A pharmacodynamic model of portal hypertension in isolated perfused rat liver

AIM: To develop a pharmacodynamic model of portal hypertension from chronic hepatitis.METHODS: Pathological changes and collagen depositions were analyzed using morphometry to confirm CCl₄-induced chronic hepatitis. At d₀, d₂₈, d₅₆ and d₈₄ of the process, the portal perfused velocities (μL/min) in isolated rat livers were exactly controlled with a quantified pump. The pressure (mmHg) was monitored with a Physiological System. The geometric concentrations of phenylephrine or acetylcholine were added to a fixed volume (300 mL) of the circulating perfusate. The equation, the median effective concentration and its 95% confidence intervals of phenylephrine or acetylcholine were regressed with Prism-4 software in non-linear fit and various slopes. In the isolated perfused rat livers with chronic hepatitis, both median effective concentrations were defined as the pharmacodynamic model of portal hypertension.RESULTS: At d₀, d₂₈, d₅₆ and d₈₄, the equations of portal pressure potency from the concentrations of phenylephrine used to constrict the portal vein in isolated perfused rat livers were Y = 0.1732 + 0.3970/[1 + 10^{(-4.3061-0.4407 X)}], Y = -0.004934 + 0.12113/[1 + 10^{(-3.1247-0.3262 X)}], Y = 0.0104 + 0.2643/[1 + 10^{(-8.8462-0.9579 X)}], and Y = 0.01603 + 0.12107/[1 + 10^{(-5.1134-0.563 X)}]; the median effective concentrations were 1.69 × 10^-10 mol/L, 2.64 × 10^-10 mol/L, 5.82 × 10^-10 mol/L, and 8.24 × 10^-10 mol/L, respectively. The equations from the concentrations of acetylcholine used to relax the portal vein were Y = -0.4548 + 0.3274/[1 + 10^{(6.1538 + 0.5554 X)}], Y = -0.05391 + 0.06424/[1 + 10^{(3.8541 + 0.3469 X)}], Y = -0.2733 + 0.22978/[1 + 10^{(3.0472 + 0.3008 X)}], and Y = -0.0559 + 0.053178/[1 + 10^{(5.6336 + 0.5883 X)}]; the median effective concentrations were 8.40 × 10^-10 mol/L, 7.73 × 10^-12 mol/L, 5.98 × 10^-11 mol/L, and 2.66 × 10^-10 mol/L, respectively.CONCLUSION: A pharmacodynamic model of portal hypertension in isolated perfused rat livers with chronic hepatitis was defined as the median effective concentrations of phenylephrine and acetylcholine.     

Aging in vivo and neuraminidase treatment of rat erythrocytes and their sequestration by the isolated perfused rat liver

The isolated perfused rat liver as a model for phagocytosis leaves the specific arrangement of macrophages in the original tissue structure intact. Therefore phagocytosis may occur under terms closely approaching physiological conditions. Separation of rat erythrocytes according to their density in 13% young, 75% mature and 12% old cells permits the establishment of differences in the mean cellular volume (MCV), mean cellular hemoglobin concentration (MCHC) and content of N-acetylneuraminic acid per red blood cell (NANA/RBC). Perfusion of these fractions does not result in significant differences of sequestration of the isolated liver. Enzymatic release of 90% of membrane-bound sialic acid causes a significant increase of 20% in the elimination of erythrocytes from the perfusate. Treatment with neuraminidase is a trigger for sequestration either by adherence or by complete phagocytosis. Incubation of neuraminidase-treated erythrocytes with plasma does not increase the removal rate of cells during perfusion.     

The metabolism of recombinant erythropoietin in the isolated perfused rat liver

ABSTRACT— Indirect evidence points to extrarenal organs, presumably the liver, as the site of degradation of erythropoietin (EPO). The metabolism of both fully glycosylated and desialated intrinsically labelled ³⁵S-Cysteine recombinant human erythropoietin (rhEPO) was therefore studied in isolated Wistar rat livers perfused in a recirculating mode for 180 min with a hemoglobin-free medium containing rhEPO. Perfusate and bile levels of rhEPO were measured by RIA. Total ³⁵S-radioactivity in liver, bile and perfusate as well as non-acid precipitable radioactivity in perfusate were determined. In addition, detection of ³⁵S-radioactivity was performed after subcellular fractionation of rat livers perfused with desialo-³⁵S-Cysteine rhEPO. While concentrations of fully glycosylated ³⁵S-Cysteine rhEPO did not exhibit any detectable decrease during perfusion, desialo-³⁵S-Cysteine rhEPO was rapidly cleared from the perfusate. After 60 min of perfusion, only 32% of the initial levels of both immunoreactive rhEPO and total radioactivity remained in the perfusate. Quantitative hepatic accumulation of desialated tracer was demonstrated. Subcellular fractionation showed extensive hepatic degradation of the desialated tracer. Furthermore, during perfusion progressively larger amounts of small molecular weight degradation products of the tracer were found in the perfusate. Bile excretion of both fully glycosylated and desialated tracer was negligible. The significance of hepatic metabolism of desialo-³⁵S-Cysteine rhEPO was supported by reduced removal of desialo-³⁵S-Cysteine rhEPO from plasma in hepatectomized rats. It is hypothesized that continuous in vivo desialation is a crucial rate-limiting step in the degradation of circulating EPO.     

