The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart

The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA) decreased control values of CF only at lower values of CPP but did not change nitrite outflow indicating antioxidant properties of NDGA. In addition, L-NAME decreased the effects induced by NDGA on CF and nitrite outflow indicating the role of NO.     

Interaction between L-arginine: no system and cyclooxygenase metabolic products of arachidonic acid in coronary autoregulation.

Coronary autoregulation (CA) is the intrinsic ability of the heart to maintain its nutritive blood supply constant over a wide range of perfusion pressure. This phenomenon is regulated through several control mechanisms, while metabolic and myogenic control mechanism have dominant effects. In last few years, endothelial control mechanism, which is part of metabolic control, was intensive investigated. Dominant topic of endothelial-investigation was bioregulatory L-arginine: NO system, with his effective product--nitric oxide (NO). On the other hand, cyclooxygenase metabolic pathway products of arachidonic acid plays an important role in the control of vasomotor tone of coronary arteries. For this purpose, the aim of our study was to evaluate role of L-arginine: NO system, cyclooxygenase metabolites of arachidonic acid, as well as, their interactions in the control of CA of the isolated rat heart.. In our study rat hearts autoregulate CF between 50 and 90 cm H2O of CPP. Basal release (at 60 cm H2O) of NO (as nitrite), cAMP, cGMP and HX+X (i.e. adenosine) amounted to 2.85+/-0.25 nmol/min/g wt, 29.45+/-2.22 pmol/min/g wt, 0.43+/-0.08 pmol/min/g wt and 37.50+/-2.89 nmol/min/g wt respectively. Release of NO, cAMP and cGMP were strictly parallel with CPP-CF curve, while release of adenosine (i.e. HX + X) was an inverse function of perfusion pressure. Inhibition of NOS (L-NAME, 30 micromol/l) significantly widened autoregulatory range (40-100 cm H2O), with significant reduction in CF and NO- and cGMP release, while release of cAMP was completely reversed in the presence of L-NAME. However, inhibition of cyclooxygenase didn't influence autoregulatory range, with similar changes of NO- and cAMP-release and completely inversed values of released adenosine. When L-NAME an indomethacin (an nonspecific COX-inhibitor), 3 micromol/l where added together, they exhibit interactions between these two enzymatic systems. Namely, when L-NAME was added first, indomethacin didn't influence hemodynamic effects of NOS-inhibitor. On the other hand, when COX-inhibitor was added first, L-NAME widened autoregulatory range in small manner as after control autoregulatory experiments (40-90 cm H2O). All hemodynamic changes were followed with similar changes in NO-release, what suggest that exist interaction between L-arginine: NO system and COX-metabolites in the regulation of coronary autoregulation.     

The effects of vitamin C and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of vitamin C (ascorbic acid) on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by N(ω)-nitro-L-arginine monomethyl ester (L-NAME) in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40-120 cm H2O). Coronary flow, nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were determined. The experiments were performed during control conditions and in presence of vitamin C (100 μM) alone or vitamin C (100 μM) + L-NAME (30 μM). Administration of vitamin C induced only increase of nitrite levels, while vitamin C + L-NAME induced significant decrease of coronary flow above autoregulatory range, i.e. especially at higher coronary perfusion pressure (CPP) values, accompanied with similar dynamic in nitrite outflow. Vitamin C + L-NAME also induced significant decrease in TBARS production. The results of our study show no significant effects of vitamin C administration either on ROS levels or on coronary flow in isolated rat heart.     

