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.     

Effect of adaptation to periodic hypoxia on post-infarction fall of blood pressure and hyperactivation of the endothelium

Acute stress concomitant to the experimental myocardial infarction has induced endothelial hyperactivation of the rat aorta exhibited in an increase of inhibition of norepinephrine-induced contractions of vascular smooth muscle, enhanced endothelium-dependent relaxation correlating with a fall of systemic blood pressure. Preliminary adaptation of rats to intermittent hypobaric hypoxia greatly prevented the stress-induced endothelial hyperactivation and beneficially affected the postinfarction time course of blood pressure.     

慢性缺氧对大鼠肺动胍内皮依赖性舒张反应及CGMP含量的影响

This experiment was designed to investigate whether chronic hypoxia affect rat pulmonary artery (PA) endothelium-dependent relaxation and the content of cGMP in PA. Both ACh and ATP could induce endothelium-dependent relaxation of PA, not prevented by indomethacin, but completely abolished by methylene blue. These results indicated that vasodilatation of PA induced by both ACh and ATP is mediated by EDRF (endothelium-derived relaxing factor). Chronic hypoxia significantly depressed PA endothelium-dependent relaxation. The percent relaxation of IPPA and EPPA by 10(-6) mol/L ACh was 61.3% and 59.2% of those in control, and the percent relaxation of IPPA and EPPA by 1.8 x 10(-5) mol/L ATP was 64.9% and 55.3% respectively of the control. Chronic hypoxia also depressed SNP-induced endothelium-independent relaxation. Chronic hypoxia significantly decreased the content of cGMP in PA. The basic level of cGMP was 51.9 +/- 5.7 (n = 14) in hypoxia group and 84.9 +/- 9.7 (n = 14) pmol/g wet wt. in control group (P less than 0.01). After treatment of PA with ACh (10(-7) mol/L), the content of cGMP was 91.4 +/- 7.3 (n = 5) pmol/g wet wt. in hypoxic group and 240.8 +/- 30.6 (n = 5) pmol/g wet wt. in control group (P less than 0.01). Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of soluble guanylate cyclase in vascular smooth muscle cells.     

Effects of Choto-san on hemorheological factors and vascular function in stroke-prone spontaneously hypertensive rats

Choto-san is a formula used for the treatment of headache and vertigo. Recently it has often also been used for hypertension and dementia. One of the mechanisms involved is thought to be the improvement of blood circulation, but the details are still unclear. In this study, the effect of Chotosan was studied on nitric oxide (NO) function, hemorheological factors and endothelial function in stroke-prone spontaneously hypertensive rats (SHR-SP). Rats were given Choto-san in drinking water for eight weeks. Body weight, blood pressure, serum NO2-/NO3-, lipid peroxides, blood viscosity, erythrocyte deformability and endothelium-dependent/-independent relaxation were measured. The results indicated that Choto-san caused a decrease in blood pressure and an increase in erythrocyte deformability and NO function. Blood viscosity was not changed. Furthermore, endothelium-dependent relaxation by acetylcholine was significantly increased as compared to control. In this study, it was supposed that Choto-san had a protective effect on the endothelium. SHR-SP is a useful model for human brain stroke, and Choto-san showed a protective effect against cerebral vascular injury in the susceptible rat.     

Role of the endothelium in the development of the contractile reactions of vascular smooth muscle during decreased oxygenation

Noradrenaline-preactivated vascular smooth muscles (VSM) of the rat thoracic aorta showed two-phase reactions in response to decreased oxygenation: significant relaxation was preceded by transient constriction. When the endothelium was removed only VSM relaxation phase was retained, with no constriction observed. The data obtained suggest an endothelium-dependent nature of VSM constriction reaction to hypoxia, in contrast to endothelium-independent VSM relaxation. Intracellular calcium is also assumed to play an essential role in the formation of endothelium-dependent constriction VSM reaction to hypoxia.     