Glucose transport and hypoxia–reoxygenation injury in the perfused rat liver

Abstract During liver transplantation, oxidative stress occurs during hypoxia and reoxygenation of the donor organ. Chemical oxidative stress impairs cell membrane transport. Therefore, in this study the influence of hypoxia and reoxygenation on hepatocellular membrane transport was investigated. Specifically, glucose transport was studied in the perfused rat liver using the multiple indicator-dilution technique. First, it was observed that in normal rat livers, glucose transport was rapid but saturable ( K _m48 ± 10 mmol/L and V _max9.4 ± 0.9 μmol/s per g of liver). To simulate hypoxia and reoxygenation, livers were perfused for 30 min with nitrogen-saturated buffer and then with oxygen-saturated buffer for 20 min. The livers from fed rats were protected from hypoxia-reoxygenation injury whereas those from fasted rats were highly susceptible to injury as determined by lactate dehydrogenase release. After reoxygenation, the rate of glucose influx decreased significantly by ∼50% in the fasted livers ( P < 0.001) but was unaffected in the fed livers. This impairment of the hepatocellular transport of glucose, which could be secondary to oxidative injury to the hepatocyte membrane, has implications for the function of donor livers that have sustained hypoxia-reoxygenation ('preservation') injury during transplantation.     

The role of the liver in the disposal of orally administered 14C-glucose in the normal rat

Summary 6-¹⁴C-glucose (specific activity 6.0 Ci/g) was administered by oesophageal tube to fasted normal rats; the load was 1.5 g/kg. Rats were killed, in groups, at 0, 10, 20, 30, 60, 120 and 180 min after administration of the load, and the blood and liver sampled. Serum insulin and serum glucose reached a peak at 20 min. The radioactive serum glucose measured by the radioactivity in the dimedone derivatives of 6-C of glucose reached a peak at 60 min. At this time, 92% of the serum glucose originated from the load. Liver glycogen started to rise after 10–20 min, reaching a maximum at 120 min. The incorporation of ¹⁴C-glucose into liver glycogen paralleled the changes in total glycogen. It is suggested that during the first hour following an oral glucose load there is a reduction in the hepatic release of glucose, accompanied by an increase in hepatic glycogen synthesis. During the second hour there is a further marked increase in hepatic glycogen. Between 120 and 180 min there is a decrease in hepatic glycogen, and hepatic glucose release increases. The results emphasize the important role of the liver in the disposal of an oral glucose load.Presented in part at the Autumn Meeting of the British Diabetic Association, Aberdeen, Scotland, 27th–28th September, 1968.     

Role of the 13C-methacetin breath test in the assessment of acute liver injury in a rat model

AIM: To evaluate the role of the ¹³C-methacetin breath test (¹³C-MBT) in the assessment of acute liver injury in a rat model.METHODS: Acute liver injury in rats was induced by a single intraperitoneal injection of D-galactosamine (D-GalN). Forty-eight male Sprague-Dawley rats were randomly assigned to a control group (n = 8) and five model groups (each n = 8), and acute liver injury was assessed at different time points (6, 12, 24, 48 and 72 h) after D-GalN injection. The ¹³C-MBT, biochemical tests, 15-min retention rate of indocyanine green (ICG_R15), and liver biopsy were performed and compared between the control and model groups. Correlations between parameters of the ¹³C-MBT (T_max, MV_max, CUM₁₂₀ and DOB_max), biochemical tests, ICG_R15 and liver necrosis score were also analyzed using Spearman’s correlation analysis.RESULTS: T_max, MV_max, CUM₁₂₀ and DOB_max, as well as most of the traditional methods, correlated with the liver necrosis score (r = 0.493, P < 0.05; r = -0.731, P < 0.01; r = -0.618, P < 0.01; r = -0.592, P < 0.01, respectively). MV_max, CUM₁₂₀ and DOB_max rapidly decreased and were lower than those in the controls as early as 6 h after D-GalN injection (3.84 ± 0.84 vs 5.06 ± 0.78, P < 0.01; 3.35 ± 0.72 vs 4.21 ± 1.44, P < 0.05; 52.3 ± 20.58 vs 75.1 ± 9.57, P < 0.05, respectively) and reached the lowest point 24 h after D-GalN injection. MV_max, CUM₁₂₀ and DOB_max returned to normal levels 72 h after D-GalN injection and preceded most of the traditional methods, including liver biopsy.CONCLUSION: The ¹³C-MBT is a sensitive tool for the timely detection of acute liver injury and early prediction of recovery in a rat model. Further clinical studies are warranted to validate its role in patients with acute liver injury.     

