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PubMed1983

Influence of hypoxia on the metabolism and excretion of misonidazole by the isolated perfused rat liver--a model system.

The isolated perfused rat liver was evaluated as a model system for the characterization of misonidazole metabolism under hypoxic conditions. Misonidazole metabolism by livers perfused under aerobic conditions was also examined. The clearance of misonidazole was more than three times greater under anaerobic compared to aerobic conditions (4.94 +/- 1.56 vs 1.27 +/- 0.22 ml/min; means +/- S.D., N = 3). Misonidazole metabolites were detected only in the bile. Analysis of these metabolites by reverse-phase high performance liquid chromatography (HPLC) demonstrated that misonidazole metabolism was also qualitatively changed when anaerobic conditions were employed. Misonidazole beta-glucuronide was the major metabolite detected under aerobic conditions, but it was a minor metabolite in anaerobically perfused livers. The three major metabolites produced under anaerobic conditions were not characterized, but desmethyl misonidazole (RO-07-9963) and the 2-amino-imidazole derivative of misonidazole (1-[2-aminoimidazol-1-yl]-3-methoxy-2-propanol) were excluded as possible structures.

Animals↗
PubMed1989

Hemodynamic and electrophysiological effects of mercury in intact anesthetized rabbits and in isolated perfused hearts.

Using intact anesthetized rabbits and isolated perfused hearts, the hemodynamic and electrophysiological effects of mercury (Hg) were examined in order to assess the role of cardiovascular dysfunction in Hg intoxication. The most consistent and prominent cardiovascular effect was a significant reduction in blood pressure. This cardiodepressive action was probably brought about by the primary action of Hg on the heart rather than by altered sympathetic activity, as evidenced by normal renal nerve activity at times when the hemodynamic actions of Hg were clearly manifest. Although the principal target organ for the toxic actions of inorganic Hg is the kidney, chronic exposure to both inorganic and organic Hg frequently results in signs and symptoms of CNS dysfunction. The profound hemodynamic effects of Hg that we have observed emphasize the potential importance of Hg cardiotoxicity and indicate the need to differentiate between the primary and the secondary effects of Hg intoxication on CNS tissues for evaluation of the toxic effects of Hg compounds.

Animals↗
PubMed1987

Oxytocin and arginine vasopressin stimulate steroid secretion by the isolated perfused rat adrenal gland.

Using the intact isolated perfused rat adrenal preparation we have shown for the first time a direct effect of oxytocin on adrenocortical steroid secretion. Oxytocin specifically stimulated aldosterone secretion in a dose-dependent manner with a threshold dose of 1 pmol. Arginine vasopressin was also shown to be a potent stimulus to aldosterone secretion and was additionally found to stimulate inner zone function. Using superfused adrenal cells, the effects of arginine vasopressin were only seen at 10,000 times higher doses than were effective in the intact perfused gland, and oxytocin had no effect at any dose. These results reinforce the hypothesis that tissue integrity is essential for full expression of steroidogenic control mechanisms. We conclude that oxytocin and vasopressin may play a role in the control of steroidogenesis.

Adrenal Cortex Hormones↗
PubMed1981

Synthesis of factor II antigen by isolated perfused rat liver.

Rat Factor II (prothrombin), isolated and purified by chromatography on Blue Dextran-agarose, was used to raise an antiserum in rabbits. On the basis of single radial immunodiffusion measurements. Factor II synthesis by isolated perfused rat liver amounted to 0.54 mg/300 cm2 body surface area of the liver donor in 10 h. Corresponding measurements of Factor II coagulant activity revealed cumulative synthesis of 802 Iowa units. Coumadin added to the liver perfusate blocked production of Factor II coagulant activity, but did not change synthesis of the immunologically measured protein. In perfusions in which either heparin or citrate was used as anticoagulant, synthesis of albumin was not affected by the choice of anticoagulant but bile production and synthesis of Factor II were significantly less in citrate perfusions.