The effects of folic acid and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart

The aim of this study was to assess the effects of folic acid on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by l-NAME in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180–200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40–120 cmH₂O). Coronary flow and markers of oxidative stress: nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were calculated. The experiments were performed during control conditions and in presence of folic acid (100 μM) alone or folic acid (100 μM) plus l-NAME (30 μM). Control values of coronary flow varied in range from 4.37 ± 0.10 ml/min/g wt at 40 cmH₂O to 12.05 ± 0.42 ml/min/g wt at 120 cmH₂O. Nitrite outflow varied from 1.68 ± 0.17 nmol/min/g wt at 40 cmH₂O to 3.56 ± 0.17 nmol/min/g wt at 120 cmH₂O and was parallel with coronary perfusion pressure-coronary flow curve. Folic acid significantly increased coronary flow (40–120 cmH₂O, 5.63 ± 0.10 ml/min/g wt and 15.2 ± 0.42 ml/min/g wt, respectively) and was accompanied by significant increase in nitrite outflow (2.28 ± 0.29 nmol/min/g wt at 40 cmH₂O to 6.66 ± 0.50 nmol/min/g wt at 120 cmH₂O). In addition, folic acid significantly decreased superoxide anion production especially at upper coronary perfusion pressure values (60% at 120 cmH₂O) and increased index of lipid peroxidation (37.16% at 120 cmH₂O), respectively. Folic acid plus l-NAME did not change control values of coronary flow significantly. However, folic acid plus l-NAME increased nitrite outflow especially at upper coronary perfusion pressure values (43.05% at 120 cmH₂O) and did not change significantly superoxide anion production or index of lipid peroxidation versus control values, respectively. The results clearly showed that on isolated rat hearts at gradually increased constant perfusion pressure, folic acid increased coronary flow, increased nitrite outflow, decreased superoxide anion production, and increased index of lipid peroxidation. These effects were reversed or blocked by l-NAME thus demonstrating mediation or at least participation of NO in the mechanism of the folic acid-induced effects.     

Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.     

Effect of carbohydrate supplements and water on exercise metabolism in the heat

Carbohydrate (CHO) supplements of different concentrations were compared with water to determine their effects on thermal regulation and plasma volume maintenance while subjects exercised for 2 h in the heat and to determine their impact on carbohydrate utilization. Trained cyclists (n = 12) rode at 48.8 +/- 0.8% maximal O2 consumption in an environmental chamber maintained at 33.0 +/- 0.1 degree C and 51.7 +/- 1.4% relative humidity on three separate occasions. During each exercise bout the subjects received 3 ml/kg body wt of H2O, a 2.0% glucose polymer (LC) solution, or an 8.5% glucose polymer (HC) solution every 15 min. Muscle biopsies from the vastus lateralis were obtained before and after the H2O and HC trials only. Rectal temperature and heart rate, but not O2 consumption, rose from the 10- to 120-min period of exercise. No differences among treatments were found for these variables. There were also no significant differences among treatments for percent changes in plasma volume and blood volume. Plasma glucose and insulin were unchanged during the H2O and LC trials but were significantly elevated during the HC trial. In addition, CHO oxidation was significantly greater during the HC trial than during the H2O trial from 60 to 120 min of exercise. However, the reduction in muscle glycogen during the HC trial (206.5 +/- 23.6 mumol/g protein) was significantly less (P less than 0.05) than during the H2O trial (342.3 +/- 41.9 mumol/g protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of tidal volume on pulmonary NO release,tissue lipid peroxidation and surfactant phospholipids

