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PubMed1998

The influence of chylomicron remnants on endothelial cell function in the isolated perfused rat aorta.

A system for the perfusion of the isolated rat aorta which allowed the study of both the uptake of chylomicron remnants by the artery wall and their effects on endothelial function was developed. Perfusion for 2 h with 125I-labelled native or oxidised (by treatment with copper sulphate) chylomicron remnants showed that small amounts became associated with the artery wall (0.111 +/- 0.034 and 0.216 +/- 0.082 ng protein/mg tissue, respectively). Tests on endothelial function were carried out in vessel rings prepared after perfusion of the aortas in the presence or absence of chylomicron remnants for 2 h. After perfusion of the vessels with oxidised chylomicron remnants, the maximum response to phenylephrine (PE) was significantly increased (from 0.34 +/- 0.06 to 0.51 +/- 0.04 g/mg tissue; P < 0.05), while the maximum % relaxation to carbachol (CCh) was significantly decreased (from 91.6 +/- 2.4 to 71.5 +/- 7.2; P < 0.05) and the response to S-nitroso-N-acetylpenicillimine (SNAP) was unaffected. Perfusion with native chylomicron remnants showed a tendency to induce similar effects, although the changes observed did not reach statistical significance. As the lipoproteins were not present in the solution bathing the vessel rings during these tests, these effects can be attributed to perfusion of the aortas with chylomicron remnants, despite only small quantities being associated with the artery wall. The results suggest that oxidised chylomicron remnants influence vascular endothelial function by interfering with the L-arginine-nitric oxide (NO) pathway. The observed potentiation of contraction to PE may be due to inhibition of the basal release of NO or to the release of contractile factors. These findings support a role for dietary lipoproteins in the modulation of endothelial cell function which occurs in the pathogenesis of atherosclerosis.

Animals↗
PubMedDate not supplied

The influence of glycerol on the Ca flux in the isolated perfused rat heart.

Perfusion of the isolated rat heart with a fluid containing 800mM glycerol in addition to its normal constituents causes a contraction height decrease of about 50%. A change to normal perfusion fluid causes a period of contracture to occur. After this period the heart is perfectly viable. Mechanical and osmotic effects could be ruled out as possible causes. No gross disturbances were seen in the electron micrographs. An effect on intermediary metabolism is equally unlikely. Presumably, we must seek the explanation in a direct effect of the glycerol on the Ca++ fluxes across the cellular and intracellular membranes.

Animals↗
PubMed1983

Tissue toxicity in experimental isolated perfusion with adriamycin.

Adriamycin is known as an antineoplastic agent with a broad spectrum of activity for human malignancies. To determine the highest tolerable concentrations of adriamycin in isolated perfusion of the extremities the hind legs of 12 dogs were isolated in an extracorporeal circuit and perfused under normothermic conditions. Dosages of about 10 mg/l perfused extremity were well tolerated by the animals, and lower concentrations did not result in any side effects. However, in most cases, dosages higher than 10 mg adriamycin/l extremity volume induced severe edema, pain, and histological changes.

Animals↗
PubMed1973

Estrogen formation by the isolated perfused rhesus monkey brain.

Perfusion of two isolated brains from immature male rhesus monkeys with [(3)H]androstenedione resulted in the identification of free and conjugated [(3)H]estrone and free [(3)H]estradiol from the perfusates. In the dissected cerebral tissues, estrogens were recovered only from the hypothalamus and limbic system. The production of estrogens from androstenedione during the 40-minute perfusions in these two experiments totaled 1.58 and 2.83 nanograms.

Androstenes↗
PubMed1976

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.

Adenosine Triphosphatases↗
PubMed2000

Metabolism of dipropyl disulfide by rat liver phase I and phase II enzymes and by isolated perfused rat liver.

