Effect of vasodilators on hepatic microcirculation in cirrhosis: a study in the isolated perfused rat liver

We studied the effects of a series of vasodilators on intrahepatic vascular resistance of isolated perfused cirrhotic rat livers in basal conditions and during norepinephrine-induced vasoconstriction. Cirrhosis was induced by repeated intraperitoneal injections of carbon tetrachloride. The vasodilators were a nonselective beta-adrenergic antagonist (propranolol), an alpha 1-adrenergic antagonist (prazosin), a nonselective beta-adrenergic agonist (isoproterenol), an alpha 2-agonist (clonidine), nitrovasodilators (nitroglycerin and nitroprusside), calcium channel blockers (verapamil, diltiazem, nifedipine), papaverine, diazoxide and pentoxifylline. In basal conditions, isoproterenol, nitroglycerin, papaverine, pentoxifylline and nitroprusside demonstrated significant vasodilatory activity. However, the response was weak and isoproterenol was the only drug active in the therapeutic range of concentrations. Propranolol, prazosin, verapamil, diltiazem, nifedipine and diazoxide were ineffective. Prazosin, papaverine and pentoxifylline reduced norepinephrine-induced vasoconstriction, whereas isoproterenol, clonidine and propranolol were ineffective. We conclude that several vasodilators can reduce resistance in the cirrhotic rat liver, but their potency is low and few are effective at therapeutic concentrations. Furthermore, their activity may be blunted when resistance is increased by norepinephrine.     

Uridine catabolism by the isolated perfused rat liver

A new approach in the treatment of gastrointestinal tumors with 5-fluorouracil involves the infusion of high doses of uridine to improve the chemotherapeutic efficiency of the former. High amounts of uracil formed from uridine can interfere with the hepatic catabolism of 5-fluorouracil and thus increase its bioavailability and toxicity. In our study, we analysed the metabolite pattern of uridine in the effluent of isolated perfused rat livers in relation to portal uridine levels. The livers were perfused hemoglobin-free without recirculation at a constant flow. In the perfusate, uridine was changed from 0.5 to 100 mumol/l. The complete degradation of [2-14C]uridine and [2-14C]uracil was monitored via the release of labeled CO2. Radioactive catabolites of uridine including uracil and the sum of dihydrouracil and beta-ureidopropionate were separated by high-performance liquid chromatography and counted using a radioactivity flow monitor. Portal uridine concentrations were increased from 0.5 to 100 mumol/l and were accompanied by a rise in the relative amount of non-metabolized uridine in the effluent from 13 to 78%. At uridine concentrations above 50 mumol/l, there was a constant release of uracil into the effluent, indicating saturation of uridine phosphorolysis or transport. The amount of 14CO2 formed by the liver reflecting complete uridine breakdown was higher than any other uridine metabolite when uridine concentration varied from 0.5 to 15 mumol/l. Saturation of 14CO2 formation was achieved at a uridine concentration of 25 mumol/l. Higher peak values of 14CO2 release were observed after direct infusion of equivalent amounts of uracil into the portal vein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of fibronectin by the isolated perfused rat liver

The major site(s) of synthesis of plasma fibronectin is unknown. Using the isolated perfused rat liver and anti-rat fibronectin antiserum, we have measured net hepatic synthesis of fibronectin during 10-hr perfusion periods. We calculated the circulating plasma fibronectin pool of a 195-g rat with body surface area of 300 sq cm to be 3.3 mg: net hepatic synthesis of 3.75 +/- 1.0 mg/300 sq cm body surface area of the rat liver donor was seen in liver perfusions of 10 hr. When the hormones cortisol and insulin were added to the liver perfusate, net synthesis of 5.05 +/- 0.82 mg/300 sq cm was seen. Synthesis of two known acute phase reactant proteins, fibrinogen and alpha-2 acute phase globulin, was also substantially greater in the presence of cortisol and insulin. Plasma fibronectin levels measured in intact rats that had abscess produced by subcutaneous turpentine injection were 0.57 +/- 0;05 mg/ml compared to 0.38 +/- 0.02 mg/ml in normal control animals. Our studies indicate that hepatic synthesis contributes substantially to the plasma fibronectin pool and show that such synthesis is enhanced by the hormones cortisol and insulin. In the intact rat, inflammation was associated with elevated levels of plasma fibronectin.     

