S) with the NO-donor S-nitrosoglutathione (GSNO) has been described to generate new reaction products, including polysulfides and nitrosopersulfide (SSNO(-)) via intermediacy of thionitrous acid (HSNO). The aim of the present work was to investigate the vascular effects of the longer-lived products of the Sulfide/GSNO interaction. Here we show that the products of this reaction relax precontracted isolated rings of rat thoracic aorta and mesenteric artery (but to a lesser degree rat uterus) with a >2-fold potency compared with the starting material, GSNO (50 nM), whereas Na2S and polysulfides have little effect at 1-5 µM. The onset of vasorelaxation of the reaction products was 7-10 times faster in aorta and mesenteric arteries compared with GSNO. Relaxation to GSNO (100-500 nM) was blocked by an inhibitor of soluble guanylyl cyclase, ODQ (0.1 and 10 µM), and by the NO scavenger cPTIO (100 µM), but less affected by prior acidification (pH 2-4), and unaffected by N-acetylcysteine (1 mM) or methemoglobin (20 µM heme). By contrast, relaxation to the Sulfide/GSNO reaction products (100-500 nM based on the starting material) was inhibited to a lesser extent by ODQ, only slightly decreased by cPTIO, more markedly inhibited by methemoglobin and N-acetylcysteine, and abolished by acidification before addition to the organ bath. The reaction mixture was found to generate NO as detected by EPR spectroscopy using N-(dithiocarboxy)-N-methyl-D-glucamine (MGD2)-Fe(2+) as spin trap. In conclusion, the Sufide/GSNO reaction products are faster and more pronounced vasorelaxants than GSNO itself. We conclude that in addition to NO formation from SSNO(-), reaction products other than polysulfides may give rise to nitroxyl (HNO) and be involved in the pronounced relaxation induced by the Sulfide/GSNO cross-talk.

Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels.

H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl(-). Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25-1 µmol kg(-1)i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca(2+) channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca(2+) ryanodine (RyR2) and Cl(-) single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25-1 µmol kg(-1) i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca(2+) current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl(-) channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby (•)NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl(-) cardiac membrane channels might be involved in its biological actions.

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.

Effect of chronic nNOS inhibition on blood pressure, vasoactivity, and arterial wall structure in Wistar rats.

While the unequivocal pattern of endothelial nitric oxide (NO) synthase (eNOS) inhibition in cardiovascular control has been recognised, the role of NO produced by neuronal NOS (nNOS) remains unclear. The purpose of the present study was to describe the cardiovascular effects of NO production interference by inhibition of nNOS with 7-nitroindazole (7-NI). Wistar rats (10 weeks old) were used: control and experimental rats were administered 7-NI 10 mg/kg b.w./day in drinking water for 6 weeks. Systolic blood pressure (BP) was measured by the tail-cuff plethysmographic method. Isolated thoracic aortas (TAs) were used to study vasomotor activity of the conduit artery in vitro. The BP response of anaesthetised animals was used to follow the cardiovascular-integrated response in vivo. Geometry of the TA was measured after perfusion fixation (120 mm Hg) by light microscopy. Expression of eNOS was measured in the TA by immunoblot analysis. Although 6 weeks of nNOS inhibition did not alter systolic BP, the heart/body weight ratio was decreased. Relaxation of the TA in response to acetylcholine (10(-9)-10(-5)mol/L) was moderately inhibited. However, no difference in the BP hypotensive response after acetylcholine (0.1, 1, 10 microg) was observed. The contraction of TA in response to noradrenaline (10(-10)-10(-5)mol/L), and the BP pressor response to noradrenaline (0.1, 1 microg) was attenuated. The inner diameter of the TA was increased, and the wall thickness, wall cross-sectional area, and wall thickness/inner diameter ratio were decreased. The expression of eNOS in the TA was increased. In summary, cardiac and TA wall hypotrophy, underlined by decreased contractile efficiency, were observed. The results suggested that two constitutive forms of NOS (nNOS, eNOS) likely participate in regulation of cardiovascular tone by different mechanisms.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang‐(1‐7)/Mas receptor axis, renin‐angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT‐PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up‐regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang‐(1‐7) in organ response to the developing hypertension in SHRs.

