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PubMed2002

Development and application of an isolated perfused rat liver model to study the stimulation and inhibition of tumor necrosis factor-alpha production ex vivo.

PURPOSE: To develop an isolated perfused rat liver (IPRL) model with low baseline levels of tumor necrosis factor (TNF)-alpha in the outlet perfusate to study the effects of immunostimulants and immunosuppressants on the release of TNF-alpha from this organ. METHODS: Isolated rat livers were perfused with a buffer containing no albumin or three different bovine serum albumin (BSA) preparations. Using the no-albumin perfusate, the inhibitory effects of methylprednisolone (MP) on lipopolysaccharide (LPS)-stimulated release of TNF-alpha were studied in livers isolated 1 or 5 h after the intravenous administration (5 mg/kg) of MP. The concentrations of TNF-a in the outlet perfusates were measured using enzyme-linked immunosorbent assay. RESULTS: In the absence of albumin, the perfusate levels of TNF-alpha were close to zero. However, when the perfusate contained BSA. the TNF-alpha levels in the perfusate reached as high as 1200 pg/ml at steady state. An injection of LPS into IPRLs perfused with a no-albumin perfusate resulted in mean (+/- SD) TNF-alpha steady-state concentrations of 825 +/- 125 pg/ml. The pretreatment of rats with MP before liver harvest attenuated the LPS-induced TNF-alpha release in the livers. However, the attenuation was substantial (>60%) and was statistically significant only 5 h after pretreatment with MP. CONCLUSIONS: Perfusates containing BSA may result in nonphysiologically high levels of TNF-alpha. An IPRL with a no-albumin perfusate is more suitable for studies of the stimulation and inhibition of TNF-alpha production by this organ.

Adjuvants, Immunologic↗
PubMed1994

Effect of potassium on the action of the KATP modulators cromakalim, pinacidil, or glibenclamide on arrhythmias in isolated perfused rat heart subjected to regional ischaemia.

OBJECTIVE: The ATP sensitive potassium channel openers cromakalim (n = 10) and pinacidil (n = 10), and a blocker of this channel, glibenclamide (n = 10), were studied in isolated perfused rat hearts subjected to regional ischaemia at varying concentrations (2 to 8 mM) of external potassium ([K+]o). METHODS: Hearts were isolated and perfused on a Langendorff apparatus. Vehicle (0.1% DMSO), cromakalim (10 microM), pinacidil (10 microM), or glibenclamide (10 microM) were given 10 min before ischaemia. The left coronary artery was then occluded for 15 min and reperfused for 5 min. RESULTS: No agent caused more than a 10% change in heart rate. Both cromakalim and pinacidil increased (30%), and glibenclamide decreased (30%) coronary flow at 4 and 6 mM [K+]o. In the vehicle group, increases in [K+]o produced concentration dependent reductions in arrhythmia scores by decreasing ventricular fibrillation. No concentration dependent effects of [K+]o on ischaemic ventricular tachycardia was observed. Under ischaemic conditions, potassium channel openers and glibenclamide more markedly reduced ischaemic ventricular tachycardia and fibrillation relative to the effects of increased [K+]o. CONCLUSIONS: Ischaemic ventricular fibrillation was inversely related to changes in [K+]o, whereas effects on ventricular tachycardia were all-or-none. Neither potassium channel openers nor glibenclamide elicited significant proarrhythmic activity despite variations in [K+]o. These data suggest that both potassium channel openers and glibenclamide display potential antiarrhythmic activity through their ability to abolish two distinct arrhythmogenic mechanisms during ischaemia. It is also suggested that the underlying mechanisms of ventricular tachycardia and fibrillation are coupled during ischaemia in the rat.

Animals↗
PubMed1993

Increase in portal flow induces c-myc expression in isolated perfused rat liver.

