Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

Endurance training does not alter the level of neuronal nitric oxide synthase in human skeletal muscle.

The effect of endurance training on neuronal nitric oxide synthase (nNOS) content and distribution in muscle was investigated. Seven male subjects performed 6 wk of one-legged knee-extensor endurance training (protocol A). Muscle biopsies, obtained from vastus lateralis muscle in the untrained and the trained leg, were analyzed for nNOS protein and activity as well as immunohistochemical distribution of nNOS and endothelial nitric oxide synthase (eNOS). Muscle biopsies were also obtained from another seven male subjects before and after 6 wk of training by endurance running (protocol B) and analyzed for nNOS protein. No difference was found in the amount of nNOS protein in the untrained and the trained muscle either with protocol A or protocol B (P > 0.05). In protocol A, the activity of nNOS was 4.76 +/- 0.56 pmol. mg protein(-1). min(-1) in the control leg, and the level was not different in the trained leg (P > 0.05). nNOS was present in the sarcolemma and cytosol of type I and type II muscle fibers, and the qualitative distribution was similar in untrained and trained muscle. The number of eNOS immunoreactive structures and the number of capillaries per muscle fiber were higher (P < 0.05) after training than before. The present findings demonstrate that, in contrast to findings on animals, nNOS levels remain unaltered with endurance training in humans. Evidence is also provided that endurance training may increase the amount of eNOS, in parallel with an increase in capillaries in human muscle.     

Endothelial dysfunction does not require loss of endothelial nitric oxide synthase

Whereas altered nitric oxide (NO.) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO. formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO. synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO. formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by the L-[(14)C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO. generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO. synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.     

RNA interference-produced autoregulation of inducible nitric oxide synthase expression

Vector-mediated delivery of short-hairpin RNA (shRNA) to regulate gene expression holds a great therapeutic promise. We hypothesize that gene expression can be autoregulated with RNA interference. We used inducible nitric oxide synthase (iNOS) as a gene model to test this hypothesis. Lipopolysaccharide dose-dependently increased iNOS in rat aortic smooth muscle cells and the nitrite production from these cells. These increases were attenuated in cells transfected with plasmids containing code for iNOS shRNA whose expression was controlled by an iNOS promoter. The production of shRNA was lipopolysaccharide dose-dependent. The lipopolysaccharide-induced iNOS expression in rat C6 glioma cells also was attenuated by transfection with plasmids containing the iNOS shRNA code. These results provide proof-of-concept evidence for using RNA interference technique to achieve autoregulation of gene expression.     

Shoot apex removal does not alter autoregulation of Modulation in soybean

The suppression of new nodule development in soybean (Glycine max (L.) Merr.) has been previously demonstrated to involve the shoot through reciprocal grafts between the wild-type cultivar Bragg and its supernodulating mutant nts382. Using the same grafting technique, but modified through the excision of the shoot apex region and emerging lateral shoots, we show here that autoregulation of nodule number still existed despite apex removal. This radical treatment lowered total nodule number per plant as well as root, shoot and nodule dry weight. Bragg shoots grafted onto nts382 roots gave wild-type nodulation (26 nodules, 15mg total nodule mass) as compared to nts382 shoots grafted onto Bragg roots (340 nodules, 277 mg total nodule mass). Specific nodule mass differed between supernodulating (about 0·5-1·0mg per nodule) and wild-type nodulating (2·3 mg per nodule) plants. In contrast to other growth characteristics, apex removal did not affect specific nodule size, except in plants with wild-type shoots and nts382 (supernodulation) roots. Apex removal only slightly affected the percentage of nodule weight per total root weight in nts382, but had a severe effect in wild type. Growth reductions varied between the normal and supernodulating plants. The fact that autoregulation of nodulation still functions in plants devoid of functional shoot apices suggests that the autoregulation signal may not be derived from the apex regions and that the leaf may be a likely source.     

Quercetin supplementation does not alter antioxidant status in humans

Abstract

This study measured the influence of ingesting quercetin on plasma measures for oxidative stress and antioxidant capacity. Male and female subjects (n = 1002) varying in age (18–85 years) and body mass index (BMI) (16.7–52.7 kg/m²) were studied. Subjects were randomized to one of three groups using double-blinded methods: placebo, 500 mg or 1000 mg quercetin/day with 125 mg or 250 mg vitamin C/day, respectively. Pre- and post-study fasting blood samples show that plasma quercetin increased in a dose-responsive manner. The pattern of change in plasma F₂-isoprostanes, oxidized low density lipoprotein, reduced glutathione, ferric reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) did not differ between supplementation groups or after adjustment for gender, age, BMI and disease status. In summary, quercetin supplementation over 12 weeks in doses of 500 mg or 1000 mg/day significantly increased plasma quercetin levels, but had no influence on several measures of oxidative stress and antioxidant capacity.     