Adaptation to periodic hypoxia decreases ethanol consumption and abstinence-related damages to the internal organs during withdrawal in chronically alcoholized animals]

Adaptation to intermittent hypoxia in a hypobaric altitude chamber reduced two-fold ethanol consumption in chronically alcoholized rats and limited or eliminated abstinence syndrome. The effect of the adaptation was evident from prevented development of abstinence analgesia, enhanced alcohol consumption following deprivation, abstinence activation of lipid peroxidation in the liver, and release of hepato-specific enzymes fructose monophosphate and gamma-glutamyl transpeptidase into blood. At the same time adaptation prevented the fall of cardiac fibrillation threshold and pronounced disturbance of ventricular contraction and relaxation. The problem is discussed of using adaptation to intermittent hypoxia in the treatment for those forms of alcoholism in which abstinence plays the key role.     

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.     

Age-dependent modulation of endothelium-dependent vasodilatation by chronic hypoxia in ovine cranial arteries.

Although abundant evidence indicates that chronic hypoxia can induce pulmonary vascular remodeling, very little is known of the effects of chronic hypoxia on cerebrovascular structure and function, particularly in the fetus. Thus the present study explored the hypothesis that chronic hypoxemia also influences the size and shape of cerebrovascular smooth muscle and endothelial cells, with parallel changes in the reactivity of these cells to endothelium-dependent vasodilator stimuli. To test this hypothesis, measurements of endothelial and vascular smooth muscle cell size and density were made in silver-stained common carotid and middle cerebral arteries from term fetal and nonpregnant adult sheep maintained at an altitude of 3,820 m for 110 days. Chronic hypoxia induced an age-dependent remodeling that led to smooth muscle cells that were larger in fetal arteries but smaller in adult arteries. Chronic hypoxia also increased endothelial cell density in fetal arteries but reduced it in adult arteries. These combined effects resulted in an increased (adult carotid), decreased (adult middle cerebral), or unchanged (fetal arteries) per cell serosal volume of distribution for endothelial factors. Despite this heterogeneity, the magnitude of endothelium-dependent vasodilatation to A23187, measured in vitro, was largely preserved, although sensitivity to this relaxant was uniformly depressed. N(G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and endothelium denudation each independently blocked A23187-induced vasodilation without unmasking any residual vasoconstrictor effect. Indomethacin did not significantly attenuate A23187-induced relaxation except in the hypoxic adult middle cerebral, where a small contribution of prostanoids was evident. Vascular sensitivity to exogenous nitric oxide (NO) was uniformly increased by chronic hypoxia. From these results, we conclude that chronic hypoxia reduced endothelial NO release while also upregulating some component of the NO-cGMP-PKG vasodilator pathway. These offsetting effects appear to preserve endothelium-dependent vasodilation after adaptation to chronic hypoxia.     

Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."     

The Role of Preventing Nitric Oxide Deficiency in the Antihypertensive Effect of Adaptation to Hypoxia

Shortage of endothelial nitric oxide (NO) manifested as decreased daily urinary excretion of nitrate and nitrite as well as attenuated endothelium-dependent relaxation of conduit and resistance vessels progresses with age-related increase of blood pressure (BP) in stroke-prone spontaneously hypertensive rats (SHRSP). Simultaneous NO-dependent suppression of vascular contractions is, apparently, due to the inducible NO synthase activity in vascular smooth muscle specific for spontaneously hypertensive rat. The adaptation of rats to hypobaric hypoxia initiated at early hypertensive stage (at the age of 5–6 weeks) decelerates hypertension progress. The antihypertensive effect of the adaptation was accompanied by stimulation of endothelial NO synthesis and prevention of impaired NO-dependent response in isolated blood vessels. Nitric oxide stores were formed in the vascular wall of SHRSP and WKY rats at the same time. The obtained data indicate that the correction of endothelial NO deficiency plays a significant role in the antihypertensive effect of adaptation to hypoxia.     

