Detection and Description of Various Stores of Nitric Oxide Store in Vascular Wall

We analyzed the possibility of the existence of various NO pools in the vascular wall. Incubation of isolated rat aorta with dinitrosyl iron complex (NO donor) led to the formation of NO stores in the vascular wall detected by vascular relaxation response induced by diethyldithiocarbamate and N-acetylcysteine. Comparison of the effects of successive application of diethyldithiocarbamate and N-acetylcysteine revealed two NO pools (one pool responded to both agents, while other responded only to N-acetylcysteine). Inhibition of guanylate cyclase with methylene blue abolished the response to diethyldithiocarbamate, while the reaction to N-acetylcysteine decreased by the value, corresponding to diethyldithiocarbamate-dependent relaxation. It is hypothesized that in the vascular wall NO is stored in the form protein-bound dinitrosyl iron complexes and S-nitrosothiols in hydrophilic and hydrophobic cell compartments.     

Regional cerebral blood flow during acute hypoxia in individuals susceptible to acute mountain sickness

Individuals susceptible to high altitude pulmonary edema show altered pulmonary vascular responses within minutes of exposure to hypoxia. We hypothesized that a similar acute-phase vulnerability to hypoxia may exist in the brain of individuals susceptible to acute mountain sickness (AMS). In established AMS and high altitude cerebral edema, there is a propensity for vasogenic white matter edema. We therefore hypothesized that increased cerebral blood flow (CBF) during acute hypoxia would also be disproportionately greater in white matter (WM) than grey matter (GM) in AMS-susceptible subjects. We quantified regional CBF using arterial spin labeling MRI during 30 min hypoxia (F(I)O(2) = 0.125) in two groups: AMS-susceptible (AMS-S, n = 6) who invariably experienced AMS at altitude, and AMS-resistant (AMS-R, n = 6) who never experienced AMS despite multiple rapid ascents to high altitude. SaO(2) during hypoxia did not differ between groups (AMS-S = 87+/-4%, AMS-R = 89+/-3%, p = 0.3). Steady-state whole-brain CBF increased in hypoxia (p<0.005), but did not differ between groups (normoxia: AMS-S = 42.7+/-14.0 ml/(100 g min), AMS-R = 41.7+/-10.1 ml/(100 g min); hypoxia: AMS-S = 47.8+/-19.5 ml/(100 g min), AMS-R = 48.2+/-10.1 ml/(100 g min), p = 0.65), and cerebral oxygen delivery remained constant. The percent change in CBF did not differ between brain regions or between groups (although absolute CBF change was greater in GM): (GM: AMS-S = 6.1+/-7.7 ml/(100 g min) (10+/-11%), AMS-R = 8.3+/-5.7 ml/(100 g min) (17+/-11%), p = 0.57; WM: AMS-S = 4.3+/-5.1 ml/(100 g min) (12+/-15%), AMS-R = 4.8+/-2.9 ml/(100 g min) (16+/-9%), p = 0.82). CONCLUSION: CBF increases in acute hypoxia, but is not different between WM and GM, irrespective of AMS susceptibility. Acute phase differences in regional CBF during acute hypoxia are not a primary feature of susceptibility to AMS.     

Skeletal muscle blood flow in humans and its regulation during exercise

Regional limb blood flow has been measured with dilution techniques (cardio-green or thermodilution) and ultrasound Doppler. When applied to the femoral artery and vein at rest and during dynamical exercise these methods give similar reproducible results. The blood flow in the femoral artery is ～0.3 L min^?1 at rest and increases linearly with dynamical knee-extensor exercise as a function of the power output to 6–10 L min^?1 (Q = 1.94 + 0.07 load). Considering the size of the knee-extensor muscles, perfusion during peak effort may amount to 2–3 L kg^?1 min^?1, i.e. ～100-fold elevation from rest. The onset of hyperaemia is very fast at the start of exercise with T_½ of 2–10 s related to the power output with the muscle pump bringing about the very first increase in blood flow. A steady level is reached within ～10–150 s of exercise. At all exercise intensities the blood flow fluctuates primarily due to the variation in intramuscular pressure, resulting in a phase shift with the pulse pressure as a superimposed minor influence. Among the many vasoactive compounds likely to contribute to the vasodilation after the first contraction adenosine is a primary candidate as it can be demonstrated to (1) cause a change in limb blood flow when infused i.a., that is similar in time and magnitude as observed in exercise, and (2) become elevated in the interstitial space (microdialysis technique) during exercise to levels inducing vasodilation. NO appears less likely since NOS blockade with L -NMMA causing a reduced blood flow at rest and during recovery, it has no effect during exercise. Muscle contraction causes with some delay (60 s) an elevation in muscle sympathetic nerve activity (MSNA), related to the exercise intensity. The compounds produced in the contracting muscle activating the group III–IV sensory nerves (the muscle reflex) are unknown. In small muscle group exercise an elevation in MSNA may not cause vasoconstriction (functional sympatholysis). The mechanism for functional sympatholysis is still unknown. However, when engaging a large fraction of the muscle mass more intensely during exercise, the MSNA has an important functional role in maintaining blood pressure by limiting blood flow also to exercising muscles.     

