Oxidative stress attenuates NO-induced modulation of sympathetic neurotransmission in the mesenteric arterial bed of spontaneously hypertensive rats

Current evidence suggests that hyperactivity of the sympathetic nervous system and endothelial dysfunction are important factors in the development and maintenance of hypertension. Under normal conditions the endothelial mediator nitric oxide (NO) negatively modulates the activity of the norepinephrine portion of sympathetic neurotransmission, thereby placing a "brake" on the vasoconstrictor ability of this transmitter. This property of NO is diminished in the isolated, perfused mesenteric arterial bed taken from the spontaneously hypertensive rat (SHR), resulting in greater nerve-stimulated norepinephrine and lower neuropeptide Y (NPY) overflow from this mesenteric preparation compared with that of the normotensive Wistar-Kyoto rat (WKY). We hypothesized that increased oxidative stress in the SHR contributes to the dysfunction in the NO modulation of sympathetic neurotransmission. Here we demonstrate that the antioxidant N-acetylcysteine reduced nerve-stimulated norepinephrine and increased NPY overflow in the mesenteric arterial bed taken from the SHR. Furthermore, this property of N-acetylcysteine was prevented by inhibiting nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester, demonstrating that the effect of N-acetylcysteine was due to the preservation of NO from oxidation. Despite a reduction in norepinephrine overflow, the nerve-stimulated perfusion pressure response in the SHR mesenteric bed was not altered by the inclusion of N-acetylcysteine. Studies including the Y(1) antagonist BIBO 3304 with N-acetylcysteine demonstrated that this preservation of the perfusion pressure response was due to elevated NPY overflow. These results demonstrate that the reduction in the bioavailability of NO as a result of elevated oxidative stress contributes to the increase in norepinephrine overflow from the SHR mesenteric sympathetic neuroeffector junction.     

Effects of long-term losartan and L-arginine treatment on haemodynamics, glomerular filtration, and SOD activity in spontaneously hypertensive rats

Recently, it has been reported that losartan, an angiotensin II receptor (ATR) antagonist, depresses the angiotensin II-induced production of superoxide radicals. Also, in spontaneously hypertensive rats (SHR) endothelial dysfunction is associated with decreased nitric oxide (NO) synthesis. In this study, we examined the effects of long-term ATR blockade and L-arginine supplementation on the haemodynamic parameters, glomerular filtration, and oxidative status in SHR. Adult male SHR were treated with losartan (10 mg/kg) and with the NO donor L-arginine (2 g/kg) for 4 weeks. The animals were divided into the following experimental groups: control (n = 7), L-arginine (n = 7), losartan (n = 7), and L-arginine + losartan (n = 7). Mean arterial pressure (MAP), regional blood flow, urea clearance, and activity of superoxide dismutase (SOD) were measured at the end of treatment. MAP was significantly reduced in the losartan group compared with the control group (133.3 +/- 7.3 vs. 161.5 +/- 14.5 mm Hg). Aortic blood flow was significantly higher and aortic vascular resistance was significantly lower in all treated groups than in the control. Urea clearance rose significantly in the L-arginine + losartan group compared with control (393.27 +/- 37.58 vs. 218.68 +/- 42.03 microL x min(-1) x 100 g(-1)) as did the activity of SOD (1668.97 +/- 244.57 vs. 1083.18 +/- 169.96 U/g Hb). Our results suggest that the antihypertensive effect of losartan and L-arginine in SHR is not primarily mediated by increased SOD activity. Also, combined treatment with ATR blockade and L-arginine supplementation has a beneficial effect on renal function that is, at least in part, mediated by increased SOD activity in SHR.     

