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PubMed1981

Effects of isoproterenol treatment of isolated perfused rat hearts on myofibrillar phosphorylation and ATPase activity.

Perfusion of isolated rat hearts with isoproterenol resulted in increases in the level of protein-bound phosphate of the myofibrils. After perfusion of the hearts with 32P, followed by SDS-polyacrylamide gel electrophoresis of the purified myofibrils, four major 32P-containing protein bands were identified. Most of the increased 32P incorporation produced by isoproterenol was localized on the troponin I and myosin light chain bands, and, to lesser extent, on the M-protein band. ATPase activity was tested in the purified myofibrils. No changes in Ca2+ requirement for activation were found after isoproterenol perfusion. However, maximal ATPase activity was markedly reduced in the myofibrils obtained from isoproterenol-treated hearts. It would appear that the myofibrillar protein phosphorylation induced by isoproterenol perfusion results in a decrease in actomyosin ATPase activity.

Animals↗
PubMed1985

Effects of hypothermic kidney preservation on the isolated perfused kidney: a comparison of reperfusion methods.

Two isolated-perfused kidney methods were used to study the effects of hypothermic preservation on renal function in dog kidneys. The isolated-machine-perfused kidney (IMPK) used an in vitro perfusion technique--the perfusate was a Krebs-bicarbonate type delivered to the kidney at 37 degrees C by a mechanical pump at a constant pressure (100 mm Hg). The isolated-blood-perfused kidney (IBPK) utilized transplantation of the preserved kidney to the femoral vasculature. Renal function (urine analysis) was determined over a 1-hr reperfusion interval and included GFR (creatinine clearance), urine formation, and Na+ reabsorption. Kidneys preserved for only 24 hr by cold storage in either Collins'--C3 solution or in hypotonic citrate and kidneys hypothermically perfused for 24 hr demonstrated greater retention of renal function when reperfused by blood (IBPK) than with the in vitro perfusate (IMPK). The GFR was reduced by 38-58% when tested with the IBPK, but by 80-90% when tested with the IMPK. Na+ reabsorption was normal (97%) with blood reperfusion but was reduced to 36-50% in cold-stored kidneys and 82% in hypothermically perfused kidneys determined by machine reperfusion (IMPK). However, kidneys perfused for 72 hr demonstrated more similar renal functions when tested by either IMPK or IBPK. GFR was reduced to 20% (IBPK) and 11% (IMPK) and Na+ reabsorption averaged 76-85% (IBPK or IMPK). These results suggest that either reperfusion method is suitable for determining the effects of renal preservation on kidney function in kidneys preserved for 72 hr but, for short-term preserved kidneys (24 hr), the IBPK model may be preferred.

Animals↗
PubMed1983

Tricarboxylic acid cycle metabolites during ischemia in isolated perfused rat heart.

Isolated rat hearts were, after a retrograde perfusion by the Langendorff procedure, rendered ischemic by lowering the aortic pressure to zero. The rate of proteolysis and temporal patterns of the changes in the concentrations of the metabolites of the tricarboxylic acid cycle, related amino acids, ammonia, and breakdown products of the adenine nucleotides were determined. The most significant change in the amino acid metabolism was a decrease of the proteolysis to one-tenth and a large accumulation of alanine, which was almost stoichiometric to the degradation of aspartate plus asparagine. The accumulation of malate and succinate was small compared with the metabolic net fluxes of aspartate and alanine. The metabolic balance sheet suggests that aspartate was converted to alanine. A prerequisite for this would be a feed in of carbon of aspartate to the tricarboxylic acid cycle as oxalacetate, reversal of the malate dehydrogenase, and production of pyruvate by the malic enzyme reaction. Alanine accumulating during ischemia is not glycolytic in origin but occurs through a concerted operation of anaplerotic reactions and tricarboxylic acid cycle metabolite disposal. The data also suggest that the potentially energy-yielding reduction of fumarate to succinate is not significant in the ischemic myocardium.