The metabolism of dipropyl disulfide (DPDS), an Allium sulfur compound, was investigated in rat liver cell subfractions and in an isolated perfused rat liver. DPDS is oxidized to dipropyl thiosulfinate (DPDSO) by rat microsomes. The contribution of cytochrome P450 enzymes (CYPs) or flavin-containing monooxygenases (FMO) to the formation of DPDSO from its precursor was investigated. In rat microsomes, the reaction followed Michaelis-Menten kinetics with a K(m) = 0.52 +/- 0.1 mM and a V(max) = 5.91 +/- 0.5 nmol/min/mg of protein, respectively (mean +/- S.E., n = 4). Both FMOs and CYPs were involved in DPDS oxidation, although the contribution of CYPs was predominant. Liver microsomes from phenobarbital-treated rats showed a 3.2-fold increase in the rate of formation of DPDSO. Among many CYP isoform-specific inhibitors, only CYP2B1/2 inhibitors decreased the formation of DPDSO and the best correlation between the rate of DPDS oxidation with specific monooxygenase activities was found with a marker of CYP2B1/2 activity. The action of phase II enzymes on DPDS metabolism was studied by incubating DPDS or DPDSO with liver cytosols or microsomes. Two metabolites were formed from DPDS: propylglutathione sulfide conjugate and propylmercaptan, whereas with DPDSO, only the glutathione conjugate was observed. No conjugate compound was detected in the presence of UDP-glucuronyl transferases. When isolated rat liver was perfused with DPDS, different metabolites were obtained in the output and in the liver tissues: propylmercaptan appeared in the output, whereas methylpropyl sulfide, methylpropyl sulfone, and propylglutathione sulfide were detected in the liver tissue.

Animals↗
PubMed1975

Study of indirect methods for measuring hepatic blood flow in the isolated perfused dog liver.

This study was undertaken to assess the reliability of the constant infusion clearance and extraction technique (CIT) and the single injection clearance method (SIT), to measure hepatic perfusate flow at different levels of liver function. In the 36 isolated perfused dog livers, hepatic perfusate flow(HPF) was monitored by a direct method (DM), and the HPF was simultaneously measured with an indirect method (IM) utilizing bromosulphophthalein BSP) as indicator (20 SIT and 16 SIT). In 16 experiments, absolute ethanol (750 mM) and sodium taurocholate (600 muM) were seperately utilized to impair BSP metabolism. By comparison with DM, in the experiments with an extraction ratio(ER) of BSP lower than 10%, the error of the IM (ml/min) was for the CIT 154.57 +/- 46.55 and for the SIT 107.25 +/- 34.20. This error was significantly greater than in the experiments with an ER greater than 10% (CIT 15.70 +/- 3.88, SIT 8.20 +/- 1.38) p less than 0.01. In the studies with ER less than 10%, no difference was found in the error detected with the SIT (107 +/- 34.20) and CIT measurements (154.37 +/- 46.55); in the experiments with an ER greater than 10%, the error with the CIT (15.70 +/- 3.88) was greater than the one attained with the SIT (8.20 +/- 1.38, p less than 0.10 greater than 0.05). It is concluded that both methods are reliable techniques, for measuring HPF in livers with normal or near normal clearance capacity, but even in these ideal experimental conditions the values for HPF are inacurate when the liver clearance function is moderate or severly impaired.

Animals↗
PubMed1975

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.

Animals↗
PubMed1982

Inhibition of glucagon secretion by exogenous glucagon in the isolated, perfused dog pancreas.

To determine if glucagon can inhibit its own secretion, exogenous glucagon was perfused in the isolated canine pancreas at concentrations ranging from 90 to 1050 pg/ml during either stimulation or suppression of endogenous glucagon secretion. When endogenous glucagon secretion was completely suppressed by the addition of 200 mg/dl of glucose to the perfusate, the concentration of glucagon in the venous effluent ranged from 80.8 to 89.9% of the level perfused; these deficits of 10-20% were attributed to uptake and/or degradation of exogenous glucagon by the pancreas. By contrast, when endogenous glucagon secretion was stimulated by perfusing with 10 mM arginine or by glucopenia of 25 mg/dl, there was a significant increase in the glucagon deficit (the efflux glucagon level during glucagon perfusion minus the sum of the pre-perfusion efflux level of endogenous glucagon and the concentration of glucagon perfused); only 60-76% of the expected glucagon concentration was present in the venous efflux. This increase in deficit is assumed to reflect suppression of endogenous glucagon. The glucagon deficit rose progressively with deficit reached the approximate level of endogenous glucagon, at which no further increase occurred, suggesting that suppression at that point was complete. To determine if the glucagon-suppressing action of glucagon was mediated by an increase in glucagon-stimulated insulin or somatostatin secretion, these hormones were measured in the various experiments. Insulin and somatostatin increased significantly when glucagon was perfused with the glucose arginine-containing solution but did not rise significantly when the glucopenic buffer was used, although suppression of endogenous glucagon was similar in each instance. It is concluded that glucagon suppresses glucagon secretion. Evidence that insulin and/or somatostatin mediate this action could not be obtained.