The degree of cerebal oxidative stress in rats during ischemia-reperfusion combined with a modulaion of L-arginine-NO system

We estimated the cerebral oxidative stress during ischemia-reperfusion of the brain in conditions of modulated L-arginine-NO system in rats. In 128 rats, the changes in lipid peroxidation products (conjugated dienes, malondialdehyde, Schiff bases) and antioxidant defense factors (retinal, a-tocopherol, SH-groups) were studied. Analysis of the inhibitor (administration of NO synthase substrate L-arginine, non-selective inhibitor Nw-nitro-L-arginine methyl ester, selective inhibitor of neuronal NO synthase 7-nitroindasole, or selective inhibitor of inducible NO synthase S-methyl-isothiourea) showed the NO-dependent nature of oxidative stress in rats with brain ischemia-reperfusion. NO generated by different NO synthase isoenzymes played different roles: hyperactivation of the neuronal NO synthase was associated with oxidative stress during both brain ischemia-reperfusion periods, and the higher inducible NO synthase activity had such association only during the later period.     

Increasing oxidative stress with molsidomine increases blood pressure in genetically hypertensive rats but not normotensive controls

Spontaneously hypertensive rats (SHR) have a higher level of oxidative stress and exhibit a greater depressor response to a superoxide scavenger, tempol, than normotensive Wistar-Kyoto rats (WKY). This study determined whether an increase in oxidative stress with a superoxide/NO donor, molsidomine, would amplify the blood pressure in SHR. Male SHR and WKY were given molsidomine (30 mg.kg(-1).day(-1)) or vehicle (0.01% ethanol) for 1 wk, and blood pressure, renal hemodynamics, nitrate and nitrite excretion (NOx), renal superoxide production, and expression of renal antioxidant enzymes, Mn- and Cu,Zn-SOD, catalase, and glutathione peroxidase (GPx), were measured. Renal superoxide and NOx were higher in control SHR than in WKY. Molsidomine increased superoxide by approximately 35% and NOx by 250% in both SHR and WKY. Mean arterial blood pressure (MAP) was also higher in control SHR than WKY. Molsidomine increased MAP by 14% and caused renal vasoconstriction in SHR but reduced MAP by 16%, with no effect on renal hemodynamics, in WKY. Renal expression of Mn- and Cu,Zn-SOD was not different between SHR and WKY, but expression of catalase and GPx were approximately 30% lower in kidney of SHR than WKY. The levels of Mn- and Cu,Zn-SOD were not increased with molsidomine in either WKY or SHR. Renal catalase and GPx expression was increased by 300-400% with molsidomine in WKY, but there was no effect in SHR. Increasing oxidative stress elevated blood pressure further in SHR but not WKY. WKY are likely protected because of higher bioavailable levels of NO and the ability to upregulate catalase and GPx.     

Hydrogen‐rich water alleviates cyclosporine A‐induced nephrotoxicity via the Keap1/Nrf2 signaling pathway

Oxidative stress induced by long‐term cyclosporine A (CsA) administration is a major cause of chronic nephrotoxicity, which is characterized by tubular atrophy, tubular cell apoptosis, and interstitial fibrosis in the progression of organ transplantation. Although hydrogen‐rich water (HRW) has been used to prevent various oxidative stress‐related diseases, its underlying mechanisms remain unclear. This study investigated the effects of HRW on CsA‐induced nephrotoxicity and its potential mechanisms. After administration of CsA (25 mg/kg/day), rats were treated with or without HRW (12 mL/kg) for 4 weeks. Renal function and vascular activity were investigated. Histological changes in kidney tissues were analyzed using Masson's trichrome and terminal deoxynucleotidyl transferase dUTP nick‐end labeling stains. Oxidative stress markers and the activation of the Kelch‐like ECH‐associated protein 1 (Keap1)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling pathway were also measured. We found that CsA increased the levels of reactive oxygen species (ROS) and malonaldehyde (MDA), but it reduced glutathione (GSH) and superoxide dismutase (SOD) levels. Such alterations induced vascular dysfunction, tubular atrophy, interstitial fibrosis, and tubular apoptosis. This was evident secondary to an increase in urinary protein, serum creatinine, and blood urea nitrogen, ultimately leading to renal dysfunction. Conversely, HRW decreased levels of ROS and MDA while increasing the activity of GSH and SOD. This was accompanied by an improvement in vascular and renal function. Moreover, HRW significantly decreased the level of Keap1 and increased the expression of Nrf2, NADPH dehydrogenase quinone 1, and heme oxygenase 1. In conclusion, HRW restored the balance of redox status, suppressed oxidative stress damage, and improved kidney function induced by CsA via activation of the Keap1/Nrf2 signaling pathway.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).     