Amino Acids↗
PubMed1987

PAF-acether induced cardiac dysfunction in the isolated perfused guinea pig heart.

PAF-acether (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) has been implicated in a variety of inflammatory and ischaemic disorders (e.g., myocardial ischemia, anaphylactic shock). Recently, the peptide leukotrienes (i.e., LTC4, LTD4) have been shown to mediate the increase in coronary vascular resistance induced by PAF-acether in the isolated perfused rat heart. In isolated perfused guinea pig hearts, PAF-acether produced a dose-dependent increase in coronary perfusion pressure (CPP) and a decrease in contractile force (CF). At 50 pmol/l, PAF increased CPP by 13 +/- 3 mm Hg and decreased CF by 47 +/- 12% in 8 hearts. Radioimmunoassay of the coronary effluent did not detect peptide leukotrienes or thromboxane B2 (TxB2) in response to PAF. Addition of a specific PAF-acether receptor antagonist, CV-6209 (25 nmol/l), blocked the increase in coronary perfusion pressure and decrease in contractile force. OKY-1581 (400 nmol/l), a thromboxane synthetase inhibitor or LY-171,883 (7.3 mumol/l) a leukotriene D4 receptor antagonist, failed to prevent the increase in CPP or the decrease in CF. These data indicate that the PAF-acether induced increase in CPP is not mediated by the peptide leukotrienes or thromboxane A2 (TxA2). Possible mechanisms for the increase in CPP induced by PAF-acether in the isolated perfused guinea pig heart include a direct receptor mediated constriction of coronary resistance vessels, release of a non-eicosanoid coronary constrictor as a mediator of the response, or via enhancement of coronary microvascular permeability.

Animals↗
PubMed1989

Estimation of physiological volumes in the isolated perfused porcine skin flap.

A requirement for pharmacokinetic modeling of isolated perfused organ systems used for in vitro to in vivo extrapolations is a measure of the organ's physiological spaces. Two methods were employed to estimate intravascular and extracellular spaces in four isolated perfused porcine skin flaps (IPPSF) from 2 pigs. One of these methods used regression techniques based on a one-compartment model, and was found to be a good estimator of intravascular volume only. The other method was a non-parametric numerical integration technique, and was found to give reasonable estimates for both intravascular and extracellular volumes. The latter method's estimate for extracellular space was 0.20 +/- 0.03 mL/g, and the estimated vascular space by both methods was 0.08 +/- 0.01 mL/g.

Animals↗
PubMed1981

Increased biliary GSSG-secretion and loss of hepatic glutathione in isolated perfused rat liver after paraquat treatment.

Perfusion of isolated rat livers with 1 mM paraquat for 3 hours led to a stimulated release of oxidized glutathione into the effluent caval perfusate and into the bile. Whereas the biliary stimulation was 245%, stimulation in to the perfusate was only 19.2%. In addition, the glutathione content in the paraquat-treated livers decreased from 2.74 +/- 0.23 to 0.80 +/- 0.07 mumol/g liver. The hepatic content of GSSG, however, was not changed by paraquat. This resulted in an elevated ratio of GSSG/GSH+2GSSG from 0.036 to 0.113. The total amount of GSSG released via bile and perfusate is less than the total loss of hepatic GSH. These findings are discussed in view of mechanisms by which paraquat decreases in GSH in the liver.

Animals↗
PubMed1990

Depressant effects of L-tyrosine on isolated perfused rat and rabbit hearts.