Cutaneous interstitial nitric oxide concentration does not increase during heat stress in humans.

Inhibition of cutaneous nitric oxide (NO) synthase reduces the magnitude of cutaneous vasodilation during whole body heating in humans. However, this observation is insufficient to conclude that NO concentration increases in the skin during a heat stress. This study was designed to test the hypothesis that whole body heating increases cutaneous interstitial NO concentration. This was accomplished by placing 2 microdialysis membranes in the forearm dermal space of 12 subjects. Both membranes were perfused with lactated Ringer solutions at a rate of 2 microl/min. In both normothermia and during whole body heating via a water perfused suit, dialysate from these membranes were obtained and analyzed for NO using the chemiluminescence technique. In six of these subjects, after the heat stress, the membranes were perfused with a 1 M solution of acetylcholine to stimulate NO release. Dialysate from these trials was also assayed to quantify cutaneous interstitial NO concentration. Whole body heating increased skin temperature from 34.6 +/- 0.2 to 38.8 +/- 0.2 degrees C (P < 0.05), which increased sublingual temperature (36.4 +/- 0.1 to 37.6 +/- 0.1 degrees C; P < 0.05), heart rate (63 +/- 5 to 93 +/- 5 beats/min; P < 0.05), and skin blood flow over the membranes (21 +/- 4 to 88 +/- 10 perfusion units; P < 0.05). NO concentration in the dialysate did not increase significantly during of the heat stress (7.6 +/- 0.7 to 8.6 +/- 0.8 microM; P > 0.05). After the heat stress, administration of acetylcholine in the perfusate significantly increased skin blood flow (128 +/- 6 perfusion units) relative to both normothermic and heat stress values and significantly increased NO concentration in the dialysate (15.8 +/- 2.4 microM). These data suggest that whole body heating does not increase cutaneous interstitial NO concentration in forearm skin. Rather, NO may serve in a permissive role in facilitating the effects of an unknown neurotransmitter, leading to cutaneous vasodilation during a heat stress.     

Nitric oxide synthase inhibition does not alter the reactive hyperemic response in the cutaneous circulation.

Reactive hyperemia is the sudden rise in blood flow after release of an arterial occlusion. Currently, the mechanisms mediating this response in the cutaneous circulation are poorly understood. The purpose of this study was to 1). characterize the reactive hyperemic response in the cutaneous circulation and 2). determine the contribution of nitric oxide (NO) to reactive hyperemia. Using laser-Doppler flowmetry, we characterized reactive hyperemia after 3-, 5-, 10-, and 15-min arterial occlusions in 10 subjects. The total hyperemic response was calculated by taking the area under the curve (AUC) of the hyperemic response minus baseline skin blood flow (SkBF) [i.e., total hyperemic response = AUC - [baseline SkBF as %maximal cutaneous vascular conductance (CVC(max) x duration of hyperemic response in s]]. For the characterization protocol, the total hyperemic response significantly increased as the period of ischemia increased from 5 to 15 min (P < 0.05). However, the 3-min response was not significantly different from the 5-min response. In the NO contribution protocol, two microdialysis fibers were placed in the forearm skin of eight subjects. One site served as a control and was continuously perfused with Ringer solution. The second site was continuously perfused with 10 mM NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NO synthase. CVC was calculated as flux/mean arterial pressure and normalized to maximal blood flow (28 mM sodium nitroprusside). The total hyperemic response in control sites was not significantly different from l-NAME sites after a 5-min occlusion (3261 +/- 890 vs. 2907 +/- 531% CVC(max. s). Similarly, total hyperemic responses in control sites were not different from l-NAME sites (9155 +/- 1121 vs. 9126 +/- 1088% CVC(max. s) after a 15-min arterial occlusion. These data suggest that NO does not directly mediate reactive hyperemia and that NO is not produced in response to an increase in shear stress in the cutaneous circulation.     