慢性缺氧大鼠肺动脉内皮依赖性舒张反应与内皮结构的动态变化

本实验对慢性减压缺氧（５０００ｍ）过程中肺动脉ＡＣｈ内皮依赖性舒张反应作了动态观察,并结合分析了其与内皮超微结构和肺动脉压演变的关系。结果表明,缺氧３─２１ｄ,平均肺动脉压（ｍＰＡＰ）显著递增（Ｐ＜０．０５─０．００１）,而缺氧４０ｄ组基本与缺氧２１ｄ组持平,未再进一步升高。缺氧１ｄ组,各ＡＣｈ浓度（１０－１０、１０－９、１０－７、１０－６、１０－５ｍｏｌ／Ｌ）引起的内皮依赖性舒张反应明显受抑（Ｐ＜０．０５─０．００１）。缺氧７ｄ组,舒张反应的受抑程度与缺氧１ｄ组基本相同;但ＡＣｈ１０－５ｍｏｌ／Ｌ引发的反应则较缺氧１ｄ时更弱。缺氧２１ｄ和４０ｄ组,ＡＣｈ１０－６和１０－５ｍｏｌ／Ｌ引起的舒张反应,尽管仍显著低于对照,但却基本上高于缺氧１ｄ和７ｄ组。其余各浓度ＡＣｈ引发的反应则已趋于恢复至对照水平。电镜观察,缺氧１─１４ｄ肺动脉内皮呈逐渐加重的水肿变性;缺氧２１─４０ｄ内皮水肿消失,代之出现渐趋活跃的内皮增生。结果提示,随缺氧时间延长,因内皮从损伤逐渐加重到出现代偿适应,可能存在相应的内皮舒张因子由释放减少到有所恢复的动态变化过程,并对整体肺动脉压有一定程度的影响。     

Effects of chelator treatment on aorta and corpus cavernosum from diabetic rats.

Transition-metal catalyzed reactions contribute to oxidative stress, which has been implicated in the pathogenesis of diabetic complications. The aim was to evaluate the effects of treatment with the transition metal chelator trientine on endothelium-dependent relaxation of aorta and corpus cavernosum from streptozotocin-induced diabetes of 8 weeks duration in rats. Effects on cavernosum autonomic innervation were also examined. Diabetes caused a 30.1 +/- 3.8% reduction in maximum aorta endothelium-dependent relaxation to acetylcholine (ACh), which was markedly attenuated (72.7 +/- 10.6%) by trientine treatment. Reversal treatment (4 weeks untreated diabetes, 4 weeks trientine) did not effect endothelium-dependent relaxation compared with aortas from rats with 4 weeks of diabetes, however, there was a 22.5 +/- 6.2% improvement compared with 8 weeks of diabetes. Eight weeks of diabetes caused a 41.5 +/- 6.6% reduction in corpus cavernosum endothelium-dependent maximum relaxation to ACh that was 70.1 +/- 16.9% prevented by trientine. Cavernosum nonadrenergic, noncholinergic (NANC) nerve stimulation caused frequency-dependent relaxation to a maximum of 40.9 +/- 2.4%, which was reduced by diabetes to 24.2 +/- 2.1%. Trientine partially prevented this deficit, maximum relaxation being 31.9 +/- 2.3%. Thus, metal chelator treatment has beneficial effects on aorta and cavernosum endothelium-dependent relaxation and on cavernosum NANC innervation.     

Insulin-induced impairment via peroxynitrite production of endothelium-dependent relaxation and sarco/endoplasmic reticulum Ca(2+)-ATPase function in aortas from diabetic rats

We designed this study to determine whether a high insulin level and a diabetic state need to exist together to cause an impairment of endothelium-dependent relaxation. In diabetic rat aortas organ-cultured with insulin [vs both control rat aortas cultured with insulin and diabetic rat aortas cultured in serum-free medium]: (1) the relaxation responses to both acetylcholine (endothelium-dependent relaxation) and Angeli's salt (nitric oxide donor) were significantly weaker, (2) acetylcholine-stimulated nitric oxide production was significantly smaller, (3) superoxide and nitric oxide production into the culture medium was greater, and (4) the levels of both nitrotyrosine and tyrosine-nitrated sarco/endoplasmic reticulum calcium ATPase (SERCA) protein were greater. The insulin-induced effects were prevented by cotreatment with either a superoxide scavenger or a peroxynitrite scavenger. After preincubation with an irreversible SERCA inhibitor, the relaxation induced by the nitric oxide donor was significantly impaired in control aortas cultured with or without insulin and in diabetic aortas cultured without insulin, but not in diabetic aortas cultured with insulin. These results suggest that the coexistence of a high insulin level and an established diabetic state may lead to an excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in SERCA function.     

