Altered Nitric Oxide Synthase Activity Research Articles

19 - Apocynin reduces pressor and vasoconstrictor effects of Ang II in SHR

NAD(P)H oxidase (NOX) activity and expression can be activated by angiotensin (Ang) II. We have previously observed that apocynin, a NOX inhibitor, prevented endotelial dysfunction and reduced blood pressure by increasing nitric oxide (NO) and reducing ROS concentrations in endothelial cells. We evaluated the effect of apocynin on the contractile responses to Ang II in resistance arteries of SHR and the mechanisms involved on these effects. SHR were treated from the 4th to the 10th week of life with apocynin (30 mg/Kg). Wistar rats were used as normotensive control. Using mesenteric arteries, we performed concentration-response curves to Ang II and determined eNOS and NOX isoforms and subunits expressions, lucigenin chemiluminescence and nitrate/nitrite levels. Data were expressed as mean ± SEM. Apocynin increased endothelium modulation and/or NOS activity on Ang II vasoconstrictor responses in mesenteric arteries of SHR. Treatment with apocynin did not alter NO synthase activity, and eNOS, NOX1, NOXO1, and NOX4 expressions, however, it decreased NOX2 and p47phox expressions in mesenteric beds of SHR treated. Moreover, treatment apocynin was able to decrease ROS production in these vessels. The lower reactivity of Ang II in resistance arteries would lead to lower peripheral vascular resistance and consequently the reduction of mean arterial pressure and Ang II pressor effect in SHR treated with apocynin, as previously observed. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. Ethics Comittee CEUA FOA 450/2015. Financial Support FAPESP 2016/22180-9, CAPES and CNPq.

Abstract 644: Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats

Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats Krystal N. Brinson and Jennifer C. Sullivan, Experimental Medicine, Georgia Health Sciences University, Augusta, GA 30912 We previously published that male spontaneously hypertensive rats (SHR) having less nitric oxide synthase (NOS) enzymatic activity in the renal inner medullary (IM) than female SHR, which cannot be explained by protein expression alone. Tetrahydrobiopterin (BH4) is an essential cofactor for NOS activity. BH4 is easily oxidized to BH2, which is not a NOS cofactor, and male SHR have greater levels of oxidative stress than females. Therefore, we hypothesized that 1) male SHR will have greater increases in NOS enzymatic activity following BH4 supplementation than females and 2) antioxidant treatment will result in greater increases in NOS enzymatic activity in male SHR regardless of BH4 supplementation. Studies measured renal IM NOS enzymatic activity in the presence and absence of BH4 from 12-13 week old control (N=5-6) or antioxidant treated (tempol, 30 mg/kg/day in drinking water) male and female SHR (N=4-5). Supplementation with BH4 increased NOS activity in male SHR (177±35 vs. 315±52 pmol/mg; p=0.04), with no effect in females (434±112 vs. 548±84 pmol/mg). Following 2 weeks of antioxidant treatment, NOS enzymatic activity in male SHR was significantly increased compared to control SHR (p=0.04), and supplementation with BH4 no longer altered NOS enzymatic activity in males (588±143 vs. 648±153 pmol/mg). Antioxidant treatment did not significantly alter renal IM NOS enzymatic activity in female SHR compared to untreated controls, and BH4 supplementation did not alter NOS activity in tempol-treated females (473±82 vs. 580±66 pmol/mg). Tempol treatment also abolished the sex difference in renal IM NOS enzymatic activity. In conclusion, our data suggest that greater levels of oxidative stress in male SHR contributes to lower levels of NOS enzymatic activity compared to females. We hypothesize that this results in greater BH4 deficiency in males contributing to decreases in NO and increases in blood pressure.

Neurochemical Plasticity of Nitric Oxide Synthase Isoforms in Neurogenic Detrusor Overactivity after Spinal Cord Injury

Nitric oxide (NO) participates in the neural pathways controlling the lower urinary tract (LUT). Expression of NO synthase (NOS) can be upregulated after spinal cord injury (SCI), and altered NOS activity may participate in resulting LUT dysfunction. To investigate distribution of NOS-immunoreactivity (NOS-IR) in neurons of rats following SCI and the possible effects of NOS inhibitors. Expression of neuronal and inducible NOS-IR in lumbosacral spinal cord was assessed in rats. Cystometry was performed to examine effects of intrathecal injection of NOS inhibitor. There was increased expression of neuronal NOS-IR after trauma. Maximum bladder capacity was increased by neuronal NOS (nNOS) inhibitors. Upregulation of nNOS may facilitate emergence of the spinal micturition reflex following SCI; nNOS inhibitor suppressed SCI-induced urinary incontinence by increasing bladder capacity. Our results indicate manipulation of NO production could help treat LUT dysfunction after SCI.