Animals↗
PubMed1984

Effect of phenylephrine on glutamate and glutamine metabolism in isolated perfused rat liver.

Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.

Animals↗
PubMed1996

Endothelial modulation of pH-dependent pressor response in isolated perfused rabbit lungs.

With the use of isolated perfused rabbit lungs (n = 152), roles of endothelium-derived relaxing factor (EDRF) in pulmonary vascular responses to hypocapnia and hypercapnia were studied. Lungs were ventilated with a gas mixture containing 1, 5, or 10% CO2 and 21% O2, adjusting the perfusate pH to 7.8, 7.4, or 7.1, respectively. Methemoglobin (MetHb), hemoglobin (Hb), methylene blue (MB), and L-argininosuccinic acid (L-ASA) were used as modulators of EDRF. To eliminate augmented shear stress, we used papaverine during hypercapnia. As a measure of EDRF, we spectrophotometrically examined nitric oxide (NO) metabolites in the perfusate. Hypocapnia and hypercapnia evoked, respectively, unsustainable vasodilatation and vasoconstriction. Hb, MB, and L-ASA, but not MetHb, produced an increase in baseline pulmonary arterial pressure (Ppa). These agents also exacerbated vasoconstriction during hypercapnia. Hypercapnia and hypocapnia caused an increase and decrease, respectively, in EDRF production. L-ASA suppressed EDRF production in hypercapnic lungs. Papaverine did not suppress EDRF production under hypercapnia. In conclusion, 1) the effects of pH on pulmonary circulation are transient, 2) the increase in Ppa caused by hypercapnia is modulated by EDRF, and 3) the pulmonary EDRF genesis is activated by hypercapnic acidosis but suppressed by hypocapnic alkalosis.

Animals↗
PubMed1976

Lactate production in isolated perfused rat lung.

The ability of the isolated perfused rat lung to produce lactate under aerobic and hypoxic conditions has been studied. The lung has been found capable of producing lactate at a rate of 53 mumol/g dry wt per h in the presence of as great as 1 mM extracellular lactate under aerobic conditions. Essentially 100% of the lactate synthesized was derived from [U-14C]glucose. When hypoxic, the rate of lactate production doubled, but only 60% was found to be derived from [U-14C]glucose. When the perfused lung was exposed to aerobic conditions following hypoxia, it was found that lactate production did not decrease. However, lactate production from [U-14C]glucose did increase. It is concluded that lung tissue possesses a high rate of lactate production under aerobic contitions from glucose in comparison to glucose oxidation to CO2. When the lung is hypoxic, lactate production increases as a probable result of amino acid catabolism with little anaerobic energy production occurring. The effect of hypoxia was not reversed within the duration of the performed experiments.

Animals↗
PubMed1987

Hypoxia-induced alterations of norepinephrine vascular reactivity in isolated perfused cat lung.

Past work in the isolated perfused cat lung has shown that acute hypoxia (H) changes the response to norepinephrine (NE) from vasoconstriction to vasodilation but has no effect on the response to serotonin (S). These results could be related to the increase in pulmonary arterial pressure or vascular resistance during the hypoxic pressor response or a direct effect of H. We addressed this question, in the same preparation, by comparing responses to NE under four conditions in each experimental animal (n = 12): 1) NE infused during normoxia; 2) NE infused after vascular resistance (Rpv) was increased with serotonin; 3) NE infused after Rpv was increased by H; 4) NE infused after lobar pressure was raised by an increase in flow (P/F). PO2 values during H were varied (27-56 Torr). S and H produced a 137 +/- 35 and 43 +/- 8% delta Rpv increase in lobar vascular resistance, respectively. P/F increased lobar pressure 91 +/- 10%. Only NE infusion during H demonstrated significant differences in lobar pressure and Rpv compared with control normoxic periods. There was no correlation between responses to NE during S, H, and P/F and degree to which each stimulus increased Rpv or lobar pressure (r = 0.003, 0.28, 0.24). A significant relationship between response to NE during H vs. PO2 during H was observed (r = 0.78; P less than 0.001). In a subset of animals, we repeated the infusion of NE during H and P/F post-beta-blockade. The decrease in vascular response to NE during H and the correlation of PO2 with NE response were abolished (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Modulation by prostaglandins of adrenergic transmission in the isolated perfused rabbit and rat kidney.