Animals↗
PubMed1982

Action of phosphorus on calcium release in isolated perfused rat tails.

A bone perfusion system using isolated rat tails was developed to study the action of inorganic phosphorus (Pi) on calcium (Ca) release in bone. Rats were either pre-labelled with 45Ca to study calcium removal from bone or 45Ca was added to the perfusate to study Ca deposition into bone. When perfusate Pi was increased from 3.5 to 5.25 mg/dl, a marked inhibition of 45Ca release was demonstrated. 85Sr microspheres, used to demonstrate differences in the flow of perfusate between bone and soft tissue, showed that the Pi effect was not due to alterations in perfusate flow to bone. Increasing the perfusate Pi concentration from 3.5 to 7 mg/dl did not increase 45Ca deposition into bone, nor did it affect baseline or PTH-stimulated cyclic AMP production. The Pi effect was inhibited by reducing the perfusate temperature to 4 degrees C, by using an anerobic perfusate, adding iodoacetate to the perfusate, and finally, by pre-treating the rats with actinomycin D prior to the experiment. Thus, in this model an increase in the Pi concentration of the perfusate inhibits Ca release from bone but does not increase Ca uptake by bone. The inhibitory effect appears to be dependent on normal cell metabolism.

Animals↗
PubMed1995

Characterisation of functional endothelin receptors in the canine isolated perfused spleen.

The endothelin receptor subtypes involved in the vasoconstriction, capsular smooth muscle contraction, prostaglandin E2 and prostacyclin release induced by endothelin-1 have been investigated in the canine isolated perfused spleen using both the endothelin ETA receptor antagonist FR 139317 and the endothelin ETB receptor agonist IRL 1620. THe isolated canine spleen was perfused with warmed (37 degrees C) and oxygenated (95% O2/5% CO2) Krebs solution at constant flow with continuous recording of splenic arterial perfusion pressure and spleen weight. Samples of splenic venous effluent were collected to determine the amounts of prostaglandin E2 and prostacyclin, measured by radioimmunoassay. Endothelin-1 (4-200 pmol) and IRL 1620 (20-1000 pmol) dose-dependently increased splenic arterial perfusion pressure but the former was more potent on a molar basis (the molar dose ratio IRL/endothelin-1 required to increase splenic arterial vascular resistance by 25% was approximately 33). The infusion of the nitric oxide inhibitor N omega-nitro-L-arginine methyl ester (10 microM), but not of the enantiomer N omega-nitro-D-arginine methyl ester, significantly potentiated the increase in splenic arterial vascular resistance induced by endothelin-1. The infusion of FR 139317 (1 microM) markedly attenuated the increased splenic arterial perfusion pressure induced by endothelin-1 without affecting that evoked by IRL 1620. At the highest dose (200 pmol), endothelin-1 induced a small but significant capsule contraction as reflected by the reduction in the spleen weight. The infusion of FR 139317 (1 microM) abolished this contractile effect. IRL 1620 (in doses up to 1000 pmol) did not significantly affect the capsule tone.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1982

The study of ovulation in the isolated perfused rabbit ovary. 1. Methodology and pattern of steroidogenesis.

A previously described technique for perfusion of the isolated rabbit ovary in a recirculating system (Ahrén et al., 1975) was modified for studies of preovulatory follicular development and ovulation. Follicular ruptures were induced either by injection of human chorionic gonadotropin (hCG) to the animal 5-6 h prior to perfusion or by adding luteinizing hormone (LH) directly to the medium during perfusion. Ovulation occurred 9-14 h after hCG injection in vivo and 7-13 h after the addition of LH in vitro. Ovulations did not occur unless the ovaries were stimulated by gonadotropin in vivo or in vitro. The patterns of release of estradiol-17 beta and progesterone into the medium are consistent with the measurements reported by others on these steroids in ovarian venous blood in vivo. This experimental model is likely to be useful in further exploration of the physiology, biochemistry and anatomy of ovulation.