缺氧时心肌血流量的变化及一氧化氮和内皮素-1的调节作用

目的探讨一氧化氮和内皮素-1在缺氧时对心肌血流量的调节作用。方法大鼠随机分为平原组和急性缺氧组,用99mTc标记蟾蜍红细胞测定心肌血流量,用Gess法和放免法分别测量血浆和心肌NO2-、内皮素-1（endothelin-1,ET-1）含量,用双波长分光光度法测量一氧化氮氧合酶（nitric oxide synthase,NOS）活性。结果急性缺氧导致左右心室心肌血流量、血浆和心肌血NO2-、ET-1含量、NOS活性明显增高（P〈0.05）,左右心室心肌血管阻力和心肌ET-1/NO2-比值明显下降（P〈0.05）,血球压积（Hct）及心室重量指数无明显变化。结论急性缺氧时,左右心室心肌血流量增加,ET-1/NO参与了急性缺氧时心肌血流量的调节,以NO的扩血管作用为主。     

新生大鼠缺血缺氧性脑病时脑微循环血流及一氧化氮的变化

目的：探讨脑微循环血流及一氧化氮（NO）在新生大鼠缺血缺氧性脑病（HIE）时的变化。方法：结扎新生大鼠右侧颈总动脉，观察脑微循环血流量、脑组织含水量、NO、丙二醛（MDA）含量的变化。结果：新生大鼠HIE动物有明显的左侧偏瘫等行为障碍，肉眼观察脑组织变白，脑微循环血流量（2 h、第2 d、第4 d）明显低于对照组（P<0.01）；脑组织含水量（2 h、第2 d）明显高于对照组（P<0.01）；NO及MDA含量（2 h、第2 d、第4 d）明显多于对照组（P<0.01或P<0.05）。结论：新生大鼠HIE时脑微循环血流量减少、NO含量及脂质过氧化的代谢产物MDA含量增高。     

Effects of endothelin receptor type A antagonism and nitric oxide synthase inhibition on cerebral blood flow in hypertensive rats

Effects of the endothelin receptor type A antagonist BQ 123 and the NO synthase inhibitor L -NMMA on cerebral blood flow were studied in vivo in anaesthetized hypertensive (SHR) and normotensive (WKY) rats. The effects of acetylcholine following pre-treatment with these drugs were also studied with the microsphere method for blood flow determination in the cortex, thalamus, caudatus, pons, medulla, cerebellum and hypophysis. BQ 123 (1 mg kg^?1) induced only minor effects on cerebral blood flow in both strains (n = 8), whereas L -NMMA (N = 8; 20 mg kg^?1) reduced regional cerebral blood flow significantly in most regions (21–54%) in the hypertensive, but not in the normotensive rat. In normotensive rats pre-treated with BQ 123 intravenous administration of acetylcholine (2 μg kg^?1 min^?1) induced a widespread significant increase (20–50%) in cerebral blood flow despite a reduction of the mean arterial blood pressure, while no significant effects were seen in hypertensive animals. Intravenous infusion of acetylcholine in animals pre-treated with L -NMMA did not affect cerebral blood flow in most regions in either of the two rat strains. In conclusion, a vasodilatory response to acetylcholine was found following endothelin receptor A antagonism in the WKY rat only, suggesting a role for endothelin in the control of cerebral blood flow in this strain. Furthermore, a higher basal vasodilating nitric oxide-tone seems to be present in the hypertensive rat compared with the normotensive rat.     

