Catecholamines,nitrogen oxide,and resistivity to stressor lesions: effect of adaptation to hypoxia

Pronouncement of stress-induced disturbance of searching behaviour (using "open field" test) and stomach ulceration were compared for the first time with activity of the catecholamine system in hypothalamus and striatum and also with activity of the stress-limiting system of nitric oxide (NO) in the rats of two strains August and Wistar, which differ in their resistance against stress-induced cardiovascular disorders. The effect of prior adaptation to hypobaric hypoxia on these disorders was also studied. August rats appeared to be more resistant than Wistar rats against stress-induced disturbance of the searching behaviour and stomach ulceration. Results of measuring the content of catecholamines in brain structures and the content of NO stable metabolites nitrate/nitrite in plasma suggested that these differences could be due to the stress activation of the nigro-striatal dopaminergic system in August rats, which was not observed in Wistar rats, and also to the higher production of NO in August than in Wistar rats. Adaptation to hypoxia considerably restricted these stress disorders in rats of both strains. Importantly, the protective effects were associated with activation of the nigro-striatal dopaminergic system in all the animals. In the result, adapted Wistar rats, as distinct from non-adapted Wistar rats, displayed a stress activation of this system. The protective effects of adaptation were also accompanied by an increased NO synthesis. Taken together, the data suggest an important role of the responsiveness of the brain dopaminergic system and NO system in the mechanism of resistance against stress-induced disturbances.     

Production and storage of nitric oxide in adaptation to hypoxia.

Adaptation to hypobaric hypoxia is known to exert multiple protective effects related with nitric oxide (NO). However the effect of adaptation to hypoxia on NO metabolism has remained unclear in many respects. In the present work we studied the interrelation between NO production and storage in the process of adaptation to hypoxia. The NO production was determined by the total nitrite/nitrate concentration in rats plasma. The volume of NO store was evaluated in vitro by the magnitude of isolated aorta relaxation to diethyldithiocarbamate. It was shown that both the nitrite/nitrate level and the NO store increased as adaptation to hypoxia developed. Furthermore, the NO store volume significantly correlated with plasma nitrite/nitrate. Therefore, adaptation to hypoxia stimulates NO production and storage and these effects can potentially underlie NO-dependent beneficial effects of adaptation.     

Role of nitric oxide in adaptation to hypoxia and adaptive defense

Adaptation to hypoxia is beneficial in cardiovascular pathology related to NO shortage or overproduction. However, the question about the influence of adaptation to hypoxia on NO metabolism has remained open. The present work was aimed at the relationship between processes of NO production and storage during adaptation to hypoxia and the possible protective significance of these processes. Rats were adapted to intermittent hypobaric hypoxia in an altitude chamber. NO production was determined by plasma nitrite/nitrate level. Vascular NO stores were evaluated by relaxation of the isolated aorta to diethyldithiocarbamate. Experimental myocardial infarction was used as a model of NO overproduction; stroke-prone spontaneously hypertensive rats (SHR-SP) were used as a model of NO shortage. During adaptation to hypoxia, the plasma nitrite/nitrate level progressively increased and was correlated with the increase in NO stores. Adaptation to hypoxia prevented the excessive endothelium-dependent relaxation and hypotension characteristic for myocardial infarction. At the same time, the adaptation attenuated the increase in blood pressure and prevented the impairment of endothelium-dependent relaxation in SHR-SP. The data suggest that NO stores induced by adaptation to hypoxia can either bind excessive NO to protect the organism against NO overproduction or provide a NO reserve to be used in NO deficiency.     

Antistress Effect of Nitric Oxide

Rat strains feature different resistances to stress. The increased production of nitric oxide (NO) in the August strain prevents the appearance of ulcerous lesions of gastric mucosa and behavioral changes induced by restraint stress. Wistar rats feature a lower level of NO production and are more sensitive to restraint stress compared to the August rats according to both the ulcerous gastric lesions and behavioral parameters. The stress-induced release of catecholamines was mimicked by experimental hyperfunction of the dopaminergic (DA) system induced by L-DOPA. The NO synthase inhibitor N-nitro-L-arginine (L-NNA) enhanced the L-DOPA-induced behavioral changes. This effect was more pronounced in the August strain. The administration of the exogenous NO donor, dinitrosyl iron complexes (DNIC), limited the behavioral disturbances induced by L-DOPA in both rat strains. The protective effect of DNIC in conditions of the DA system hyperfunction is similar to the effect of a D₂blocker sulpiride. Thus, NO has a central antistress effect apparently mediated by limiting the release of catecholamines.     