Effects of PAF on cardiac function and eicosanoid release in the isolated perfused rat heart: comparison between normotensive and spontaneously hypertensive rats

The aim of this study was (a) in isolated perfused rat heart to characterize the effects of platelet-activating factor (PAF) on coronary flow, ventricular contractility, and eicosanoid release and (b) to determine whether PAF effects are altered in hearts from spontaneously hypertensive rats (SHR). PAF (10^–10–10^–7 mol) dose-dependently decreased coronary flow and ventricular contractility; concomitantly, coronary effluent concentrations of thromboxane (TX)B₂ and prostaglandin F₂ (PGF₂) were elevated but not those of prostacyclin. The PAF receptor antagonist WEB 2086 (10^–7–10^–5 mol/l) concentration-dependently antagonized these PAF effects. In addition, the cyclo-oxygenase inhibitor indomethacin (5×10^–5 mol/l) prevented PAF (10^–9–10^–7 mol) induced eicosanoid release; in the presence of indomethacin PAF caused coronary constriction and ventricular depression only at the highest dose (10^–7 mol) but had no effect at 10^–9 or 10^–8 mol. Moreover, the TXA₂ antagonist SQ 29,548 (10^–6 mol/l) completely inhibited 10^–8 mol PAF induced ventricular depression but did not effect coronary constriction. In SHR PAF (10^–9–10^–7 mol) evoked decreases in coronary flow and ventricular contractility did not differ from those in normotensive Wistar-Kyoto rats while PAF induced TXA₂ and PGF₂ release was markedly enhanced. In addition, decreases in coronary flow and ventricular contractility induced by the TXA₂ agonist U 46619 (10^–7 mol/l) were markedly depressed in SHR. We conclude that in isolated perfused rat heart PAF causes coronary constriction and depression of ventricular function mainly indirectly through released TXA₂ and/or PGF₂. Moreover, the fact that in SHR the PAF effects on coronary flow and ventricular function are not altered despite markedly enhanced TXA₂ and PGF₂ release supports the view that in the SHR the receptors mediating TXA₂ and/or PGF₂ effects are desensitized.     

Metabolic and Functional Consequences of Successive No-flow and Sustained Low-flow Ischaemia; aP MRS Study in Rat Hearts

Recently, a model of acute hibernation, based on successive no-flow and low-flow ischaemia in the isolated rabbit heart has been described. In the present study this model was used in isolated rat hearts.³¹P NMR was used to follow the time course of intracellular pH (pH_i) and high-energy phosphates; mechanical activity of the heart was assessed simultaneously. Control hearts were subjected to 180 min of low-flow ischaemia and 60 min of reperfusion (group A). In the acute hibernation group, low-flow was preceded by 5 min of no-flow ischaemia (group B). In group A contracture developed during low-flow. The time to onset of contracture was 51 min (range: 28 to 123 min). In group B, contracture did not occur during low-flow ischaemia (P<0.01); recovery of left ventricular developed pressure and end-diastolic pressure was significantly better during the first 15 min of reperfusion (P<0.05). In group A pH_idecreased from 7.06±0.04 to 6.64±0.14 during the first 30 min of low-flow. After contracture developed in this group two pH_ivalues were measured amounting to 6.33±0.15 and 6.86±0.05 at the end of low-flow. At the end of reperfusion pH_iwas 6.29±0.05 and 7.09±0.06. In group B, pH_idecreased from 7.08±0.03 to 6.55±0.03 during no-flow ischaemia. During low-flow ischaemia, pH_iincreased to 6.73±0.05 and remained constant. During reperfusion pH_irecovered to 7.06±0.03. In group A and B phosphocreatine (PCr) levels at the end of low-flow ischaemia amounted to 13±8% and 26±6% of pre-ischaemic levels, respectively. During reperfusion, PCr recovery was better in group B: 67±12%v23±11% (P<0.05). In group A and B, ATP levels at the end of low-flow ischaemia were 5±10% and 19±9%, respectively. The rate of ATP depletion during low-flow ischaemia was initially similar in both groups, but between 45 and 90 min ATP depletion still continued in group A, while this had leveled off in group B (P<0.01). During reperfusion no significant changes in ATP were observed. We propose that increased glucose transport and glycolytic flux are able to maintain ionic homeostasis and diastolic function when low-flow ischaemia is preceded by a short period of no-flow ischaemia.     