Nitric oxide regulates ovarian blood flow in the rat during the periovulatory period

BACKGROUND: The aim of this study was to characterize the roles of nitric oxide (NO) on the rat ovarian blood flow (OBF) during the preovulatory period. METHODS AND RESULTS: Immature Sprague-Dawley rats were primed with pregnant mares' serum gonadotrophin (PMSG, 15 IU) and given hCG (15 IU) 48 h later. The ovary was exposed 48-56 h after PMSG, a laser Doppler probe was attached to the ovarian surface and OBF was measured at two time periods: preovulatory (PO) 48 h after PMSG and ovulatory (OV) 6-8 h after hCG. A non-selective NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), was injected i.v. (4 and 10 mg/kg) or intrabursally (1 mg/kg). Intravenous administration of L-NAME to OV rats rapidly increased blood pressure and reduced OBF by 30%, which returned to the pretreatment level within 30 min. L-NAME given into the ovarian bursa of both PO and OV rats did not affect blood pressure and reduced OBF by nearly 40%, which remained low throughout the experiment. Intravenous injection of hCG to PO rats increased OBF to 116.1% at 5 min and 133.5% at 30 min in relation to the pretreatment level. When L-NAME was given intrabursally, subsequent hCG injection was without effect. CONCLUSIONS: These results indicate that locally produced NO is important for the maintenance and increase of rat OBF during the preovulatory period.     

Effects of inhibition of neuronal nitric oxide synthase on NMDA-induced changes in cerebral blood flow and oxygen consumption

This study was performed to determine whether neuronal nitric oxide synthase (nNOS) is involved in altering regional cerebral blood flow (rCBF) and oxygen consumption during N-methyl-D-aspartate (NMDA) receptor stimulation. A craniotomy was performed in rats, under isoflurane anesthesia, to expose the cerebral cortex. For the control group (n=7), an NMDA patch (10^–3 M) was applied to the exposed cortex (ipsilateral cortex, IC) for 10 min before determining rCBF and O₂ consumption. The patch was changed every 5 min. To block nNOS, 7-nitroindazole (7-NI, 25 mg/kg i.p.) was administered 30 min before NMDA application (7-NI group, n=7). The autoradiographic technique was used to determine rCBF and regional O₂ consumption was measured using cryomicrospectrophotometry. Blood pressure, heart rate, blood gases, and hemoglobin were similar between the two groups. In the control group, rCBF (108±32 ml/100 g per min) and O₂ consumption (4.8±0.8 ml O₂/100 g per min) of the IC where NMDA was applied were higher than those of the contralateral cortex (CC) (78±16 ml/100 g per min and 3.1±0.4 ml O₂/100 g per min, respectively). Neither rCBF nor O₂ consumption of the IC of the 7-NI group was statistically different from that of the CC. However, O₂ consumption of the IC of the 7-NI group was lower (3.9±1.0 ml O₂/100 g per min) than that of the IC of the control group. Our data demonstrated that a direct cortical application of NMDA increased O₂ consumption and rCBF, and that pretreatment with 7-NI not only attenuated the effects of NMDA on rCBF but also decreased the O₂ consumption during NMDA receptor stimulation. Electronic Publication     

Disturbance of the cerebral blood flow during ischemia and its correction by leu-enkephalin

Laboratory of General Pathology of the Microcirculation, Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 8, pp. 117–120, August, 1991.     

Contribution of nitric oxide to cerebral blood flow regulation under hypoxia in rats

This study was designed to clarify whether nitric oxide (NO) participates in the regulation of local cerebral blood flow (CBF) during hypoxia (inhalation of 15% O₂ in N₂). The CBF response to hind-paw stimulation (evoked CBF) of Sprague-Dawley (SD) rats was measured by laser-Doppler flowmetry. Physiological variables, such as heart rate, mean blood pressure, and PaCO₂ during hypoxia, were identical to those under normoxic conditions. Hypoxia increased the baseline CBF (17.5 ± 14.3%) and the normalized peak amplitude of evoked CBF (31.1 ± 18.5%) relative to those during normoxia. When an NOS inhibitor was infused intravenously, these differences were abolished in both the baseline CBF or evoked CBF between normoxic and hypoxic conditions, whereas the heart rate decreased and the mean blood pressure increased during hypoxia in comparison with these during normoxia. The field potential was constant under all experimental conditions. These results suggest that NO plays a major role in the regulation of baseline and evoked CBF during hypoxia.     