Effect of nafenopin on the uptake of bilirubin and sulfobromophthalein by isolated perfused rat liver

Hepatic uptake of bilirubin and sulfobromophthalein has kinetic characteristics suggesting facilitated diffusion. Because these compounds demonstrate mutual competition for uptake, a shared uptake mechanism has been presumed. Previous studies in isolated perfused regenerating liver revealed depressed uptake of bilirubin, sulfobromophthalein, and asialoorosomucoid, a desialylated glycoprotein which enters hepatocytes by receptor-mediated endocytosis. This study was designed to determine whether or not depressed transport seen in liver regeneration occurs in other states of hepatocellular proliferation. Rats were pretreated with nafenopin (200 mg/kg . day x 2), a drug that causes rapid hepatocellular proliferation similar to that seen in regeneration. Twenty-four hours after nafenopin treatment, liver weight increased by 40%. Influx, efflux, and sequestration rate constants in isolated perfused liver were quantitated by a computer fit to the model of Goresky. Results 1 day after nafenopin treatment revealed no change in transport parameters for bilirubin and asialoorosomucoid, but 55% and 49% reductions in influx of sulfobromophthalein and conjugated bilirubin, respectively. These studies suggest that hepatocellular proliferation alone is not responsible for the transport alterations seen during liver regeneration. Nafenopin effectively unmasks differences in uptake of bilirubin and other more water soluble organic anions such as sulfobromophthalein and conjugated bilirubin, suggesting that their uptake mechanisms are partially independent.     

Effect of acute uremia on insulin removal by the isolated perfused rat liver and muscle

The effect of acute uremia on insulin removal by liver and muscle was investigated by measuring the extraction of porcine insulin during recycling perfusion of isolated liver and hindlimb from nephrectomized rats. Insulin removal by the liver was not affected by uremia, and hepatic extraction of insulin by normal and uremic livers averaged 38% at perfusate insulin concentrations ranging from 50 to 600 μU/ml. In contrast, insulin extraction by normal hindlimb averaged 13.4% at comparable insulin concentrations, and this was reduced to 4.3% as a result of acute uremia. A similar reduction in insulin extraction was noted when hindlimb from uremic animals was perfused with normal blood media or when normal hindlimb was perfused with uremic blood. These findings indicate that the efficiency of insulin removal by the liver is unaffected by uremia and is approximately threefold higher than that for hindlimb muscle. On the other hand, it is apparent that skeletal muscle, by virtue of its mass and relatively high blood flow, is also an important organ system for insulin removal. Furthermore, the data suggest that the 68% reduction in insulin extraction by hindlimb from uremic rats may contribute to the prolongation of insulin removal from plasma that is characteristic of patients with renal failure.     

Effect of insulin in vitro on the isolated,perfused alloxan-diabetic rat liver

Summary Withdrawal of exogenous insulin and a subsequent fast (24 h) of alloxan diabetic rats stimulated rates of gluconeogenesis, ureogenesis, ketogenesis, and amino acid release by in situ perfused livers when compared to those from normal, fasted rats. The contribution of liver glycogen to the high rates of gluconeogenesis observed with the diabetic liver could be excluded. Perfusate lactate concentrations remained constant during the period when the elevated rate of gluconeogenesis was observed with diabetic liver. Addition of insulin as a bolus (750 mU) and continuous infusion (12.5 mU/min) to the perfusion medium of diabetic livers resulted in constant perfusate levels of glucose, urea and -amino nitrogen indicating a suppression of the catabolic processes present in the fasted, diabetic liver. The rate of ketogenesis was also slowed by insulin to about half the rate prior to addition of the hormone. These data indicate that insulin has an immediate anti-catabolic effect in the perfused, diabetic liver.     