Upregulation of platelet (L)-arginine: nitric oxide pathway after exercise training in hypertension

We investigated whether physical exercise can affect platelet L-arginine?- nitric oxide pathway in spontaneously hypertensive rats (SHR). Sixteen male SHR and 16 Wistar Kyoto rats (WKY) were divided among exercise (EX) and sedentary (SED) groups. After 20?weeks of treadmill training, systolic blood pressure (mm?Hg) was significantly lower in exercised spontaneously hypertensive rats (SHR/EX; 138?± 8) than in sedentary spontaneously hypertensive rats (SHR/SED; 214?± 9). Exercise significantly increased platelet L-arginine transport (pmol L-arginine·(10(9) cells)(-1)·min(-1)), assessed by incubation with L-[(3)H]-arginine, in both WKY (SED, 0.196?± 0.054 compared with EX, 0.531?± 0.052) and SHR (SED, 0.346?± 0.076 compared with EX, 0.600?± 0.049). Nitric oxide synthase (NOS) activity (pmol L-citrulline·(10(8) cells)(-1)), measured by the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, was significantly increased in SHR/EX (0.072?± 0.007) compared with SHR/SED (0.038?± 0.007), but no changes were observed in WKY. The iNOS and eNOS protein levels assessed by Western blot were not affected by exercise. This upregulation of the platelet L-arginine-NO pathway may attenuate the risk of thromboembolic events, supporting the role of exercise in hypertension management.     

Influence of NG-monomethyl-L-arginine on endothelium-dependent relaxations in the perfused mesenteric vascular bed of the rat

Endothelium-dependent relaxation mediated by the formation of nitric oxide (NO) from L-arginine, is prevented by the arginine analog NG-monomethyl L-arginine (L-NMMA) (Palmer et al., Biochem. Biophys. Res. Comm. 153:1251-1256 (1988)). In the rat mesenteric arterial bed, incubation with L-NMMA did not prevent acetylcholine-induced relaxation, which, however, was reversed when L-NMMA was added during its maximum effect. A similar profile of action was observed with methylene blue, an inhibitor of guanylate cyclase. Methylene blue, but not L-NMMA, increased basal perfusion pressure. These data indicate that in the mesenteric arterial bed, NO formation via the L-NMMA-sensitive pathway occurs during stimulation with acetylcholine, but not under basal conditions.     

Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor potential canonical (TRPC) channels, in the present study we tested the hypothesis that increased vasomotion in hypertension is directly linked to increased TRPC expression. Using a small vessel myograph we observed significantly increased norepinephrine‐induced vasomotion in mesenteric arterioles from SHR compared to normotensive Wistar–Kyoto (WKY) rats. Using immunoblottings we obtained significantly increased expression of TRPC1, TRPC3 and TRPC5 in mesenteric arterioles from SHR compared to WKY, whereas TRPC4 and TRPC6 showed no differences. Norepinephrine‐induced vasomotion from SHR was significantly reduced in the presence of verapamil, SKF96365, 2‐aminoethoxydiphenylborane (2‐APB) or gadolinium. Pre‐incubation of mesenteric arterioles with anti‐TRPC1 and anti‐TRPC3 antibodies significantly reduced norepinephrine‐induced vasomotion and calcium influx. Control experiments with pre‐incubation of TRPC antibodies plus their respective antigenic peptide or in the presence of anti‐β‐actin antibodies or random immunoglobulins not related to TRPC channels showed no inhibitory effects of norepinephrine‐induced vasomotion and calcium influx. Administration of candesartan or telmisartan, but not amlodipine to SHR for 16 weeks significantly reduced either the expression of TRPC1, TRPC3 and TRPC5 as well as norepinephrine‐induced vasomotion in mesenteric arterioles. In conclusion we gave experimental evidence that the increased TRPC1, TRPC3 and TRPC5 expression in mesenteric arterioles from SHR causes increased vasomotion in hypertension.     

