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.     

The effects of nimodipine and L-NAME on coronary flow and oxidative stress parameters in isolated rat heart

The aim of this study was to assess the effects of Ca2+ channel antagonist nimodipine (in concentration which competitive inhibited phosphodiesterase 1--PDE1) on oxidative stress alone or under inhibition of nitric oxide synthase by L-NAME in isolated rat heart. The hearts from male Wistar albino rats (n=18, BM about 200 g, age 8 weeks) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure conditions (CPP, 40-120 cm H2O). The experiments were performed under control conditions, in the presence of Nimodipine (2 microM) or Nimodipine (2 microM) plus L-NAME (30 microM). Coronary flow (CF) varied in the autoregulatory range from 3.7 +/- 0.4 ml/min/g wt at 50 cm H2O to 4.35 +/- 0.79 at 90 cm H2O. Basal nitrite outflow, index of lipid peroxidation (measured as TBARS release) and superoxide anion release (O2-) (at 60 cm H2O) were 0.64 +/- 0.18 nmol/min/g wt, 0.55 +/- 0.13 micromol/min/g wt and 19.72 +/- 3.70 nmol/min/g wt, respectively. Nimodipine induced significant vasodilation at all values of CPP (from 26% at 40 cm H2O to 36% at 120 cm H2O) accompanied with significant decrease of nitrite outflow (from 59% at 40 cm H2O to 40% at 120 cm H2O), significant increase of TBARS above autoregulatory range (about 40%) and significant increase of O2- release (from 186% at 40 cm H2O to 117% at 120 cm H2O). However, perfusion with L-NAME completely reversed the effects of Nimodipine. Nimodipine-induced flow changes were decreased under L-NAME (from 3% at 40 cm H2O to 11% at 120 cm H2O) without changes in the autoregulatory range, accompanied with significantly increased nitrite outflow (from 69% at 40 cm H2O to 36% at 120 cm H2O) and TBARS release (almost 50%), as well as significantly decreased O2- release (from 50% at 40 cm H2O to 43% at 120 cm H20). Our findings show that effect of nimodipine on coronary flow should be significantly influenced by NO, TBARS and O2- release in isolated rat heart.     

The effects of cyclooxygenase and nitric oxide synthase inhibition on oxidative stress in isolated rat heart

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas still exist about potential impact of these drugs on cardiovascular system. The present study was aimed to estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA], and SC-560) on oxidative stress in isolated rat heart, with special focus on l-arginine/NO system. The hearts of male Wistar albino rats (total number n = 96, each group 12 rats, 8 weeks old, body mass 180–200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40–120 cmH₂O). After control experiments the hearts were perfused with the following drugs: 100 μmol/l ASA (Aspirin), alone or in combination with 30 μmol/l l-NAME, 0.3 μmol/l meloxicam (movalis) with or without 30 μmol/l l-NAME, 3 μmol/l meloxicam (alone or in combination with 30 μmol/l l-NAME), 30 μmol/l l-NAME, and administration of 0.25 μmol/l SC-560. In samples of coronary venous effluent the following oxidative stress markers were measured spectrophotometrically: index of lipid peroxidation (measured as thiobarbituric acid reactive substances [TBARS]), superoxide anion radical release (O₂ ^?), and hydrogen peroxide (H₂O₂). While ASA was found to have an adverse influence on redox balance in coronary circulation, and coronary perfusion, meloxicam and SC-560 do not negatively affect the intact model of the heart. Furthermore, all effects were modulated by NOS inhibition. It seems that interaction between COX and l-arginine/NO system truly exists in coronary circulation, and can be one of the possible causes for achieved effects. That means: those effects induced by different inhibitors of COX are modulated by subsequent inhibition of NOS.     

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.     

Sustained inflation and incremental mean airway pressure trial during conventional and high-frequency oscillatory ventilation in a large porcine model of acute respiratory distress syndrome

Background

To compare the effect of a sustained inflation followed by an incremental mean airway pressure trial during conventional and high-frequency oscillatory ventilation on oxygenation and hemodynamics in a large porcine model of early acute respiratory distress syndrome.