In the isolated perfused rabbit kidney prostaglandins (PGS) E1 (0.02-0-1 ng/ml), E2 (0.02-0.1 ng/ml), and A2 (1-5 ng/ml) inhibited the vasoconstrictor responses to sympathetic nerve stimulation by 21-44%, 31-39%, and 20-23%, respectively, without alerting those to injected norepinephrine. In contrast, in the rat kidney PGE1 (0.5 ng/ml), PGE2 (0.5 ng/ml), and PGA2 (5 ng/ml) enhanced the vasoconstrictor responses to sympathetic nerve stimulation by 41%, 27%, and 11%, respectively; the equiconstrictor responses to injected norepinephrine remained unaltered. Higher concentrations of these agents produced vasodilation in the rabbit kidney and vasoconstriction in the rat kidney. In both species PGF2alpha produced vasoconstriction and enhanced the response to both adrenergic stumuli. In the rabbit kidney inhibitors of PG synthesis augmented the responses to sympathetic nerve stimulation without altering those to injected norepinephrine, whereas in the rat kidney inhibition of the responses to both adrenergic stimuli occurred. Arachidonic acid inhibited the vasoconstrictor responses to sympathetic nerve stimulation in the rabbit kidney, but in the rat kidney it caused augmentation of these responses. Since these effects of arachidonic acid were reduced by indomethacin, they appear to be mediated through the acid's conversion to PGS. We conclude that PGS of the E series modulate adrenergic transmission in the kidney and that their modulatory actions are species dependent.

Animals↗
PubMed1976

Isolated perfused rabbit lung: a critical appraisal.

The isolated perfused lung (IPL), when compared to available in vitro and in vivo pulmonary systems, is a preparation that fulfills a majority of the ideal criteria for studying metabolism, binding and/or physiological response to xenobiotics. The IPL is an exceptionally useful method when there is a need for concurrent administration of multiple agents in different physical forms. Various details such as physiological and biochemical parameters and the construction of a small animal tracheal valve system are discussed.

Animals↗
PubMed1985

Glutamine metabolism in isolated perfused rat liver. The transamination pathway.

In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.

Alanine↗
PubMed1975

Insulin sensitivity of isolated perfused rat liver.

The responsiveness of the isolated perfused rat liver to different metabolic effects of insulin was investigated during recycling perfusion. Infusion of porcine insulin at rates of 6, 9, 16 and 33 mU/hr. resulted in stable perfusate insulin levels averaging 41, 72, 120 and 229 muU/ml., respectively. Since the portal vein insulin concentration in the intact rat averaged 48 muU/ml. after a twenty-six-hour fast and 125 muU/ml. two hours after removal of food, the studies were conducted at insulin levels within the physiological range. The effect of each insulin concentration on the net accumulation of K+, AMINO ACID NITROGEN, UREA NITROGEN AND GLUCOSE IN The perfusing medium was assessed against the net accumulation of perfusate constituents during perfusion of control livers and livers perfused with perfusate insulin levels greater than 500 muU/ml. The results indicate that essentially maximal suppression of amino acid nitrogen outflow and retention of K+ OCCURRED AT INSULIN CONCENTRAtions of 72 muU/ml., with lesser effects being noted at 41 muU/ml. Inhibition of ureogenesis was demonstrated at insulin levels above 120 muU/ml. However, significant effects of insulin on suppressing net glucose outflow was not observed until insulin levels had reached 500 muU/ml. due presumably to the absence of a sustained rate of glycogenolysis by control livers. The observation that perfused livers from normal rats are extremely sensitive to several metabolic effects of insulin at physiological concentrations suggests that this experimental approach can provide useful information as to the role of the liver in the pathogenesis of various insulin resistant states.

