Microbial production and uptake of nitric oxide in soil

Abstract Fluxes of NO from three different soils have been studied by a flow-through system in the laboratory as a function of gas flow rate, of NO mixing ratio, and of incubation conditions. The dependence of net NO fluxes on gas flow rates and on NO mixing ratios could be described by a simple model of simultaneous NO production and NO uptake. By using this model, rates of gross NO production, rate constants of NO uptake, and NO compensation mixing ratios could be determined as function of the soil type and the incubation condition. Gross NO production rates were one to two orders of magnitude larger under anaerobic than under aerobic conditions. NO uptake rate constants, on the other hand, were only 5–8 times larger so that the compensation mixing ratios of NO were in a range of about 1600–2200 ppbv under anaerobic and of about 50–600 ppbv under aerobic conditions. The different soils exhibited similar NO uptake rate constants, but the gross NO production rate and compensation mixing ratio was significantly higher in an acidic (pH 4.7) sandy clay loam than in other less acidic soils. Experiments with autoclaved soil samples showed that both NO production and NO uptake was mainly due to microbial metabolism.     

L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^?/? mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.     

L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.

Previously, our laboratory found that pulmonary hypertension developed and lung nitric oxide (NO) production was reduced when piglets were exposed to chronic hypoxia (Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung Cell Mol Physiol 274: L517-L526, 1998). The purposes of this study were to determine whether L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10% O(2) (chronic hypoxia) for 10-12 days. Lung NO production was assessed in isolated lungs from both groups by measuring the perfusate accumulation of nitrites and nitrates (collectively termed NO(-)(x)) before and after addition of L-arginine (10(-2) M) to the perfusate. The rate of perfusate NO(-)(x) accumulation increased by 220% (from 0.8 +/- 0.4 to 2.5 +/- 0.5 nmol/min, P < 0.05) after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 +/- 0. 8 before vs. 3.3 +/- 0.4 nmol/min after L-arginine) in control lungs. In the second series of studies, L-arginine uptake was evaluated by measuring the perfusate concentration of L-[(3)H]arginine at fixed time intervals. The perfusate concentration of L-[(3)H]arginine at each time point was less (P < 0.05) in control than in chronic hypoxic lungs. Thus L-arginine uptake was impaired and may underlie in part the reduction in lung NO production that occurs when piglets are exposed to 10-12 days of chronic hypoxia. Moreover, these findings in isolated lungs lead to the possibility that L-arginine supplementation might increase in vivo lung NO production in piglets with chronic hypoxia-induced pulmonary hypertension.     

Electric fields caused by blood flow modulate vascular endothelial electrophysiology and nitric oxide production

Endothelial cells are exposed to a ubiquitous, yet unexamined electrical force caused by blood flow: the electrokinetic vascular streaming potential (EVSP). In this study, the hypothesis that extremely low frequency (ELF) electric fields parameterized by the EVSP have significant biological effects on endothelial cell properties was studied by measuring membrane potential and nitric oxide production under ELF stimulation between 0 and 2 Hz and 0–6.67 V/m. Using membrane potential and nitric oxide sensitive fluorescent dyes, bovine aortic endothelial cells (BAECs) in culture were studied in the presence and absence of EVSP‐modeled electric fields. The transmembrane potential of BAECs was shown to depolarize between 1 and 7 mV with a strong dependency on both the magnitude and frequency of the isolated ELF field. The findings also support a field interaction with a frequency‐dependent tuning curve. The ELF field complexly modulates the nitric oxide response to adenosine triphosphate stimulation with potentiation seen with up to a sevenfold increase. This potentiation was also frequency and magnitude dependent. An early logarithmic phase of NO production is enhanced in a field strength‐dependent manner, but the ELF field does not modify a later exponential phase. This study shows that using electric fields on the order of those generated by blood flow influences the essential biology of endothelial cells. The inclusion of ELF electric fields in the paradigm of vascular biology may create novel opportunities for advancing both the understanding and therapies for treatment of vascular diseases. Bioelectromagnetics 34:22–30, 2013. © 2012 Wiley Periodicals, Inc.     

In vivo assessment of microvascular nitric oxide production and its relation with blood flow

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.     