Methods

Severe lung injury (Ali) was induced in 18 healthy pigs (55.3 ± 3.9 kg, mean ± SD) by repeated saline lung lavage until PaO₂ decreased to less than 60 mmHg. After a stabilisation period of 60 minutes, the animals were randomly assigned to two groups: Group 1 (Pressure controlled ventilation; PCV): FIO₂ = 1.0, PEEP = 5 cmH₂O, V_T = 6 ml/kg, respiratory rate = 30/min, I:E = 1:1; group 2 (High-frequency oscillatory ventilation; HFOV): FIO₂ = 1.0, Bias flow = 30 l/min, Amplitude = 60 cmH₂O, Frequency = 6 Hz, I:E = 1:1. A sustained inflation (SI; 50 cmH₂O for 60s) followed by an incremental mean airway pressure (mPaw) trial (steps of 3 cmH₂O every 15 minutes) were performed in both groups until PaO₂ no longer increased. This was regarded as full lung inflation. The mPaw was decreased by 3 cmH₂O and the animals reached the end of the study protocol. Gas exchange and hemodynamic data were collected at each step.

Results

The SI led to a significant improvement of the PaO₂/FiO₂-Index (HFOV: 200 ± 100 vs. PCV: 58 ± 15 and T_Ali: 57 ± 12; p < 0.001) and PaCO₂-reduction (HFOV: 42 ± 5 vs. PCV: 62 ± 13 and T_Ali: 55 ± 9; p < 0.001) during HFOV compared to lung injury and PCV. Augmentation of mPaw improved gas exchange and pulmonary shunt fraction in both groups, but at a significant lower mPaw in the HFOV treated animals. Cardiac output was continuously deteriorating during the recruitment manoeuvre in both study groups (HFOV: T_Ali: 6.1 ± 1 vs. T₇₅: 3.4 ± 0.4; PCV: T_Ali: 6.7 ± 2.4 vs. T₇₅: 4 ± 0.5; p < 0.001).

Conclusion

A sustained inflation followed by an incremental mean airway pressure trial in HFOV improved oxygenation at a lower mPaw than during conventional lung protective ventilation. HFOV but not PCV resulted in normocapnia, suggesting that during HFOV there are alternatives to tidal ventilation to achieve CO₂-elimination in an "open lung" approach.     

Activation of Rac-1 and Cdc42 stabilizes the microvascular endothelial barrier

We have demonstrated previously that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in mouse microvascular myocardial endothelial (MyEnd) cells in vitro as well as in rat mesenteric microvessels in vivo. In this study, we tested the hypothesis that specific activation of Rac-1 would stabilize microvascular endothelial barrier functions. For this purpose we used Escherichia coli Cytotoxic necrotizing factor (CNF-1) under conditions (300 ng/ml, 120 min) where it strongly activated Rac-1 and Cdc42 but not Rho A in MyEnd cells. Under these conditions, CNF-1 induced translocation of the actin-binding proteins cortactin and vasodilator-stimulated phosphoprotein (VASP) to cell junctions, increased the junction-associated actin filament belt, and reduced monolayer permeability. We also tested the effect of CNF-1 on endothelial barrier properties in vivo using single-perfused mesenteric microvessels. In contrast to cultured microvascular monolayers, CNF-1 did not reduce baseline barrier functions assayed as hydraulic conductivity (Lp). However, following 120 min pretreatment, CNF-1 significantly attenuated the peak Lp increase in response to platelet-activating factor (PAF, 10 nM) to 12.6±4×10⁻⁷ cm/(s cmH₂O) compared to 46.2±10×10⁻⁷ cm/(s cmH₂O) in experiments using PAF alone. These experiments indicate that activation of Rac-1 and Cdc42 stabilizes microvascular endothelial barrier functions in vitro and in vivo, likely by increasing the junction-associated actin cytoskeleton.     

