Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Interaction of glyceryl trinitrate and sodium nitroprusside with bovine pulmonary vein homogenate and 10,000 x g supernatant: biotransformation and nitric oxide formation

The current proposed mechanism of action of nitrovasodilator drugs involves biotransformation to nitric oxide, which is postulated to be the active vasodilator substance. Our objective was to determine whether nitric oxide was formed from two prototype nitrovasodilator drugs, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), after incubation with bovine pulmonary vein (BPV) preparations. GTN or SNP was incubated in an argon atmosphere with phosphate buffer, BPV homogenate, or the 10,000 x g supernatant fraction of the homogenate. Nitric oxide formation, as determined by a chemiluminescence-headspace gas method, was measurable following the incubation of SNP with BPV homogenate and 10,000 x g supernatant. There was no detectable formation of nitric oxide from the incubation of GTN with the two BPV preparations, although GTN was biotransformed to glyceryl dinitrate, as determined by gas-liquid chromatography. There was decreased recovery of nitric oxide during the incubation of authentic nitric oxide with the two BPV preparations as compared with buffer. In conclusion, formation of nitric oxide was measured for the interaction of SNP, but not GTN, with BPV preparations. However, the data do not exclude the possible formation of nitric oxide from GTN, as nitric oxide was shown to be sequestered or transformed by the BPV preparations.     

Biotransformation of glyceryl trinitrate by rat brain homogenate.

Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biotransformation of the organic nitrate vasodilator drug to a mixture of glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN). Heating of the RBH at 100 degrees C for 5 min and (or) pretreatment with 5 mM N-ethylmaleimide at 37 degrees C for 10 min demonstrated that about two-thirds of the GTN biotransformation activity was due to a sulfhydryl-dependent enzymatic process resulting in the predominant formation of 1,2-GDN, and that the remaining biotransformation activity was due to a sulfhydryl-dependent nonenzymatic process resulting in the selective formation of 1,3-GDN. In a preliminary experiment, nitric oxide formation was observed during the incubation of GTN with RBH under anaerobic conditions. These data support the idea that some of the therapeutic and adverse effects of GTN are mediated through its action in the central nervous system.     

Chemiluminescent simultaneous determination of phosphatidylcholine hydroperoxide and phosphatidylethanolamine hydroperoxide in the liver and brain of the rat.

The quantification of phospholipid hydroperoxides in biological tissues is important in order to know the degree of peroxidative damage of membrane lipids. For this purpose, optimal conditions for the chemiluminescent simultaneous assay of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in rat liver and brain were determined. A chemiluminescence detection-high performance liquid chromatography (CL-HPLC) method that incorporates cytochrome c and luminol as a post-column hydroperoxide-specific luminescent reagent was used (Miyazawa et al. 1987. Anal. Lett. 20: 915-925; Miyazawa. 1989. Free Radical Biol. Med. 7: 209-217). An n-propylamine-bound silica column with hexane-2-propanol-methanol-water 5:7:2:1 (v/v/v/v) (flow rate 1.0 ml/min) as eluant was used to determine both PCOOH and PEOOH, which were separated from each other and from other lipids and lipid-soluble antioxidants. High reproducibility and sensitivity as low as 10 pmol hydroperoxide-O2 were observed with a mixture of 10 micrograms/ml cytochrome c and 2 micrograms/ml luminol in 50 mM borate buffer (pH 10.0, flow rate 1.1 ml/min) as luminescent reagent and a post-column mixing joint temperature of 40 degrees C. Using the established analytical conditions, it was confirmed that both PCOOH (1324 +/- 122 pmol/g liver, 114 +/- 18 pmol/g brain, mean +/- SD) and PEOOH (728 +/- 89 pmol/g liver, 349 +/- 60 pmol/g brain, mean +/- SD) are present in the liver and brain of Sprague-Dawley rats bred on a slightly modified AIN-76A semisynthetic diet for 3 months. The phospholipid hydroperoxide content in the rat liver was shown to be affected by dietary oils, but not significantly affected in the brain.     