Vascular protective actions of a nitric oxide aspirin analog in both in vitro and in vivo models of diabetes mellitus

BACKGROUND: Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals. MATERIAL AND METHODS: Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae. RESULTS: Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue. CONCLUSIONS: Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.     

Divergent actions of chronic insulin treatment in vivo versus acute treatment ex vivo on diabetic-induced endothelial dysfunction

Streptozotocin-induced diabetes is associated with hyperglycemia and hypoinsulinemia. The role of insulin in diabetes-induced endothelial dysfunction is unknown. In the present study, we evaluated the effect of chronic insulin treatment in vivo versus acute insulin administration ex vivo on endothelium-dependent relaxation in aortic rings of the streptozotocin-induced diabetic rat. Relaxation to acetylcholine (but not A23187) was impaired in diabetic compared to control rings. This defect was prevented by chronic insulin treatment but was not reversed by acute insulin administration ex vivo. Thus, endothelial dysfunction in the streptozotocin-induced diabetic rat is specific for the chronic diabetic state and not to vascular toxicity of streptozotocin. Nevertheless, it is apparent that insulin at a physiological concentration does not cause an acute direct effect on facilitating endothelium-dependent relaxation in diabetic blood vessels.     

Effects of cilazapril on the retinal vessels in spontaneously hypertensive rats: corrosion cast and scanning electron microscopic study

The effects of the long-term oral angiotensin-converting enzyme inhibitor, cilazapril, on retinal circulation in stroke-prone spontaneously hypertensive (SHR-SP) rats were assessed by scanning electron microscopy (SEM), corrosion casts and transmission electron microscopy (TEM). Two groups of 20 male SHR-SP rats were compared. One group was treated with 10 mg/kg/day of cilazapril from 4 to 40 weeks of age, and the other group received no treatment. A third group of male Wistar-Kyoto (WKY) rats served as age-matched controls. At regular intervals the rats were weighed, and their systolic blood pressure was measured. Cilazapril normalized systolic arterial pressure to 121+/-2.7 mm Hg (SD) in the treated SHR-SP rats. There was no significant difference in body weight between the two groups of SHR-SP. In the 40-week-old SHR-SP rats without treatment corrosion cast and SEM revealed hypertensive retinal vascular changes. In the 40-week-old SHR-SP rats treated with cilazapril, these changes were markedly decreased to the level seen in WKY rats. The differences in caliber of retinal capillaries between the treated SHR-SP and untreated SHR-SP rats were statistically significant (p<.0001). TEM in the cilazapril-treated SHR-SP rats revealed intact basement membranes (0.29+/-0.057 microm) of the endothelial cells and pericytes, but in the untreated SHR-SP rats the basement membrane was thickened (0.51+/-0.123 microm) (p<.0001) and the pericytes damaged. Our results show that the long-term administration of cilazapril decreased systolic arterial pressure to a nearly normal level and prevented hypertensive retinal vascular changes, probably by improving endothelial function. The effects of cilazapril on the retinal vasculature are described for the first time. SEM of corrosion casts is a valuable technique for showing the effects of some drugs on the vasculature easily, precisely and three-dimensionally.     

