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PubMed1988

Direct 19F NMR spectroscopic observation of 5-fluorouracil metabolism in the isolated perfused mouse liver model.

A direct analysis of 5-fluorouracil (FUra) metabolism in the isolated perfused mouse liver was carried out using 19F NMR spectroscopy. In livers treated with low (30 mg/kg body weight (b.w.] or high (180 mg/kg b.w.) doses of FUra, with (180 mg/kg b.w.) or without thymidine (dThd), FUra was rapidly catabolized to alpha-fluoro-beta-ureidopropionic acid (FUPA) and alpha-fluoro-beta-alanine (FBAL), the latter usually becoming by far the major catabolite. The first catabolite of FUra, 5,6-dihydro-5-fluorouracil (5FUH2), was never detected both in the liver and in perchloric acid (PCA) extracts. In the latter, it was generally split into three signals, the major one being attributed to 5-fluorouridine-5'-monophosphate. Moreover, when the liver was treated with high doses of FUra and dThd, a signal for fluoronucleoside was observed in the liver, in PCA extracts and in perfusate where it was attributed to 5-fluoro-2'-deoxyuridine (FdUrd). The injection of concurrent dThd with the high dose of FUra led to a marked reduction of catabolism due to competitive inhibition of dihydrouracil dehydrogenase. Moreover, a significant formation of FdUrd was noticed whereas 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) was not detected. dThd therefore inhibits the conversion of FdUrd to FdUMP.

Animals↗
PubMed1993

Inactivation of peptidoleukotrienes in the isolated perfused rat lung.

Inactivation of peptidoleukotrienes by alveolar transfer and metabolism was investigated in isolated perfused rat lungs. [3H]Leukotriene (LT)C4 or [3H]LTE4 was instilled into the airways and 25 min later the distribution of tritiated metabolites in bronchoalveolar lavage fluid, lung, and perfusate was determined. Metabolism of LTC4 was found to occur in both the alveolar and vascular space in contrast to metabolism of LTE4, which occurred exclusively on the alveolar side. The alveolar transfer of leukotrienes into the perfusate was slow with only 25-29% transfer occurring during the 25-min perfusion. Only a small percentage of instilled LTC4 was transferred out of the airways unmetabolized, suggesting that this leukotriene is not the major one that is removed from the airways. In contrast, LTE4 either added directly into the airways or produced in the alveolar space from LTC4 metabolism was more readily transferred out of the lung. LTE4 was transported into alveolar cells by a nonenergy requiring, receptor-mediated mechanism inhibited by LTD4/LTE4 receptor antagonists and by probenecid. Cysteine-containing compounds (cysteine, cystine, and N-acetylcysteine) added to the perfusate caused a significant increase in LTE4/N-acetyl-LTE4 transport possibly due to a coupling of cyst(e)ine (in) and LTE4 (out) transport. Metabolism of LTC4 by gamma-glutamyltransferase appears to be the rate limiting step of peptidoleukotriene inactivation in the rat airways, being necessary for both metabolic inactivation and the formation of products which can be eliminated by transfer.

Animals↗
PubMed1993

Effects of aging on gastrin and somatostatin secretion from isolated perfused rat stomach.

We have examined the release of gastrin and somatostatin from the isolated perfused stomach of rats of three different age groups (4 months, 12 months, and 24 months old) in response to bombesin and carbachol. The basal release of gastrin was diminished in 24-month-old rats. Basal somatostatin release showed an age-related decrease. Bombesin (10(-10) and 10(-9) M) and carbachol (10(-8) and 10(-7) M) stimulated gastrin release in each age group. The integrated release of gastrin in response to bombesin (10(-10) and 10(-9) M) or carbachol (10(-8) M) did not differ among the three age groups, although integrated gastrin release in response to carbachol (10(-7) M) decreased in 24-month-old rats. Bombesin-stimulated release of somatostatin decreased in 12- and 24-month-old rats. Carbachol inhibited release of somatostatin in each age group. Compared with 4-month-old rats, the inhibition of somatostatin release by carbachol was less in 24-month-old rats at 10(-8) and 10(-7) M, and less in 12-month-old rats at 10(-7) M. The decreased basal gastrin secretion and well-preserved gastrin response were further confirmed in conscious aged rats tested by means of oral gavage with 10% peptone. Our findings indicate that gastrin response to the stimuli is well preserved with aging, whereas the response of somatostatin diminishes in an age-related manner. Aging has different effects on the release of gastrin and somatostatin from the rat stomach.

Aging↗
PubMed1995

Verapamil and flunarizine protect the isolated perfused rat liver against warm ischemia and reperfusion injury.