Amino Acids↗
PubMed1975

Creatine release from the isolated perfused rat heart.

Creatine release from the isolated perfused rat heart decreases during the first 40 min of perfusion to a constant rate which is maintained for at least a further 50 min. Reoxygenation after a period of 15 min of anoxia is accompanied by an increase in creatine release. The highest rate of release occurs during preperfusion after the excision of the heart. These losses might contribute to impairment of function after anoxia.

Aerobiosis↗
PubMed1986

[Action of [3,14-L-selenocysteine, 8-D-tryptophan]-somatostatin on insulin and glucagon secretion of the isolated perfused pancrease of the Wistar rat].

By isolated perfused pancreas of Wistar rats the glucose (11 mmol/l) and arginine (10 mmol/l) stimulated insulin (IRI) and glucagon (IRG) secretion was measured in order to investigate the inhibitory activities of somatostatin-14 (SS 14) and the somatostatin analogue [3,14-L-seleno-cysteine, 8-D-tryptophan]-somatostatin (SeSS). SS-14 or SeSS (152.8 nmol/l) inhibit the glucose stimulated IRI secretion by 75 and 65%, respectively. Only the second phase of the biphasic arginine stimulated insulin secretion pattern by 40%. SeSS has under these conditions no effect, whereas 58 nmol/l SS-14 or SeSS show a suppressing effect on the first (20 and 55%, respectively) and second phase (65 and 85%, respectively) of the insulin secretion. Using 5.8 nmol/l SS-14 or SeSS the arginine stimulated IRG secretion was inhibited only in the second phase of the biphasic glucagon secretion pattern by about 40%. 58 nmol/l SS-14 or SeSS show an inhibiting effect on the first and on the second phase of secretion, in both cases about 50%. It is concluded that in the SS-14 molecule the sulfur of cysteine in position 3 and 14 can be exchanged by selenium without modifying the biological activities measured in the glucose or arginine stimulated IRI and IRG secretion in vitro. The D-Trp8 in the SeSS analogue does not show the typical better inhibitory action of D-Trp8-SS-14 on insulin and glucagon secretion compared with SS-14. Possibly the selenium in the SeSS analogue abolishes this effect.

Animals↗
PubMedDate not supplied

The relation between sodium transport and oxygen consumption in isolated perfused rat kidney.

A non-filtering isolated perfused rat kidney model was developed to determine renal oxygen consumption in the absence of tubular reabsorption. The oxygen consumption in the absence of filtration was 3.1 +/- 0.8 micron of O2/min/g. This "basal" value was substantially higher than that of 2.0 micron of O2/min/g calculated by extrapolation of a plot of sodium reabsorption vs. oxygen consumption in filtering kidneys. It was also found that ouabain 10(-4) M decreased oxygen consumption in non-filtering kidneys and acetazolamide increased it in a dose dependent fashion. Furosemide did not change "basal" oxygen consumption. In filtering kidneys, the ratio of sodium reabsorbed to oxygen consumed averaged 36.8 muEq of Na/mumole of O2. It is concluded that basal oxygen consumption is not a fixed quantity and can be changed by experimental manipulations thereby changing the relation between transport activity and oxygen consumption.

Acetazolamide↗
PubMed1978

Immunologic block against antigen absorption from isolated perfused rabbit lungs.