We examined expression of the c-myc oncogene in isolated perfused livers to elucidate the mechanisms involved in triggering the proliferation of hepatocytes after partial hepatectomy (PH). During perfusion with a 1:1 mixture of Dulbecco's modified Eagle's medium and the oxygen transport fluid FC-43, rat livers were two-thirds resected (PH), and further perfused for 1 1/2 hours at the physiological portal flow throughout the perfusion. Expression of c-myc in the perfused livers with PH(+) was ten times higher than in those with PH(-). Furthermore, expression of c-myc in the PH(-) livers perfused with a threefold volume of the physiological portal flow was 5-10 times higher than that in the livers perfused with the physiological portal flow. The perfusates that passed through the livers did not induce DNA synthesis of primary cultured hepatocytes. These results suggest that an increase in the portal flow volume may act as a trigger for hepatocyte proliferation after PH.

Animals↗
PubMed1989

Definition of a physiologic pharmacokinetic model of cutaneous drug distribution using the isolated perfused porcine skin flap.

A physiologic pharmacokinetic model describing drug disposition in the isolated perfused porcine skin flap (IPPSF) is derived. The IPPSF is well suited for experimental studies of dynamic drug distribution into skin because arterial and venous drug fluxes can be continuously monitored. The system parameters of the model are uniquely identifiable and describe the cutaneous efflux profile as a function of arterial input flux and tissue partitioning or extraction. This model allows experimental results obtained from an in vitro preparation to serve as a quantitative input to an in vivo, whole animal pharmacokinetic system. Experimental infusion applications of the cancer chemotherapeutic agents cisplatin and carboplatin and the antimicrobials tetracycline and doxycycline are reported herein.

Animals↗
PubMed2000

A new system for the metabolic investigation of the isolated, perfused rat heart.

NMR spectroscopy is an invaluable technique in metabolic investigations of isolated, perfused hearts. Most studies employ global perfusion methods together with an NMR coil that surrounds the heart and thus detects signals from its entirety. The present report describes the construction and testing of a novel, two surface-coil probe, in combination with a dual-perfused heart preparation, that enables spectra to be collected independently from the two coronary beds of the rat heart.(31)P NMR spectra of perfused rat hearts in which the septum and right ventricle have been made ischaemic, while the free left ventricular wall is fully perfused, demonstrate the powerful potential of this new system.

Animals↗
PubMed1982

Direct renal action of captopril (SQ 14225): dissociation of natriuretic and vascular actions in isolated perfused rat kidney.

Direct effects of captopril on renal function were examined in isolated perfused rat kidneys. Captopril induced a significant increase in urinary volume and urinary sodium excretion (1.8- and 1.7-fold, respectively; both p less than 0.005), whereas urinary potassium excretion and renovascular resistance were not significantly changed. Because the perfusion medium lacks angiotensinogen, kininogen and aldosterone, the natriuretic action in perfused kidneys may not be related to its inhibitory action on angiotensin I converting enzyme or kininase II. Because the natriuresis was not accompanied by changes in renovascular resistance, it is suggested that captopril possesses a direct natriuretic action and that this property may partly explain the mechanism of captopril-induced natriuresis clinically observed.

Animals↗
PubMed1982

Trichlorethylene and halothane inhibit uptake of 5-hydroxytryptamine in the isolated perfused rat lung.

Lung uptake of 5-hydroxytryptamine (5-HT) was determined in isolated perfused and ventilated rat lung, and was found to decrease with time according to a two-compartmental model. When the lungs were exposed to either trichlorethylene (TRI) or halothane, the uptake of 5-HT was drastically reduced. Both TRI and halothane gave log dose inhibition curves, which were superimposed, i.e. they were equally potent to inhibit lung uptake of 5-HT. At a concentration TRI of 18,000 ppm, the extraction of 5-HT was inhibited by 80 +/- 2 (X +/- S.E.M.) per cent, at 8500 ppm the inhibition was 65 +/- 6 per cent and 25 +/- 1 per cent at 3000 ppm. When the lungs were exposed to halothane, the inhibition was 85 +/- 6 per cent at 40,000 ppm, 48 +/- 1 per cent at 6000 ppm, and 15 +/- 0.3 per cent at 2000 ppm. When exposure to the solvent was discontinued, extraction of 5-HT was rapidly normalized. There was no detectable displacement of [3H]-5-HT from lungs saturated with the amine when they subsequently were exposed to solvent-containing atmosphere. This inhibition of lung uptake of 5-HT from the circulation is therefore postulated as to be an effect dependent on concentration solvent in the tissue, and is probably due to a reversible membrane stabilization of the endothelium.