Using the model of the isolated perfused rat liver, we investigated the influence of the two pharmacologically different calcium channel blockers, verapamil and flunarizine, on changes of ion homeostasis, liver weights, pH deviations and enzyme activities during warm ischemia (37 degrees C) and reperfusion. The LDH and GLDH activities were determined and the calcium, potassium, and sodium concentrations were measured in the effluent. Warm ischemia (180 min) caused an increased enzyme release, a high influx of calcium and sodium into the liver and a massive potassium efflux current. Normoxic reperfusion led to a further increase in hepatic enzyme release and although the loss of potassium ceased, the calcium influx into the liver continued. By the end of reperfusion the liver weight had increased significantly (P < 0.01) in the control group. The two calcium entry blockers were added to the perfusate in various concentrations. Both substances protected the liver against warm ischemia and normoxic reperfusion damage, but they did not inhibit calcium inflow. However, the potassium efflux was significantly reduced by all concentration tasted (P < 0.001). After reperfusion the liver weights were significantly lower in the treated groups (P < 0.001) than in control animals. Thus, the calcium entry blockers verapamil and flunarizine protect liver cells against damage caused by warm ischemia and reperfusion. Furthermore, they prevent the disruption of intracellular potassium homeostasis, which seems to be related to improved volume regulation of liver cells.

Animals↗
PubMedDate not supplied

Metabolism of 14C-busulfan in isolated perfused rat liver.

1,4-14C Busulfan gave one main metabolite in the isolated perfused rat liver during 4 hr cyclic perfusion. The cumulative bile excretion contained about 38% of the total radioactivity. About 1% of unchanged 14C-busulfan was excreted in the bile. The metabolite was identified as gamma-glutamyl-beta-(S-tetrahydrothiophenium) alanyl-glycine (sulfonium ion of glutathione) by 252Cf-plasma desorption time-of-flight mass spectrometry. The formation of the metabolite was drastically decreased when the glutathione-S-transferase was inhibited, which indicates that the major reaction of busulfan with glutathione is enzymatic in nature. The sulfonium ion was more stable in the perfusate (t1/2 = 22.4 hr, 37 degrees C) than in the bile (t1/2 = 3.2 hr, 37 degrees C) at pH 7.4.

Animals↗
PubMed1982

Thromboxane B2 uptake and metabolism in isolated perfused lung. Identification and comparison with prostaglandin F2 alpha.

Thromboxane B2 was metabolised in isolated perfused guinea-pig lungs to a product identified by negative ion chemical ionisation mass spectrometry as 13,14-dihydro-15-ketothromboxane B2. Conversion was measured by radio TLC and was greater in guinea-pig than rat lungs (29.1 vs. 13.8% at 10 ng/ml), but similar in lungs from normal and sensitized guinea-pigs. Thromboxane B2 metabolism was less than that of prostaglandin F2 alpha but, like it, was prevented at 5 degrees C and reduced by cycloheximide pretreatment. Tissue to medium ratio in perfused guinea-pig lungs was 3.4 for thromboxane B2, but 0.2 for insulin (showing that thromboxane B2 is accumulated within the lung) and was altered after experimental manipulations. Neither lung slices, crude homogenates, cytosolic and microsomal fractions nor purified prostaglandin 15-hydroxydehydrogenase metabolised thromboxane B2 in vitro, although prostaglandin F2 alpha was extensively inactivated. Quantitative partition coefficient and albumin-binding data confirm that thromboxane B2 lacks prominent lipophilicity, implying that cellular uptake in lung must be carrier-mediated. We conclude that thromboxane B2 is a substrate for pulmonary degradation which may form a route for the biological inactivation of thromboxane A2. Its resistance to prostaglandin 15-hydroxydehydrogenase as conventionally tested remains paradoxical and is discussed.

Animals↗
PubMed1988

Nitrofurazone: kinetics and oxidative stress in the singlepass isolated perfused rat liver.