Tyrosine exerts potent cardiovascular effects: smaller doses induce tachycardia and hypertension while higher doses induce bradycardia and hypotension. However, the direct cardiac effects of this amino acid have not been characterised. In the present study increasing doses of L-tyrosine were administered to the perfusate of isolated rat (0.01-10.0 mg) and rabbit (0.5-40.0 mg) hearts. Heart rate and isometric force of contraction or amplitude of contractions, and either perfusion pressure or flow of perfusate were recorded. In rat hearts L-tyrosine decreased heart rate and isometric force of contraction. In rabbit hearts L-tyrosine also decreased heart rate and amplitude of contractions. The effects on coronary vasculature were variable. In rat hearts, high doses of L-tyrosine induced bi-phasic changes with initial coronary dilatation, followed by vasoconstriction. In rabbit hearts the predominant effect of L-tyrosine was coronary artery constriction. These results show that the inhibitory cardiovascular effects of L-tyrosine in vivo may be at least in part, explained by direct cardiac effects of this amino acid.

Animals↗
PubMed1990

Effect of natriuretic agents, vasoactive agents and of the inhibition of metabolism on sodium handling in the isolated perfused kidney of the nephrotic rat.

1. The kidney taken from a rat rendered nephrotic by exposure to puromycin aminonucleoside retains sodium abnormally when perfused in isolation and has an abnormally low vascular resistance (J. D. Firth et al., Clin. Sci. 1989; 76, 387-95). In this study the relation of oxygen consumption to sodium reabsorption has been examined in the isolated nephrotic organ, which has also been exposed to a variety of natriuretic agents and to the effect of inhibition of metabolism by cooling, in an attempt to discern the transport process, or processes, responsible for abnormal tubular handling of sodium. In addition, the effects of three endogenous vasoconstrictors, noradrenaline, angiotensin II and endothelin, on the function of the isolated nephrotic kidney have been examined. 2. The ratio of mol of sodium reabsorbed by the tubules of the isolated nephrotic kidney to mol of oxygen consumed was reduced in comparison with the control kidney (means +/- SEM): 9.22 +/- 0.97 versus 15.43 +/- 1.55 (P less than 0.002). 3. In the presence of ouabain (1 mmol/l), acetazolamide (1 mmol/l), frusemide (200 mumol/l), the combination of these three agents together, hydroflumethiazide (100 mumol/l), benzamil (100 nmol/l) or atrial natriuretic peptide (1000 pmol/l), a lesser increment in sodium excretion was induced in the isolated nephrotic kidney than in the control kidney and the nephrotic organ continued to excrete less sodium in both absolute and fractional terms. 4. This suggests that enhanced tubular sodium reabsorption in the isolated nephrotic kidney does not depend upon abnormally increased activity of the Na+/K(+)-adenosine triphosphatase, bicarbonate-dependent sodium transport, Na+/K+/2Cl- co-transport, electrically neutral proportionate reabsorption of sodium and chloride (distal tubule), epithelial sodium channel (distal tubule) or atrial natriuretic peptide-sensitive sodium transport processes. 5. When isolated nephrotic kidneys and normal kidneys were cooled to 8-10 degrees C the handling of sodium became virtually identical in the two groups. On re-warming to 37 degrees C, the original differences in sodium handling between nephrotic and control kidneys were restored. This implies that the mechanism responsible for the abnormal tendency to retain sodium is temperature-sensitive; as yet it remains otherwise undefined. 6. The sensitivity of the renal vessels to noradrenaline, angiotension II and endothelin, as judged by the percentage reduction in perfusate flow rate produced by a given concentration of any of these agents, was not substantially altered in the nephrotic kidney compared with the control kidney. Increase in vascular tone was not associated with amelioration of the tendency of the isolated nephrotic organ to retain sodium. Increasing concentrations of angiotensin II caused the filtration rate to increase in the nephrotic kidney. This effect was unexpected: in the control preparation, as anticipated, angiotensin II caused the filtration rate to decrease.

Acetazolamide↗
PubMed1995

Influence of physicochemical parameters and perfusate flow rate on the distribution of solutes in the isolated perfused rat hindlimb determined by the impulse-response technique.