Glycolysis and glucose oxidation during reperfusion of ischemic hearts from diabetic rats

Stimulationg of glucose oxidation by dichloroacetate (DCA) treatment is beneficial during recovery of ischemic hearts from non-diabetic rats. We therfore determined whether DCA treatment of diabetic rat hearts (in which glucose use is extremely low), increases recovery of function of hearts reperfused following ischemia. Isolated working hearts from 6 week streptozotocindiabetic rats were perfused with 11 mM [2-³H/U-¹⁴C]glucose, 1.2 mM palmitate, 20 μU/ml insulin, and subjected to 30 min of no flow ischemia followed by 60 min reperfusion. Heart function (expressed as the product of heart rate and peak systolic pressure), prior to ischemia, was depressed in diabetic hearts compared to controls (HR × PSP × 10^?3 was 18.2 ± 1 and 24.3 ± 1 beats/mm Hg/min in diabetic and control hearts respectively) but recover to pre-ischemic levels following ischemia, whereas recovery of control of control hearts was significantly decreased (17.8 ± 1 and 11.9 ± 3 beats/mm Hg/min in diabetic and control hearts respectively). This enhanced recovery of diabetic rat hearts occurred even though glucose oxidation during reperfusion was significantly reduced as compared to controls (39 ± 6 and 208 ± 42 nmol/min/g dry wt, in diabetic and control hearts respectively). Glycolytic rate (³G₂O production) during reperfusion were similar in diabetic and control hearts (1623 ± 359 and 2071 ± 288 nmol/min/g dry wt, respectively). If DCA (1 mM) was added at reperfusion, hearts from control animals exhibited a significant improvement in function (HR × PSP × 10^? recovered to 20 ± 4 beats/mm Hg/min) that was accompanied by a 4-fold increase in glucose oxidation (from 208 ± 42 to 753 ± 111 nmol/min/g dry wt). DCA was without effect on functional recovery of diabetic rat hearts during reperfusion but did significantly increase glucose oxidation from 39 ± 6 to 179 ± 44 nmol/min/g dry wt). These data suggests that, unlike control hearts, low glucose oxidation rates are not an important factor in reperfusion recovery of previouskly ischemic diabetic rat hearts.     

Blood-endothelial cell and blood-brain transport ofl-proline, α-aminoisobutyric acid,andl-alanine

We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [¹⁴C]l-proline, [¹⁴C]l-alanine, and [¹⁴C] α-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (K_in) of 0.55 ± 0.15 × 10⁻⁴ ml/s/g and had no reversible compartment. AIB had a low K_in of 0.68±0.14×10⁻⁴ ml/s/g and a significant reversible volume of 4.34±0.51×10⁻³ ml/g in parietal cortex.l-alanine had the highest transfer coefficient, 3.11±0.26 × 10⁻⁴ ml/s/g, and a reversible volume of 10.03±0.93×10⁻³ ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport forl-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma andl-leucine and unchanged by sodium-free buffer, confirming its passage by the L₁ system.l-alanine uptake was sodium-independent and not reduced by plasma.l-serine,l-cysteine,l-leucine andl-phenylalanine produced similar inhibition (66%) whilel-alanine produced a lower inhibition (41%).l-arginine increased alanine uptake in cortex and thalamus. Addingl-serine tol-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest thatl-alanine is transported by another L transport system different from the L₁ system at the luminal membrane.     

Effect of aluminum on the blood-brain barrier permeability during nitric oxide-blockade-induced chronic hypertension in rats

We examined the effect of aluminum on the permeability of the blood-brain barrier (BBB) during nitric oxide-blockade-induced chronic hypertension in rats. Animals were given the inhibitor of nitric oxide synthase, l-NAME (N ^ω-nitro-l-arginine methyl ester), for 4 wk to induce chronic hypertension. Two groups of rats were given an intraperitoneal injection of aluminum chloride. The integrity of the BBB was assessed by a quantitative measurement for Evans blue (EB) dye. The arterial blood pressure in l-NAME- and l-NAME plus aluminum-treated animals was significantly elevated from 115±2.8 and 110±1.7 mm Hg to 174±5.2 and 175±4.8 mm Hg, respectively (p<0.01). The EB dye content in the brain regions of the rats in the l-NAME group was increased, but there was no statistical significance compared to the saline group. The extravasation of EB dye was significantly increased in the brain regions of the animals treated with aluminum compared to the rats treated with saline (p<0.05). A significantly higher EB dye content in the brain regions was observed in the l-NAME plus aluminium group compared to l-NAME, aluminum, and saline groups (p<0.01). These findings indicate that exposure to a high level of aluminum leads to an additional increase in BBB permeability where nitric oxide-blockade-induced chronic hypertension potentiates the effect of aluminum to enhance BBB permeability to EB dye.     