Animals↗
PubMed1979

Synthesis of procoagulant factor VIII, factor VIII related antigen and other coagulation factors by the isolated perfused rat liver.

The synthesis of factor VIII and other coagulation factors has been studied using an isolated, perfused rat liver. Synthetic function of the liver was validated by adding [35S]L-methionine to the perfusion medium and performing two-dimensional crossed immunoelectrophoresis and autoradiography on samples obtained during perfusion. Progressive incorporation of radioactivity into plasma proteins was demonstrated. This was inhibited by cycloheximide. Coagulation factor assays demonstrated synthesis of factors II, IX and X and of factor V and procoagulant factor VIII (VIIIC). Synthesis of factor VIII related antigen (VIIIRAg), measured in an immunoradiometric assay, was not significantly demonstrated. Addition of warfarin to the perfusion medium inhibited the synthesis of factors II, IX and X but not of factors V and VIII. Cycloheximide completely inhibited synthesis of all coagulation factors but actinomycin acted only after a latent period. Reticuloendothelial cell blockade was attempted by adding ethionine to the perfusion medium or by administration of Indian ink to the donor animals prior to removal of the livers. In these instances synthesis of factor V and factor VIIIC was inhibited but not that of factors II, IX and X. The results confirmed the functional capacity of the isolated liver for synthesizing proteins and the vitamin K dependent coagulation factors, and suggested similar kinetic features for the synthesis of factors V and VIIIC. Failure to detect significant VIIIRAg synthesis in these experiments is consistent with the hypothesis that this protein is released by vascular endothelial cells throughout the body and is activated or joined to VIIIC or stimulates its production in the liver.

Animals↗
PubMed1998

The effect of hepatocyte enlargement on the hemodynamic characteristics of the isolated perfused rat liver preparation.

The influence of hepatocyte enlargement on intrahepatic hemodynamics was assessed in the isolated perfused rat liver preparation (IPRL) using two experimental models: hypotonic liver cell swelling and phenobarbitone-induced hepatocyte hypertrophy. The analysis of pressure-flow data obtained from the portal vascular bed over a flow range of 0 to 70 mL/min in the presence of a maximally-effective concentration of the vasodilator agent papaverine hydrochloride (6 x 10(-4) mol/L) enabled the calculation of P0, an estimate of the pressure required to passively distend the intrahepatic vasculature, and Gmax, the maximal portal vascular conductance. By comparison with an isotonic perfusion medium (Krebs-Henseleit buffer [KH] containing 2.5% bovine serum albumin [BSA]), perfusion with a hypotonic medium induced a significant increase in mean hepatocyte cross-sectional area (H(A)) (590 +/- 21 vs. 324 +/- 23 microm(-2), p < .05), a fall in Gmax (0.39 +/- 0.08 vs. 2.02 +/- 0.18 mL/min/g/mm hg, P < .001), and an increase in P0 (2.96 +/- 0.38 vs. 1.58 +/- 0.07 mm hg, P < .001). Phenobarbitone administered in drinking water (0.5 g/L) over a period of 60 days also induced a significant degree of hepatocyte enlargement (HA, 510 +/- 29 microm2, P < .05). On day 7, portal pressure measured in vivo in this group was significantly elevated compared with untreated controls (10.5 +/- 0.3 vs. 8.4 +/- 0.2 mm hg, P < .001), while in the IPRL Gmax was reduced (0.48 +/- 0.01 mL/min/g/mm hg, P < .001), and P0 was increased (2.23 +/- 0.17 mm hg, P < .05). However, with continued phenobarbitone treatment portal pressure, Gmax and P0 returned toward control values. The results confirm that hepatocyte enlargement is associated with a significant disturbance of intrahepatic hemodynamics but also that some adaptation occurs if hepatocyte enlargement is sustained over a prolonged period of time.

Animals↗
PubMed1996

Temperature dependence of NMR relaxation times of nucleoside triphosphates and inorganic phosphate in the isolated perfused rat liver. Effect on Pi compartmentation.