Animals↗
PubMed1985

The disposition of pyrimethamine in the isolated perfused rat liver.

We have investigated the disposition of pyrimethamine base in the isolated perfused rat liver (IPRL) preparation after the administration of pyrimethamine (0.5 mg, 5 microCi). In the first half hour of the study, pyrimethamine underwent marked hepatic uptake, thereafter perfusate plasma drug levels declined monoexponentially with a half life (t 1/2) of 3.0 +/- 1.0 hr. Area under the perfusate plasma concentration/time curve (AUC)0----infinity was 6.9 +/- 1.9 microgram/hr/ml. Pyrimethamine was found to be a low clearance compound (78.4 +/- 25.3 ml/hr identical to 8.6% of liver perfusate flow) with a large volume of distribution (267.5 +/- 55.3 ml) in the IPRL. The combined AUCS(0----5hr) for pyrimethamine (AUC 4.8 +/- 0.5 microgram/hr/ml) and pyrimethamine 3-N-oxide (AUC0----5hr 0.9 +/- 0.6 microgram/hr/ml) accounted for 57% of the total AUC0----5hr of [14C] radioactivity (10.0 +/- 2.6 micrograms/hr/ml). This indicates the presence of metabolites of pyrimethamine as yet unidentified in the perfusate. Biliary excretion of [14C] during the course of the IPRL preparations was extensive (29.0 +/- 10.3%) though only a small proportion was due to pyrimethamine and the 3-N-oxide metabolite. The majority of radioactivity in the bile was attributable to highly polar, but unidentified metabolites of pyrimethamine. At the conclusion of each experiment (5 hr), a significant proportion of [14C] radioactivity was recovered from the livers (22.9 +/- 5.3%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged pyrimethamine, with trace levels of the 3-N-oxide metabolite. Sub-cellular fractionation of the homogenized livers revealed the most pronounced localisation of pyrimethamine to be in the lipid rich 10,000 g pellet (13.0 +/- 2.6%), the remainder being distributed equally between the 105,000 g pellet and supernatant. Neither pyrimethamine, [14C] radioactivity, nor pyrimethamine 3-N-oxide were extensively taken up by red cells throughout the study. Therefore, the large volume of distribution (267.5 +/- 55.3 ml) underlines the extent of pyrimethamine localisation in the liver.

Animals↗
PubMed1981

Characterization of the isolated perfused mouse brain as a system for neurochemical studies.

The preparation of the isolated perfused mouse brain (IPMB) is described along with its electrophysiological, morphological, biochemical and pharmacological properties. Using high performance liquid chromatography with electrochemical detection, the primary metabolite of mammalian central nervous system norepinephrine, 3-methoxy-4-hydroxyphenethyleneglycol (MHPG), was measured in the perfusate at 15-min intervals. The rate of MHPG production was similar to literature values of the rate of norepinephrine turnover in mouse brain. MHPG production rate in the IPMB was blocked by pretreatment with 6-hydroxydopamine and was increased by pretreatment with reserpine.

Animals↗
PubMed1982

Metabolism of 1,2-dimethylhydrazine in isolated perfused rat liver.