Role of the oestrogen receptor in liver regeneration in the male rat

Partial hepatectomy in male rats results in raised serum oestrogen levels, nuclear binding of oestrogen receptor (ER) and feminization of certain aspects of hepatic metabolism. It has been proposed that these changes may have an important role in liver regeneration. The present study was performed to ascertain the effects of the oestrogen agonist diethylstilbestrol (DES), 2 mg/kg, and the oestrogen antagonist tamoxifen (TAM), 2 mg/kg, on liver regeneration induced by partial hepatectomy in the male rat. Regenerative activity was determined by incorporation of [³H]-thymidine into hepatic DNA as well as by measurement of liver remnant weight. Following partial hepatectomy, there was a trend towards an increase in liver remnant weight at 24 h in rats treated with DES (DES, 5.95 ± 1.52 g; vehicle, 4.87 ± 0.66 g; P= 0.06) though by 48 h no effect was found. Tamoxifen treatment did not significantly affect liver weight at 24 h but by 48 h there was a highly significant reduction in liver remnant weight (TAM, 5.41 ± 0.85 g; vehicle, 7.31 ± 1.43 g; P < 0.001). Neither DES nor TAM treatment influenced liver regeneration as determined by [³H]-thymidine incorporation into hepatic DNA. We conclude that pharmacologic manipulation of oestrogens does not influence the initiation of the regenerative process but that oestrogen may facilitate later phases of hepatic growth.     

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.     

Analysis of the daily changes of melatonin receptors in the rat liver

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10–200 mg/kg) increased in a dose‐dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.     

Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver

Summary The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10^–7mol/l islet amyloid polypeptide affected neither basal nor glucagon (10^–9 mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91±12.07 vs control: 0.07±0.03 pmol·100 g body weight^–1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7×10^–11 mol/l) increased integral hepatic glucose release within 84 min (763.4±161.7 vs –25.7±73.2 mol · 100 g body weight^–1 in the control group, p<0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2±180.0 mol·100 g body weight^–1, p<0.01). Simultaneous infusion of 10^–7 mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0±102.5 mol·100 g body weight^–1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10^–9 mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60±3.36 vs 1.67±1.28 mol·min^–1·100 g body weight^–1). These results suggest that islet amyloid polypeptide neither directly affects hepatic glycogenolysis nor causes insulin resistance to hormone-sensitive glucose production, but may increase the size of the hepatic glycogen pool by enhancing gluconeogenesis.     

Receptor-mediated endocytosis of chemically modified albumins by sinusoidal endothelial cells and Kupffer cells in rat and human liver

Human serum albumin (HSA), formaldehyde-treated HSA (FHSA), and HSA polymerized with glutaraldehyde (pHSA) were conjugated with colloidal gold (15 (15G) or 50 (50G) nm in diameter). The labeled proteins were injected into the portal veins of rats and followed by electron microscopy. Both 15G-FHSA and 15G-pHSA were taken up by sinusoidal endothelial cells (Ec) and Kupffer cells (Kc). Five minutes after injection, gold particles were observed on the surface of Ec and Kc. At 10 min, most gold particles were gathered in the coated pits and vesicles of Ec. In Kc, gold particles were observed in both coated vesicles and macropinocytotic vesicles. At 15 min, the gold particles were localized mainly in the endosomes and some lysosomes of Ec and in the large vacuoles of Kc. At 30 min, the gold particles had been gathered into the secondary lysosomes and condensed. At 60 min, some gold particles were observed in the cytoplasm of Ec. The fate of 15G-pHSA was the same as that of 15G-FHSA. Simultaneous injection of 15G-pHSA and 50G-FHSA revealed that particles of both sizes were taken up together into the coated pits and vesicles of Ec. Preperfusion of livers with unlabeled FHSA, pHSA, or formaldehyde-treated bovine serum albumin (FBSA) inhibited the uptake of 15G-FHSA or 15G-pHSA by Ec. In a human liver biopsy specimen, both 15G-FHSA and 15G-pHSA were taken up by Ec and Kc through coated vesicles, as in the rat liver. These results suggest that aldehyde-modified albumin is taken up by both Ec and Kc, and that both FHSA and pHSA share a common receptor on Ec.     