Sitting and Supine Esophageal Pressures in Overweight and Obese Subjects

Esophageal pressure (P_Es) can be used to approximate pleural pressure (P_pl) and might be clinically useful, particularly in the obese e.g., to guide mechanical ventilator settings in critical illness. However, mediastinal artifact (the difference between true P_pl and P_Es) may limit acceptance of the measurement, and reproducibility of P_Es measurements remains unknown. Therefore, we aimed to assess the effect of body posture on P_Es in a cohort of obese, but healthy subjects, some of whom had multiple measurements, to address the clinical robustness of esophageal manometry. Twenty‐five overweight and obese subjects (BMI > 25 kg/m²) and 11 control lean subjects (BMI < 25 kg/m²) underwent esophageal manometry with pressures measured seated and supine. Twenty overweight and obese subjects had measurements repeated after ～1 to 2 weeks. Anthropometric data and sitting and supine spirometry were recorded. The average end‐expiratory P_Es sitting and supine were greater in the overweight and obese group than the lean group (sitting ?0.1 ± 2.1 vs. ?3.3 ± 1.2 cmH₂O, supine 9.3 ± 3.3 vs. 6.9 ± 2.8 cmH₂O, respectively). The mean differences between repeated measurements were small (?0.3 ± 1.7 cmH₂O sitting and ?0.1 ± 1.5 cmH₂O supine). P_Es correlated with a number of anthropometric and spirometric variables. In conclusion, P_Es are slightly greater in overweight and obese subjects than lean subjects; but changes with position are similar in both groups. These data indicate that mediastinal weight and postural effects on P_Es are within a clinically acceptable range, and suggest that esophageal manometry can be used to inform clinical decision making across wide range of body types.     

Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri

This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7 mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt solution with 7 mm CaCl₂ ^.2H₂O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks. Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997     

The steady-state rate of ATP synthesis in the perfused rat heart measured by 31P NMR saturation transfer

The steady-state rate of ATP synthesis in the isolated, Langendorff-perfused rat heart was determined using a ³¹P NMR saturation transfer method. At 37°C and a perfusion pressure of 70 cm H₂O the value is 2.8 ± 0.3 (n=5 ± S.E.M.) μmol.s^?1. (g. dry wt.)^?1. The activity of creatine phosphokinase measured in the same experiments was 14.6 ± 1.0 μ mol.s^?1 .(g. dry wt.)^?1. From the rate of ATP synthesis and the separately measured oxygen consumption we calculated an apparent mitochondrial ADP:O ratio of 3.5 ± 0.8 in the intact tissue.     

Specific changes in the in vitro production of prostanoids by the ovine cervix at parturition

A method of tissue superfusion has been used to measure $\text{in vitro}$ prostanoid production by the ovine cervix during late pregnancy and at parturition. In late pregnancy (105–135 days of gestation) cervical tissue produced relatively large amounts of prostaglandin E (PGE); in comparison, the production rates of prostaglandin F (PGF), 13, 14-dihydro-15-oxo-prostaglandin F (PGFM) and 6-oxo-prostaglandin F_1α were generally low. Thromboxane B₂ (TXB₂) production was minimal and often unmeasurable. There were significant increases in the production rates of PGE and 6-oxo-PGF_1α by cervical tissue taken immediately after delivery, when compared to late pregnancy. Mean production rates of PGE increased from 19.8 ± 4.1 to 43.8 ± 7.4 ng/g. dry wt./min; 6-oxo-PGF_1α production rates increased more than three-fold from 10.0 ± 2.7 to 34.6 ± 9.8 ng/g. dry wt./min (means ± S.E.M.). There were no significant differences in the rates of production of PGF, PGFM and TXB₂ by the two groups.     