Extracellular pH affects inflammatory cell production of superoxide and nitric oxide

Previous research has described how high cellular metabolism creates an acidic environment in inflammatory cells during respiratory burst. The aim of our work was to describe the acid-base dependence of exudate in superoxide (O2.-) and nitric oxide (NO.) generation by inflammatory cells from a carrageenan-granuloma. Although the carrageenan solution was alkaline (pH 7.74 when equilibrated with air) the exudate showed an acidification that stabilised at around 7 units of pH. A notable hypercapnia, but not hypoxia, was found in the exudate at up to 24 h. The effect of extracellular acidosis on O2.- and NO. production by inflammatory cells was also studied. The maximum O2.- production and the lowest levels of NO. were found at pH 7, which was closer to the pH of the granuloma-pouch. These results suggest that experiments with inflammatory cells ex vivo should be carried out at an identical pH to that found in vivo in order to reproduce the physiological mechanisms of free radical generation during inflammatory processes.     

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.     

Effect of uterine blood flow occlusion on shear stress-mediated nitric oxide production and endothelial nitric oxide synthase expression during ovine pregnancy

During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 +/- 1 days' gestation [mean +/- SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO(2)/NO(3) (NO(x)) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% +/- 2.1%, uterine perfusion pressure only fell 12.2% +/- 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced approximately 28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO(x) levels were reduced 22.1% +/- 6.7% and 22.6% +/- 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial ( approximately 2.5 h) increase in systemic arterial and uterine venous NO(x) levels by 116% +/- 30% and 97% +/- 49%, respectively. Despite a decline in NO(x) levels after 6 h, no significant differences versus preocclusion NO(x) levels were detected by 24 h of occlusion in this experimental group. In contrast, NO(x), UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO(x).     

Sex differences in renal injury and nitric oxide production in renal wrap hypertension

To investigate the faster rate of renal disease progression in men compared with women, we addressed the following questions in the renal wrap (RW) model of hypertension: 1) Do sex differences exist in RW-induced renal injury, which are independent of sex differences in blood pressure? 2) Do sex differences in nitric oxide (NO) production exist in RW hypertension? Male (M) and female (F) rats underwent sham-operated (M-Sham, n = 7; F-Sham, n = 10) or RW (M-RW, n = 13; F-RW, n = 14) surgery for 9 wk. Markers of renal injury, including the glomerulosclerosis index (F-RW, 0.70 +/- 0.1 vs. M-RW, 2.2 +/- 0.6; P < 0.05), mean glomerular volume (F-RW, 1.05 +/- 0.050 x 10(6) vs. M-RW, 1.78 +/- 0.15 x 10(6) microm(3); P < 0.001), and proteinuria (F-RW, 68.7 +/- 15 vs. M-RW, 124 +/- 7.7 mg/day; P < 0.001) were greater in RW males compared with RW females. Endothelial NO synthase protein expression was elevated in the renal cortex (3.2-fold) and medulla (2.2-fold) 9 wk after RW in males, whereas no differences were observed in females. Neuronal NO synthase protein expression was unchanged in the renal cortex in males and in both the renal cortex and medulla in females, whereas in the male medulla, neuronal NOS was decreased by 57%. These data suggest the degree of renal injury is greater in male compared with female rats in RW hypertension despite similar degrees of hypertension and renal function and may involve sex differences in renal NO metabolism.     

Antioxidant treatment normalizes nitric oxide production, renal sodium handling and blood pressure in experimental hyperleptinemia