Mechanical stress during ventilation may cause or aggravate acute lung injury. This study investigates the influence of low vs. high tidal volume (V(t)) on factors known to play key roles in acute lung injury: nitric oxide release, eNOS and iNOS gene expression, lipid peroxidation (LPO), and surfactant phospholipids (PL). Isolated rabbit lungs were subjected to one of three ventilation patterns for 135 min (V(t)-PEEP): 6 ml/kg-0 cm H(2)O. 12 ml/kg-0 cm H(2)O 6 ml/kg-5 cm H(2)O, 12 ml/kg-0 cm H(2)O, and 6 ml/kg-5 cm H(2)O resulted in comparable peak inspiratory pressure (PIP). This allowed comparing low and high V(t) without dependence on PIP. Ventilatory patterns did not induce changes in pulmonary artery pressure, vascular permeability (K(f,c)), PIP or pulmonary compliance. High V(t) in comparison with both of the low V(t) groups caused an increase in BALF-nitrite (30.6+/-3.0* vs. 21.4+/-2.2 and 16.2+/-3.3 microM), BALF-PL (1110+/-19* vs. 750+/-68 and 634+/-82 microg/ml), and tissue LPO product accumulation (0.62+/-0.051* vs. 0.48+/-0.052 and 0.43+/-0.031 nmol/mg), *P<0.05 each. Perfusate nitrite and BALF-PL composition (assessed by use of 31P-NMR spectroscopy and MALDI-TOF mass spectrometry) did not differ among the groups. High V(t) ventilation reduced eNOS gene expression but did not affect iNOS expression. The increased release of NO and the accumulation of LPO products may represent early lung injury while elevated BALF-PL may reflect distension-induced surfactant secretion.     

High in comparison with low tidal volume ventilation aggravates oxidative stress-induced lung injury

Ventilator settings influence the development and outcome of acute lung injury. This study investigates the influence of low versus high tidal volume (V(t)) on oxidative stress-induced lung injury.Isolated rabbit lungs were subjected to one of three ventilation patterns (V(t)-positive end-expiratory pressure, PEEP): LVZP (6 ml/kg-0 cm H(2)O), HVZP (12 ml/kg-0 cm H(2)O), LV5P (6 ml/kg-5 cm H(2)O). These ventilation patterns allowed a comparison between low and high V(t) without dependence on peak inspiratory pressure (PIP). Infusion of hypochlorite (1000 nmol/min) or buffer (control) was started at t=0 min. Pulmonary artery pressure (PAP), PIP and weight were continuously recorded. Capillary filtration coefficient [K(f,c) (10(-4) ml s(-1) cm H(2)O(-1) g(-1))] was gravimetrically determined (-15/30/60/90/120 min).PIP averaged 5.8+/-0.6/13.9+/-0.6/13.9+/-0.4 cm H(2)O in the LVZP, HVZP and LV5P groups. PIP, K(f,c) or PAP did not change in control groups, indicating that none of the ventilation patterns caused lung injury by themselves. Hypochlorite-induced increase in K(f,c) but not hypochlorite-induced increase in PAP, was significantly attenuated in the LVZP-/LV5P- versus the HVZP-group (K(f,c,max.) 1.0+/-0.23/1.4+/-0.40 versus 3.2+/-1.0*). Experiments with hypochlorite were terminated due to excessive edema (>50 g) at 97+/-2.2/94.5+/-4.5 min in the LVZP-/LV5P-group versus 82+/-3.8* min in the HVZP-group (*: P<0.05).Low V(t) attenuated oxidative stress-induced increase in vascular permeability independently from PIP and PEEP.     

Hydrogen peroxide increases the activities of soxRS regulon enzymes and the levels of oxidized proteins and lipids in Escherichia coli

The effects of hydrogen peroxide treatments on Escherichia coli KS400 and AB1157 cells were assessed by monitoring the accumulation of oxidative damage products, carbonyl proteins and thiobarbituric acid-reactive substances (TBARS), as well as the activities of selected antioxidant enzymes. H(2)O(2) treatment stimulated increases in both TBARS and carbonyl protein levels in dose- and time-dependent manners in KS400 cells. The accumulation of TBARS was much more variable with H(2)O(2) treatment; TBARS content was significantly increased in response to 5 microM H(2)O(2), whereas a significant increase in carbonyl protein content occurred at 100 microM H(2)O(2). Similarly, treatment with 20 microM hydrogen peroxide for different lengths of time resulted in peak TBARS accumulation by 20 min, whereas carbonyl protein levels were significantly elevated only after 60 min. In AB1157 cells, treatment with 20 microM hydrogen peroxide for 20 min led to strong increases in both carbonyl protein and TBARS levels. This treatment also triggered increased activities of enzymes of the oxyR regulon (catalase, peroxidase, and glutathione reductase) in both strains. In the AB1157 strain, H(2)O(2) exposure also increased the activities of two enzymes of the soxRS regulon (superoxide dismutase and glucose-6-phosphate dehydrogenase) by 50-60%. The data show differential variability of lipids versus proteins to oxidative damage induced by H(2)O(2,) as well as strain-specific differences in the accumulation of damage products and the responses by antioxidant enzymes to H(2)O(2) stress.     