Erigeron breviscapus prevents defective endothelium-dependent relaxation in diabetic rat aorta

We examined the endothelium-dependent relaxation response to acetylcholine (Ach) in streptozotocin-induced diabetic rat aorta at the stages of 2- and 6-wks' duration in vitro, and compared with another two groups which were treated with dietary supplement of 0.1% Aminoquanidine (AG) and 0.5% Erigeron breviscapus (EB) from 1-week of diabetes induction. At the stage of 2-wks' duration of diabetes, relaxation responses to lower concentrations of Ach in 0.3 uM phenylepherine-precontracted aortas were diminished significantly (P<0.05) compared with age-matched control, but the maximal relaxation of Ach remained unchanged. At the stage of 6-wks' duration, diabetes caused an approximately 60% (P<0.001) deficit in maximum relaxation, and this was significantly (P<0.001) prevented in AG and EB treated groups. There was an approximately 40% enhancement in the maximum contractile response to phenylepherine with diabetes (P<0.05), which was unaffected significantly by AG and EB treatments. The data suggest that the defective endothelium-dependent relaxation in diabetic rat aorta occurred as early as 2-wks' duration of diabetes, and the treatments of AG and EB could protect vascular endothelium although the deficits in vascular smooth muscle contractile responses were not protected.     

Effects of diabetes and evening primrose oil treatment on responses of aorta,corpus cavernosum and mesenteric vasculature in rats

Diabetes causes endothelial dysfunction, with deleterious effects on nitric oxide (NO) mediated vasodilatation. However, in many vessels other local vasodilators such as endothelium-derived hyperpolarizing factor (EDHF), prostacyclin, epoxides or endocannabinoids are also important. Several of these factors may be derived from omega-6 essential fatty acids via arachidonate metabolism. Diabetes inhibits this pathway, a defect that may be bypassed by diets enriched with omega-6 gamma-linolenic acid-containing oils such as evening primrose oil (EPO). The aim was to examine the effects of preventive EPO treatment on endothelium-dependent and neurally mediated vasorelaxation. Diabetes was induced by streptozotocin in rats; duration was 8 weeks. Vascular responses were examined in vitro on thoracic aorta, corpus cavernosum and perfused mesenteric bed preparations. Diabetes caused 25% and 35% deficits, respectively, in aorta and corpus cavernosum NO-mediated endothelium-dependent relaxation to acetylcholine that were largely unaffected by EPO treatment. Moreover, a 44% reduction in maximum corpus cavernosum vasorelaxation to nitrergic nerve stimulation was not prevented by EPO. However, for the mesenteric vascular bed, a 29% diminution of responses to acetylcholine, mediated by both NO and EDHF, was 84% attenuated by EPO treatment. When the EDHF component was isolated during NO synthase inhibition, a 76% diabetic deficit was noted. This was completely prevented by EPO treatment, which also caused supernormal EDHF responses in nondiabetic rats. EPO treatment prevented the development of deficits in endothelium-dependent relaxation in diabetic rats. Effects were particularly marked on the resistance vessel EDHF system, which may have potential therapeutic relevance for diabetic microvascular complications.     

Dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta?

Endothelial cells modulate the function of their underlying smooth muscle. Thus, altered endothelial behavior could be important in the pathogenesis of vascular and lymphatic diseases, including human and animal filariasis. Endothelium-dependent relaxation is depressed in both in vivo canine femoral artery of dogs infected with Dirofilaria immitis and in vitro rat aorta exposed to adult D. immitis. The experiments reported here were designed to determine if filarial cyclooxygenase products could depress endothelium-dependent relaxation in vitro. Pretreatment of the parasites, but not the vascular ring, with either indomethacin or aspirin, prevented filarial-induced depression of relaxation. Analysis of heartworm-conditioned medium by gas chromatography--mass spectrometry revealed two peaks in the biologically active medium that were not present in the control. One peak had a retention time and chromatographic profile characteristic of derivatized PGD2 standard, and the other was not identified. Incubation of the vascular ring with PGD2 mimicked filarial-induced depression of endothelium-dependent relaxation at low, but not high, concentrations of acetylcholine. Thus, filarial PGD2 may be involved in altered endothelium-dependent relaxation seen in heartworm-infected dogs.     

Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia

As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.