The effect of tolbutamide on cerebral blood flow during hypoxia and hypercapnia in the anaesthetized rat

The increase in blood flow in the cerebral cortex of the anaesthetized rat during hypoxia and hypercapnia was investigated. Cerebral blood flow (CBF) was measured using the hydrogen clearance method with acutely implanted platinum electrodes. Hypoxia (PaO₂ 35.3±2.4 Torr) and hypercapnia (PaCO₂ 68.1±5.1 Torr) increased basal CBF from 76.3±9.0 ml/100g/min to 168.1±20.1 ml/100g/min and 162.4±31.9 ml/100g/min respectively. The sulphonylurea tolbutamide (1mM in 1%DMSO) had no significant effect on CBF in hyperoxia or in hypercapnia. However, it attenuated the increase of CBF during hypoxia by 66 ±11% (P<0.01). This suggests that opening of tolbutamide-sensitive potassium channels may be involved in the process of hypoxic vasodilation in the rat cerebral cortex.     

In Vivo Accumulation of nitric oxide in blood vessels

Against the background of NO-synthase blockade, diethyldithiocarbamate had no effect on the tone of isolated rat aorta, but induced relaxation of aorta preparations isolated afterin vivo NO accumulation and isolated aorta incubated with dinitrosyl iron complex. Guanylate cyclase inhibitor methylene blue prevented the relaxation induced by diethyldithiocarbamate. These data suggest that accumulation of NO in the organism can result in its accumulation in the vessel wall. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 629–632, June, 1999     

Brain oxidation is an initial process in sleep induction

CNS activity is generally coupled to the vigilance state, being primarily active during wakefulness and primarily inactive during deep sleep. During periods of high neuronal activity, a significant volume of oxygen is used to maintain neuronal membrane potentials, which subsequently produces cytotoxic reactive oxygen species (ROS). Glutathione, a major endogenous antioxidant, is an important factor protecting against ROS-mediated neuronal degeneration. Glutathione has also been proposed to be a sleep-promoting substance, yet the relationship between sleep and cerebral oxidation remains unclear. Here we report that i.c.v. infusion of the organic peroxide t-butyl-hydroperoxide at a concentration below that triggering neurodegeneration (0.1 μmol/100 μl/10 h) promotes sleep in rats. Also, microinjection (2 nmol, 2 μl) or microdialysis (100 μM, 20 min) oft-butyl-hydroperoxide into the preoptic/anterior hypothalamus (POAH) induces the release of the sleep-inducing neuromodulators, nitric oxide and adenosine, without causing neurodegeneration. Nitric oxide and adenosine release was inhibited by co-dialysis of the N-methyl-d-aspartate receptor antagonist, d(−)-2-amino-5-phosphonopentanoic acid (D-AP5; 1 mM), suggesting that glutamate-induced neuronal excitation mediates the peroxide-induced release of nitric oxide and adenosine. Indeed, Ca²⁺ release from mitochondria and delayed-onset Ca²⁺ influx via N-methyl-d-aspartate receptors was visualized during peroxide exposure using Ca²⁺ indicator proteins (YC-2.1 and mitochondrial-targeted Pericam) expressed in organotypic cultures of the POAH. In the in vitro models, t-butyl-hydroperoxide (50 μM) causes dendritic swelling followed by the intracellular Ca²⁺ mobilization, and D-AP5 (100 μM) or glutathione (500 μM) inhibited t-butyl-hydroperoxide-induced intracellular Ca²⁺ mobilization and protected POAH neurons from oxidative stress.

These data suggest that low-level subcortical oxidation under the control of an antioxidant system may trigger sleep via the Ca²⁺-dependent release of sleep-inducing neuromodulators in the POAH, and thus we propose that a moderate increase of ROS during wakefulness in the neuronal circuits regulating sleep may be an initial trigger in sleep induction.     

Changes in cerebral blood flow during and after 48 h of both isocapnic and poikilocapnic hypoxia in humans