IL-2 induces pulmonary edema and vasoconstriction independent of circulating lymphocytes

We investigated the effect of IL-2 in the isolated guinea pig lung perfused with phosphate-buffered Ringer's solution (containing 0.5 g/100 ml albumin and 5.5 mM dextrose) to determine the mechanism of IL-2-induced pulmonary edema. IL-2 (0 to 10,000 U/ml) was added to the perfusate following a 10 min baseline steady-state period. Pulmonary arterial pressure (Ppa), pulmonary capillary pressure (Ppc), and change in lung weight (as a measure of developing pulmonary edema) were recorded at 0, 10, 30, 40, and 60 min. The capillary filtration coefficient (Kf.c), an index of vascular permeability to water, was measured at 30 and 60 min. Infusion of IL-2 increased Ppc (from 3.9 +/- 0.1 cm H2O at baseline to 8.8 +/- 1.1 cm H2O at 60 min for IL-2 at 2000 U/ml, p less than 0.01; and from 3.8 +/- 0.1 cm H2O at baseline to 8.9 +/- 0.6 cm H2O at 60 min for IL-2 at 10,000 U/ml, p less than 0.01. The lung weight also increased (32% at IL-2 concentration of 2000 U/ml, and 26% at IL-2 concentration of 10,000 U/ml) The capillary filtration coefficient did not change with IL-2 infusion. The IL-2 response was prevented using the pulmonary vasodilator, papaverine. The infusion of IL-2 was associated with the generation of thromboxane A2(TxA2) in the effluent perfusate. Inhibition of TxA2 synthetase using Dazoxiben prevented the pulmonary vasoconstriction and edema response to IL-2. In addition, IL-2 had no effect on the transendothelial clearance of 125I-albumin. The results indicate that IL-2 causes pulmonary edema secondary to an increase in Ppc. The response is mediated by IL-2 stimulation of TxA2 generation from the lung.     

Endothelium-dependent hypotensive and vasorelaxant effects of the essential oil from aerial parts of Mentha x villosa in rats.

Cardiovascular effects of an essential oil from the aerial parts of Mentha x villosa (OEMV) were tested in rats using a combined in vivo and in vitro approach. In non-anesthetized normotensive rats, OEMV (1, 5, 10, 20, 30 mg kg(-1) body wt., i.v.) induced a significant and dose-dependent hypotension (-3 +/- 1.8%; -6 +/- 0.7%; -40 +/- 6.7%; -58 +/- 3.8%; -57 +/- 2.1%, respectively) associated with decreases in heart rate (-1 +/- 0.3%; -9 +/- 0.9%; -17 +/- 3.2%; -72 +/- 3.1%; -82 +/- 1.4%, respectively). The hypotensive and bradycardic responses evoked by OEMV were attenuated and blocke by pre-treatment of the animals with atropine (2 mg kg(-1) body wt., i.v.). In isolated rat atrial preparations, OEMV (10, 100, 300, 500 microg ml(-1)) produced concentration-related negative chronotropic and inotropic effects (IC50 value = 229 +/- 17 and 120 +/- 13 microg ml(-1), respectively). In isolated rat aortic rings, increasing concentrations of OEM (10, 100, 300, 500 microg ml(-1)) were able to antagonize the effects of phenylephrine (1 microM), prostaglandin F2alpha (10 microM) and KCl (80 mM)-induced contractions (IC50 value = 255 +/- 9, 174 +/- 4 and 165 +/- 14 microg ml(-1), respectively). The vasorelaxant activity induced by OEMV was attenuated significantly by either endothelium removal (IC50 value = 304 +/- 9 microg ml(-1)), NG-nitro L-arginine methyl ester (L-NAME) 100 microM (IC50 value=359 +/- 18 microg ml(-1)), L-NAME 300 microM (IC50 value = 488 +/- 20 microg ml(-1)) or indomethacin 10 microM (IC50 value = 334 +/- 18 microg ml(-1)). However, it was not affected by atropine 1 microM (IC50 value = 247 +/- 12 microg ml(-1)). Furthermore, the hypotensive response induced by OEMV was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg kg(-1) body wt., i.v.), while bradycardia was not altered. The results suggest that the hypotensive effect induced by OEMV is probably due to its direct cardiodepressant action and peripheral vasodilation, which can be attributed to both endothelium-dependent (via EDRFs, at least NO and prostacyclin) and endothelium-independent mechanisms (such as Ca2+ channel blockade).     