Role of nitric oxide during early phase myocardial ischemic preconditioning in rats

To date, there have been no reports on altered nitric oxide (NO) content in ischemia/reperfusion with regard to in vivo preconditioning procedures. These studies are important for understanding the mechanisms of NO during early myocardial ischemic preconditioning. The aim of the present study was to investigate the mechanisms of NO during early myocardial ischemic preconditioning by measuring levels of NO and cyclic guanosine monophosphate (cGMP), as well as activity of nitric oxide synthase (NOS) in ischemia/reperfusion with respect to preconditioning in rats. Sixty-six female Sprague-Dawley rats were randomly divided into four groups: ischemic preconditioning group (IP), ischemia/reperfusion group (I/R), control group (CON), and preconditioning procedure group (PC). In the PC group, rats were further divided into PC1-, PC1 +, PC2-, PC2 +, PC3-, and PC3 + subgroups. Rats underwent left coronary artery occlusion and reperfusion, and subsequently, NOS activity and levels were assessed with spectrophotometric analysis. cGMP contents were measured with radioimmunoassay. The level of NO and cGMP, as well as the activity of NOS, were significantly higher in the IP group compared to the I/R and CON groups (P < 0.05). During preconditioning prior to prolonged ischemia, NO and cGMP levels varied markedly with ischemia and reperfusion. The levels of NO repeatedly increased when the heart was exposed to three episodes of 5-minute ischemia, and were almost completely reversed during each reperfusion period. NO and cGMP levels were significantly different between the 5-minute period of ischemia and the same period of reperfusion during preconditioning. NO plays an important role during early phase myocardial ischemic preconditioning in rats. NO and cGMP could be triggers and mediators of early phase myocardial ischemic preconditioning. Altered NOS activity following ischemic stress could be the primary inducer of higher NO levels detected. NO and cGMP fluctuations might be the trigger for protection during early phase myocardial ischemic preconditioning.

Posttranslational regulation of NO synthase activity in the renal medulla of diabetic rats

Shear stress increases nitric oxide (NO) production by endothelial cells, inner medullary collecting duct cells, and thick ascending limb. We postulated that the osmotic diuresis accompanying type 1 diabetes is associated with increased NO synthase (NOS) activity and/or expression in the renal medulla. Diabetes was induced by injection of streptozotocin, with insulin provided to maintain moderate hyperglycemia (Hyp) or euglycemia (Eug) for 3 wk. Sham rats received vehicle treatments. A separate group of rats (Phz) received phlorizin to produce a glucose-dependent osmotic diuresis. Renal medullary NOS1 and NOS2 activities did not differ between groups, whereas NOS3 activity was significantly increased in Hyp. Neither NOS1 nor NOS3 protein levels differed significantly between groups. Reduced phosphorylation of NOS3 at Thr(495) and Ser(633) was evident in medullary homogenates from Hyp rats, with no difference apparent at Ser(1177). Immunohistochemical analysis indicated prominent expression of pThr(495)NOS3 in the thick ascending limb and collecting duct of Sham and Phz rats. Hyp rats displayed staining in the collecting duct but minimal thick ascending limb staining. Immunostaining with anti-pSer(1177)NOS3 was evident only in the thick ascending limb, with no apparent differences between groups. In summary, glucose-dependent osmotic diuresis alone did not alter NOS activity or expression in the renal medulla. Diabetic hyperglycemia increased medullary NOS3 activity without a concomitant increase in NOS3 protein levels; however, NOS3 phosphorylation was reduced at Thr(495) and Ser(633). Thus changes in the phosphorylation of NOS at known regulatory sites might represent the primary mechanism underlying increased renal medullary NOS activity in diabetic hyperglycemia.

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells.

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.

Intrauterine hypoxia–ischemia alters nitric oxide synthase expression and activity in fetal and neonatal rat brains

The effects of intrauterine hypoxia–ischemia (HI) on nitric oxide synthase (NOS) activity and on expression of NOS isoforms were investigated in fetal and neonatal rat brains. Rat fetuses were subjected to either a 30-min intrauterine HI insult or a sham operation (SH) on gestational day 17 (G17). NOS activity in the homogenate of the rat brain was detectable on G17 and increased with age. NOS activity in the HI group was 20–30% higher than in the SH group from 6 to 48 h after the HI, but was 30% lower than in the SH group from postnatal day 8 to 14. Expression of the inducible NOS (iNOS) mRNA, as examined by RT-PCR, was increased as compared to the SH group from 6 to 24 h after the HI surgery. Expression of the constitutive neuronal NOS (nNOS) mRNA was reduced in the HI group from 24 h after the HI surgery up to postnatal day 14. Immunoblotting data have shown that alterations in NOS isoform protein expression caused by the intrauterine HI were consistent with the mRNA expression data. The overall results indicate that prenatal HI has long-lasting effects on function and expression of NOS in fetal and neonatal rat brains and that the altered NOS activity may be associated with prenatal HI-induced neurological abnormalities.