During acclimatization to the hypoxia of altitude, the cerebral circulation is exposed to arterial hypoxia and hypocapnia, two stimuli with opposing influences on cerebral blood flow (CBF). In order to understand the resultant changes in CBF, this study examined the responses of CBF during a period of constant mild hypoxia both with and without concomitant regulation of arterial P(CO2). Nine subjects were each exposed to two protocols in a purpose-built chamber: (1) 48 h of isocapnic hypoxia (Protocol I), where end-tidal P(O2) (P(ET,O2)) was held at 60 Torr and end-tidal P(CO2) (P(ET,CO2)) at the subject's resting value prior to experimentation; and (2) 48 h of poikilocapnic hypoxia (Protocol P), where P(ET,O2) was held at 60 Torr and P(ET,CO2) was uncontrolled. Transcranial Doppler ultrasound was used to assess CBF. At 24 h intervals during and after the hypoxic exposure CBF was measured and the sensitivity of CBF to acute variations in P(O2) and P(CO2) was determined. During Protocol P, P(ET,CO2) decreased by 13% (P < 0.001) and CBF decreased by 6% (P < 0.05), whereas during Protocol I, P(ET,CO2) and CBF remained unchanged. The sensitivity of CBF to acute variations in P(O2) and P(CO2) increased by 103% (P < 0.001) and 28% (P < 0.01), respectively, over the 48 h period of hypoxia. These changes did not differ between protocols. In conclusion, CBF decreases during mild poikilocapnic hypoxia, indicating that there is a predominant effect on CBF of the associated arterial hypocapnia. This fall occurs despite increases in the sensitivity of CBF to acute variations in P(O2)/P(CO2) arising directly from the hypoxic exposure.     

大鼠局灶性脑缺血后皮层和尾壳核NOS变化

目的：了解局灶性脑缺血再灌注过程中一氧化氮合酶（NOS）变化与脑缺血损伤的关系，探索治疗时间窗。材料和方法：用线栓法建立局灶性脑缺血模型，大脑中动脉阻塞（MCAO）的时间分别为15、30、60、90、120min，再灌注24h。用NADPH－d组织化学法，检测局灶性脑缺血再灌注后缺血半暗区（额叶皮层）和中心区（顶叶皮层和尾壳核）NOS活性变化。结果：半暗区NOS阳性神经元数量于60min达峰值；中心区NOS阳性神经元数量于30min达峰值，90－120min急剧减少。结论：局灶性脑缺血再灌注过程中NOS参与脑缺血损伤，在中心区与半暗区NOS活性变化不一致，半暗区NOS达峰值时间较中心区延长。推测中心区运用NOS抑制剂最佳时机在30min内，半暗区最佳时机在60min内。     

Renal arterial infusion of tempol prevents medullary hypoperfusion,hypoxia, and acute kidney injury in ovine Gram-negative sepsis

Aim

Renal medullary hypoperfusion and hypoxia precede acute kidney injury (AKI) in ovine sepsis. Oxidative/nitrosative stress, inflammation, and impaired nitric oxide generation may contribute to such pathophysiology. We tested whether the antioxidant and anti-inflammatory drug, tempol, may modify these responses.

Methods

Following unilateral nephrectomy, we inserted renal arterial catheters and laser-Doppler/oxygen-sensing probes in the renal cortex and medulla. Noanesthetized sheep were administered intravenous (IV) Escherichia coli and, at sepsis onset, IV tempol (IVT; 30 mg kg⁻¹ h⁻¹), renal arterial tempol (RAT; 3 mg kg⁻¹ h⁻¹), or vehicle.

Results

Septic sheep receiving vehicle developed renal medullary hypoperfusion (76 ± 16% decrease in perfusion), hypoxia (70 ± 13% decrease in oxygenation), and AKI (87 ± 8% decrease in creatinine clearance) with similar changes during IVT. However, RAT preserved medullary perfusion (1072 ± 307 to 1005 ± 271 units), oxygenation (46 ± 8 to 43 ± 6 mmHg), and creatinine clearance (61 ± 10 to 66 ± 20 mL min⁻¹). Plasma, renal medullary, and cortical tissue malonaldehyde and medullary 3-nitrotyrosine decreased significantly with sepsis but were unaffected by IVT or RAT. Consistent with decreased oxidative/nitrosative stress markers, cortical and medullary nuclear factor-erythroid-related factor-2 increased significantly and were unaffected by IVT or RAT. However, RAT prevented sepsis-induced overexpression of cortical tissue tumor necrosis factor alpha (TNF-α; 51 ± 16% decrease; p = 0.003) and medullary Thr-495 phosphorylation of endothelial nitric oxide synthase (eNOS; 63 ± 18% decrease; p = 0.015).

Conclusions

In ovine Gram-negative sepsis, renal arterial infusion of tempol prevented renal medullary hypoperfusion and hypoxia and AKI and decreased TNF-α expression and uncoupling of eNOS. However, it did not affect markers of oxidative/nitrosative stress, which were significantly decreased by Gram-negative sepsis.