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PubMed1954

Synthesis of all plasma protein fractions except gamma globulins by the liver; the use of zone electrophoresis and lysine-epsilon-C14 to define the plasma proteins synthesized by the isolated perfused liver.

Lysine-epsilon-C(14)-labeled plasma proteins produced by the normal rat and the isolated perfused rat liver have been fractionated by preparative zone electrophoresis. The isolated perfused liver incorporates lysine-epsilon-C(14) into the plasma albumin, alpha globulin, and beta globulin (including fibrinogen) fractions. No significant C(14) incorporation into the trichloracetic acid-precipitable proteins of the gamma globulin fraction was observed. Presumptive evidence indicates that the alpha globulins turn over more rapidly than any other major plasma protein fraction. The increased production of gamma globulins in liver disease is discussed.

Animals↗
PubMed1984

P-31 nuclear magnetic resonance analysis of brain: II. Effects of oxygen deprivation on isolated perfused and nonperfused rat brain.

Phosphatic metabolite (perchloric acid extractable) concentrations of cerebral tissues were analyzed by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy following external perfusion of the isolated rat brain (30 min or 60 min) under the following conditions: (a) constant perfusion pressure with either fluorocarbon- or erythrocyte-based medium, and (b) constant perfusate flow rate (3 ml/min) with the erythrocyte-based medium. Metabolite concentrations of control perfused brains were compared with those in nonperfused controls to provide a basis for detecting any qualitative or quantitative changes in cerebral metabolite composition. Metabolic responses of perfused brains to ischemia (incomplete ischemia, 83% reduction in flow for 10 min; transient complete ischemia for 1.5 or 2 min) were evaluated immediately after the ischemic episode and at selected time points during reperfusion (3 and 15 min). Alterations in cerebral metabolite levels induced by hypoxia were analyzed using a nonperfused rat brain model. Irrespective of the perfusion method employed, the phosphatic metabolites of control perfused rat brains were identical quantitatively to those of the nonperfused controls. Cerebral ischemia resulted in significantly increased levels of ADP, AMP + IMP, Pi, fructose 1,6-diphosphate, and glycerol 3-phosphate (global ischemia only), whereas ATP and phosphocreatine (PCr) levels declined significantly. The magnitude of these changes varied with the severity of the ischemia; however, following 15 min of control reperfusion metabolite levels had reverted to preischemic values. Significant perturbations in tissue phosphoethanolamine (3.84 delta resonance) content were evident at various time points during ischemia and postischemic recovery, which varied according to the perfusion conditions. In contrast to the changes observed in response to ischemia, hypoxia affected only cerebral high-energy phosphate levels. ATP and PCr levels were reduced, while a concomitant, essentially equimolar, increase in Pi and ADP was observed. The present studies indicate that in terms of phosphatic metabolites, the control equilibrated isolated perfused rat brain is quantitatively and qualitatively indistinguishable from the nonperfused rat brain in vivo regardless of the perfusion conditions (constant flow versus constant pressure). The metabolic responses to ischemia and hypoxia, as measured by P-31 NMR, were consistent with the pattern of changes reported elsewhere. Overall, P-31 NMR spectroscopic evaluation of the intact rat brain provides a potential experimental context for dynamic measures of cerebral metabolism under exogenously controlled conditions. Th

Animals↗
PubMed1988

pH and compartmentation of isolated perfused rat liver studied by 31P and 19F NMR.

The phosphorus NMR spectra of isolated perfused rat liver displays a prominent inorganic phosphate peak at 2.76 +/- 0.05 ppm relative to liver glycerolphosphocholine at 0.49 ppm. From titration curves of phosphorus-containing compounds this corresponds to a pH of 7.4. The spectra also display a shoulder on the prominent inorganic phosphate peak at 2.22 +/- 0.55 ppm corresponding to a pH of 7.0. Fructose is phosphorylated in the C1 position by the liver and the resulting fructose-1-phosphate is located in the cytosol. From the titration curves, this compound was at pH 7.0. When inorganic phosphate was added to the perfusate, 13 of 25 livers showed two inorganic phosphate peaks resolved in the difference spectra, one originating from the perfusate, the other at pH 7.0. When large (50 mM) fructose doses were administered to the liver, the prominent peak decreased allowing two peaks to be resolved. FCCP treatment of the liver caused the two peaks to coalesce with the final pH of both the fructose-1-phosphate and inorganic phosphate being the same. 19F NMR of difluoromethylalanine gave an intracellular pH of 7.4 for the isolated perfused liver. The data presented do not lend themselves to satisfactory interpretation, and call into question the correctness of the traditional assignment of liver inorganic phosphate being cytosolic in origin.