Reliability of Single-Use PEEP-Valves Attached to Self-Inflating Bags during Manual Ventilation of Neonates – An In Vitro Study

Introduction

International resuscitation guidelines suggest to use positive end-expiratory pressure (PEEP) during manual ventilation of neonates. Aim of our study was to test the reliability of self-inflating bags (SIB) with single-use PEEP valves regarding PEEP delivery and the effect of different peak inflation pressures (PIP) and ventilation rates (VR) on the delivered PEEP.

Methods

Ten new single-use PEEP valves from 5 manufacturers were tested by ventilating an intubated 1kg neonatal manikin containing a lung model with a SIB that was actuated by an electromechanical plunger device. Standard settings: PIP 20cmH₂O, VR 60/min, flow 8L/min. PEEP settings of 5 and 10cmH₂O were studied. A second test was conducted with settings of PIP 40cmH₂O and VR 40/min. The delivered PEEP was measured by a respiratory function monitor (CO₂SMO⁺).

Results

Valves from one manufacturer delivered no relevant PEEP and were excluded. The remaining valves showed a continuous decay of the delivered pressure during expiration. The median (25^th and 75^th percentile) delivered PEEP with standard settings was 3.4(2.7–3.8)cmH₂O when set to 5cmH₂O and 6.1(4.9–7.1)cmH₂O when set to 10cmH₂O. Increasing the PIP from 20 to 40 cmH₂O led to a median (25^th and 75^th percentile) decrease in PEEP to 2.3(1.8–2.7)cmH₂O and 4.3(3.2–4.8)cmH₂O; changing VR from 60 to 40/min led to a PEEP decrease to 2.8(2.1–3.3)cmH₂O and 5.0(3.5–6.2)cmH₂O for both PEEP settings.

Conclusion

Single-use PEEP valves do not reliably deliver the set PEEP. PIP and VR have an effect on the delivered PEEP. Operators should be aware of these limitations when manually ventilating neonates.     

Effect of triethylenepentaminehexaacetic acid on the renal damage in cadmium-treated Syrian hamsters

Cadmium (Cd)-induced nephropathy was treated by triethylene-pentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 mg/kg body wt CdCl₂ showed proteinuria, urinaryN-acetyl-β-d-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 mg/kg body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7±2.2 to 4.3±0.5 mg/d) and NAG (0.17±0.06 to 0.04±0.02 U/d). Urinary excretion of Cd was significantly increased (from 87±51.3 to 3052±1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4±3.0 to 21.8±2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1±1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041±105 vs 1104±358 nM/g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5±2.1 to 9.8±2.0 μg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cd-induced nephropathy in hamsters. Although the precise mechanism of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue.     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Role of Endogenous ET‐1 in the Regulation of Myocardial Blood Flow in Lean and Obese Humans

Endothelin is an important determinant of peripheral vascular tone, and increased endogenous endothelin activity contributes to peripheral vascular dysfunction in human obesity. The contributions of endothelin to the regulation of coronary vascular tone in health in humans have not been well studied. We hypothesized that the contribution of endothelin to the regulation of myocardial perfusion would be augmented in human obesity. Using [NH₃]ammonia positron emission tomography (PET), we measured myocardial perfusion under resting and adenosine‐stimulated conditions on two separate days, with and without concurrent exposure to BQ123, an antagonist of type A endothelin receptors (1 µmol/min IV beginning 90 min before measurement). We studied 10 lean and 9 obese subjects without hypertension, hyperlipidemia, or diabetes mellitus. We observed a BQ123‐induced increase in resting myocardial perfusion of ～40%, not different between lean and obese subjects (BQ123‐induced increase in flow: lean 0.12 ± 0.20, obese 0.32 ± 0.51 ml/g/min, P = 0.02 BQ123 effect, P = 0.27 comparing response across groups). Although basal flow rates varied by region of the myocardium, the BQ123 effect was seen in all regions. BMI and cholesterol were significantly related to BQ123‐induced increases in basal tone in multivariable analysis. There was no baseline difference in the adenosine‐stimulated increase in blood flow between lean and obese subjects, and BQ123 failed to augment these responses in either group. These observations suggest that endothelin is an important contributor to the regulation of myocardial perfusion under resting conditions in healthy lean and obese humans, with increased contributions in proportion to increasing obesity.     