Phagocytosis activity of the isolated perfused rat liver

The reticulohistiocytary system was tested with colloidal carbon. Isolated livers were perfused at 20 degrees C, 28 degrees C, 37 degrees C and 45 degrees C with a hemoglobin and protein free medium. It was shown that the perfusion at 28 degrees C revealed a significantly better phagocytosis as at temperatures of 37 degrees C and 45 degrees C. The control parameters of the liver function got, gpt lactate-pyruvate, beta-hydroxybutyrate and acetoacetate displayed the best results at 28 degrees C.     

Effect ofS-adenosyl-l-methionine on ethanol cholestasis and hepatotoxicity in isolated perfused rat liver

We investigated whetherS-adenosyl-l-methionine (SAMe) influences the inhibitory effect of ethanol on bile secretion and ethanol hepatotoxicity in the isolated perfused rat liver. SAMe (25 mg/kg intramuscularly three times a day) was administered for three days consecutively. Liver was then isolated and perfused with taurocholate to stabilize bile secretion and exposed to 1% ethanol for 70 min. The effect of ethanol on bile flow, bile salt biliary secretion, oxygen liver consumption, AST and LDH release in the perfusate, and hepatic concentration of glutathione, malondialdehyde, and diene conjugates was compared between SAMe-treated livers (N=11) and paired controls (N=11). Control experiments without ethanol were also performed (N=6). Exposure to 1% ethanol induced a significantly (P<0.03) higher inhibition of bile flow (–35% vs 17%) and bile salt secretion (–28% vs 16%) in untreated compared with SAMe-treated livers. During 1% ethanol exposure, the release of LDH and AST in the perfusate was significantly lowre (P<0.02) in SAMe-treated livers. Oxygen liver consumption was markedly inhibited by 1% ethanol administration (P<0.02 vs controls without ethanol), an effect almost totally prevented by SAMe treatment (P<0.02 vs ethanol controls). The hepatic concentration of total glutathione was significantly (P<0.02) decreased by 1% ethanol exposure, but this effect was less pronounced in SAMe-treated than in untreated controls (P<0.02). The hepatic levels of malondialdehyde and diene conjugates were not significantly changed by ethanol exposure in either SAMe-treated or control livers in comparison to ethanol-free controls. To evaluate if SAMe protection against ethanol cholestasis was related to an effect on vesicular exocytosis, the biliary excretion of the fluid-phase marker horseradish peroxidase was analyzed. Ethanol inhibited (P<0.04) horseradish peroxidase excretion, which, however, remained unaffected by SAMe (N=6). In conclusion, SAMe counteracted, in the IPRL, the inhibitory effect of ethanol acute administration on bile flow and bile salt secretion while failing to influence vesicular exocytosis. SAMe counteracted ethanol acute hepatotoxicity.     

The elimination of aged glycoproteins by the isolated perfused rat liver

The isolated livers of Sprague-Dawley rats aged ten months were perfused with nine different glycoproteins (coeruloplasmine, haptoglobin, transferrin, alpha2-mocroglobulin, apolipoprotein A, plasminogen, acid alpha1-glycoprotein, alpha1-antitrypsin, beta2-glycoprotein), which were suspended in a basic medium free of hemoglobin and other proteins. Besides a control group two other groups were formed to give a model for aging: one group of glycoproteins was desialysed, another was desilysed and incubated with immunoglobulins before the perfusion. In spite of the application of two procedures which are supposed to produce a model of old glycoproteins, neither treatment resulted in the recognition, binding and elimination of the glycoproteins from the perfusion medium.     