Animals↗
PubMedDate not supplied

Adequacy of oxygenation of isolated perfused rat heart.

Detailed arguments are presented for the adequacy of oxygenation of the isolated perfused rat heart preparation when subjected to a work load according to the model of Neely. Lactate discharge from a heart perfused with glucose does not equal hypoxia (although lactate output is a prime sign of hypoxia in the heart in situ where numerous other substrates are available). Adequacy of oxygenation does not mean normality of oxygenation. There is still an outstanding question as to whether the high oxygen tensions might not promote lipid peroxide formation, thereby sensitizing the hearts to the potentially harmful effects of fatty acids. Bearing in mind the above reservations, the isolated perfused heart model has made major contributions to the study of normal myocardial metabolic patterns. Such a rapid pace of advance would not have been possible without the use of the Krebs-Henseleit bicarbonate buffer medium.

Adenosine Diphosphate↗
PubMed1988

Metabolism of intact parathyroid hormone in isolated perfused rat liver and kidney.

Metabolism of synthetic human parathyroid hormone (PTH) 2 X 10(-10) to 5 X 10(-9) M was studied in 16 isolated perfused rat kidneys and 12 isolated perfused rat livers. Organ clearances were measured by assays specific for intact PTH. Production of fragments was analyzed by high-performance liquid chromatography (HPLC) and radioimmunoassays specific for NH2-terminal, midmolecule, and COOH-terminal PTH. The livers cleared intact PTH and NH2-terminal immunoreactive PTH (iPTH) at the same rate. Midmolecule iPTH was cleared significantly (P less than 0.001) slower, as was COOH-terminal iPTH (P less than 0.005), and HPLC studies demonstrated production of midmolecule/COOH-terminal PTH fragments, while no NH2-terminal fragments were found. Clearance in the kidneys of intact PTH and of NH2-terminal, midmolecule, and COOH-terminal iPTH was not significantly different from clearance of inulin. No clearance of intact PTH was found in nonfiltering kidneys. HPLC studies did not demonstrate release of any PTH fragments from the kidneys. In conclusion, the liver was not selective for intact PTH, and differential hepatic clearance, possibly together with direct glandular secretion, may contribute to the predominance of COOH-terminal PTH fragments in plasma.

Animals↗
PubMed1987

Ketoconazole impairs biliary excretory function in the isolated perfused rat liver.

The effects of ketoconazole (KT) on the hepatic excretory function was investigated in the isolated perfused rat liver. KT, at the concentrations of 5 X 10(-5) M or 10(-4) M caused dose-dependent decreases of the biliary bile acid concentration and excretion rate, with no significant effect on bile flow rates. Neither dose altered perfusate flow through the liver. Furthermore, at the same two concentrations, KT impaired the sulfobromophthalein transport in a dose-dependent manner. In contrast, the drug did not alter 14C-sucrose bile to perfusate ratio and did not cause enzyme release from the liver into the perfusate. The study demonstrates that KT possesses an intrinsic toxicity in the isolated perfused rat liver and suggests caution in the use of this drug in hepatopathic patients.

Animals↗
PubMed2000

The circulating hormonal milieu of the endocrine pancreas in healthy individuals, organ donors, and the isolated perfused human pancreas.