Gluconeogenesis from serine in rabbit hepatocytes

L-Serine alone is not gluconeogenic in isolated rabbit hepatocytes, whereas in rat liver this amino acid has been reported to yield as much glucose as does L-lactate itself. The current study has been an investigation into the explanation of the difference between the two species. Hepatocytes were isolated from 48-h-starved, 750- to 1000-g male rabbits, and the viability of each preparation was judged by ATP levels (2.4 +/- 0.2 mumol/g wet wt) at the beginning and end of the incubation as well as gluconeogenesis from 10 mM L-lactate (0.83 +/- 0.08 mumol/min/g wet wt). L-Serine alone produced virtually no glucose or pyruvate accumulation above baseline. Hydroxypyruvate, however, did appear in the incubation mixture. When L-serine and pyruvate were combined to test the functional activity of L-serine:pyruvate aminotransferase (EC 2.6.1.51), however, gluconeogenesis remained at the rate produced by pyruvate alone (0.61 +/- 0.04 mumol/min/g wet wt). On the other hand, the combination of L-serine and L-lactate produced rates of glucose accumulation 35% above that of L-lactate alone. The combination of L-lactate plus hydroxypyruvate produced nearly maximal rates (1.39 +/- 0.08 mumol/min/g wet wt), approaching those achieved by a physiologic ratio (10:1) of L-lactate and pyruvate. Hydroxypyruvate itself was only moderately gluconeogenic (0.44 +/- 0.04 mumol/min/g wet wt). That a reduction of the cytoplasmic free [NAD+]/[NADH] ratio by L-lactate was not its only contribution to L-serine utilization was suggested by the fact that ethanol completely eliminated gluconeogenesis from virtually all precursors (or combinations) tested, with the exception of hydroxypyruvate. It has been concluded from the data that, probably in contrast to the rat, the major pathway for the entrance of L-serine into gluconeogenesis in rabbit hepatocytes is through the pathway initiated by L-serine: pyruvate aminotransferase and that L-lactate is an important participant (i) by generating cytoplasmic reducing equivalents (NADH), (ii) by supplying pyruvate for the transaminating reaction itself, and, perhaps, (iii) by preventing hydroxypyruvate from being reduced by L-lactate dehydrogenase (EC 1.1.1.27) to L-glycerate.     

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.     

Effects of solution of low pH and taurocholate on release of beta-endorphin-like immunoreactivity from human duodenal mucosa in vitro

The presence of beta-endorphin-like immunoreactivity (beta-EpLI) in human duodenum and its release were studied. beta-EpLI was detected in the duodenum (mucosa, 26.7 +/- 6.3 pmol/g wet weight, mean +/- SEM; remaining tissue 23.1 +/- 5.3 pmol/g wet weight) and the stomach (7.1 pmol/g wet weight). The two activities gave similar curves for inhibition of beta-Ep radioimmunoassay of synthetic beta-Ep. On gel-filtration chromatography of a duodenal extract, two components of beta-EpLI were separated. When human duodenal mucosa was perfused with a solution of pH2 or 1mM or 5mM taurocholate, the release of beta-EpLI from mucosa into the perfusate increased 2-4 fold. These results indicate that beta-EpLI present in human duodenal is released by the direct action of low pH or taurocholate on the duodenal mucosa and suggest that it may have a physiological role.     

In vivo assessment of microvascular nitric oxide production and its relation with blood flow

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

Downregulation of Na+-K+-ATPase pumps in skeletal muscle with training in normobaric hypoxia.