The disposition of the antibiotic nitrofurazone was studied in the singlepass isolated perfused rat liver. Both the effects of the steady-state level of drug and the composition of the perfusate were evaluated. The higher level (120 micrograms/ml) of nitrofurazone in a perfusion medium lacking the glutathione (GSH) precursors, glycine, glutamic acid and cysteine, caused a marked increase in bile flow (from 1.01 +/- 0.07 to 2.33 +/- 1.07 microliters/min/g), massive biliary efflux of glutathione disulfide (GSSG) (from 0.55 +/- 0.07 to 60.6 +/- 25.4 nmol/min/g) and a sharp decline in the caval efflux of GSH (to undetectable levels) and the tissue level of GSH (from 5.74 +/- 0.20 to 2.68 +/- 0.13 mumol/g). Even after the drug was discontinued, these parameters were not restored to control levels. The lower level (30 micrograms/ml) of nitrofurazone with or without amino acid supplementation and the higher level with supplementation induced less dramatic effects. Using [35S]methionine, a new conjugated metabolite of nitrofurazone and glutathione was detected. The data suggest that the toxicity of the reactive oxygen species generated by the redox cycling of the nitro group and the reactive metabolites generated by further reduction of nitrofurazone can be mitigated by adequate glutathione levels, but that livers lacking sufficient glutathione to scavenge these reactive species may be damaged.

Animals↗
PubMed1996

Central hemodynamic effects of recombinant human relaxin in the isolated, perfused rat heart model.

OBJECTIVE: To determine the cardiac effects of relaxin in the isolated, perfused rat heart model, and to see if pregnancy modifies the hormone's actions. METHODS: Hearts were excised from 18 female Sprague-Dawley rats (ten pregnant, eight nonpregnant) and attached to a Langendorff apparatus. Left ventricular systolic pressure, heart rate, and contractility were measured. Hearts were exposed serially to 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 ng/mL concentrations of recombinant human relaxin. RESULTS: Hearts from pregnant rats had lower heart rates than those from nonpregnant animals. Relaxin increased heart rate, left ventricular systolic pressure, and contractility in a dose-dependent fashion. Pregnancy did not modify this response. CONCLUSION: Recombinant human relaxin is a potent inotropic and chronotropic agent. The effects coupled with the physiologic increase of relaxin during human pregnancy indicate that relaxin may be involved in the cardiovascular changes of pregnancy.

Animals↗
PubMed1978

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats.

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.

Aerobiosis↗
PubMed2006

Relative contribution of OAT1 and OAT3 transport activities in isolated perfused rabbit renal proximal tubules.

The expression of both OAT1 and OAT3 along the isolated rabbit renal proximal tubule (RPT) was determined using RT-PCR. They were found to be very strong in S2 segment and weak in S1 and S3 segments. We further examined the relative transport activity of these transporters in isolated perfused rabbit RPT using [(3)H]para-aminohippurate ([(3)H]PAH), and estrone sulfate ([(3)H]ES) as specific substrates for rbOAT1 and rbOAT3, respectively. The transport activity of OAT1 was in the order S2>S1=S3 segments and that of OAT3 was in the order S1=S2>>S3 segments. The addition of alpha-ketoglutarate (100 muM) in the bathing medium increased both OAT1 and OAT3 transport activities in all segments of proximal tubule. The kinetics of [(3)H]succinic acid transport, used to measure the activity of sodium dicarboxylate transporter 3 (NaDC3), were examined. The J(max) for succinic acid was in the order S2>S3 and unmeasurable in the S1 segment. Our data indicate that both OAT1 and OAT3 play quantitatively significant roles in the renal transport of organic anions along the proximal tubule but predominately in S2 segment. The relative contribution of both transporters depends on their relative expression levels and may possibly be affected by the activity of NaDC3 in RPT.

Animals↗
PubMed1991

Stereoselective disposition of ibuprofen enantiomers in the isolated perfused rat kidney.

The renal clearance of ibuprofen enantiomer was studied separately in the isolated perfused rat kidney at initial perfusate concentrations of 10 micrograms/ml (n = 4) and 100 micrograms/ml (n = 4). Perfusate and urine samples were measured for R(-) and S(+)-ibuprofen using a stereospecific HPLC assay; urine samples were also analyzed after alkaline hydrolysis. Functional viability of the kidney was assured by determining the fractional excretion of glucose and glomerular filtration rate (GFR) at similar perfusion pressures. The clearance of ibuprofen was equivalent to the apparent formation clearance of conjugated enantiomer since unchanged ibuprofen could not be detected in the urine. At 10 and 100 micrograms/ml, the clearance (+/- SD) of R(-)-ibuprofen was 2.50 +/- 1.28 and 2.19 +/- 1.42 microliters/min, respectively. At 100 micrograms/ml, the clearance of S(+)-ibuprofen was 0.805 +/- 0.290 microliters/min. The protein binding of ibuprofen was found to be concentration dependent and favored the R(-)-enantiomer. The excretion ratio (clearance corrected for free fraction and GFR) of R(-)-ibuprofen was 0.398 +/- 0.209 and 0.295 +/- 0.209 for perfusate concentrations of 10 and 100 micrograms/ml, respectively. The excretion ratio of S(+)-ibuprofen was 0.0886 +/- 0.0335 for perfusate concentrations of 100 micrograms/ml. These results demonstrate that the sum of renal mechanisms involved for the clearance of R(-)- and S(+)-ibuprofen was net reabsorption. Ibuprofen was recovered in the urine solely as conjugated material and no evidence of R(-) to S(+) conversion was observed. In addition, the data suggest that R(-)-ibuprofen is cleared through the kidney faster than its S(+)-enantiomer.