Although basal circulating levels of individual islet cell hormones have been measured, few studies compared the molar ratios of the major hormones secreted by the endocrine pancreas. This study examined the basal levels of four major islet hormones: insulin, C-peptide (C-P), glucagon (G), and pancreatic polypeptide (PP) in normal subjects, in organ donors with brain death, and in the isolated perfused human pancreas. Basal blood samples were taken from normal, fasted control subjects (NCs). Pancreata were obtained from 17 organ donors (ODs) with donor portal vein (DPV) and radial arterial (DRA) blood samples taken before organ procurement. Single-pass perfusion was performed on the procured pancreata, and after rewarming and equilibration, basal samples were collected from the splenic vein (SV) for 30 min. Radioimmunoassays of insulin, C-P, G, and PP were performed on all samples, and basal levels of all hormones were expressed as a common unit, femtomoles per milliliter. The data suggest that in the basal state, these four major islet hormones circulate in a relatively constant molar ratio. The ratio of the hormones is altered in brain death and with in vitro perfusion of the pancreas. The isolated perfused human pancreas secretes a relatively constant molar ratio of these hormones; however, this ratio is markedly different from the circulating ratio seen in either the NC group or the OD group. We conclude that a relatively constant hormonal milieu is secreted from the normal endocrine pancreas, and this hormonal milieu is altered after brain death and with isolation and perfusion of the human pancreas.

Adult↗
PubMed1993

Cholestatic potentials of alpha-naphthylisothiocyanate (ANIT) and beta-naphthylisothiocyanate (BNIT) in the isolated perfused rat liver.

Previous studies in rats have shown that a single oral dose of alpha-naphthylisothiocyanate (ANIT), but not the regioisomer beta-naphthylisothiocyanate (BNIT), results in intrahepatic cholestasis. The present studies were designed to evaluate the intrinsic cholestatic potential of ANIT and BNIT in the isolated perfused rat liver. Livers from male Sprague-Dawley rats (300-450 g) were isolated and perfused with Krebs-Henseleit buffer supplemented with 50 microM taurocholate and ANIT or BNIT (0, 5, 15 or 50 microM). Rates of bile flow, bile acid uptake and bile acid excretion were monitored for up to 70 min. Permeability of tight junctions also was evaluated. At concentrations of 5 microM, neither ANIT nor BNIT altered hepatobiliary function or tight junction permeability. In contrast, perfusion with 50 microM ANIT or BNIT for 35 min resulted in decreases in bile flow rates of 19 +/- 8 and 13 +/- 4%, respectively. After 70 min of perfusion with ANIT or BNIT, rates of bile flow were decreased by 78 +/- 5 and 71 +/- 4%, respectively. Bile acid excretion also was decreased following perfusion with 50 microM ANIT or BNIT. Perfusion with 50 microM ANIT or BNIT decreased bile acid uptake by 51 +/- 13 and 46 +/- 6%, respectively, at 60 min. Bile/plasma (B/P) ratios of [3H]sucrose were not affected by ANIT or BNIT at any time during perfusion, indicating that changes in bile flow and bile acid excretion in the isolated perfused liver were not associated with increased hepatocyte tight junction permeability. These data demonstrate that the direct portal infusion of a 50 microM concentration of either ANIT or BNIT produced marked decreases in bile flow, indicating that these isomers have a comparable intrinsic cholestatic potential in the isolated perfused liver.

1-Naphthylisothiocyanate↗
PubMed2000

Sulphonylurea drugs reduce hypoxic damage in the isolated perfused rat kidney.

Sulphonylurea drugs have been shown to protect against hypoxic damage in isolated proximal tubules of the kidney. In the present study we investigated whether these drugs can protect against hypoxic damage in a whole kidney preparation. Tolbutamide (200 microM) and glibenclamide (10 microM) were applied to the isolated perfused rat kidney prior to changing the gassing from oxygen to nitrogen for 30 min. Hypoxic perfusions resulted in an increased fractional excretion of glucose (FE % glucose 14.3+/-1.5 for hypoxic perfusions vs 4.9+/-1.6 for normoxic perfusions, mean +/- s.e. mean, P<0.05), which could be completely restored by 200 microM tolbutamide (5.7+/-0.4 for tolbutamide vs 14.3+/-1.5 for untreated hypoxic kidneys, P<0.01). Furthermore, tolbutamide reduced the total amount of LDH excreted in the urine (220+/-100 mU for tolbutamide vs. 1220+/-160 mU for untreated hypoxic kidneys, P<0.01). Comparable results were obtained with glibenclamide (10 microM). In agreement with the effect on functional parameters, ultrastructural analysis of proximal tubules showed increased brush border preservation in tolbutamide treated kidneys compared to untreated hypoxic kidneys. We conclude that glibenclamide and tolbutamide are both able to reduce hypoxic damage to proximal tubules in the isolated perfused rat kidney when applied in the appropriate concentrations.