The relationship between solute distribution, physicochemical properties, and tissue physiology was determined by the impulse-response (IR) technique and statistical moment analysis in the isolated perfused rat hindlimb. The concentration of bovine serum albumin (BSA; 2, 4.7, and 7%, w/v), perfusate flow rate (4 and 8 mL/min), and solute physicochemical properties (lipophilicity, P, fraction unbound to protein, fu; fraction ionized, fi; and molecular weight MW) were varied to better understand the underlying determinants of solute distribution. An apparent low availability was found for a number of the solutes as a consequence of tissue sequestration. This low availability precludes the estimation of an apparent volume of distribution (V) for these solutes. The V of solute and tissue (skin, fat, and muscle) blood flow increased with perfusion flow rate (p < 0.01). The unbound distribution volume (Vu) of basic solutes was significantly linear with respect to P. Multiple linear regression analysis showed that the distribution volume of solute in tissue was significantly related to fu (p < 0.01), but not improved by including relationships to P, MW, and fi. Data obtained with this IR technique yield results consistent with in vivo studies in terms of the importance of fu as a determinant of V. This work has shown that the estimations of solute V by the IR technique in a single-pass preparation are unreliable for solutes with a low availability due to apparent solute sequestration into tissue. The parameter V may also be affected by changes in the perfused limb physiology associated with the perfusion conditions used. The Vs for lidocaine and diazepam vary with fu in accordance with deductions based on the results of steady-state studies.

Adrenergic beta-Antagonists↗
PubMed2006

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver.

Copper is one of the major metals causing environmental contamination. Previous studies showed that copper induced toxic effects in isolated perfused rat liver models and these effects were associated with lipid peroxidation. Here we investigated whether effects of copper (at concentrations of 0.01, 0.03, and 0.1 mM of Cu(2+) in Krebs-Henseleit buffer perfusing the isolated rat liver for 60 min), were associated with biliary epithelial cell injury, as well as protein oxidation and oxidative DNA damage. The highest concentration of copper in perfusate (0.1 mM) did not allow complete evaluation of all parameters because it blocked portal flow within 30 min of perfusion, indicating severe microcirculatory disturbances. Further, copper decreased secretion of bile and it increased lactate dehydrogenase, aspartate transaminase, and alanine transaminase leakage into perfusate as well as liver weight in a dose-dependent manner. Biliary gamma-glutamyltransferase, an index of biliary epithelial cell integrity increased similarly at 0.01 and 0.03 mM copper concentrations in perfusate. Compared to controls, 0.01 and 0.03 mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative DNA damage. The results suggest that toxic effects of copper in the isolated perfused rat liver may involve biliary epithelial cells and they are associated with lipid peroxidation, protein oxidation, and oxidative DNA damage.

Alanine Transaminase↗
PubMed1976

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.

Amino Acids, Essential↗
PubMed1993

An automatized computer-method utilizing Procomm Plus and DataEase (4.2) PC and SAS (6.06) mainframe software for isolated, perfused guinea-pig heart studies.

A powerful, time sharing and automatized method of a comprehensive data analysis for isolated, perfused guinea-pig heart studies is described. Data are collected using DataEase PC software (version 4.2) into forms with data fields specified for vital parameters consistently recorded in isolated, perfused heart studies (HR, CBF, PEAKPRESSURE, DPDT, MVO2). After running, DataEase reports the data and information is uploaded to an IBM 3081D mainframe computer on each day of heart experiment and data collection. The uploading process, the data archival and the statistical analyses are automatized by Procomm Plus commands written in Aspect Source Program (.ASP) Files for logging, data transforming and file management procedures. The ASPCOMP.EXE compiler compiles these .ASP files into Aspect Script eXecutable (.ASX) programs, which run on the PC in our laboratory and activate WYLBUR (IBM 3081D Batch-job service and Command file processor) edited files in the mainframe's electronic devices then upload, backup and save data into these files. SAS EXE files containing program instructions for the data analyzing system are then forced by Procomm Plus to operate over the data just uploaded. SAS reads the DATA files by its INFILE facility and performs comprehensive statistical analyses and produces hard output including graphics and JOB reports of dose-response- and logaritmic scale curves for delivery to team members. This computerized and automatized method developed for isolated, perfused guinea-pig heart studies is capable of performing multiple file transfer, sophisticated statistical analyses and graphic procedures after one keystroke on the PC (Alt-F5 in Procomm Plus section) and also facilitates a consistent and convenient method for planning, controlling and standardizing experiments. The method is based on an interactive computer conversation between the PC in the laboratory and the remote's WYLBUR editor. No human presence is needed; however, in case of failure, Procomm Plus gives one of the team members supervising the system a phone call in order to get human help.