In previous studies with isolated perfused rabbit lungs, we observed that inhaled human serum albumin (HSA) or ovalbumin (OA) entered the pulmonary circulation antigenically intact. The inhaled proteins were also metabolized in the lung. Immunization reduced the amount of intact protein and increased the amount of metabolites absorbed. In the present study, we have begun to characterize the immune mechanisms responsible for reduced antigen absorption. A humoral immune mechanism appeared to be involved because the phenomenon could be passively transferred to normal animals by administering immune serum either 18 hr or immediately before antigen inhalation. The reduction was also observed when lungs from immunized rabbits were perfused with normal rabbit blood, indicating that antibodies in both lung and blood may be involved. Experiments, in which lungs from immunized rabbits were simultaneously insufflated with the immunizing antigen and with a nonspecific protein, demonstrated that the block against antigen absorption was specific for the immunizing antigen and was not due to some antigen-induced nonspecific changes in lung physiology.

Absorption↗
PubMed1997

Disposition of morphine in the rat isolated perfused kidney: concentration ranging studies.

The rat isolated perfused kidney was used to investigate the linearity of the renal disposition of morphine and its potential oxidative and glucuronidative metabolism by the kidney. In a set of single-dose experiments, morphine was administered to recirculating perfusion medium to achieve initial concentrations of 0.2, 2 and 20 microM (n = 4 at each concentration). In a set of multiple-dose experiments, morphine was administered to perfusate as sequential bolus doses to achieve concentrations of 0.2, 2, 20 and 200 microM (n = 6). HPLC was used to determine the concentration of morphine in perfusate and urine. Normorphine, morphine-3-glucuronide and morphine-6-glucuronide could not be detected in perfusate or urine, a result that suggests an absence of oxidative and glucuronidative metabolism of morphine by the rat kidney. The volume of distribution of morphine within the kidney was high (31 +/- 3 ml/g at 0.2 microM), which indicates extensive accumulation, and remained constant with increasing perfusate concentration. The ratio of unbound renal excretory clearance to glomerular filtration rate was always greater than unity for all kidneys, which indicates that the renal excretion of morphine involves net tubular secretion. This ratio was constant (P > .05) over the 100-fold concentration range of the single-dose study. In the multiple-dose study, the ratio was marginally but significantly (P < .05) higher at concentrations of 2, 20 and 200 microM than at 0.2 microM, a difference that cannot be explained by saturation of tubular secretion. The results suggest that the tubular secretion of morphine is not saturated over a wide range of concentrations (0.2-200 microM).

Analgesics, Opioid↗
PubMed1987

A simple and accurate new method for cytostatics dosimetry in isolation perfusion of the limbs based on exchangeable blood volume determination.

Current methods for cytostatic dosimetry in isolation perfusion of the limbs are based on either limb tissue volume (LTV) or body weight. None of them take into account the actual blood volume intra- and extracorporal, including even the blood leakage if any, in which the pharmacokinetics take place. The present study describes a method which allows the assessment of the actual exchangeable blood volume. The latter is calculated by a formula based on three hematocrit measurements. Thirty-one cases entered the study. Exchangeable limb blood volume representing the limb vascular bed was found to average 340 +/- 148 (SD) ml for upper limb perfusion and 768 +/- 279 and 621 +/- 454 ml for iliac and femoropopliteal perfusion, respectively. There was a good correlation between exchangeable limb blood volume and limb tissue volume (LTV, r = 0.7), a poor one with body weight (r = 0.3), and no correlation at all with body surface. Melphalan dosage was calculated per ml of blood and applied at 20 to 40 micrograms/ml. Comparison between calculated dose and concentration measured by high performance liquid chromatography showed a high correlation (r = 0.963). Since there was a correlation between exchangeable limb blood volume and LTV, it was possible to derive a conversion for melphalan dosage where 13 mg/liter corresponds to 20 micrograms/ml in upper limb perfusion and 10 mg/liter corresponds to 40 micrograms/ml in lower limb perfusion. Comparison between calculated melphalan dosage based on our method and the LTV method showed a large dispersion of values in the latter (12 to 18% coefficient of variation) while the dispersion given by the body weight-based method increased 2-fold (16 to 31% coefficient of variation). It is concluded that the present dosimetry method is the most suitable up to the present for accurate prediction of cytostatic concentration in isolation perfusion.

Blood Volume↗