ATP-Binding Cassette Transporters↗
PubMed1997

Rhodamine 123 accumulates extensively in the isolated perfused rat kidney and is secreted by the organic cation system.

Rhodamine 123 has been shown to be a substrate for P-glycoprotein in multidrug resistant cells. In the present investigation the disposition of rhodamine 123 was studied in the isolated perfused rat kidney. After exposing the kidneys to perfusate concentrations ranging from 10 to 1000 ng/ml, the renal clearance was 4-1 times the clearance by glomerular filtration, respectively, indicating active and saturable secretion of rhodamine 123. The rate-limiting step in secretion was found to be membrane passage from cell to tubular lumen. Suprisingly, renal clearance was not influenced by the P-glycoprotein inhibitors cyclosporin A or digoxin. However, pretreatment of the kidneys with verapamil and quinidine (inhibitors of both P-glycoprotein and organic cation transport) or cimetidine (organic cation transport inhibitor) resulted in a significantly reduced rhodamine 123 clearance, indicating that the renal organic cation carrier may be involved in active secretion. Rhodamine 123 accumulated extensively in the isolated perfused rat kidney; tissue concentrations of 270-360 times the perfusate concentration were determined. Similar accumulation ratios at different perfusate concentrations were found, suggesting that the compound enters the tubular cells by (facilitated) diffusion. In conclusion, rhodamine 123 accumulated extensively in the isolated perfused rat kidney and active renal secretion appears to be preferentially mediated by the organic cation carrier and not by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Glycosaminoglycan biosynthesis in the isolated perfused rat lung.

The suitability of an isolated lung, perfused under carefully monitored conditions, for the study of the biosynthesis of glycosaminoglycans was examined for the rat lung using either [35S]-sulfate or [6-3H]glucosamine. Metabolic and electron-microscopic studies after 3 h of perfusion showed that under the conditions of this study the isolated lung showed no anatomical or metabolic derangements. All glycosaminoglycans normally synthesized in the intact lung were identified. The predominant glycosaminoglycan was heparan sulfate (40% of total). Approximately 14% of the glucosamine incorporated into the glycosaminoglycans was found in hyaluronic acid. Less than 5% of either label was in heparin. The remainder of the synthesized glycosaminoglycans, with the exception of 10% which could not be identified, consisted of the chondroitin sulfates and dermatan sulfate. The relative proportions of the newly synthesized glycosaminoglycans, including the low amounts of heparin, are similar to those found in isolation of endogenous lung glycosaminoglycans. The isolated perfused rat lung appears to be a useful model for the study of glycosaminoglycan biosynthesis by the intact lung.

Animals↗
PubMed2003

Multinuclear NMR investigations on the metabolic recovery of the isolated perfused mouse liver after cold preservation.

This paper describes multinuclear NMR investigations on the isolated perfused mouse liver to optimize its recovery after cold preservation and normothermic reperfusion. The recovery of livers from fed is better than that from 24 h fasted animals. This better recovery is not due to a higher glycogen content before cold preservation. The recovery of livers from fasted animals is specifically enhanced by the presence of 8 mM alanine in the rinsing solution after cold preservation and in the perfusate of reperfusion. This property is not due to the ability of alanine to compensate for the lack of endogenous substrates since the amount, before cold preservation, of these substrates, is not significantly different in livers from fed and fasted animals. Furthermore, the beneficial effect of alanine is not due to an enhancement of the pyruvate dehydrogenase (PDH) activity in livers from fasted animals. In fact these livers have indeed a smaller PDH activity than the livers from fed animals but dichloroacetate, a known PDH activator has a rather deleterious effect on the recovery of fed and fasted livers. Furthermore alanine protects the fasted livers against this effect. So the beneficial effect of alanine should be due to other causes. Furthermore, we have found on a parallel model of rat isolated perfused liver, that the recovery of steatotic livers which is lower than that of normal fed livers is enhanced by a known vasodilator, pentoxifylin but not by alanine. So alanine does not either play its role through its action on microcirculation. The interaction of alanine with some membrane sodium transporters like that already reported for another protective aminoacid, glycine, is thus possible. A novel NMR method of (23)Na observation in living cells or organs should be of great interest to investigate this hypothesis.