Animals↗
PubMed1991

Uptake and reutilization of surfactant phospholipids by type II cells of isolated perfused lung.

To investigate the role of type II alveolar cells in surfactant uptake and reutilization while cells are in situ, the combination of isolated perfused rat lung and type II cell isolation has been used. After an equilibration period, isolated perfused rat lungs received an intratracheal injection of labeled rat surfactant or a labeled bovine-derived preparation, and the perfusion continued. At time intervals less than or equal to 3 h, lungs were lavaged and type II cells isolated. Freshly isolated cells from perfused lungs were highly viable as judged by trypan blue exclusion and by linear incorporation of labeled leucine into trichloroacetic acid precipitable protein during perfusion. After cell isolation, total phospholipid or phosphatidylcholine was extracted from cells. Incorporation of surfactant phosphatidylcholine label into cellular phosphatidylcholine was shown to be linear with time whether lungs received natural rat surfactant or bovine-derived surfactant preparation. Uptake of natural surfactant from alveoli into type II cells was approximately three times greater than uptake of bovine-derived material. Results of administration of 0.5 mumol surfactant phospholipid or 2.0 mumol were similar except that administration of 2.0 mumol of natural rat surfactant caused uptake to become saturated at 1 h of perfusion. An increase in cell-associated phosphatidylcholine occurred whether results were expressed on basis of cellular DNA or specific activity of cellular phosphatidylcholine. Majority of administered surfactant label remained lavage fluid associated at 3 h, whereas remainder was associated with isolated cells or debris from cell preparation. Little label was detected in perfusion medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Abnormal sodium handling occurs in the isolated perfused kidney of the nephrotic rat.

1. In order to examine the handling of sodium by the nephrotic kidney when separated from the immediate influences of renal nerves and humoral factors, kidneys were taken from nephrotic rats (puromycin aminonucleoside) and studied over a range of perfusion pressures using the isolated perfused kidney technique. 2. When perfused with medium containing 6.7 g/dl albumin, the nephrotic kidneys performed differently from controls with a reduction in sodium excretion at all pressures [(mean +/- SEM) 1.14 +/- 0.43 vs 4.20 +/- 0.69 mumol/min at 105 mmHg (14 kPa); 6.32 +/- 1.56 vs 44.60 +/- 5.30 mumol/min at 150 mmHg (20 kPa)]. Renal vascular resistance, inulin clearance, fractional sodium excretion and fractional lithium excretion were also reduced. 3. When kidneys were perfused without oncotic agent these differences between nephrotic and control kidneys remained. Perfusion with medium containing 10 g/dl albumin, designed to prevent glomerular filtration, abolished the difference in vascular resistance between the two groups. Captopril had no effect on the sodium retention or vascular resistance of nephrotic kidneys. 4. It was concluded that (a) the isolated nephrotic kidney demonstrates increased avidity for sodium, (b) the abnormality of sodium handling is not dependent on the presence of altered oncotic forces, and (c) the alteration in vascular resistance is conditional upon glomerular filtration.

Animals↗
PubMed1996

Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart.

