Nitric Oxide Synthesis L-arginine Research Articles

Сhanges in subcellular distribution of lysosomal cysteine proteinases activity in parenchymatous organs of rats under the action of nitric oxide synthesis modulators

Aim. To study the effect of non-selective inhibitor of NO-synthase N-nitro-L-arginine methyl ester (L-NAME) and substrate of nitric oxide synthesis L-arginine on the activity of cathepsins B, L, H and its subcellular distribution in liver, kidney and lung tissues.Materials and methods. The object of study – male rats Wistar line, the material was the cytoplasmic and lysosomal fraction of homogenates of liver, kidney, lung tissues. A non-selective inhibitor of inducible NO-synthase N-nitro-L-arginine methyl ester (L-NAME) was applied at a dose of 25 mg/kg, the substrate of NO synthesis L-arginine – at a dose of 500 mg/kg. Activity of cathepsins B, L, H was defined separately in the cytoplasmic and lysosomal fractions by spectrofluorometry quantitative determination of the specific substrate cleavage product 7-amido-4-methylcoumarin.Results. Suppression of nitric oxide synthesis by non-selective inhibitor of NO-synthase L-NAME (25 mg/kg, 7 days) in the kidney tissue leads to a decrease in the activity of cathepsins В, L, H in lysosomal fraction with a parallel increase in non-lysosomal activity of cathepsin L, in the liver tissue leads to an increase in lysosomal activity of cathepsin H and a decrease in non-lysosomal activity of cathepsin L. The substrate of nitric oxide synthesis L-arginine (500 mg/kg, 10 days) only causes increased activity of cathepsin L in non-lysosomal fraction of liver tissue, leads to increased lysosomal activity of cathepsin H in kidney tissue, the lung tissue shows a significant increase in the activity of the all studied cathepsins in non-lysosomal fraction, accompanied by an increase in lysosomal activity of cathepsins B and H. The revealed changes are associated with the signs of changes in the ratio of pro-enzyme and active forms of cathepsins.Conclusion. The effects of non-selective inhibitor and substrate of nitric oxide synthesis on the total activity of cathepsins B, L and H in parenchymatous organs and its subcellular distribution are tissue-specific and multidirectional in some cases and are accompanied by signs of changes in the ratio of pro-enzyme and enzymatically active forms mainly due to an increase of pro-enzyme forms.

Hypoargininemia exacerbates airway hyperresponsiveness in a mouse model of asthma

BackgroundAsthma is a chronic respiratory condition, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. The hypothesis that the substantially increased expression of arginase 1 in activated macrophages limits the availability of L-arginine for nitric oxide synthesis, and thus increases AHR in lungs of mice with experimentally induced allergic asthma was recently refuted by several studies. In the present study, we tested the hypothesis that, instead, a low circulating concentration of arginine aggravates AHR in the same murine asthma model. Female FVB F/A2tg/tg transgenic mice, which overexpress rat arginase 1 in their enterocytes, exhibit a ~ 50% decrease of their plasma L-arginine concentration.MethodsAdult female F/A2tg/tg mice and their wild-type littermates (F/A2wt/wt) were sensitized and challenged with ovalbumin (OVA/OVA). Lung function was assessed with the flexiVent™ system. Adaptive changes in the expression of arginine-metabolizing or -transporting enzymes, chemokines and cytokines, and lung histology were quantified with qPCR, ELISA, and immunohistochemistry, respectively.ResultsReduction of circulating L-arginine concentration significantly increased AHR in OVA/OVA-treated mice and, to a lesser extent, even in PBS/OVA-treated mice. The pulmonary inflammatory response in OVA/OVA-treated F/A2tg/tg and F/A2wt/wt mice was comparable. OVA/OVA-treated F/A2tg/tg mice differed from similarly treated female mice, in which arginase 1 expression in lung macrophages was eliminated, by a complete absence of an adaptive increase in the expression of arginine-metabolizing or –transporting enzymes.ConclusionA reduction of the circulating L-arginine concentration rather than the macrophage-mediated increase of arginine catabolism worsens AHR.

Functional evidence of nitrergic neurotransmission in the human urinary bladder neck

Nitric oxide (NO) is involved in the non-adrenergic non-cholinergic (NANC) inhibitory neurotransmission of the lower urinary tract. However, functional evidence of this involvement in the human urinary bladder neck has not been consistently demonstrated. Therefore, the current study investigates the relaxations to endogenously released and/or exogenously added NO, in the human bladder neck. Urothelium-denuded bladder neck strips were dissected and mounted in isolated organ baths, containing a physiological saline solution (PSS) at 37 °C and continuously gassed with 5% CO 2 and 95% O 2, for isometric force recording. The relaxations to transmural nerve stimulation (EFS) or to exogenously applied NO, as an acidified solution of NaNO 2 were carried out on strips precontracted with phenylephrine, and treated with guanethidine and atropine, to block noradrenergic neurotransmission and muscarinic receptors, respectively. EFS (0.5–16 Hz) and exogenous NaNO 2 (1 μM to 1 mM) evoked frequency- and concentration-dependent relaxations, respectively. The nerve responses were abolished by the blockade of neuronal voltage-activated Na + channels with tetrodotoxin, indicating their neurogenic character. N G-nitro- l-arginine ( l-NOARG), a NO synthase inhibitor, abolished the relaxations to nerve stimulation, which were partially reversed by the substrate of NO synthesis l-arginine. l-NOARG failed to modify the relaxations to exogenous NaNO 2. These results suggest that NO is the major NANC inhibitory neurotransmitter in the human urinary bladder neck. Blockers of NO synthase could be useful in therapy for the urinary incontinence produced by intrinsic sphincteric deficiency.