Alanine↗
PubMed1998

[Effect of CD11b/CD18 on burn-activated PMN-mediated permeability of pulmonary microvascular in isolated perfused lung].

OBJECTIVE: To determine the effect of intercellular adhesion molecule (ICAM-1) CD11b/CD18 in burn-activated PMN-mediated permeability of microvascular in isolated perfused lung. METHOD: Isolated lungs were distributed into 7 groups according to the different contents of perfused fluid: normal perfused fluid, normal rat serum, burn rat serum, normal rat PMN, burn rat PMN, normal rat PMN blocked by monoclonal antibody to adhesion molecule CD11b/CD18 and burn rat PMN blocked by antibody. The extent of isolated lung edema, vascular permeability to small molecules (fluid) and vascular permeability to large molecules (albumin) were expressed by lung weight gain (LWG), fluid filtration coefficient (Kf) and pulmonary albumin permeability-surface area product (PS) respectively. RESULT: Burn serum could increase LWG, Kf PS, so did burn-activated PMN. The latter could make PS increase more obviously. Monoclonal antibody to CD11b/CD18 on PMN could obviously decrease PMN sequestration in isolated perfused lung. The protective effect of antibody on increasing LWG and Kf, and particularly PS was demonstrated. CONCLUSION: (1) The effect of mediators from burn-activated PMN to EC was mediated by PMN adhesion to EC. (2) Some mediators from burn-activated PMN increase mainly the pulmonary vascular permeability to small molecules, and PMN adhesion to EC mediated by CD11b/CD18 may increase directly the permeability to large molecules. (3) Adhesion molecule CD11b/CD18 perhaps have an ability to regulate directly EC by combining its receptor ICAM-1 on EC.

Animals↗
PubMed1991

The uptake of bupivacaine in an in situ isolated perfused rabbit lung preparation.

A double indicator technique has been used in an in situ isolated perfused rabbit lung model to examine the first pass effect of the lung on systemic bupivacaine concentrations. Bupivacaine (0.5 mg/kg) was given in two consecutive boluses to six in situ isolated perfused New Zealand White rabbit lung preparations. The mean recovery (first bolus) of bupivacaine was 62.6% +/- 6.3 (S.E.M.), and 63.7% +/- 10.2 (second bolus), suggesting bupivacaine accumulation in the lung. The average mean transit time for bupivacaine was 280.5% +/- 24.1 and 264.8% +/- 36.7 longer than ICG (Indocyanine Green) following the first and second boluses respectively (P less than 0.01). There were no differences in the first pass effect of the lung between the first and second boluses of bupivacaine. The profiles of the bupivacaine concentrations suggest that uptake is followed by accumulation and later back diffusion. This has implications for conditions that decrease the uptake and therefore increase the risk of systemic toxicity.

Animals↗
PubMed1998

Salidiuretic action by genistein in the isolated, perfused rat kidney.

The urinary isoflavonoid genistein inhibits membrane Na-K-Cl cotransporters at similar concentrations as furosemide, but the significance of this action is unknown. Genistein was therefore investigated in rats for its potential salidiuretic actions. In the isolated, perfused rat kidney, genistein induced a maximal salidiuretic action similar to that of furosemide but was 3 to 5 times less potent than furosemide in terms of active doses (natriuresis EC50, 237+/-92 versus 56+/-20 micromol/L for genistein and furosemide, respectively). Genistein and furosemide had no additive salidiuretic actions. Genistein had no significant effect on glomerular filtration rate but was able to significantly reduce renal vascular resistance with respect to vehicle isolated perfused kidney. Indomethacin (10 micromol/L), a blocker of prostaglandin biosynthesis, reduced salidiuresis and renal vasorelaxation by genistein. Subcutaneous genistein (15 mg/kg) induced a statistically significant increase in diuresis and natriuresis with respect to vehicle during the first 6 hours of administration in rats. In conclusion, genistein compares well with furosemide in vitro for its salidiuretic profile and potency in the isolated perfused rat kidney and is also natriuretic by the subcutaneous route in the rat. Further studies are required to investigate potential natriuretic and perhaps hypotensive actions of dietary genistein.

Animals↗
PubMed1980

Studies on the linkage of energy metabolism and neuronal activity in the isolated perfused rat brain.