Nitric oxide plays a role in the protective effects of short-term adaptation to hypoxia on the stress-induced disorders in rats of the Krushinsky-Molodkina strain

The NO-synthase inhibitor L-NNA (2.5 mg/100 g) abolished the protective effects of short-term adaptation to hypoxia (1 h, 5000 m above sea level) on the development of stress-induced disorders on the model of acoustic stress in the Krushinskii-Molodkina rats genetically predisposed to audiogenic seizures. Using the electronic spine resonance method (ESR) we also demonstrated an increase in NO production during short-term hypoxia in the blood and spleen. The results suggest that NO plays a positive role in protective effects of short-term adaptation to hypoxia.     

Nitric oxide is involved in the protective effects of short-term adaptaion to hypoxia in the course of stress-induced disorders in Krushinsky-Molodkina rats

A possible involvement of nitric oxide in the protective effect of short-term adaptation of Krushinsky-Molodkina rats to mild hypoxia simulating 5000 m above sea level was studied. Nitric oxide proved to have a considerable protective effect on stress-induced disorders in Krushinsky-Molodkina rats as demonstrated using NO-synthase inhibitors and NO monitoring by electron spin resonance under different experimental conditions.     

Transforming growth factor alpha-mediated gastroprotection against stress ulceration in the rat: involvement of capsaicin-sensitive sensory neurons

Exogenously administered TGF alpha has been shown to protect rodent gastric mucosa against injury caused by acid-dependent and acid-independent injury. The present study examined whether the gastroprotective effects of TGF alpha on stress-induced gastric ulceration in the rat involves activation of capsaicin-sensitive sensory neurons. Fasted male SD rats were subjected to water restraint stress (WRS) for four hours. Thereafter, rats were euthanized; the stomach opened and macroscopic areas of gastric ulceration quantitated (mm(2)). Gastric tissue contents of TGF alpha and the sensory neuropeptide, calcitonin gene-related peptide (CGRP) were determined by radioimmunoassay. Prior to stress rats received TGF alpha 50, 100 or 200 microg/kg by intraperitoneal injection. Sensory denervation was accomplished by high dose capsaicin treatment. WRS caused severe ulceration in the gastric corpus; 46.1 + 6.6 mm(2). Parenteral administration of TGF alpha caused dose-dependent reduction in gastric injury: 34.7 + 4.9 mm(2) with 50 microg/kg (p < 0.05); 25.4 + 3.6 mm(2) with 100 microg/kg (p < 0.001) and 9.4 + 0.8 mm(2) with 200 microg/kg (p < 0.001). The gastroprotective action of TGF alpha (200 microg/kg, i.p.) was abolished by capsaicin-induced sensory denervation. In addition, WRS ulceration was associated with significant reduction in gastric CGRP (-42%) and TGF alpha (-48%) content. Reduction in CGRP content was prevented by TGF alpha pretreatment. We conclude that: 1) TGF alpha caused dose-dependent gastroprotection against WRS ulceration, 2) TGF alpha-mediated gastric mucosal protection was prevented by capsaicin-induced sensory denervation and, 3) stress-induced injury was associated with significant reduction in gastric content of both TGF alpha and CGRP.     

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.     

Role of gastric microcirculation in the gastroprotection by glucocorticoids released during water-restraint stress in rats

Our previous investigations demonstrated that glucocorticoids released in response to stress protect gastric mucosa against stress-induced ulceration. This study was designed to determine whether gastric microcirculation is involved in the mechanism of gastroprotective glucocorticoid action. For this we evaluated the effects of deficiency of glucocorticoid production during 3 hr water-restraint stress and corticosterone replacement on the stress-induced gastric erosions, gastric microcirculation and arterial pressure in rats. The stress was produced in awake rats and gastric microcirculation and arterial pressure were evaluated in animals anesthetized in 3 hr after the onset of water-restraint stress. An in vivo microscopy technique for the direct visualization of gastric microcirculation was employed. The gastric submucosal and the superficial mucosal microvessels were monitored on television screen through a microscope and the pictures were stored by microfilming for the analysis of red blood cell velocity and vessel diameter. Gastric microcirculation was estimated on the base of both the volume blood flow velocity in submucosal microvessels and the diameter of superficial mucosal venous microvessels. Gastric erosions were quantitated by measuring the area of damage. Plasma corticosterone levels were also measured after 3 hr stress by fluorometry. Water-restraint stress induced an increase in corticosterone level, an appearance of gastric erosions, a decrease in volume blood flow velocity of submucosal microvessels, a dilatation of superficial mucosal microvessels, a decrease in arterial pressure. The deficiency of glucocorticoid production during water-restraint stress promoted the stress-induced gastric ulceration, a dilatation of mucosal microvessels, a decrease of blood flow velocity in submucosal microvessels and of arterial pressure. Corticosterone replacement eliminated the effects of deficiency of glucocorticoid production on all of the parameters under study. Thus, the stress-induced corticosterone rise decreased gastric ulceration, restricted both the reduction of blood flow velocity in submucosal microvessels and a dilatation of superficial mucosal venous microvessels during water-restraint stress. These data suggest that the gastroprotective action of glucocorticoids during stress may be provided by the maintenance of gastric blood flow.     