Vitamin E attenuates acute lung injury in sheep with burn and smoke inhalation injury

Abstract

Introduction: A decrease in α-tocopherol (vitamin E) plasma levels in burn patients is typically associated with increased mortality. We hypothesized that vitamin E supplementation (α-tocopherol) would attenuate acute lung injury induced by burn and smoke inhalation injury.

Materials and Methods: Under deep anesthesia, sheep (33 ± 5 kg) were subjected to a flame burn (40% total body surface area, third degree) and inhalation injury (48 breaths of cotton smoke, < 40°C). Half of the injured group received α-tocopherol (1000 IU vitamin E) orally, 24 h prior to injury. The sham group was neither injured nor given vitamin E. All three groups (n = 5 per group) were resuscitated with Ringer's lactate solution (4 ml/kg/%burn/24 h), and placed on a ventilator (PEEP = 5 cmH₂O; tidal volume = 15 ml/kg) for 48 h.

Results: Plasma α-tocopherol per lipids doubled in the vitamin E treated sheep. Vitamin E treatment prior to injury largely prevented the increase in pulmonary permeability index and moderated the increase in lung lymph flow (52.6 ± 6.2 ml/min, compared with 27.3 ± 6.0 ml/min, respectively), increased the PaO₂/FiO₂ ratio, ameliorated both peak and pause airway pressure increases, and decreased plasma conjugated dienes and nitrotyrosine.

Conclusions: Pretreatment with vitamin E ameliorated the acute lung injury caused by burn and smoke inhalation exposure.     

l-NAME inhibits tumor cell progression and pulmonary metastasis of r/m HM-SFME-1 cells by decreasing NO from tumor cells and TNF-α from macrophages

Highly metastatic ras/myc-transformed serum-free mouse embryo (r/m HM-SFME-1) cells were injected subcutaneously to mice and the effects of Nω-nitro-l-arginine methyl ester (l-NAME) on the tumor progression and pulmonary metastasis were investigated. In addition, production of nitric oxide (NO), matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-α) in the tumor cells and in a mouse macrophage-like cell line, J774.1 cells, was analyzed. The increase in footpad thickness was significantly smaller in the mice which were fed the l-NAME containing water (4.24 ± 0.39 mg/day/mouse). The number of the tumor cells metastasized to the lungs was smaller in the l-NAME treated mice, although statistical significance was not found. Co-treatment of r/m HM-SFME-1 cells with interferon-gamma (IFN-γ; 100 U/ml) and lipopolysaccharide (LPS; 0.5 μg/ml) significantly enhanced NO production, and the presence of l-NAME at 1 mM significantly decreased this response. In r/m HM-SFME-1 cells, MMP-2 was undetectable and MMP-9 was also very little in the basal level, and both MMPs were unaffected by the IFN-γ and/or LPS treatments, not to mention by the l-NAME treatment. In J774.1 cells, any treatment including LPS appeared to enhance MMP-9 production, however, this upregulation was not inhibited by the additional presence of l-NAME. Production of TNF-α by J774.1 cells was markedly enhanced with LPS treatment, and this enhancement was significantly reduced in the presence of l-NAME. These results indicate that the inhibitory effects of l-NAME on the tumor cell progression and pulmonary metastasis could be due to suppression of NO from tumor cells and TNF-α from macrophages (Mol Cell Biochem, 2007). Hideaki Yamaguchi and Yumi Kidachi contributed equally to this work.     

l-Proline transport into renal OK epithelial cells: a second renal proline transport system is induced by amino acid deprivation

Influx of [³H]-l-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted for 8 h to amino acid deprivation, uptake of l-proline was now dominated by a low-affinity system with an apparent K _m of 4.4 ± 0.6 mM and a V _max of 10.2 ± 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K _m of 0.12 ± 0.01 mM and a V _max of 0.28 ± 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of amino acid deprivation.