Chronic renal failure alters endothelial function in cerebral circulation in mice

We examined structure, composition, and endothelial function in cerebral arterioles after 4 wk of chronic renal failure (CRF) in a well-defined murine model (C57BL/6J and apolipoprotein E knockout female mice). We also determined quantitative expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (on serine 1177 and threonine 495), and caveolin-1; quantitative expression of markers of vascular inflammation or oxidative stress [Rock-1, Rock-2, VCAM-1, and peroxisome proliferator-activated receptor-γ (PPARγ)]; and the plasma concentration of L-arginine and asymmetric dimethylarginine (ADMA). Our hypothesis was that endothelial function would be impaired in cerebral arterioles during CRF following either a decrease in NO production (through alteration of eNOS expression or regulation) or an increase in NO degradation (due to oxidative stress or vascular inflammation). Endothelium-dependent relaxation was impaired during CRF, but endothelium-independent relaxation was not. CRF had no effect on cerebral arteriolar structure and composition. Quantitative expressions of eNOS, eNOS phosphorylated on serine 1177, caveolin-1, Rock-1, Rock-2, and VCAM-1 were similar in CRF and non-CRF mice. In contrast, quantitative expression of PPARγ (which exercises a protective role on blood vessels) was significantly lower in CRF mice, whereas quantitative expression of eNOS phosphorylated on the threonine 495 (the inactive form of eNOS) was significantly higher. Lastly, the plasma concentration of ADMA (a uremic toxin and an endogenous inhibitor of eNOS) was elevated and plasma concentration of L-arginine was low in CRF. In conclusion, endothelial function is impaired in a mouse model of early stage CRF. These alterations may be related (at least in part) to a decrease in NO production.     

Increased microparticle production and impaired microvascular endothelial function in aldosterone-salt-treated rats: protective effects of polyphenols

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h⁻¹), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg⁻¹.day⁻¹), or spironolactone (30 mg.kg⁻¹.day⁻¹) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.     

Systemic arterial pressure response to two weeks of Tempol therapy in SHR: involvement of NO, the RAS, and oxidative stress

The roles of nitric oxide (NO) and plasma renin activity (PRA) in the depressor response to chronic administration of Tempol in spontaneously hypertensive rats (SHR) are not clear. The present study was done to determine the effect of 2 wk of Tempol treatment on blood pressure [mean arterial pressure (MAP)], oxidative stress, and PRA in the presence or absence of chronic NO synthase inhibition. SHR were divided into four groups: control, Tempol (1 mmol/l) alone, nitro-L-arginine methyl ester (L-NAME, 4.5 mg x g(-1).day(-1)) alone, and Tempol + L-NAME or 2 wk. With Tempol, MAP decreased by 22%: 191 +/- 3 and 162 +/- 21 mmHg for control and Tempol, respectively (P < 0.05). L-NAME increased MAP by 16% (222 +/- 2 mmHg, P < 0.01), and L-NAME + Tempol abolished the depressor response to Tempol (215 +/- 3 mmHg, P < 0.01). PRA was not affected by Tempol but was increased slightly with L-NAME alone and 4.4-fold with L-NAME + Tempol. Urinary nitrate/nitrite increased with Tempol and decreased with L-NAME and L-NAME + Tempol. Tempol significantly reduced oxidative stress in the presence and absence of L-NAME. In conclusion, in SHR, Tempol administration for 2 wk reduces oxidative stress in the presence or absence of NO, but in the absence of NO, Tempol is unable to reduce MAP. Therefore, NO, but not changes in PRA, plays a major role in the blood pressure-lowering effects of Tempol. These data suggest that, in hypertensive individuals with endothelial damage and chronic NO deficiency, antioxidants may be able to reduce oxidative stress but not blood pressure.     

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low K(m) arachidonic acid omega-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 +/- 3 to 121 +/- 4 mmHg (P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC(50) 0.69 +/- 0.17 vs. 1.77 +/- 0.40 microM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.     