Recent studies suggest that adipose tissue hormone, leptin, is involved in the pathogenesis of arterial hypertension. However, the mechanism of hypertensive effect of leptin is incompletely understood. We investigated whether antioxidant treatment could prevent leptin-induced hypertension. Hyperleptinemia was induced in male Wistar rats by administration of exogenous leptin (0.25 mg/kg twice daily s.c. for 7 days) and separate groups were simultaneously treated with superoxide scavenger, tempol, or NAD(P)H oxidase inhibitor, apocynin (2 mM in the drinking water). After 7 days, systolic blood pressure was 20.6% higher in leptin-treated than in control animals. Both tempol and apocynin prevented leptin-induced increase in blood pressure. Plasma concentration and urinary excretion of 8-isoprostanes increased in leptin-treated rats by 66.9% and 67.7%, respectively. The level of lipid peroxidation products, malonyldialdehyde + 4-hydroxyalkenals (MDA+4-HNE), was 60.3% higher in the renal cortex and 48.1% higher in the renal medulla of leptin-treated animals. Aconitase activity decreased in these regions of the kidney following leptin administration by 44.8% and 45.1%, respectively. Leptin increased nitrotyrosine concentration in plasma and renal tissue. Urinary excretion of nitric oxide metabolites (NO(x)) was 57.4% lower and cyclic GMP excretion was 32.0% lower in leptin-treated than in control group. Leptin decreased absolute and fractional sodium excretion by 44.5% and 44.7%, respectively. Co-treatment with either tempol or apocynin normalized 8-isoprostanes, MDA+4-HNE, aconitase activity, nitrotyrosine, as well as urinary excretion of NO(x), cGMP and sodium in rats receiving leptin. These results indicate that oxidative stress-induced NO deficiency is involved in the pathogenesis of leptin-induced hypertension.     

VEGF(121)- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production

The angiogenic proteins basic fibroblast growth factor (bFGF; FGF-2) and vascular endothelial growth factor 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). Growth factors were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic growth factors.     

Modelling blood flow regulation by nitric oxide in psoriatic plaques

Psoriasis is a common skin disease, with a clinical appearance of red, scaly lesions, known as plaques. Recent experimental research has shown that the ubiquitous cell-signalling molecule nitric oxide (NO) is actively synthesized within these plaques by the iNOS enzyme. In contrast, NO production from normal, healthy skin is a by-product of the reduction of nitrite in sweat. Measurement of NO release rates at the skin surface are 100 times greater from psoriatic lesions than normal skin. We propose a mathematical model for the dynamics of NO within psoriatic plaques, that incorporates diffusion, production in the basal epidermis, decay within the plaque, and active scavenging by red blood cell haemoglobin; this last effect introduces a key nonlinearity into the model. We present numerical simulations of the model in two space dimensions, and then describe an approximation that reduces the model to two coupled ordinary differential equations. This reduced system can be solved exactly, giving an approximation for the NO release rate as an explicit function of model parameters. We use this approximation to explain some recent, surprising experimental results.     

Effects of asphyxia on arterial blood pressure, formation of nitric oxide in medulla and blood parameters in the cat

The rostral ventrolateral medulla (RVLM) plays an important role in the integration of cardiovascular functions. We examined the effect of asphyxia on cardiovascular responses, on sympathetic vertebral nerve activity (VNA) and nitric oxide (NO) formation in the RVLM, on hemodynamics, and on plasma concentrations of catecholamines, blood gas partial pressures and carbohydrate metabolites. Using 16 anesthetized cats we found that the systemic arterial pressure (SAP), VNA, NO formation and the release of plasma catecholamine components of norepinephrine and epinephrine were increased during asphyxia. The onset of NO production was significantly earlier than that of SAP and VNA. The venous partial pressure of O2 decreased, while the partial pressure of CO2 increased. Furthermore, metabolism of glucose and lactate increased, as did the blood concentrations of white and red blood cells, hemoglobin and platelets. Thus, asphyxia increased SAP, VNA and NO formation. It increased the plasma catecholamines, blood gases, carbohydrate metabolites and blood cells.     