The effect of temperature on 31P NMR spectra from isolated perfused rat livers was studied at 9.4 T. Relaxation times (T1 and T2) of uncleoside triphosphates (NTP) and inorganic phosphate (Pi) were determined at 37, 25, 15, and 4 degrees C. Under hypothermic conditions, an unexpected apparent line sharpening in the Pi spectral region and a clear emergence of an additional Pi resonance were observed. This additional signal was assigned to mitochondrial Pi. T1 values obtained for cytosolic and mitochondrial Pi at 4 degrees C were 1.14 +/- 0.24 s (n = 5) and 0.71 +/- 0.18.s (n = 5), respectively. No significant mitochondrial contribution to the Pi resonance was observed at 37 degrees C. Quantification of Pi and NTP liver contents at 37 and 4 degrees C was performed by comparing the perfused liver spectrum and the corresponding perchloric acid extract spectrum. Under experimental conditions of low external Pi (0.12 mM), it was concluded that intracellular Pi was completely NMR-visible at 4 and 37 degrees C. The observation of the mitochondrial Pi signal at 4 degrees C was well explained by an increase in the Pi level within the matrix, in response to the mitochondrial swelling induced by hypothermia, as observed by electron microscopy. T2 values for the cytosolic Pi at 37 and 4 degrees C were 17 +/- 4 ms (n = 8) and 22 +/- 4 ms (n = 10), respectively. Comparison with measured linewidths indicated that line broadening for the main phosphorylated metabolites--including matrix Pi--was the result of B0 field inhomogeneity. The additional broadening of the cytosolic Pi resonance at 4 and 37 degrees C was attributed to pH heterogeneity within the liver.

Animals↗
PubMed1995

Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills.

Uptake rate constants of different classes of hydrophobic organic chemicals have been determined in isolated perfused gills of rainbow trout (Oncorhynchus mykiss) as an alternative for studies in vivo. The uptake rate constants have been compared to those determined in guppy, Poecilia reticulata, in vivo. The organic chemicals which have been used are anthracene, hexabromobenzene, octachloronaphthalene, octachlorodibenzo-p-dioxin, phenol, polychlorinated anisoles, polychlorinated benzenes, polychlorinated biphenyls, and tetrachloroveratrole. Uptake rate constants in guppy are higher than those in rainbow trout gills, and show relatively high variation in both gills and guppy. When uptake rate constants in each study are normalized for that of pentachlorobenzene (pCBz), variation is significantly reduced both in perfused gills and in guppy. All allometric relationship is derived between weight and uptake rate constant. Uptake rate constants determined in one fish can thus be used for prediction of those in other fishes. When a reference chemical, such as pCBz, is included, the gill perfusion experiments can be highly suitable to determine uptake rate constants of organic chemicals, which can be extrapolated to fish of different sizes.

Animals↗
PubMed1995

Nitric oxide mediates microvascular permeability in the isolated perfused rat mesentery?

The permeability influencing effect of nitric oxide was studied by means of an isolated perfused mesentery preparation in combination with a intravital/fluorescence microscopic set up. As a fluorescent marker served FITC-dextran mol wt 70,000 daltons. Tissue treatment was performed by application of NO-donors like SNP or GTN. Additionally the effects of bradykinin, L-nitroarginine or the combination of both were tested in regard to a permeability modulation. The results point out that NO-donors did not enhance microvascular permeability, whereas the permeability increasing effect of bradykinin is mediated by an NO-independent pathway.

Animals↗
PubMed1984

Mitoxantrone metabolism in the isolated perfused rat liver.

The hepatobiliary pharmacokinetics of mitoxantrone, a new anthracenedione derivative, was studied in the isolated perfused rat liver. Mitoxantrone was administered in doses of 0.2 and 0.4 mg/kg body weight. Multiple bile samples were obtained for 4 hours. Mitoxantrone and three metabolites were separated by high-performance thin-layer chromatography (HPTLC) and measured at 610 nm. Following 0.2 mg mitoxantrone/kg body wt, 25.8% +/- 2.6% of the administered dose was excreted in the bile during 4 h, the major metabolite M1 accounting for 80% of this. After 0.4 mg mitoxantrone/kg body wt the amounts excreted were lower and light microscopic examination showed disseminated areas of cell necrosis.

Animals↗