Animals↗
PubMed1990

Effect of endothelin on the function of the isolated perfused working rat heart.

1. The effect of endothelin on the performance of the isolated perfused working rat heart has been examined. 2. A low concentration of endothelin (60 pmol/l) produced a gradual but sustained increase in cardiac output; coronary vascular resistance was unaffected. 3. A high concentration of endothelin (600 pmol/l) produced a rapid increase in cardiac output, followed by a marked fall in cardiac output as progressive, severe coronary vasoconstriction developed. 4. The coronary vasoconstriction induced by endothelin (600 pmol/l) was partially blocked by nicardipine (0.5 mumol/l). 5. In the presence of either nicardipine (0.5 mumol/l) or verapamil (0.2 mumol/l), the increment in cardiac output induced by endothelin (600 pmol/l) was greater than that induced by the addition of the same concentration of endothelin to hearts which had not been exposed to calcium-entry blockers. 6. The effect of endothelin on myocardial contractility has a different time course, concentration dependence and response to calcium-entry blockade than the effect on the coronary vasculature. This suggests that different mechanisms are involved in the generation of the myocardial and vascular responses to endothelin.

Animals↗
PubMed1995

Transfer of immunoglobulin G across the isolated perfused human placental lobule.

This study demonstrates that IgG transfer in vitro across the isolated perfused human placental lobule can be successfully studied by using natural forms of IgG. The transfer of anti-RhD IgG (anti-D) was measured in the presence and absence of intravenous immunoglobulin (IVIgG). When anti-D and IVIgG were present alone each crossed the placenta at about the same rate, but when both forms were present at the same time the movement of one interfered with the movement of the other. This pattern of transfer is consistent with receptor-mediated transcytosis. The interactions of IgG with trophoblastic transporters may therefore be studied without the complications that might arise from the use of conventionally labelled molecules.

Biological Transport↗
PubMed1993

The evaluation of the isolated perfused liver as a model for the assessment of liver preservation.

An ex vivo isolated perfused porcine liver model was tested to assess its suitability for rapid, reliable and relatively cheap testing of organ preservation solutions for liver transplantation. The model consists of a machine driven recirculating system incorporating an organ chamber, blood pump and membrane oxygenator. Autologous blood was used for perfusion for a period of 2 h at a temperature of 37 degrees C. The model was tested with five groups of livers which had sustained varying degrees of injury ranging from minimally damaged to those known to be incapable of sustaining life when used for liver transplantation. The groups of livers were: (i) controls; (ii) preserved in University of Wisconsin solution (UW) for 6 h; (iii) preserved in an albumin-based extracellular fluid (ALB) for 6 h; (iv) preserved in UW for 18 h; and (v) preserved in ALB for 18 h. Bile production was found to be a reliable parameter of preservation damage. Changes in perfusate levels of aspartate aminotransferase, potassium, glucose and calcium also occurred in relationship to preservation damage. In contrast, weight gain of the liver, sequestration of the white cells and platelets in the liver, urea production and oxygen consumption were unreliable predictors of liver damage. Histology of biopsy specimens revealed apparently well preserved livers in all cases after preservation but before perfusion, but serious abnormalities after perfusion in long preserved livers, with features in these suggestive of damage to the sinusoidal endothelium. We believe that the model is a worthwhile adjunct to research into liver preservation.

Adenosine↗
PubMed1994

Analysis of the pressure-flow characteristics of isolated perfused rat kidneys with inhibited tubular reabsorption.

The renal hemodynamics were studied in an isolated perfused rat kidney model modified for investigations of the glomerular permeability characteristics. The tubular reabsorptive activity was inhibited by perfusion at low temperature (8 degrees C) in the presence of furosemide and nitroprusside resulting in a dramatic increase in the filtered load of fluid and solute reaching the tubules and hence in tubular pressure. The glomerular filtration rate (GFR), arterial pressure (PA) and needle pressure (intrarenal tissue pressure, PiR) were continuously recorded and the glomerular hydrostatic pressure was estimated by an arterial occlusion technique. The pre- to postglomerular resistance ratio was calculated from the pressure vs. GFR relationships for two perfusates having differing oncotic pressures (pi = 5.5 and pi = 20 mmHg), from which estimations of glomerular hydrostatic pressures (PGC) were concomitantly made. Thus, increases in delta pi could be exactly counterbalanced by equally large increases in PGC for any given GFR, the needle and Bowman's capsule pressures being dependent on GFR but not on plasma colloid oncotic pressure. The experimental interventions resulted in a pronounced elevation of PiR as compared with in vivo conditions, while the PGC values were in a normal range, resulting in reduced glomerular filtration pressures. Furthermore, the clearance of albumin varied with the oncotic pressure in agreement with the notion of heteroporosity.