Effects of serotonin and stress on the state of the gastric mucosa in rats with the intact and transsected vagus nerve

In experiments on vagotomized and intact rats with the use of two models of experimental gastric ulceration (injection of serotonin and stress) it was demonstrated that the inhibitory action of vagotomy on haemorrhagic gastric effectiveness was more pronounced in stress than after serotonin application. Vagotomy decreased stress-induced erosive lesions but increased serotonin-induced erosions that may be a result of the increase of gastric tissue sensitivity to this amine which developed simultaneously with significant decrease of its level in gastric wall after vagotomy. Serotonin-antagonist peritol decreased stress-induced gastric disturbances in vagotomized rats more significantly than in intact rats; this suggested the great role of serotonin in anti-ulcerogenic effect of vagotomy.     

Chemotherapeutic potential of diazeniumdiolate-based aspirin prodrugs in breast cancer

Diazeniumdiolate-based aspirin prodrugs have previously been shown to retain the anti-inflammatory properties of aspirin while protecting against the common side effect of stomach ulceration. Initial analysis of two new prodrugs of aspirin that also release either nitroxyl (HNO) or nitric oxide (NO) demonstrated increased cytotoxicity toward human lung carcinoma cells compared to either aspirin or the parent nitrogen oxide donor. In addition, cytotoxicity was significantly lower in endothelial cells, suggesting cancer-specific sensitivity. To assess the chemotherapeutic potential of these new prodrugs in treatment of breast cancer, we studied their effect both in cultured cells and in a nude mouse model. Both prodrugs reduced growth of breast adenocarcinoma cells more effectively than the parent compounds while not being appreciably cytotoxic in a related nontumorigenic cell line (MCF-10A). The HNO donor also was more cytotoxic than the related NO donor. The basis for the observed specificity was investigated in terms of impact on metabolism, DNA damage and repair, apoptosis, angiogenesis and metastasis. The results suggest a significant pharmacological potential for treatment of breast cancer.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

Nitrogen oxide storage as a factor of adaptive defence

Adaptation to environmental factors possesses multiple NO-dependent protective effects and stimulates the NO storage. An adaptation to a mild stress was shown to reduce the death rate in rats from 57% to 8% and to prevent a heat shock-induced hypotension and endothelial overactivation. Treatment of the rats with the NO-synthase inhibitor L-NNA interfered with the NO storage and formation of protective effects, while the NO donor dinitrosyl iron complex facilitated the NO storage and simulated the adaptive defence. The data obtained suggest an important role of the NO storage in adaptive defence of the organism.     

Dual action of nitric oxide in pathogenesis of indomethacin-induced small intestinal ulceration in rats.

We investigated the pathogenic role of nitric oxide (NO) in indomethacin-induced intestinal ulceration in rats. Nonfasting animals responded to a single administration of indomethacin (10 mg/kg, s.c.), resulting in multiple hemorrhagic lesions in the small intestine, mostly the jejunum and ileum. The damage was first observed 6 hr after indomethacin, the severity increasing progressively with time up to 24 hr later, accompanied with the gene expression of inducible NO synthase (iNOS) and the increase of nitrite and nitrate (NOx) contents in the mucosa. The ocurrence of damage was significantly prevented when iNOS induction was inhibited by dexamethasone given either once 0.5 hr before or twice 0.5 hr before and 6 hr after indomethacin. Likewise, aminoguanidine (a relatively selective iNOS inhibitor) reduced the severity of damage, irrespective whether given twice or as a single injection 6 hr after indomethacin. By contrast, the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) exhibited a biphasic effect, depending on the time of administration; the pre-administration worsened the damage, while the later administration reduced the severity of these lesions, yet both responses occureed in a L-arginine-sensitive manner. Pre-administration of L-NAME, but not aminoguanidine, significantly decreased NOx production in the intestinal mucosa of normal rats, while the increase of NOx production following indomethacin was significantly suppressed by the later administration of aminoguanidine as well as L-NAME. These results suggest that NO exerts a dual action in the pathogenesis of indomethacin-induced intestinal ulceration; NO generated by cNOS is protective against indomethacin, by maintaining the integrity of intestinal mucosa, while NO derived by iNOS plays a key pathogenic role in the ulcerogenic process.     

Evaluation of gastric anti-ulcer activity of fixed oil of Ocimum basilicum Linn. and its possible mechanism of action

Fixed oil of O. basilicum was found to possess significant antiulcer activity against aspirin, indomethacin, alcohol, histamine, reserpine, serotonin and stress-induced ulceration in experimental animal models. Significant inhibition was also observed in aspirin-induced gastric ulceration and secretion in pylorus ligated rats. The lipoxygenase inhibiting, histamine antagonistic and antisecretory effects of the oil could probably contribute towards antiulcer activity. O. basilicum fixed oil may be considered to be a drug of natural origin which possesses both antiinflammatory and anti-ulcer activity.     