H2O2causes endothelial barrier dysfunction without disrupting the arginine-nitric oxide pathway

We have previously demonstrated that nitric oxide (.NO) donors attenuate and that inhibition of endogenous nitric oxide synthase (NOS) enhances hydrogen peroxide (H2O2)-mediated porcine pulmonary artery endothelial cell (PAEC) injury. The current study investigates the hypothesis that oxidant-mediated inhibition of NOS contributes to PAEC injury. PAEC barrier function, measured as the transmonolayer clearance of albumin, was significantly impaired by H2O2 (10-100 microM) in the absence of cytotoxicity. Treatment with H2O2 did not alter NOS activity, measured as the conversion of [3H]arginine to [3H]citrulline in PAEC lysates, either immediately after treatment with 0-250 microM H2O2 for 30 min or for up to 120 min after treatment with 100 microM H2O2. H2O2 had little effect on NOS activity in intact PAECs, measured as 1) the formation of [3H]citrulline in [3H]arginine-loaded PAECs, 2) PAEC guanosine 3',5'-cyclic monophosphate content, and 3) PAEC.NO release to the culture media. These results indicate that the arginine-.NO pathway remains intact after exposure to oxidant conditions sufficient to promote functional derangements of vascular endothelial cells.

Time course of the changes in central nitric oxide synthase activity following chronic treatment with morphine in the mouse: Reversal by naltrexone

1. 1. The time course of the effect of chronic administration of morphine on the activity of nitric oxide synthase (NOS) in the brain regions and spinal cord of the mouse was determined. The effect of naltrexone by itself on the NOS activity and that induced by morphine also were determined. 2. 2. Male Swiss Webster mice were implanted subcutaneously with a pellet containing 25 mg of morphine free base for 4 days. Placebo pellet implanted mice served as controls. 3. 3. Twenty-four hours after treatment with morphine, NOS activity decreased in the cerebellum, midbrain, cortex and remainder of the brain as well as in the spinal cord. Forty-eight and 72 hr after the treatment with morphine, NOS activity increased in the cerebellum and cortex, but no change was observed in the other brain regions and spinal cord. Twenty-four hours after morphine pellet removal (withdrawal), NOS activity in all brain regions and the spinal cord had returned to normal. 4. 4. Implantation of a pellet containing 10 mg of naltrexone did not alter NOS activity in any brain region or spinal cord for 24, 48 and 72 hr or 24 hr after removal of the pellet. 5. 5. Implantation of a naltrexone pellet in conjunction with a morphine pellet blocked the changes in NOS activity in the brain region and spinal cord induced by morphine. 6. 6. It is concluded that the initial decrease in NOS activity by morphine may be related to enhanced motor activity, whereas the increase in NOS activity in certain brain regions may be associated with tolerance-physical dependence development. Additionally, the changes in central NOS activity by morphine appear to be mediated by opioid receptors because they were blocked by concurrent treatment with naltrexone.

Neuropathic pain in rats is associated with altered nitric oxide synthase activity in neural tissue

Peripheral nerve injury may lead to a chronic neuropathic pain state that results from an increase in excitability of central neurons. This central sensitization is mediated via an N-methyl- d-aspartic acid (NMDA) receptor and may involve the production of nitric oxide (NO). As NO is suggested to play a role in nociceptive transmission following nerve injury, we examined for altered NO synthase activity at multiple levels of peripheral and spinal neural tissue in a rat model of neuropathic pain. Peripheral neuropathy was induced in rats ( N = 12) by ligation of the left L5 and L6 nerve roots. Six other rats had sham surgery. An ipsilateral decrease in paw withdrawal threshold to mechanical stimuli confirmed the presence of a neuropathic pain state. Samples of the lumbar and thoracic spinal cords, L4, L5, and L6 dorsal root ganglia (DRGs), and the sciatic nerves were obtained from the lesioned and contralateral sides at 2 and 4 weeks after neuropathic surgery ( N = 6 per group). In the lumbar spinal cord, a bilateral decrease in nitric oxide synthase (NOS) activity was observed 2 and 4 weeks after neuropathic surgery. NOS activity was increased in the ipsilateral L5 and 6 DRGs 2 weeks following neuropathic surgery. An increase in NOS activity in the DRG may be an early mechanism for inducing more central changes. The bilaterally decreased NOS activity in the lumbar spinal cord may be secondary to a negative feedback mechanism resulting from increased NO production in the spinal dorsal root ganglia. Multiple alterations in expression of NOS activity that occur in both peripheral and central processing may play a role in the pain behavior resulting from peripheral nerve injury. (Preliminary results of these studies have been presented in abstract form at the annual meetings of the Society for Neuroscience, 1994, and the American Society of Anesthesiologists, 1994.)

Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine

The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO 2; 30 μM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 μM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO 2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 μM) and N-methyl-N′-nitro-N-nitrosoguanidine (200 μM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.