Angiotensin II and nitric oxide in neural control of intrarenal blood flow

We investigated the roles of the renin-angiotensin system and the significance of interactions between angiotensin II and nitric oxide, in responses of regional kidney perfusion to electrical renal nerve stimulation (RNS) in pentobarbital sodium-anesthetized rabbits. Under control conditions, RNS (0.5-8 Hz) reduced total renal blood flow (RBF; -89 +/- 3% at 8 Hz) and cortical perfusion (CBF; -90 +/- 2% at 8 Hz) more than medullary perfusion (MBF; -55 +/- 5% at 8 Hz). Angiotensin II type 1 (AT(1))-receptor antagonism (candesartan) blunted RNS-induced reductions in RBF (P = 0.03), CBF (P = 0.007), and MBF (P = 0.04), particularly at 4 and 8 Hz. Nitric oxide synthase inhibition with N(G)-nitro-L-arginine (L-NNA) enhanced RBF (P = 0.003), CBF (P = 0.001), and MBF (P = 0.03) responses to RNS, particularly at frequencies of 2 Hz and less. After candesartan pretreatment, L-NNA significantly enhanced RNS-induced reductions in RBF (P = 0.04) and CBF (P = 0.007) but not MBF (P = 0.66). Renal arterial infusion of angiotensin II (5 ng.kg(-1).min(-1)) selectively enhanced responses of MBF to RNS in L-NNA-pretreated but not in vehicle-pretreated rabbits. In contrast, greater doses of angiotensin II (5-15 ng.kg(-1).min(-1)) blunted responses of MBF to RNS in rabbits with intact nitric oxide synthase. These results suggest that endogenous angiotensin II enhances, whereas nitric oxide blunts, neurally mediated vasoconstriction in the renal cortical and medullary circulations. In the renal medulla, but not the cortex, angiotensin II also appears to be able to blunt neurally mediated vasoconstriction.     

Cholecystokinin secretagogue-induced gastroprotection: role of nitric oxide and blood flow

This study was done to examine the role of CCK in gastric mucosal defense and to assess the gastroprotective roles of nitric oxide and blood flow. In rats, the CCK secretagogues oleate and soybean trypsin inhibitor augmented gastric mucosal blood flow and prevented gastric injury from luminal irritants. Type A CCK receptor blockade negated CCK secretagogue-induced gastroprotection and exacerbated gastric injury from bile and ethanol but did not block adaptive cytoprotection. CCK secretagogue-induced gastroprotection and hyperemia were negated by nonselective nitric oxide synthase (NOS) inhibition (N(G)-nitro-L-arginine methyl ester) but not by selective inducible NOS inhibition (aminoguanidine). Gastric mucosal calcium-dependent NOS activity, but not calcium-independent NOS activity, was increased following CCK and CCK secretagogues. The release of endogenous CCK plays a role in the intrinsic gastric mucosal defense system against injury from luminal irritants. The protective mechanism appears to involve increased production of nitric oxide from primarily the constitutive isoforms of NOS and a resultant increase in blood flow.     

Real-time monitoring of nitric oxide and blood flow during ischemia-reperfusion in the rat testis

In the present study, we attempted to clarify the role of nitric oxide (NO) and its release during the ischemia-reperfusion rat testis. Eight-week-old male Sprague-Dawley rats were divided into seven groups: age-matched control rats, ischemia (30 minutes)-reperfusion (30 minutes) rats without N^G-nitro-L-arginine methyl ester (L-NAME) and L-arginine (L-Arg) treatment, ischemia (30 minutes)-reperfusion (30 minutes) rats treated with L-NAME (10, 30, and 100 mg/kg), ischemia-reperfusion rats treated with L-Arg (10 and 30 mg/kg). Sixty minutes prior to induction of ischemia, L-NAME or L-Arg was administrated intraperitoneally. Real-time monitoring of blood flow and NO release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. NO₂-NO₃ and malonaldehyde (MDA) concentrations were measured in the experimental testes. Furthermore, we investigated possible morphological changes in the testis. Clamping of the testicular artery decreased blood flow to 5–20% of the basal level measured before clamping. Immediately following clipping of the artery, NO release rapidly increased. After removing the clip, NO release gradually returned to the basal level. This phenomenon was enhanced by treatment with L-Arg and inhibited by treatment with L-NAME. NO₂-NO₃ concentrations were increased by treatment with L-Arg and decreased by treatment with L-NAME, while MDA concentrations were increased by treatment with L-NAME and were decreased by treatment with L-Arg. In histological studies, the ischemia-reperfusion caused infiltration of leukocytes and a rupture of microvessels in the testis. Our data suggest that NO has cytoprotective effects on ischemia-reperfusion injury in the rat testis.     

Increased nitric oxide production in platelets from severe chronic renal failure patients

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.