To investigate the effects of training in normoxia vs. training in normobaric hypoxia (fraction of inspired O2 = 20.9 vs. 13.5%, respectively) on the regulation of Na+-K+-ATPase pump concentration in skeletal muscle (vastus lateralis), 9 untrained men, ranging in age from 19 to 25 yr, underwent 8 wk of cycle training. The training consisted of both prolonged and intermittent single leg exercise for both normoxia (N) and hypoxia (H) during a single session (a similar work output for each leg) and was performed 3 times/wk. Na+-K+-ATPase concentration was 326 +/- 17 (SE) pmol/g wet wt before training (Control), increased by 14% with N (371 +/- 18 pmol/g wet wt; P < 0.05), and decreased by 14% with H (282 +/- 20 pmol/g wet wt; P < 0.05). The maximal activity of citrate synthase, selected as a measure of mitochondrial potential, showed greater increases (P < 0.05) with H (1.22 +/- 0.10 mmol x h-1 x g wet wt-1; 70%; P < 0.05) than with N (0.99 +/- 0.10 mmol x h-1 x g wet wt-1; 51%; P < 0.05) compared with pretraining (0.658 +/- 0.09 mmol x h-1 x g wet wt-1). These results demonstrate that normobaric hypoxia induced during exercise training represents a potent stimulus for the upregulation in mitochondrial potential while at the same time promoting a downregulation in Na+-K+-ATPase pump expression. In contrast, normoxic training stimulates increases in both mitochondrial potential and Na+-K+-ATPase concentration.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorff's perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.     

Nitric oxide synthase and cGMP-mediated stimulation of renin secretion

The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.     

Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen

This study examined vascular function and the role of superoxide in mice that chronically express human renin (R+) and human angiotensinogen (A+). Responses of aortas from R+/A+ mice and from their normotensive littermates (RA- mice) were examined in vitro. Endothelium-dependent relaxation to acetylcholine was impaired in vessels from R+/A+ mice (e.g., maximal relaxation to 100 microM acetylcholine was 45 +/- 5% and 65 +/- 3% in R+/A+ and RA- mice, respectively; P < 0.05). Relaxation was also impaired to the endothelium-independent dilators authentic nitric oxide and nitroprusside in vessels from R+/A+ mice. Maximal vasorelaxation to the endothelium-independent, non-nitric oxide dilator papaverine was similar in R+/A+ and RA- mice. Incubation of vessels from R+/A+ mice with Tiron (1 mM), a superoxide scavenger, improved relaxation to acetylcholine, nitric oxide, and nitroprusside. In contrast, incubation with diethyldithiocarbamate (1 mM), an inhibitor of copper-containing SODs, reduced acetylcholine- and nitroprusside-induced relaxation in vessels from both R+/A+ and RA- mice. Basal superoxide levels, measured with lucigenin-enhanced chemiluminescence (5 microM lucigenin) and hydroethidine-based fluorescent confocal microscopy, were higher in vessels from R+/A+ mice and were Tiron and polyethylene glycol-SOD sensitive. These results suggest that increased superoxide contributes to impaired nitric oxide-mediated relaxation in this genetic model of chronic angiotensin II-dependent hypertension.     

High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells.

Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.     

Phospholipid hydroperoxide accumulation in liver of rats intoxicated with carbon tetrachloride and its inhibition by dietary alpha-tocopherol

The formation and accumulation of phospholipid hydroperoxides, especially of phosphatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phosphatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-performance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett. 20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120 g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160 +/- 20 pmol/100 mg protein (mean +/- SD) and the PCOOH/PC molar ratio was 1.1 +/- 0.1 X 10(-5). In CCl4 (0.1 ml/100 g body wt.)-dosed rats, the liver PCOOH was 289 +/- 65 pmol/100 mg protein (PCOOH/PC = 2.4 +/- 0.4 X 10(-5], 764 +/- 271 pmol/100 mg protein (PCOOH/PC = 5.2 +/- 1.7 X 10(-5], and 856 +/- 165 pmol/100 mg protien (PCOOH/PC = 6.0 +/- 0.8 X 10(-5] at 6 h, 24 h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100 pmol/100 mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-alpha-tocopherol (5 mg/100 g body wt./day) for a week before CCl4 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CCl4 (0.25 ml/100 g body wt.) rather than with the small amount of CCl4 (0.1 ml/100 g body wt.).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

A sensitive method for the assay of sparteine oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km PM = 3033 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).     

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.