Inhibition of inducible nitric oxide synthase in persistent pain

The present study was undertaken to investigate the role of inducible nitric oxide synthase in a rat model of persistent pain. The effects of L-N6 (1-iminoethyl) lysine (L-NIL), a relatively potent and relatively selective inhibitor of inducible nitric oxide synthase, were investigated in carrageenan induced hyperalgesia L-NIL (0.1 microMole) injected intraplantar or intrathecal markedly enhanced carrageenan induced hyperalgesia. These effects were reversed during the third hour by co-administration of L-arginine (900 mg/kg i.p.) but not D-arginine. Methylene blue (MB), a soluble guanylate cyclase inhibitor, administered intrathecally (0.1 microg) had no effect on L-NIL potentiation of carrageenan hyperalgesia but abolished antinociception induced by L-arginine. Obtained results suggest that nitric oxide derived from inducible nitric oxide synthase play an inhibitory role in carrageenan produced hyperalgesia in rat.     

Cyclic GMP-phosphodiesterase inhibition does not alter cerebral oxygen consumption

The effect of zaprinast, a cyclic guanosine monophosphate inhibitor, on the level of cyclic GMP and cerebral O₂ consumption was determined. Anesthetized male Long-Evans rats were divided into a control group (n=15) and a zaprinast treated group (n=15). Vehicle was applied topically to the left cortex and 3·10⁻³ M zaprinast was applied to the right cortex. A saline treated control group was also studied. Regional cerebral blood flow was determined by [¹⁴C]-iodoantipyrine and regional O₂ extraction was determined by microspectrophotometry. The level of cyclic GMP was measured by radioimmunoassay. There were no hemodynamic or blood gas differences between groups. The level of cyclic GMP was not significantly different between the right and left cerebral cortex of the control group (17.0±4.3 and 17.7±4.6 pmol/g). In the zaprinast treated group, there was a significant (46%) increase in the level of cyclic GMP in the zaprinast treated cortex (20.5±8.1) in comparison to the vehicle treated cortex (14.0±5.7). Zaprinast did not significantly alter cerebral blood flow. There were no significant differences in regional O₂ extraction. The O₂ consumption of the zaprinast treated cortex (8.0±3.3 ml O₂·min⁻¹·100 g⁻¹) was not different from that of the vehicle treated cortex (7.0±2.9) or those of the control group. Thus, our data indicated that the increased level of cyclic GMP had no significant effect on cerebral oxygen consumption.     

Inhibition of nitric oxide synthase by cobalamins and cobinamides

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -l-[¹⁴C]arginine-to-l-[¹⁴C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN₂-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN₂-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN₂-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN₂-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with l-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the “base-off” conformation of the lower axial dimethylbenzimidazole ligand. CN₂-Cbi inhibited interferon-γ-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.     

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.     

Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney

To evaluate the role of a potential interaction between superoxide anion (O(2)(-)) and nitric oxide (NO) in regulating kidney function, we examined the renal responses to intra-arterial infusion of a superoxide dismutase mimetic, tempol (0.5 mg.kg(-1).min(-1)), in anesthetized dogs treated with or without NO synthase inhibitor, N(omega)-nitro-l-arginine (NLA; 50 microg.kg(-1).min(-1)). In one group of dogs (n = 10), tempol infusion alone for 30 min before NLA infusion did not cause any significant changes in renal blood flow (RBF; 5.2 +/- 0.4 to 5.0 +/- 0.4 ml.min(-1).g(-1)), glomerular filtration rate (GFR; 0.79 +/- 0.04 to 0.77 +/- 0.04 ml.min(-1).g(-1)), urine flow (V; 13.6 +/- 2.1 to 13.9 +/- 2.5 microl.min(-1).g(-1)), or sodium excretion (U(Na)V; 2.4 +/- 0.3 to 2.2 +/- 0.3 micromol.min(-1).g(-1)). Interestingly, when tempol was infused in another group of dogs (n = 12) pretreated with NLA, it caused increases in V (4.4 +/- 0.4 to 9.7 +/- 1.4 microl.min(-1).g(-1)) and in U(Na)V (0.7 +/- 0.1 to 1.3 +/- 0.2 micromol.min(-1).g(-1)) without affecting RBF or GFR. Although NO inhibition caused usual qualitative responses in both groups of dogs, the antidiuretic (47 +/- 5 vs. 26 +/- 4%) and antinatriuretic (67 +/- 4 vs. 45 +/- 11%) responses to NLA were seen much less in dogs pretreated with tempol. NLA infusion alone increased urinary excretion of 8-isoprostane (13.9 +/- 2.7 to 22.8 +/- 3.6 pg.min(-1).g(-1); n = 7), which returned to the control levels (11.6 +/- 3.4 pg.min(-1).g(-1)) during coadministration of tempol. These data suggest that NO synthase inhibition causes enhancement of endogenous O(2)(-) levels and support the hypothesis that NO plays a protective role against the actions of O(2)(-) in the kidney.     