The effects of cold and restraint on the glucose level, bilirubin level and the production of stressed ulcers in rats

The effect of restraint and cold on the glucose level, bilirubin level and production of stress ulcers were studied in white, adult male, Sprague-Dawley rats of the Holtzman strain. In addition, the role of mucin in reducing incidence of gastric lesions produced by stress was investigated. The highest incidence and depth of ulceration was produced by the stress factor combination of fasting for 24 h followed by physical restraint and cold exposure for 2 h. Under these conditions only one of the experimental rats died throughout the experimentation period. Fasting alone did not produce ulceration. The stress factor combination of fasting for 24 h, followed by physical restraint and cold exposure for 2 h resulted in an increase in glucose level. Increase in bilirubin level was highest in the stress factor combination of fasting, physical restraint and cold exposure acting synergistically. It can thus be hypothesized that the higher the bilirubin level the greater is the rate of red blood cell destruction. Introduction of 2 ml mucin directly into the stomach of the stressed rats reduced ulceration incidence by more than 50%.     

Distribution and metabolism of ingested NO3- and NO2- in germfree and conventional-flora rats.

Germfree and conventional-flora Sprague-Dawley rats were fed sodium nitrate or sodium nitrite in their drinking water (1,000 microgram/ml), and various organs, tissues, and sections of the intestinal tract were assayed for nitrate (NO3-) and nitrite (NO2-) by a spectrophotometric method. When fed NO3-, germfree rats had chemically detectable levels of NO3- (only) in the stomach, small intestine, cecum, and colon. Conventional-flora rats fed NO3- had both NO3- and NO2- in the stomach, but only NO3- in the small intestine and colon. When fed NO2-, germfree rats had both NO3- and NO2- in the entire gastrointestinal tract. Conventional-flora rats fed NO2- had both ions in the stomach and small intestine, but only NO3- in the large intestine. Conventional-flora rats fed NO3- or NO2- had lower amounts of these ions in the gastrointestinal tract than comparably fed germfree rats. Control (non-NO3- or NO2--fed) germfree and conventional-flora rats had trace amounts of NO3- (only) in their stomachs and bladders. These results, in conjunction with various in vitro studies with intestinal contents, suggest that NO3- or NO2- reduction is a function of the normal bacterial flora, whereas NO2- oxidation is attributable to the mammalian host. In addition, the distribution of these ions after their ingestion appears more widespread in the body than previously thought.     

NO-dependent mechanisms of adaptation to hypoxia.

In studying NO-dependent mechanisms of resistance to hypoxia, it was shown that (1) acute hypoxia induces NO overproduction in brain and leaves unaffected NO production in liver of rats; (2) adaptation to hypoxia decreases NO production in liver and brain; and (3) adaptation to hypoxia prevents NO overproduction in brain and potentiates NO synthesis in liver in acute hypoxia. Dinitrosyl iron complex (DNIC, 200 microg/kg, single dose, iv), a NO donor, decreases the resistance of animals to acute hypoxia by 30%. Nomega-nitro-L-arginine (L-NNA, 50 mg/kg, single dose, ip), a NO synthase inhibitor, and diethyl dithiocarbamate (DETC, 200 mg/kg, single dose, iv), a NO trap, increases this parameter 1.3 and 2 times, respectively. Adaptation to hypoxia developed against a background of accumulation of heat shock protein HSP70 in liver and brain. A course of DNIC reproduced the antihypoxic effect of adaptation. A course of L-NNA during adaptation hampered both accumulation of HSP70 and development of the antihypoxic effect. Therefore, NO and the NO-dependent activation of HSP70 synthesis play important roles in adaptation to hypoxia.     

Gastrointestinal nitric oxide generation in germ-free and conventional rats

Nitric oxide (NO) is a central mediator of various physiological events in the gastrointestinal tract. The influence of the intestinal microflora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats, we measured gaseous NO directly in the gastrointestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied by using an NO synthase (NOS) inhibitor, and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastrointestinal tract with the greatest concentrations in the stomach [>4,000 parts per billion (ppb)] and cecum (approximately 200 ppb) and lower concentrations in the small intestine and colon (< or =20 ppb). Cecal NO correlated with the levels in incubated luminal contents. NOS inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the cecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low (< or =30 ppb) throughout the gastrointestinal tract and absent in the incubated luminal contents. NO also remained low after a nitrate load. Our results demonstrate a pivotal role of the intestinal microflora in gastrointestinal NO generation. Distinctly compartmentalized qualitative and quantitative NO levels in conventional and germ-free rats reflect complex host microbial cross talks, possibly making NO a regulator of the intestinal eco system.