The metabolism of 1,2-dimethylhydrazine (DMH) was investigated in isolated perfused rat liver. The separation of [14C]DMH and its metabolites azomethane (AM), azoxymethane (AOM) and methylazoxymethanol (MAM) was performed by high pressure liquid chromatography (HPLC). The fractions were quantitatively detected by liquid scintillation counting of the radioactivity. It was demonstrated that DMH is highly metabolized by the liver. After 1 h of perfusion, the median amounts of DMH and its metabolites in the medium were: DMH, 16%; AM, 3%; AOM, 42%; MAM, 30% of the given dose. During this time 9.9% and 0.7% of the total radioactivity were eliminated as AM and CO2 in the oxygen stream of the perfusion apparatus; 0.6% were excreted by bile and 12.3% stored in hepatic tissue. The reaction rate (t1/2) of each individual metabolic step was estimated by means of a mathematical kinetic model as follows: DMH leads to AM, 21.8 min; AM leads to AOM, 1.5 min; AOM leads to MAM, 41.5 min; MAM leads to, 611 min. The results are discussed in comparison to in vivo experiments.

1,2-Dimethylhydrazine↗
PubMed1990

Amino acid metabolism in isolated perfused rat liver.

Conflicting evidence concerning hepatic amino acid (AA) metabolism in the isolated perfused rat liver (IPRL) led us to investigate the response of IPRL using perfusates with various AA contents. Perfusion (n = 4) with whole rat blood diluted in Krebs buffer (1:3, v/v) led to acute proteolysis on account of AA deprivation, as shown by the large release of AA (approximately 1400 mumoles in 120 min), especially branched-chain AA (BCAA) (e.g., Leu, 35.4 +/- 10.4 nmole.min-1.g-1 the first hour, 34.3 +/- 5.5 nmole.min-1.g-1 the second hour). In a first attempt to prevent proteolysis, livers (n = 4) were perfused with the previous medium supplemented with AA known for their antiproteolytic activity, at twice their physiological concentrations. Results during the first hour showed uptake of several AA (mainly alanine, glutamine, and proline), reduced release of BCAA (leucine, 12.5 +/- 6.3 nmole.min-1.g-1), and an increase in glucose and urea production. However, during the second hour, because of the use of a recirculating system, progressive AA depletion induced a reappearance of proteolysis. A two-step AA loading technique, i.e., the addition of antiproteolytic AA at the beginning of the perfusion and the addition of a balanced AA mixture at 60 min caused a further decrease in proteolysis during the 2 hr of perfusion (n = 6). Under these conditions, most AA were taken up by the liver with uptake values comparable to those observed in vivo.

Amino Acids↗
PubMed1982

Effect of prostacyclin infusion on active and inactive renin release in the isolated perfused kidney.

The effect of prostacyclin infusion into the renal artery of the isolated perfused hog kidney on the release of active and inactive renin was investigated. Infusion of prostacyclin at a rate of 0.1 microgram/min resulted in a significant increase (p less than 0.01) in active renin and a significant fall (p less than 0.01) in inactive renin. Prostacyclin also increased urinary kallikrein excretion (p less than 0.05). The results indicate that the kidney secretes not only active renin but also inactive renin, and suggest that prostacyclin stimulates the conversion of inactive renin to the active form through the activation of the renal kallikrein system.

Animals↗
PubMed1983

Dipeptide metabolism in the isolated perfused rat kidney.

Renal handling of glycyl-proline was studied in the isolated perfused rat kidney. Glycyl-proline disappeared from the perfusate as a function of time. The dipeptide was freely filtered at the glomerulus but only 6% of the filtered load was excreted in the urine as the intact peptide. More than 90% of the filtered dipeptide was reabsorbed as the intact peptide and/or its hydrolytic products. Non-filtration mechanisms were also involved to a significant extent in the clearance of the peptide. Hydrolysis at intratubular, intracellular and peritubular sites all contribute to the disappearance of the dipeptide from the perfusate, though the relative contributions of each mechanism are not known. Significant metabolic conversions, especially the conversion of glycine to serine, were also observed during perfusion.

Animals↗
PubMed1987

The effects of substance P, histamine and histamine antagonists on somatostatin and gastrin release from the isolated perfused rat stomach.