Inhibition of neuronal nitric oxide synthase by N-phenacyl imidazoles.

Nitric oxide (NO) mediates a series of physiological processes, including regulation of vascular tone, macrofage-mediated neurotoxicity, platelet aggregation, learning and long-term potentiation, and neuronal transmission. Although NO mediates several physiological functions, overproduction of NO can be detrimental and play multiple roles in several pathological diseases. Accordingly, more potent inhibitors, more selective for neuronal nitric oxide synthase (nNOS) than endothelial NOS (eNOS) or inducible NOS (iNOS), could be useful in the treatment of cerebral ischemia and other neurodegenerative diseases. We recently described the synthesis of a series of imidazole derivatives. Among them N-(4-nitrophenacyl) imidazole (A) and N-(4-nitrophenacyl)-2-methyl-imidazole (B) were considered selective nNOS inhibitors. In the present study the action mechanism of compounds A and B was analyzed. Spectral changes observed in the presence of compound A indicate that this inhibitor exerts its effect without interaction with heme iron. Moreover compounds A and B, inhibit nNOS "noncompetitively" versus arginine, but "competitively" versus BH(4).     

Cyclooxygenase and nitric oxide synthase dependence of cutaneous reactive hyperemia in humans

We tested the hypothesis that cyclooxygenases (COXs) or COX products inhibit nitric oxide (NO) synthesis and thereby mask potential effects of NO on reactive hyperemia in the cutaneous circulation. We performed laser-Doppler flowmetry (LDF) with intradermal microdialysis in 12 healthy volunteers aged 19-25 yr. LDF was expressed as the percent cutaneous vascular conduction (%CVC) or as the maximum %CVC (%CVC(max)) where CVC is LDF/mean arterial pressure. We tested the effects of the nonisoform-specific NO synthase inhibitor nitro-L-arginine (NLA, 10 mM), the nonspecific COX inhibitor ketorolac (Keto, 10 mM), combined NLA + Keto, and NLA + sodium nitroprusside (SNP, 28 mM) on baseline and reactive hyperemia flow parameters. We also examined the effects of isoproterenol, a beta-adrenergic agonist that causes prostaglandin-independent vasodilation to correct for the increase in baseline flow caused by Keto. When delivered directly into the intradermal space, Keto greatly augments all aspects of the laser-Doppler flow response to reactive hyperemia: peak reactive hyperemic flow increased from 41 +/- 5 to 77 +/- 7%CVC(max), time to peak flow increased from 17 +/- 3 to 56 +/- 24 s, the area under the reactive hyperemic curve increased from 1,417 +/- 326 to 3,376 +/- 876%CVC(max).s, and the time constant for the decay of peak flow increased from 100 +/- 23 to 821 +/- 311 s. NLA greatly attenuates the Keto response despite exerting no effects on baseline LDF or on reactive hyperemia when given alone. Low-dose NLA + SNP duplicates the Keto response. Isoproterenol increased baseline and peak reactive flow. These results suggest that COX inhibition unmasks NO dependence of reactive hyperemia in human cutaneous circulation.     

Exercise distributed across day and night does not alter circadian period in humans

In rodents, increased activity due to running-wheel access is associated with a change in observed circadian period. In humans, exposure to exercise has failed to demonstrate similar effects on period. Methodological issues with prior studies such as light exposure during exercise, length of study, and method of measuring period confounded those evaluations of the effect of exercise on period in humans. In the present experiment, the authors examined the effect of exercise on period in 8 subjects using a 44-day within-subjects inpatient study. They used a 20-h forced desynchrony protocol, in which subjects were exposed to exercise across circadian phases under dim light conditions. Exercise consisted of three 45-min sessions per wake period on an ergometer. Target exercise intensity was ~65% of maximal heart rate. Intrinsic circadian period was measured using both core body temperature and hourly plasma melatonin samples. Consistent with previous reports, the authors find no effect of exercise on endogenous circadian period as measured by either core body temperature or melatonin. Exercise distributed across biological day and night does not appear to affect circadian period.     

Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans

There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKalpha2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKalpha2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 +/- 1% V(O2 peak). In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKalpha2 activity, AMPKalpha2 Thr(172) phosphorylation and acetyl-CoA Ser(222) phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.