Hydrocortisone attenuates cyclosporin A-induced nephrotoxicity in rats

Cyclosporin A (CsA) is the prototype of immunosuppressant drugs that have revolutionized the management of all transplantation and autoimmune diseases. Side effects of CsA mainly affecting the kidney but also observed in liver and heart, limit the therapeutic use of this drug after organ transplantation. The renal toxicity of CsA is attributed to reduced renal blood flow which leads to hypoxia-reoxygenation injury accompanied by excessive generation of oxygen-derived free radicals. In several therapeutic protocols, CsA is used in association with corticosteroids to obtain better therapeutic results. Recently, our studies showed that hydrocortisone (HY) has a protective effect on CsA-induced cardiotoxicity. In fact our previous results demonstrated that in rat cardiomyocytes, CsA toxicity is due to a calcium overload, which in turn induce lipid peroxidation and determines oxidative stress-induced cell injury. Treatment with HY effectively inhibits CsA-induced toxicity, decreasing lipid peroxidation as well as calcium intracellular concentration. In this study we evaluated in vivo the effects of CsA, used alone or in association with HY, on some parameters of renal dysfunction (blood urea nitrogen; BUN, creatinine, and cholesterol), malondialdheyde (MDA) levels, antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and apoptosis. CsA administration for 24 days resulted in a marked renal oxidative stress, which significantly deranged the renal functions. Treatment with CsA in association with HY significantly improved the renal dysfunction and renal oxidative status. This study clearly suggests the role of oxidative stress in the pathogenesis of CsA-induced nephrotoxicity.     

Role of oxidative stress in age-related reduction of NO-cGMP-mediated vascular relaxation in SHR

The incidence of hypertension increases during the late stages of aging; however, the vascular mechanisms involved are unclear. We investigated whether the late stages of aging are associated with impaired nitric oxide (NO)-mediated vascular relaxation and enhanced vascular contraction and whether oxidative stress plays a role in the age-related vascular changes. Aging (16 mo) male spontaneously hypertensive rats (SHR) nontreated or treated for 8 mo with the antioxidant tempol (1 mM in drinking water) or vitamin E (E; 5,000 IU/kg chow) and vitamin C (C; 100 mg. kg-1. day-1 in drinking water) and adult (12 wk) male SHR were used. After the arterial pressure was measured, aortic strips were isolated from the rats for measurement of isometric contraction. The arterial pressure and phenylephrine (Phe)-induced vascular contraction were enhanced, and the ACh-induced vascular relaxation and nitrite/nitrate production were reduced in aging compared with adult rats. In aging rats, the arterial pressure was nontreated (188 +/- 4), tempol-treated (161 +/- 6), and E + C-treated (187 +/- 1 mmHg). Phe (10-5 M) caused an increase in active stress in nontreated aging rats (14.3 +/- 1.0) that was significantly (P < 0.05) reduced in tempol-treated (9.0 +/- 0.7) and E + C-treated rats (9.8 +/- 0.6 x 104 N/m2). ACh produced a small relaxation of Phe contraction in nontreated aging rats that was enhanced (P < 0.05) in tempol- and E + C-treated rats. l-NAME (10-4 M), inhibitor of NO synthase, or ODQ (10-5 M), inhibitor of cGMP production in smooth muscle, inhibited ACh relaxation and enhanced Phe contraction in tempol- and E + C-treated but not the nontreated aging rats. ACh-induced vascular nitrite/nitrate production was not different in nontreated, tempol- and E + C-treated aging rats. Relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was smaller in aging than adult rats but was not different between nontreated, tempol- and E + C-treated aging rats. Thus, during the late stages of aging in SHR rats, an age-related inhibition of a vascular relaxation pathway involving not only NO production by endothelial cells but also the bioavailability of NO and the smooth muscle response to NO is partially reversed during chronic treatment with the antioxidants tempol and vitamins E and C. The data suggest a role for oxidative stress in the reduction of vascular relaxation and thereby the promotion of vascular contraction and hypertension during the late stages of aging.     

