Effects of nitric oxide synthase inhibition and endothelin ETA receptor blockade on haemodynamics in hypertensive rats

1. The objectives of the present study were to study regional differences in haemodynamics between spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats induced by the nitric oxide synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) and the endothelin ETA receptor antagonist BQ 123 in vivo in tissues known to be important for blood pressure (BP) regulation (heart, kidney and skeletal muscle). Furthermore, the effect of acetylcholine (ACh) infusion (2 micrograms/kg per min) was examined after L-NMMA or BQ 123. The microsphere method was used for determinations of cardiac index (CI) and regional haemodynamics. 2. NG-Monomethyl-L-arginine (20 mg/kg) increased BP (26-48%; P < 0.01) and reduced CI in both rat strains. BQ 123 (1 mg/kg) reduced BP slightly (-4 to 11%; P < 0.05). 3. NG-Monomethyl-L-arginine significantly increased myocardial and skeletal muscle vascular resistance in SHR only; however, in the kidney, L-NMMA reduced blood flow and increased vascular resistance in both rat strains. 4. BQ 123 induced minor changes in regional haemodynamics that were not significantly different between the two strains. 5. Acetylcholine following BQ 123 induced an increase in myocardial blood flow in WKY rats, but decreased blood flow in SHR. Acetylcholine following L-NMMA reduced myocardial blood flow in both strains. 6. Acetylcholine following BQ 123 induced renal vasodilation in WKY rats but, following L-NMMA, ACh did not induce renal vasodilation in either rat strain. In contrast, L-NMMA did not abolish the vasodilation of acetylcholine in skeletal muscle in WKY rats. 7. In conclusion, the contribution of nitric oxide to basal vessel tone was not impaired in the heart, skeletal muscle and kidney in SHR. Antagonism of ETA receptors caused similar haemodynamic responses in both rat strains in these organs. Furthermore, NOS inhibition, but not ETA blockade, blunted the expected ACh-induced vasodilation in the heart and kidney in WKY rats, but not in skeletal muscle in both strains.     

Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats

The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L-NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross-sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide.     

Differential effect of nitric oxide synthase inhibitors on acetylcholine-induced relaxation of rat pulmonary and celiac artery rings

BN rats are well-known for their high capacity for IgE production and hyperresponsiveness to exposure to allergens or other chemicals. We examined the histological changes in the nasal cavity, trachea and lungs of BN and F344 rats after the inhalation of aerosol formaldehyde (HCHO), which exerts direct toxic effects on the respiratory system. The incidence of clinical signs such as sneezing and abnormal respiration in HCHO-treated F344 rats was higher than that in HCHO-treated BN rats. The mean body weight of HCHO-treated F344 rats apparently decreased in comparison with control F344 rats, but that of HCHO-treated BN rats was not significantly different from that of control BN rats. Changes such as squamous metaplasia, stratification, degeneration and desquamation were observed by light microscopy in nasal, tracheal and bronchial mucosa in the lungs of the HCHO-treated F344 rats. In the HCHO-treated BN rats, similar but milder lesions were restricted to the nasal mucosa. Scanning electron microscopic observation supported these light microscopic observations. These results suggest that BN rats have lower sensitivity to HCHO inhalation than F344 rats.     

Chronic morphine increases biosynthesis of nitric oxide synthase in the rat spinal cord

The present study was undertaken to determine the influence of chronic morphine treatment on the biosynthesis of nitric oxide synthase (NOS) in the rat spinal cord using in situ hybridization and immunohistochemical methods. Repeated administration of morphine (20-100 mg/kg/day; 10 days) increased the NOS mRNA level in laminae I-IV and X 3 h after the last injection. That effect was accompanied by an increase in both the number of NOS-positive cells (24 h) and the optical density of NOS-immunoreactivity (3 and 24 h). The results indicate that repeated morphine administration increases NOS biosynthesis in the rat spinal cord, which may reflect adaptive changes accounting for development of opiate tolerance and dependence.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

The effect of ketotifen on nitric oxide synthase activity

1. We studied the effect of ketotifen, a second generation H1-receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo. 2. Ketotifen (100 micrograms ml-1) increased human colonic NOS activity from 3.7 +/- 0.6 to 14.5 +/- 1.3 nmol g-1 min-1 (P < 0.005, ANOVA). In rat renal cortical and medullary tissues ketotifen increased NOS activity by 55% and 86%, respectively (P < 0.001). The stimulation of NOS activity was attenuated by NADPH deletion and by the addition of N omega nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, but not by [Ca2+] deprivation. NOS activity was unaffected by two other H1-antagonists, diphenhydramine and astemizole, or by the structurally related cyproheptadine. Renal cortical NOS activity was also significantly stimulated 90 min after intravenous administration of ketotifen to anaesthetized rats. 3. Ketotifen administration to anaesthetized rats induced modest declines in blood pressure and reduced total renal, cortical and outer medullary vascular resistance. This is in contrast to diphenhydramine, which did not induce renal vasodilatation. 4. We conclude that ketotifen stimulates NOS activity by mechanisms other than H1-receptor antagonism. The association of this effect with therapeutic characteristics of ketotifen and the clinical implications of these findings are yet to be defined.     