Myocardial uptake of thiopental enantiomers by an isolated perfused rat heart preparation was examined after perfusion with protein-free perfusate. Outflow perfusate samples were collected at frequent intervals for 20 min during single-pass perfusion with 10 micrograms/ml racemic thiopental (washin phase) and for another 45 min during perfusion with drug-free perfusate (washout phase). (+)- and (-)-thiopental concentrations were assayed by chiral high-performance liquid chromatography. Heart rate, perfusion pressure, and electro-cardiogram were also monitored. During the washin phase, there was no significant difference between the mean values of the equilibration rate constants of (+)- and (-)-thiopental enantiomers (0.44 +/- 0.07 min-1 and 0.43 +/- 0.09 min-1, respectively, P > 0.05). Mean volumes of distribution of (+)- and (-)-thiopental enantiomers were similar (6.34 +/- 1.20 and 6.45 +/- 1.29 ml/g for the washin phase and 7.22 +/- 0.71 and 7.47 +/- 0.81 ml/g for the washout phase, respectively, P > 0.05). This indicates that tissue accumulation of thiopental enantiomers in the isolated perfused rat heart was not stereoselective. Uptake of thiopental by the heart was perfusion flow rate-limited and independent of capillary permeability. These findings suggest that myocardial tissue concentration of racemic thiopental should be an accurate predictor of myocardial drug effect.

Anesthetics, Intravenous↗
PubMed1994

Effect of ethanol on hepatobiliary transport of cationic drugs. A study in the isolated perfused rat liver, rat hepatocytes and rat mitochondria.

The effect of ethanol on the hepatic uptake of various cationic drugs was studied in isolated perfused rat livers, isolated rat hepatocytes and isolated rat liver mitochondria. In isolated rat hepatocytes and in isolated perfused rat livers, the uptake of the model organic cation tri-n-butylmethylammonium was found to be markedly stimulated by ethanol in a concentration-dependent fashion. The uptake of tri-n-butylmethylammonium at 1 microM was increased to 120% and 137% at 0.5% (v/v, (= 87 mM)) and 1% (v/v. (= 174 mM)) ethanol, respectively. At 25 microM, tri-n-butylmethylammonium uptake was increased to 124% and 152% at 0.5% (v/v) and 1% (v/v) of ethanol, respectively. The uptake of the organic cations azidoprocainamide methoiodide, vecuronium, ORG 9426 and ORG 6368, the anionic compound taurocholate and the uncharged compound ouabain was not markedly increased at these ethanol concentrations. The mechanism of action of ethanol on the uptake of tri-n-butylmethylammonium was further studied. Competitive inhibitors for the type I organic cation uptake system, procainamide ethobromide and verapamil, almost completely blocked uptake of tri-n-butylmethylammonium (1 microM) in the presence of 1% (v/v) ethanol, indicating that carrier-mediated uptake is still involved and that additional passive diffusion is unlikely. Neither the plasma membrane potential nor the accumulation of the cation in mitochondria was altered after ethanol treatment, suggesting that potential driving forces for uptake and sequestration were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1987

Adaptive changes of H+ and/or bicarbonate transport in chronic metabolic acidosis: its quantitative evaluation by using isolated perfused kidney.

We have recently improved the technique of the isolated perfused rat kidney by addition of amino acids to the perfusion solution. Utilizing this improved preparation, we examined to what extent chronic acid-base status can alter H+ and/or bicarbonate transport in the whole kidney and the effect of acetazolamide (10(-4) M) on this process. Kidneys from rats that had been drinking tap water (control), 1.5% NH4Cl (acidotic), or 1.5% NaHCO3 (alkalotic) were perfused with a solution of identical pH and PCO2. During the control period, the fractional bicarbonate reabsorption was higher in acidotic rats than in control rats by 1.0% and urine pH was lower than those in control. On the other hand, the two parameters were not different between control and alkalotic rats. Acetazolamide increased urine pH and decreased the fractional bicarbonate reabsorption in all, but these parameters in acidotic and alkalotic rats were not different compared with those in control. We conclude that adaptive changes of H+ and/or bicarbonate transport in chronic metabolic acidosis corresponds to about 1.0% as fractional bicarbonate reabsorption by the isolated perfused kidney and is largely carbonic anhydrase dependent.

Absorption↗