Hyperglycemia-induced cholestasis in the isolated perfused rat liver

In a previous report we showed that cholestasis in diabetic rats is due in part to hyperglycemia. To gain information about the mechanism responsible for this phenomenon, bile flow was studied in isolated perfused rat livers. The perfusion media were modified erythrocyte-free Krebs-Henseleit solutions. Under these experimental conditions, no cholestasis was observed in isolated rat livers obtained from rats treated with streptozotocin (6 mg/100 gm body wt) 6 days before the experiments. We then proceeded to use normal animals. The composition of the perfusion media was modified to maintain the osmolality even after increasing D-glucose concentrations from 0 to 35 mmol/L. Bile flow was not affected with doses up to 15 mmol/L D-glucose. Beyond a threshold value for plasma D-glucose concentrations between 15 and 20 mmol/L, cholestasis was observed. Using D-glucose analogs such as L-glucose and 3-O-methyl-D-glucose, bile flow was also reduced (by 0.54 and 0.53 microliters/min/gm liver, respectively). Isosmotic sucrose-containing perfusion media were also observed to impair bile flow (by 0.66 microliters/min/gm liver). However, i-erythritol and D-mannitol failed to inhibit bile formation. The study of bile/plasma concentration ratios determined using tracer amounts of radioactive sugars indicated that this value was much lower for cholestatic sucrose (0.11) and L-glucose (0.31) than for noncholestatic i-erythritol (0.99) and D-mannitol (0.98). Cholestasis was partly reversed after induction by 35 mmol/L D-glucose if perfusion media were replaced by sugar-free ones, but also by media containing 25 mmol/L D-glucose. Insulin given during the perfusion with sugar-free media was observed to have no effect on bile flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of free choline by isolated perfused rat liver.

The uptake of free choline by isolated perfused rat liver was characterized. A saturable uptake mechanism [Ka = 0.17 +/- 0.07 mM (SD); Vmax = 0.84 +/- 0.16 mumol/min X g dry weight] and a nonsaturable mechanism (through which uptake is proportional to choline concentration in the perfusate) were identified. Most of the choline transported into hepatocytes was converted to betaine, phosphorylcholine, or lecithin. Free choline also accumulated within the intracellular space, suggesting that choline oxidase activity does not always limit choline's uptake by the liver.     

Uptake and catabolism of gamma-aminobutyric acid by the isolated perfused rat liver

Serum concentrations of gamma-aminobutyric acid (GABA) are increased in liver failure, possibly because of decreased hepatic GABA catabolism. To study in detail the role of the liver in GABA metabolism, uptake and catabolism of GABA by isolated perfused liver from normal rats and rats with galactosamine- or carbon tetrachloride-induced liver failure were measured. Hepatic GABA uptake was almost complete at GABA concentrations of up to 10 microM and approached saturation at a concentration of 50 microM. The apparent affinity of hepatic GABA uptake was 38 microM and the apparent maximal velocity was 158 nmol/g.min. Hepatic GABA uptake was sodium-dependent. gamma-Aminobutyric acid taken up by the liver was rapidly catabolized as measured by 14CO2 formation from [U-14C]GABA. Aminooxyacetic acid, a GABA transaminase inhibitor, completely and irreversibly inhibited hepatic GABA catabolism and thereby also inhibited hepatic GABA uptake. Although uptake of GABA by livers of carbon tetrachloride- or galactosamine-treated rats was decreased (apparent maximal velocity, 103 and 98 nmol/g.min, respectively), at physiologic GABA concentrations in the perfusate GABA uptake and catabolism was not different from that of untreated controls. The observed impairment of hepatic GABA uptake or catabolism by the diseased liver would be expected to contribute to increased GABA levels in peripheral blood plasma in liver failure. However, the magnitude of the observed impairment would be insufficient to account for a 10-fold increase in such levels.     

Production and release of plasminogen by isolated perfused rat liver.