环加氧酶-2抑制剂尼美舒利通过改善内皮功能和增加NO含量对抗大鼠心肌氧化应激损伤

本文旨在研究冠状动脉内皮和NO在选择性环加氧酶2（cyclooxygenase2,COX-2）抑制剂尼美舒利（nimesulide）对抗心肌氧化损伤中的作用。离体大鼠心脏行Langendorff灌流,给予H2O2（140Bmol／L）观察心脏收缩功能。用U-46619灌流心脏,使冠状动脉预收缩后,观察冠状动脉对内皮依赖性舒张因子5-HT和内皮非依赖性舒张因子硝普钠（sodiumnitroprusside,SNP）的反应。结果显示：（1）与空白对照组（100％）相比,H202灌流20min后,左心室发展压[left ventriculardevelo pedpressure,LVDP,（54．8±4．0）％],和心室内压最大变化速率【±dp／dtmax（50．8±3．1）％和（46．2±2．9）％]明显降低。H2O2灌流前尼美舒利（5μmol/L）预处理10min,能够显著抑制H2O2引起的LVDP和μdp/dtmax下降[（79．9±2．8）％,（80．3±2．6）％和（81．4±2．6）％,P〈0．0l]。（2）与空白对照组相比,H2O2灌流后,5-HT和SNP引起内皮依赖性和内皮非依赖性血管舒张功能均明显下降;而尼美舒利预处理10min能明显对抗内皮依赖性血管舒张功能的下降[（-22．2±4．2）％vsH2O2组（-6．0±2．5）％,P〈0．0l],但对其内皮非依赖性血管舒张功能的下降没有明显作用[（-2．0±1．8）％vsH202组（-7．0±3．5）％,P〉0．05]。（3）一氧化氮合酶（nitric oxide synthase,NOS）抑制剂L-NAME能够部分取消尼美舒利预处理对H20,应激心脏心功能指标的改善作用ILVDP和±dp/dtmax分别为（60．2±2．1）％,（63．9±2．4）％和（63．1±2．9）％,P〈0．01]。同时尼美舒利预处理10min能使H202应激心肌NO含量增加[（2．63±0．40）vs（1．36±0．23）nmol／gprotein,P〈0．051,而L-NAME抑制此作用。（4）选择性COX-1抑制剂吡罗昔康（piroxicam）预处理不能抑制H202引起的LVDP和±dp/dtmax下降,但促进左心室舒张末压（1eftventricular end diastolicpressure,LVEDP）升高;吡罗昔康对H202引起的内皮依赖性和内皮非依赖性血管舒张功能下降无显著作用。以上结果提示,选择性COX-2抑制剂尼美舒利能够对抗大鼠离体心肌氧化应激损伤,其机制可能是通过改善内皮依赖性血管舒张功能和增加心肌NO含量起作用。     

Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells

BACKGROUND: Empirical observations have shown that ozonated autohemotherapy markedly improves the symptoms of chronic limb ischemia (muscular pain at rest, intermittent claudication, etc) in atherosclerotic patients, but mechanisms of action remain unclear. AIMS: Human endothelial cells (HUVECs) are known to release nitrogen monoxide (NO) and we investigated the biological effects of human ozonated serum on HUVECs in culture. METHODS: We assessed the relevance of peroxidation, the release of NO as nitrite and of three classical cytokines. RESULTS: The treatment of HUVECs with ozonated serum yields a dose dependent increase of thiobarbituric acid reactive substances (TBARS) and of hydrogen peroxide (H2O2) and a decrease of protein thiol groups (PTG). Concomitantly, in comparison to either the control or the oxygenated sample, there is a significant and steady increase of nitric oxide (NO) production; this is markedly enhanced by the addition of L-arginine (20 microM) and inhibited in the presence of the NO inhibitor, L-NAME (20 mM). The main mediator of ozone action is H2O2 as it has been shown either after its direct measurement or by the addition of 20, 40 and 100 microM. Moreover, during 24 hours incubation we have investigated the production of endothelin 1 (ET-1), E-selectin and Interleukin 8 (IL-8) and it appears that ozonation enhances IL-8, inhibits E-selectin and hardly modifies ET-1 production. CONCLUSIONS: It appears that reinfusion of ozonated blood, by enhancing release of NO, may induce vasodilation in ischemic areas and reduce hypoxia.     

Pathophysiological consequences of enhanced intracellular superoxide formation in isolated perfused rat liver.

The potential toxicity of enhanced intracellular reactive oxygen formation was investigated in isolated perfused livers of male Fischer rats. The presence of the redox-cycling agent diquat in the perfusate (200 microM) increased the basal efflux of glutathione disulfide (GSSG) into bile (2.65 +/- 0.26 nmol GSH-equivalents/min per g liver wt.) and perfusate (0.55 +/- 0.15 nmol/min per g) approximately 10-fold. Since no evidence was found for degradation of GSSG in the biliary tract of these animals, it could be estimated that diquat induced a constant O2- generation of approximately 1000 nmol/min per g liver wt for 1 h. Thus, reactive oxygen formation under these conditions was 1-2 orders of magnitude higher than under various pathophysiological conditions. Only minor liver injury (release of lactate dehydrogenase activity) was observed. To increase the susceptibility of the liver to the oxidant stress, animals were pretreated in vivo with 200 mg/kg body wt. phorone, which caused a 90% depletion of the hepatic glutathione content, 100 mg/kg ferrous sulfate, a combination of phorone and ferrous sulfate, or 40 mg/kg BCNU, which caused a 60% inhibition of hepatic GSSG reductase. Only the combined treatment of phorone + ferrous sulfate or BCNU caused a significant increase of the diquat-induced liver injury. Our results demonstrated an extremely high resistance of the liver against intracellular reactive oxygen formation (even with impaired detoxification systems) and can serve as reference for the evaluation of potential contributions of reactive oxygen to liver injury in various disease states.     

Manipulation of dietary carbohydrates after prolonged effort modifies muscle sarcoplasmic reticulum responses in exercising males