Decreased interleukin-2 production by rat uterine artery, aorta and uterine tissues during pregnancy

Changes in size and function during pregnancy are unique to the uterine artery. The aim of this study was to determine the interleukin (IL)-6 activity of the uterine artery wall tissue in pregnant rats. A total of 18 Charles River white rats (nine virgin and nine in midpregnancy) were used for the study. Bilateral uterine arteries were obtained, together with reference tissues from aorta and uterus. IL-6 production was measured as optical density (OD)/mg protein, in control culture media, and in the presence of stimulants including IL-1, tumour necrosis factor alpha and lipopolysaccharide. Polyclonal rabbit anti-human IL-6 antibodies were used to assess IL-6 activity. In control culture medium, uterine artery tissue samples from virgin rats produced significantly higher concentrations of IL-6 than samples obtained from pregnancy animals (1.8 +/- 0.3 versus 0.9 +/- 0.25 OD/mg protein respectively (mean +/- SE, P = 0.001). Stimulation by lipopolysaccharide increased IL-6 activity of the uterine artery wall. In comparison with the uterine artery, the aorta produced higher activities of IL-6, and its production in virgin animal samples was higher than during pregnancy. Stimulants increased IL-6 production by both aorta and uterus tissues. Neutralization of IL-6 activity was obtained in a range of 77-93% in all samples. The lower level of IL-6 activity during pregnancy in the uterine artery and in reference tissues including aorta and uterus, may be related to acceptance of pregnancy by maternal tissues.     

Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.     

Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery: role of platelets

There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.     

Inhibition of nitric oxide synthase attenuates osmotic thirst in the rat

Changes in water intake after intraperitoneal injection of a nitric oxide synthase (NOS) inhibitor was studied in the rat. Administration of NW-nitro-L arginine methyl ester (L-NAME) at a dose of 50 mg/kg attenuated osmotic thirst induced by intraperitoneal injection of hypertonic saline, but did not affect spontaneous intake of water and thirst induced by subcutaneous injection of angiotension II. Pretreatment with L-arginine significantly attenuated the inhibition of osmotic thirst evoked with subsequent L-NAME. Administration of NW-nitro-D-arginine methyl ester (D-NAME) altered neither the spontaneous nor the osmotic drinking behavior. These findings suggest that NO may affect the osmotically induced drinking.     

Ultrastructural localization and comparative distribution of nitric oxide synthase and N-methyl-D-aspartate receptors in the shell of the rat nucleus accumbens

Nitric oxide (NO), the diffusible gas formed by nitric oxide synthase (NOS) has been implicated in the enhanced locomotor activity attributed mainly to increased dopamine release in the shell of the nucleus accumbens (Acb). Furthermore, the release of both NO and dopamine are known to be altered by agonists of N-methyl-D-aspartate (NMDA) type glutamate receptors in this region. We examined the cellular sites of NO synthesis and the sites of potential relevancy for functional associations between neurons containing NOS and the NMDA receptor in the shell of the Acb. This was achieved by dual ultrastructural immunogold and immunoperoxidase labeling of antisera raised against the brain form of NOS and the NMDAR1 subunit of the NMDA receptor in this region of rat brain. NOS-like immunoreactivity (NOS-LI) was seen throughout the cytoplasm of isolated medium-large somata, aspiny dendrites and axon terminals. In 217 NOS-labeled profiles, NMDAR1-like immunoreactivity (NMDAR1-LI) was colocalized in 17% of somata and dendrites. Additionally, 35% of NOS-labeled dendrites apposed glial processes containing NMDAR1-LI, and 29% apposed axon terminals containing NMDARI-LI. NOS-labeled terminals more rarely colocalized NMDAR1 or apposed NMDAR1-labeled glial processes or dendrites. These results provide anatomical evidence that, in the shell of the Acb, NMDA receptors are localized so as to directly modulate the output of neurons producing NO as well as to influence other neurons and glia having the greatest access to the released gas.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Influence of hyperthyroidism on the activity of liver nitric oxide synthase in the rat

Previous studies of associations of metabolic polymorphisms with the occurrence of malignant brain tumors have suggested that there is a significantly increased risk of development of adult gliomas in individuals who carry a poor metabolizer CYP2D6 variant allele and the GSTT1 null genotype. To investigate this further, a population-based case control study of adult glioma in the San Francisco Bay area was conducted. Patients (n = 188) diagnosed with brain tumors and controls (n = 166) were enrolled using random digit dialing and were frequency matched for age, ethnicity and gender. Genotyping for the polymorphisms was performed using standard PCR-based techniques. The analysis of the data was restricted to Caucasians because the prevalence of these traits is known to vary by ethnicity. No overall association of either the GSTT1 null genotype or CYP2D6 homozygous variant PM genotype was observed with the occurrence of brain tumors. However, when stratified by histopathologic subtype, there was a significantly increased risk for oligodendroglioma associated with the GSTT1 null genotype, with an OR of 3.2 (95% CI 1.1-9.2). These data suggest that the GSTT1 polymorphism may play a role in the development of a subset of malignant brain tumors in adults, and indicate the need for further studies.