An isolated rat brain preparation was perfused using glucose-free (= aglycemic) media. The high-energy phosphates, substrates of the glycolytic pathway, free amino acids, acetylcholine as well as the intracellular distribution of hexokinase activity were determined in brain tissues. The EEG was evaluated visually. The levels of glycolytic substrates, glutamate, and glutamine in cortical tissue decreased after aglycemic perfusion whereas the asparte level increased and the GABA level remained unchanged. The high-energy phosphate content seemed to be unaffected for about 15 min of aglycemic perfusion and fell significantly after 20 min. The EEG of the isolated brain changed rapidly after starting aglycemic perfusion and became isoelectric after 12--15 min. Hyperglycemic perfusion (35 mmol glucose per liter perfusion medium) did not alter the energy metabolism of the isolated brain. The breakdown of cerebral energy metabolism and of EEG activity was postponed when thiopental was added to the perfusion medium. The soluble hexokinase activity measured in cortical tissue was reduced after aglycemic perfusion and was enhanced after thiopental. Hyperglycemic perfusion did not influence the intracellular hexokinase distribution. The acetylcholine level in the striatum of the isolated rat brain was significantly decreased by aglycemia and was increased in hypothalamus by thiopental. It was suggested that hexokinase bound to the mitochondrial membrane may play an important role in the relationship of energy metabolism and neuronal activity.

Acetylcholine↗
PubMed1985

Effect of gallopamil on energy metabolism of the isolated perfused rat brain in the postischemic period.

The isolated perfused rat brain was used to demonstrate an effect of gallopamil on energy metabolism affected by ischemia. After a perfusion period of 30 min and 10 min of ischemia the isolated brain preparation was reperfused. From the onset of perfusion onwards, gallopamil (1 or 10 mumol/l) was present in the medium. The higher concentration of gallopamil accelerated significantly the restoration of the high-energy phosphates in the recovery stage: after 2 min of recirculation the ATP and the creatine-P levels were higher and the AMP level was lower in cortical tissue of drug-treated brains than in untreated controls. These results suggest that gallopamil protected brain energy metabolism against ischemic damage.

Adenosine Monophosphate↗
PubMed1995

Evaluation of a preservation solution containing fructose-1,6-diphosphate and mannitol using the isolated perfused rat kidney. Comparison with Euro-Collins and University of Wisconsin solutions.

The renal preservation ability of a flushing solution (F-M) with fructose-1,6-diphosphate (1 g/dl) and mannitol (2 g/dl) during cold ischaemia was studied with the isolated perfused rat kidney model and compared with the Euro-Collins (EC) and University of Wisconsin (UW) solutions. Kidneys were stored in hypothermia for 4 and 18 h after initial flushing with the solution being tested, and then reperfused at 37 degrees C in an isolated perfusion circuit for 90 min with a Krebs-Henseleit solution containing 4.5% albumin. Forty-four kidneys were studied and divided in a control group and six study groups according to the cold ischaemia time and flushing solution used. Renal functional parameters of plasma flow rate (PFR), renal vascular resistance (RVR), urine flow rate (UFR) glomerular filtration rate (GFR), fractional (FRNa) and net (TNa) sodium reabsortion were assessed during reperfusion. Conventional histology and malondialdehyde tissue levels (MDA) were also evaluated. Our results show that PFR, RVR, and UFR were similar in all study groups. After 4 and 18 h of cold ischaemia, GFR, FRNa and TNa were better, and conventional histology worse in F-M than in EC flushed kidneys. After 4 and 18 h of cold ischaemia, GFR, FRNa and TNa, in fact, were not different between F-M and UW flushed kidneys. After 4 h of cold ischaemia, conventional histology was similar in F-M and UW flushed kidneys. Nevertheless, after 18 h of cold ischaemia, UW flushed kidneys showed worse histological parameters than F-M flushed kidneys. After 4 h of cold ischaemia, MDA was similar in kidneys flushed with three solutions. After 18 h of cold ischaemia MDA was higher in EC than in F-M or UW flushed kidneys. In summary, our newly developed cold storage solution shows promising results in renal preservation and its ability to preserve is at least as good as UW solution assessed in the isolated perfused rat kidney.

Adenosine↗