Effects of PPARγ Agonist Pioglitazone on Redox-Sensitive Cellular Signaling in Young Spontaneously Hypertensive Rats

PPARγ receptor plays an important role in oxidative stress response. Its agonists can influence vascular contractility in experimental hypertension. Our study was focused on the effects of a PPARγ agonist pioglitazone (PIO) on blood pressure regulation, vasoactivity of vessels, and redox-sensitive signaling at the central (brainstem, BS) and peripheral (left ventricle, LV) levels in young prehypertensive rats. 5-week-old SHR were treated either with PIO (10 mg/kg/day, 2 weeks) or with saline using gastric gavage. Administration of PIO significantly slowed down blood pressure increase and improved lipid profile and aortic relaxation after insulin stimulation. A significant increase in PPARγ expression was found only in BS, not in LV. PIO treatment did not influence NOS changes, but had tissue-dependent effect on SOD regulation and increased SOD activity, observed in LV. The treatment with PIO differentially affected also the levels of other intracellular signaling components: Akt kinase increased in the the BS, while β-catenin level was down-regulated in the BS and up-regulated in the LV. We found that the lowering of blood pressure in young SHR can be connected with insulin sensitivity of vessels and that β-catenin and SOD levels are important agents mediating PIO effects in the BS and LV.

Lipids modulate H2S/HS− induced NO release from S-nitrosoglutathione

Recently we observed that a gas messenger H(2)S/HS(-) released NO from S-nitrosoglutathione (Ondrias et al., Pflugers Arch. 457 (2008) 271-279). However, the effect of biological compounds on the release is not known. Measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that unsaturated fatty acid-linoleic acid and lipids having unsaturated fatty acids: asolectin, dioleoylphosphocholine and dioleoylphosphoserine depressed the H(2)S/HS(-) induced NO release from S-nitrosoglutathione. On the other hand, a depression effect of the saturated fatty acid-myristic acid and lipids having saturated fatty acids, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine was less pronounced. The inhibition effect increased with the decreasing gel-to-liquid phase transitions temperature of the fatty acids and lipids. We suggest that lipid composition of biological membranes modulates NO release from nitrosoglutathione induced by H(2)S/HS(-), assuming that a reaction of H(2)S/HS(-) with unsaturated bonds of fatty acids may be partially responsible for the effect.

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings.

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys >> GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 µmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.

Long-term effect of prazosin and losartan administration on blood pressure, heart, carotid artery, and acetylcholine induced dilation of cardiovascular system of young Wistar rats and SHR.

The long-term effects of prazosin and losartan administration on blood pressure, trophicity of the heart and carotid arteries, and responses of the cardiovascular system to acetylcholine, were studied in Wistar rats and spontaneously hypertensive rats (SHRs). Four-week-old rats were treated with prazosin (10 mg/kg b.w./day in tap water) or losartan (20 mg/kg b.w./day in tap water) for 5-6 weeks. BP was measured by plethysmographic method. Ten animals of each group were subjected to in vivo studies and subsequent to morphological investigations. The right jugular vein was cannulated for administration of acetylcholine (0.1, 1, and 10 µg). After perfusion with a glutaraldehyde fixative (120 mmHg), the carotid arteries were embedded in Durcupan ACM, and the inner diameter (ID), wall thickness (WT) (tunica intima and media), cross sectional area (CSA) (tunica intima and media), and WT/ID ratio were calculated. In Wistar rats and SHRs, prazosin and losartan administration produced a decrease in the blood pressure and trophicity of the heart. In Wistar rats, both drugs decreased the WT, CSA, and the WT/ID ratio. In addition, these drugs increased the circumferential stress of the artery without affecting the ID. In contrast, in the SHRs, only losartan administration produced these effects. Importantly, both the drugs improved the responses to acetylcholine in SHRs.

The Effect of Chronic NO Synthase Inhibition on the Vasoactive and Structural Properties of Thoracic Aorta, NO Synthase Activity, and Oxidative Stress Biomarkers in Young SHR

Although the role of nitric oxide (NO) in essential hypertension is still unclear, the effects of long-term NO deficiency have not yet been investigated during the critical juvenile period in spontaneously hypertensive rats (SHR). We aimed to analyze the effects of chronic NO synthase (NOS) inhibition on systolic blood pressure (sBP), vasoactivity, morphological changes and superoxide level in the thoracic aorta (TA), NOS activity in different tissues, and general biomarkers of oxidative stress in plasma of young SHR. Four-week-old SHR were treated with NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day, p.o.) for 4-5 weeks. L-NAME treatment induced a transient sBP increase only, and surprisingly, slightly inhibited endothelium-dependent relaxation of TA. Hereby, the inhibition of NOS activity varied from tissue to tissue, ranging from the lowest in the TA and the kidney to the highest in the brain stem. In spite of an increased sensitivity of adrenergic receptors, the maximal adrenergic contraction of TA was unchanged, which was associated with changes in elastin arrangement and an increase in wall thickness. The production of reactive oxygen species in the TA was increased; however, the level of selected biomarkers of oxidative stress did not change. Our findings proved that the TA of young SHR responded to chronic NO deficiency by the development of adaptive mechanisms on the functional (preserved NO-derived vasorelaxation, unincreased contraction) and molecular (preserved NOS activity) level.