In view of conflicting evidence for a major hepatic role in the synthesis of circulating plasminogen, the precursor of the fibrinolytic enzyme plasmin, we carried out the present study with a sensitive assay in order to measure the accumulation of small quantities of plasminogen in a recycling rat liver perfusion system. We have purified plasminogen from Sprague-Dawley rat plasma and have raised a monospecific antiserum against it in rabbits. Isolated rat liver perfusions were performed with an oxygenated recycling perfusate consisting of a perfluorotributylamine/Pluronic F-68 emulsion (Fluosol 43) free of plasma proteins and blood cells. The system was shown to be capable of synthesizing albumin and transferrin. The cumulative appearance of plasminogen in the perfusate was measured by a sensitive, specific radioimmunoassay. Plasminogen concentration increased progressively during the first 2 hr of perfusion; the observed average net synthesis in five separate experiments was approximately 35 microgram/hr per 100 g of body weight. Exposure of the perfused liver to 18 microM cycloheximide inhibited additional increase in the titer of plasminogen. Evidence for de novo synthesis was provided by the incorporation of 14C-labeled leucine into specific immunoprecipitates of plasminogen and the inhibition of this incorporation by cycloheximide. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a single peak of radioactivity corresponding to Mr of 82,000. These data indicate that the liver is a major site of plasminogen production.     

Effect of physical training on insulin uptake by the perfused rat liver

Hepatic uptake of insulin by the in situ perfused rat liver has been measured in rats subjected for 9 wk to physical training (T rats) on a treadmill. Three sedentary groups, one food-restricted (FR) to have the same weight as T rats, one freely-eating (FE), and one sucrose-fed (SF) were also studied. Each liver was cyclically perfused for 30 min with three different insulin concentrations in the medium (～80, ～500, and ～2000 μU/ml). Plasma insulin concentrations in T rats were 50 and 17, in FR 55 and 20, in FE 71 and 30 and in SF 84 and 42 μU/ml in portal and peripheral venous plasma, respectively. Insulin decay curves followed first order kinetics. Taking FE rats as controls physical training enhanced hepatic clearance (expressed in ml/g/min) by 37%–60% whereas sucrose feeding reduced it by 15%–31% during the three perfusion periods. Food restriction in the FR rats caused only minor changes. Similar results were obtained with respect to hepatic extraction ratio and insulin removal. There was a significant negative correlation between portal insulin concentration and hepatic clearance rate. The results indicate that the hepatic extraction of insulin depends on portal insulin concentration. The mean net insulin uptake of the liver was similar in the groups, utilizing portal insulin concentrations and the hepatic clearance rate for calculations. As previously shown, the low peripheral plasma insulin levels after physical training are due mainly to a decreased insulin secretion from the pancreas. This is thus magnified by the efficient trapping of insulin in the liver.     

Effect of oleic acid on insulin secretion by the isolated perfused rat pancreas

The isolated perfused rat pancreas was utilized to investigate the effect of oleic acid on insulin secretion. In the absence of glucose, a continuous infusion of oleic acid (1500 micromol/l) induced a biphasic insulin release. This effect was reduced at low extracellular calcium concentration. In the presence of oleic acid 1500 micromol/l, the insulin response to 10 mmol/l arginine occurred earlier, the total amount of insulin released in response to the amino acid being unchanged. Such an effect was not obtained when oleic acid in the medium was 750 micromol/l, but it was observed in the presence of oleic acid 1500 micromol/l when the concentration of albumin in the perfusate was increased from 2 g/100 ml to 4 g/100 ml. The insulin response to a continuous infusion of glucose (4.4 mmol/l and 16.7 mmol/l) was potentiated by the presence of oleic acid 1500 micromol/l in the perfusate. No modification of the biphasic pattern of insulin response to glucose 16.7 mmol/l was observed. These results demonstrate that high concentrations of oleic acid stimulate insulin release from the isolated perfused rat pancreas and modulate the insulin response to arginine or glucose.