UNLABELLED: Secretion of somatostatin-like immunoreactivity (SLI) from the isolated perfused rat stomach has been shown to be inhibited by substance P. The present study was initiated to examine the possibility that this action of substance P was mediated via release of histamine. Substance P (1 microM) reduced basal secretion of SLI in agreement with earlier studies. Neither pyrilamine nor cimetidine influenced this action. Basal immunoreactive gastrin (IRG) secretion was unaffected by substance P. Addition of pyrilamine during substance P perfusion increased IRG secretion whereas addition of cimetidine resulted in a delayed decrease on removal of both compounds. Histamine (1 and 10 microM) increased SLI secretion and reduced IRG secretion. Pyrilamine increased and cimetidine decreased IRG secretion but neither drug influenced SLI secretion. Pyrilamine had no effect on histamine-stimulated SLI secretion but inhibition of IRG secretion by histamine was converted to stimulation. Cimetidine potentiated histamine stimulation of SLI secretion and inhibition of IRG secretion. IN CONCLUSION: (1) substance P inhibition of SLI secretion is unlikely to be mediated via release of histamine. (2) The gastrin cell appears to have both H1- and H2-receptors which mediate opposite actions but H1-receptor-mediated inhibition is predominant. (3) Histamine weakly stimulates SLI secretion but there may be both inhibitory and stimulatory pathways acting via H2- and H1-receptors, respectively.

Animals↗
PubMed1982

Dynamics of insulin and glucagon secretion by the isolated perfused cat pancreas.

1. The dynamics of insulin and glucagon secretion by the isolated perfused cat pancreas was studied in response to glucose and amino acid stimuli. 2. Insulin secretion is biphasic in response to either glucose or amino acid stimuli. 3. Glucose caused a much more pronounced first phase release than did a complete amino acid mixture; whereas glucose and the amino acid mixture stimulated late second phase insulin secretion equipotently. 4. The same amino acid mixture produced a biphasic secretion of glucagon characterized by a rapid and transient spike-release which was followed by a substantial, relatively steady-state, second secretory phase. 5. Various combinations of glucose and amino acids resulted in insulin secretion which suggests a simple additive stimuli phenomenon; whereas no such additive effect was observed with respect to amino acid stimulation of glucagon secretion. 6. Instead, various of the amino acids appear to stimulate maximal glucagon secretion independently. 7. Glucose failed to cause a suppression of amino acid induced glucagon release. 8. The quantitative aspects of this study suggest that the cat pancreas is very responsive to amino acid modulation of hormone release, a fact which may well reflect the high amino acid content of the cat's diet.

Amino Acids↗
PubMed1983

Effects of hyperthermia on plasma glycoprotein catabolism by the isolated perfused rat liver.

Clearance and degradation of the glycoprotein, asialofetuin (AF), by the isolated perfused rat liver at supranormal temperatures were investigated. The half-life for disappearance of AF was similar at 37, 41, and 42 degrees C, P greater than 0.05. There was a significant difference between the amount of hydrolysis of AF at 37, 41, and 42 degrees C, P less than 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor endocytosis by the hepatocyte at elevated temperatures.

Animals↗
PubMed2004

The action of extracellular NAD+ on Ca2+ efflux, hemodynamics and some metabolic parameters in the isolated perfused rat liver.

The action of NAD+ on hemodynamics and metabolism of the isolated perfused rat liver was investigated. Extracellular NAD+ (20-100 microM) stimulated glycogen breakdown (glucose release) and inhibited oxygen uptake. Lactate production was predominantly increased, and pyruvate production was predominantly inhibited. NAD+ also increased the portal perfusion pressure. All metabolic effects were strictly Ca2+-dependent. The effects were absent when Ca2+ was excluded, and reintroduction of the cation restored the effects. In preloaded livers, NAD+ accelerated 45Ca2+ efflux. The action of NAD+ was sensitive to three inhibitors of eicosanoid synthesis, suggesting that this action is mediated by these compounds, which are known to be produced and released by Kupffer and endothelial cells. It is impossible to infer from the available data if NAD+ exerts all these effects by itself or if they are caused by one or more of its extracellular hydrolysis products. Nicotinamide was ineffective and can be excluded, but especially cyclic ADP-ribose and ADP-ribose are possibilities that should be considered in future work.

Animals↗