Angiotensin II-induced contraction is attenuated by nitric oxide in afferent arterioles from the nonclipped kidney in 2K1C

Two-kidney, one-clip (2K1C) is a model of renovascular hypertension where we previously found an exaggerated intracellular calcium (Ca(i)(2+)) response to ANG II in isolated afferent arterioles (AAs) from the clipped kidney (Helle F, Vagnes OB, Iversen BM. Am J Physiol Renal Physiol 291: F140-F147, 2006). To test whether nitric oxide (NO) ameliorates the exaggerated ANG II response in 2K1C, we studied ANG II (10(-7) mol/l)-induced calcium signaling and contractility with or without the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME). In AAs from the nonclipped kidney, l-NAME increased the ANG II-induced Ca(i)(2+) response from 0.28 +/- 0.05 to 0.55 +/- 0.09 (fura 2, 340 nm/380 nm ratio) and increased contraction from 80 +/- 6 to 60 +/- 6% of baseline (P < 0.05). In vessels from sham and clipped kidneys, l-NAME had no effect. In diaminofluorescein-FM diacetate-loaded AAs from the nonclipped kidney, ANG II increased NO-derived fluorescence to 145 +/- 34% of baseline (P < 0.05 vs. sham), but not in vessels from the sham or clipped kidney. Endothelial NOS (eNOS) mRNA and ser-1177 phosphorylation were unchanged in both kidneys from 2K1C, while eNOS protein was reduced in the clipped kidney compared with sham. Cationic amino acid transferase-1 and 2 mRNAs were increased in 2K1C, indicating increased availability of l-arginine for NO synthesis, but counteracted by decreased scavenging of the eNOS inhibitor asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolase 2. In conclusion, the Ca(i)(2+) and contractile responses to ANG II are blunted by NO release in the nonclipped kidney. This may protect the nonclipped kidney from the hypertension and elevated ANG II levels in 2K1C.

Modulation of the l-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.

Sickle cell anemia is associated with reduced nitric oxide bioactivity in peripheral conduit and resistance vessels.

The purpose of the study was to examine endothelium-dependent and -independent vasodilation of conduit and resistance vessels in sickle cell anemia (HbSS). We measured the effects of intra-arterial infusion of methacholine, sodium nitroprusside, and NG-monomethyl-L-arginine (L-NMMA) on forearm blood flow using venous occlusion plethysmography in eight patients with HbSS and 11 HbAA controls. We assessed vasodilation of the conduit brachial artery using ultrasound in 17 patients with HbSS and 41 nonanemic controls. Forearm blood flow response to methacholine was similar in HbSS and HbAA, while the response to sodium nitroprusside was greater in those with HbSS. Nitric oxide synthase inhibition with L-NMMA attenuated methacholine-induced forearm blood flow to a lesser extent in HbSS compared to HbAA, suggesting diminished nitric oxide (NO) contribution to endothelium-dependent vasodilation. Flow-mediated and nitroglycerin-induced dilation were impaired in HbSS compared to controls and were not affected by the antioxidant vitamin C or the precursor substrate for NO synthesis L-arginine. NO bioactivity is reduced but differs in peripheral conduit and resistance vessels in HbSS. Resistance vessels have preserved response to exogenous NO donors but have diminished contribution of NO to endothelium-dependent vasodilation. Conduit vessels demonstrate impaired vasodilation to endogenous and exogenous NO.

Effects of nitric oxide on hemodynamic changes and metabolism following acute hepatic inflow occlusion in pigs

The purpose of this study was to examine the effects of nitric oxide on systemic hemodynamics and oxygen metabolism following acute hepatic inflow occlusion. Fourteen mongrel pigs received solvent (control group, n = 4), the nitric synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME group, n = 5), or the substrate for nitric oxide synthesis L-arginine (L-arginine group, n = 5) 30 min before hepatic inflow occlusion. Following 30 min of hepatic ischemia, all livers were reperfused. While all pigs in the control group and L-arginine group survived more than 7 days after reperfusion, two of five pigs in the L-NAME group died during hepatic ischemia period. However, venous oxygen saturation was significantly lower during and after ischemic period, oxygen extraction ratio was significantly higher during hepatic ischemic period in L-NAME group, and systemic vascular resistance was also significantly higher 5 and 15 min after hepatic inflow occlusion. Furthermore, lactate/pyruvate ratio was significantly higher during hepatic ischemic period in L-NAME group. In summary, inhibition of nitric oxide synthesis resulted in maintaining higher arterial blood pressure, but aggravated tissue oxygenation during and after hepatic inflow occlusion. We conclude that nitric oxide appears to decrease arterial blood pressure, but improve the tissue oxygenation in acute hepatic inflow occlusion and reperfusion.

Inhibition of arginase by [formula omitted] in alveolar macrophages: implications for the utilization of l-arginine for nitric oxide synthesis

The hypothesis was investigated that the nitric oxide (NO) synthase intermediate, N G - hydroxy- l-arginine (HOArg), is an arginase inhibitor in rabbit or rat alveolar macrophages. Exogenously applied HOArg strongly inhibited the arginase activity present in these cells (IC 50 ≥ 15 μM), and attenuated l-[ 3H]arginine transport (IC 50 ≥ 500 μM) in rabbit alveolar macrophages. Moreover, up to 37 μM HOArg were detected in the conditioned medium, but not in the lysate, of rat alveolar macrophages exposed to bacterial lipopolysaccharide for 18 h. HOArg may thus be a potent endogenous arginase inhibitor in these cells which increases the availability of l-arginine for NO biosynthesis.