The hypothesis tested was that disturbances in the sarcoplasmic reticulum (SR) Ca2+-cycling responses to exercise would associate with muscle glycogen reserves. Ten untrained males [peak O2 consumption (VO2 peak) = 3.41 +/- 0.20 (SE) l/min] performed a standardized cycle test (approximately 70% VO2 peak) on two occasions, namely, following 4 days of a high (Hi CHO)- and 4 days of a low (Lo CHO)-carbohydrate diet. Both Hi CHO and Lo CHO were preceded by a session of prolonged exercise designed to deplete muscle glycogen. SR Ca2+ cycling in crude homogenates prepared from vastus lateralis samples indicated higher (P < 0.05) Ca2+ uptake (microM x g protein(-1) x min(-1)) in Hi CHO compared with Lo CHO at 30 min (2.93 +/- 0.10 vs. 2.23 +/- 0.12) and at 67 min (2.77 +/- 0.16 vs. 2.10 +/- 0.12) of exercise, the point of fatigue in Lo CHO. Similar effects (P < 0.05) were noted between conditions for maximal Ca2+-ATPase (microM x g protein(-1) x min(-1)) at 30 min (142 +/- 8.5 vs. 107 +/- 5.0) and at 67 min (130 +/- 4.5 vs. 101 +/- 4.7). Both phase 1 and phase 2 Ca2+ release were 23 and 37% higher (P < 0.05) at 30 min of exercise and 15 and 34% higher (P < 0.05), at 67 min during Hi CHO compared with Lo CHO, respectively. No differences between conditions were observed at rest for any of these SR properties. Total muscle glycogen (mmol glucosyl units/kg dry wt) was higher (P < 0.05) in Hi CHO compared with Lo CHO at rest (+36%), 30 min (+53%), and at 67 min (+44%) of cycling. These results indicate that exercise-induced reductions in SR Ca2+-cycling properties occur earlier in exercise during low glycogen states compared with high glycogen states.     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Comparison of cerebrospinal fluid transport in fetal and adult sheep

We quantified cerebrospinal fluid (CSF) transport (conductance) and CSF outflow resistance in late-gestation fetal and adult sheep using two methods, a constant pressure infusion method and a bolus injection technique into the lateral ventricles. No significant differences in CSF conductance (fetus 0.013 +/- 0.002, adult 0.014 +/- 0.003 ml x min(-1) x cm H(2)O(-1)) or CSF outflow resistance (fetus 83.7 +/- 9.8, adult 84.7 +/- 19.7 cm H(2)O x ml(-1) x min) were observed. To confirm CSF transport to plasma in fetal animals, (125)I- or (131)I-labeled human serum albumin (HSA) was injected into the lateral ventricles. The tracer entered fetal plasma with an average mass transport rate of 1.91 +/- 0.47% injected/h (n = 9). In two fetuses, we monitored the tracer appearance in plasma and cervical and thoracic duct lymph after injection of radioactive HSA into the ventricular CSF. As was the case in adult animals, fetal tracer concentrations increased in all three compartments over time, with the highest concentrations measured in lymph collected from the cervical lymphatics. These results 1) indicate that global CSF transport parameters in the late-gestation fetus and adult sheep are similar and 2) suggest an important role for extracranial lymphatic vessels in CSF transport before birth.     

Temporal mismatch between induction of superoxide dismutase and ascorbate peroxidase correlates with high H2O2 concentration in seawater from clofibrate-treated red algae Kappaphycus alvarezii

Algal cells have developed different strategies to cope with the common environmentally promoted generation of H(2)O(2), which include induction of catalase (CAT) and ascorbate peroxidase (APX), massive H(2)O(2) release in seawater, and synthesis of volatile halocarbons by specific peroxidases. The antioxidant adaptability of the economically important carrageenophyte Kappaphycus alvarezii (Doty) Doty (Gigartinales: Rhodophyta) was tested here against exposure to clofibrate (CFB), a known promoter of peroxisomal beta-oxidation in mammals and plants. Possibly as a consequence of CFB-induced H2O2 peroxisomal production, the maximum concentration of H(2)O(2) in the seawater of red algae cultures was found to occur (120+/-17 min) after the addition of CFB, which was followed by a significant decrease in the photosynthetic activity of PSII after 24 h. Interestingly, 4 h after the addition of CFB, the total SOD activity was about 2.5-fold higher than in the control, whereas no significant changes were observed in lipoperoxidation levels (TBARS) or in CAT and APX activities. The two H(2)O(2)-scavenging enzymes were only induced later (after 72 h), whereupon CAT showed a dose-dependent response with increasing concentrations of CFB. A more pronounced increase of TBARS concentration than in the controls was evidenced when a 50 microM Fe(2+/3+) solution (3:2 ratio) was added to CFB-treated cultures, suggesting that the combination of exacerbated H(2)O(2) levels in the seawater-in this work, caused by CFB exposure-and Fenton-reaction catalyst (ferric/ferrous ions), imposes harsh oxidative conditions on algal cultures. The bulk of data suggests that K. alvarezii possesses little ability to promptly induce CAT and APX compared to the immediately responsive antioxidant enzyme SOD and, to avoid harmful accumulation of H(2)O(2), the red alga presumably releases H(2)O(2) into the surrounding medium as an alternative mechanism.     