Absorption↗
PubMed1977

The absorption of phenformin and its effects on glucose and water absorption in isolated perfused rat small intestine.

The effects of phenformin on glucose and water absorption from isolated perfused rat small intestine were studied. Luminal phenformin inhibited glocose and water absorption progressively as its concentration was increased from 0-1-1-0 mg.ml-1. At 0-5 mg phenformin ml-1, inhibition increased with time of exposure to phenformin up to 15 min and thereafter remained constant. Arterial infusion of phenformin (1-0 mg-ml-1) produced less inhibition of glucose and water absorption. The site of phenformin's action appeared to be intracellular. Phenformin absorption from a luminal perfusate (0-5 mg-ml-1) was measured. Although it was rapidly absorbed (22 microgram.cm intestine-1.h-1) from the lumen, less than 2 microgram.cm-1.h-1 appeared at the serosal surface of the intestine. In subsequent phenformin-free perfusion, only 25% of the absorbed phenformin was recovered in the luminal and serosal effluents.

Animals↗
PubMed1993

Catabolism of cysteine, cystine, cysteinesulfinate, and OTC by isolated perfused rat hindquarter.

The metabolism of cysteine and related compounds was investigated in the isolated perfused hindquarter of the rat. An erythrocyte-based perfusion medium was used; use of a perfluorochemical emulsion, FC-43, resulted in apparent chemical oxidation of cysteine, whereas bovine erythrocytes did not appear to contribute significantly to the metabolism of cysteine. Rat skeletal muscle perfused with L-[35S]cysteine, L-[35S]cystine, L-[35S]cysteinesulfinate, or L-2-oxo-[35S]thiazolidine-4-carboxylate (OTC) for 2 h produced [35S]sulfate and [35S]taurine. In all cases, the partitioning of cysteine or cysteinesulfinate between metabolism to taurine and sulfate was similar, suggesting that cysteine metabolism in hindquarter may occur via formation and catabolism of cysteinesulfinate by either cysteinesulfinate decarboxylase or aspartate (cysteine-sulfinate) aminotransferase. However, the activity of cysteine dioxygenase was extremely low, suggesting that the conversion of cysteine to cysteinesulfinate may have been non-enzymatic.

Adenine Nucleotides↗
PubMed1992

Regulatory volume decrease stimulates bile flow, bile acid excretion, and exocytosis in isolated perfused rat liver.

To study the effect of volume regulation on bile secretory function, isolated perfused rat livers (IPRL) were exposed to hypotonic stress (45 mM NaCl) while bile flow and the biliary excretion of bile acids and horseradish peroxidase (HRP) were assessed. Hypotonic stress induced a biphasic increase in bile flow, which rose in the first minute from 1.1 +/- 0.2 to 1.7 +/- 0.1 microliter.min-1.g liver-1 (P less than 0.01), an effect attributed to rapid osmotic equilibration of water, then increased further between 3 and 5 min to 1.6 +/- 0.1 microliter.min-1.g liver-1 (P less than 0.01, followed by a subsequent return to baseline. HRP excretion in bile increased during the second peak of bile flow from 0.9 +/- 0.2 to 1.1 +/- 0.2 ng.min-1.g liver-1, P less than 0.01. Pretreatment with colchicine but not lumicolchicine completely abolished the latter increase in bile flow and HRP excretion as did BaCl2 (1 mM), an inhibitor of both K+ channels and regulatory volume decrease (RVD) in hepatocytes. When sodium taurocholate was infused (1 mumol/min), hypotonic stress induced an even larger increase in the second peak of bile flow (5.1 +/- 0.7 microliters/g liver, P less than 0.01) and higher rates of bile acid excretion than in control perfusions with bile acid (126.2 +/- 21.0 vs. 99.0 +/- 17.1 nmol.min-1.g liver-1, P less than 0.05). These data suggest that both bile flow and bile acid excretion are stimulated during RVD by mechanisms that involve both K+ channels and microtubule-dependent exocytosis at the canalicular (apical) membrane domain.

Animals↗