Predictive Usefulness of Urinary Biomarkers for the Identification of Cyclosporine A-Induced Nephrotoxicity in a Rat Model

The main side effect of cyclosporine A (CsA), a widely used immunosuppressive drug, is nephrotoxicity. Early detection of CsA-induced acute nephrotoxicity is essential for stop or minimize kidney injury, and timely detection of chronic nephrotoxicity is critical for halting the drug and preventing irreversible kidney injury. This study aimed to identify urinary biomarkers for the detection of CsA-induced nephrotoxicity. We allocated salt-depleted rats to receive CsA or vehicle for 7, 14 or 21 days and evaluated renal function and hemodynamics, microalbuminuria, renal macrophage infiltration, tubulointerstitial fibrosis and renal tissue and urinary biomarkers for kidney injury. Kidney injury molecule-1 (KIM-1), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), fibronectin, neutrophil gelatinase-associated lipocalin (NGAL), TGF-β, osteopontin, and podocin were assessed in urine. TNF-α, IL-6, fibronectin, osteopontin, TGF-β, collagen IV, alpha smooth muscle actin (α -SMA) and vimentin were assessed in renal tissue. CsA caused early functional renal dysfunction and microalbuminuria, followed by macrophage infiltration and late tubulointerstitial fibrosis. Urinary TNF-α, KIM-1 and fibronectin increased in the early phase, and urinary TGF-β and osteopontin increased in the late phase of CsA nephrotoxicity. Urinary biomarkers correlated consistently with renal tissue cytokine expression. In conclusion, early increases in urinary KIM-1, TNF-α, and fibronectin and elevated microalbuminuria indicate acute CsA nephrotoxicity. Late increases in urinary osteopontin and TGF-β indicate chronic CsA nephrotoxicity. These urinary kidney injury biomarkers correlated well with the renal tissue expression of injury markers and with the temporal development of CsA nephrotoxicity.     

NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from approximately 50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 +/- 241 vs. 1,059 +/- 160 nM, P < 0.01; and 26 +/- 7 vs. 7 +/- 1 microM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.     

Alpha-lactorphin and beta-lactorphin improve arterial function in spontaneously hypertensive rats

alpha-lactorphin (Tyr-Gly-Leu-Phe) lowers blood pressure in conscious adult SHR. This tetrapeptide is originally released from milk protein alpha-lactalbumin by enzymatic hydrolysis. In order to evaluate the antihypertensive mechanisms of alpha-lactorphin, the effects of the tetrapeptide on vascular function were investigated in (30-35 weeks old) spontaneously hypertensive rats (SHR) with established hypertension and age-matched normotensive Wistar-Kyoto (WKY) rats in vitro. In addition, we studied the vascular effects of another structurally related tetrapeptide, beta-lactorphin (Tyr-Leu-Leu-Phe), which originates from milk protein beta-lactoglobulin. Endothelium-dependent relaxation to acetylcholine (ACh) was reduced in mesenteric arterial preparations of SHR as compared to those of WKY. In SHR, the ACh-induced relaxation was augmented by alpha-lactorphin or beta-lactorphin. The role of nitric oxide (NO) is suggested, since this improvement was abolished by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Simultaneous potassium channel inhibitor tetraethylammonium (TEA) elicited no additional effect on the ACh-induced relaxation. The cyclooxygenase inhibitor diclofenac did not attenuate the augmented ACh relaxation induced by alpha-lactorphin or beta-lactorphin, suggesting that endothelial vasodilatory prostanoids were not involved in the effect of the tetrapeptides. Endothelium-independent relaxation to the NO donor sodium nitroprusside (SNP) was augmented in mesenteric arterial preparations of SHR by simultaneous beta-lactorphin. The tetrapeptides did not alter vascular responses in mesenteric arteries from WKY. In conclusion, both alpha-lactorphin and beta-lactorphin improved vascular relaxation in adult SHR in vitro. The beneficial effect of alpha-lactorphin was directed towards endothelial function, whereas beta-lactorphin also enhanced endothelium-independent relaxation.     

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.     

Evidence that renal arginine transport is impaired in spontaneously hypertensive rats

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 μmol·kg(-1)·min(-1); 30 min) and subsequent superimposition of l-arginine (100 μmol·kg(-1)·min(-1); 30 min), the NO synthase inhibitor N(G)-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 μg·kg(-1)·min(-1)) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (-15 ± 7%; P = 0.07) and reduced medullary NO concentration (-9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[(3)H]arginine transport was less in SHR compared with SD rats (P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.