Pressure-dependent vasoactive effects of histamine in the coronary circulation

Twenty-one isolated, perfused, spontaneously rhythmic guinea pig hearts (Langendorff preparation) were used to investigate the effects of coronary perfusion pressure (CPP) on the coronary vasoactive response to a continuous infusion of histamine. Heart rate (HR), coronary perfusate flow (CPF), left ventricular pressure, dp/dtmax, oxygen extraction, and myocardial oxygen consumption (MVO2) were measured at constant CPP of 40 (n = 9), 53 (n = 6), and 65 cm H2O (n = 6) in the absence and presence of continuous intracoronary infusion of histamine [0.9 +/- 0.2 microgram/(min X g)]. At 40 cm H2O histamine caused significant coronary vasodilation. At 65 cm H2O histamine caused significant coronary vasoconstriction. At an intermediate pressure of 53 cm H2O histamine had no effect on CPF. At all three pressures HR, left ventricular pressure, dp/dtmax, and oxygen extraction increased significantly in response to histamine. MVO2 was unchanged by histamine at 65 cm H2O (flow was reduced but extraction increased. MVO2 increased modestly but significantly at 53 cm H2O (12% increase; flow unchanged but extraction increased), and increased prominently at 40 cm H2O (50% increase; flow and extraction increased). We conclude that the coronary vascular effects of continuously infused histamine are dependent on the preexisting, steady-state level of CPP in the isolated perfused guinea pig heart.     

Intestinal absorption of copper: influence of carbohydrates.

Macronutrients can modulate the intestinal absorption of trace elements by binding the metal or altering mucosal function. We investigated whether certain simple and complex carbohydrates modify copper (Cu) absorption, using an in vivo perfusion technique in the rat. Corn syrup solids, which contain a mixture of glucose polymers of diverse length, added at either 20 or 50 mosm/kg enhanced Cu absorption from a 31.5 microM (2 mg/liter) Cu solution (128 +/- 11 and 130 +/- 11 pmol/min x cm, respectively, vs 101 +/- 4 pmol/min x cm, P less than 0.05, in the absence of carbohydrate). This was concomitant with a stimulation of net water absorption (1.05 +/- 0.08 and 0.84 +/- 0.08 microliter/min x cm, respectively, vs 0.63 +/- 0.02 microliter/min x cm with no carbohydrate, P less than 0.05). Glucose, fructose, lactose, or sucrose had no influence on Cu absorption, although they altered water exchanges, an effect attributable to a reduction of the outflow component of fluid recirculation. Low concentrations of lactose resulted in a greater accumulation of Cu in the intestinal mucosa (8.75 +/- 0.71 micrograms/g vs 5.77 +/- 0.68 micrograms/g for controls, P less than 0.05). Hence, solutes that moderately stimulate mucosa-to-serosa fluid influx in a progressive manner, such as glucose polymers, may contribute to functionally increase Cu absorption. Conversely, conditions which tend to reduce water inflow or increase water outflow across the small intestinal mucosa, as may occur with high lactose diets or in cases of chronic diarrhea, may have negative effects.