O6-Methylguanine repair of methylated DNA in vitro: Cell cycle-dependence of rat liver methyltransferase activity

Summary O⁶-Methylguanine DNA transferase activity was investigated in liver proteins obtained at various intervals after partial hepatectomy and/or after hydroxyurea-induced synchronization of the liver cell cycle. Liver proteins were incubated with ³H-methylated calf thymus DNA as previously described by Pegg et al. (1981). The loss of O⁶-methylguanine was measured by radiochromatography of DNA hydrolysates. The extent of O⁶-methylguanine repair differed during the cell cycle: the activity increased in late G₁, reached a maximum in early S phase and declined in late S phase and G₂M. These results indicate that hepatocytes are endowed with an increased DNA repair capacity for this promutagenic lesion during the period of highest transformation sensitivity in the cell cycle. Though increased, however, this repair potential does not, because of its exhaustibility, appear to be sufficient to prevent initiation of transformation after high doses of alkylating carcinogens.Supported by grants from Deutsche Forschungsgemeinschaft     

Amelioration of tumor necrosis factor release by cyclosporine in warm ischemia/reperfusion injury of the rat liver: with special reference to hepatic ultrastructure

Mechanisms by which an immunosuppressant (cyclosporine, CsA) ameliorates warm ischemic injury of the liver were studied. Female Sprague-Dawley rats were subjected to 60-min normothermic liver ischemia. Animals were assigned to one of two groups: group I, controls with vehicle treatment; group II, treatment with CsA (10 mg/kg). CsA was given orally for 4 consecutive days prior to the induction of hepatic ischemia. In addition to a survival study, plasma levels of endotoxin, serum activity of tumor necrosis factor-α (TNF), and serum levels of aminotransferases were measured in blood samples collected from the suprahepatic vena cava, and hepatic ultrastructural alterations were examined under an electron microscope. The 7-day survival rate was significantly higher in the CsA-treated animals. In the control group, serum TNF levels were elevated following reperfusion and peaked at 3 h. When the values at 3 h post reflow were compared, the animals given CsA had significantly lower levels of TNF (170.0 ± 30.5 pg/ml for group I, 67.6 ± 13.7 for group II, mean ± SEM;P < 0.05). The sinusoidal lining cells and hepatocytes were drastically destroyed at 6 h post reflow in the control group, although the degree of injury at 1—3 h was less severe. On the other hand, the endothelium and parenchymal liver cells in the CsA-treated group were well preserved at 6 h in comparison with those in the control group. Our data suggest that modulation of TNF production is one of the mechanisms through which CsA prevents the exacerbation of ischemia/reperfusion injury of the liver.     

Biological effects and catabolic metabolism of 3',5'-cyclic nucleotides and derivatives in rat adipose tissue and liver

Experiments on the catabolism of N⁶, O^2′-dibutyryl cyclic AMP and N⁶-monobutyryl cyclic AMP in rat liver and epididymal adipose tissue were described. During the regulation by the dibutyryl cyclic nucleotide of several parameters of the metabolism of epididymal adipose tissue adipocytes (lipolysis and glucose metabolism), neither of the butyryl groups was removed enzymatically, and nonenzymatic formation of butyric acid amounted to no more than 4 per cent over 2 hours of incubation. Therefore, intact adipocytes exhibited no deacylase activities. Furthermore, cyclic nucleotide phosphodiesterases, isolated from subcellular fractions derived from homogenates of liver and adipose tissue, which actively hydrolyzed cyclic AMP, GMP, IMP, UMP, and TMP, but not cyclic CMP, were sterically hindered by substituent groups on the N⁶ and O^2′ positions of cyclic AMP; that is, the enzyme was completely inactive against N⁶, O^2′-dibutyryl cyclic AMP and exhibited only a trace amount of activity against N⁶-monobutyryl cyclic AMP. However, mechanisms do exist in these tissues for the removal of one or both butyryl groups; subcellular fractions derived from tissue homogenates contained an N-acyl hydrolase active against N⁶, O^2′-dibutyryl and N⁶-monobutyrl cyclic AMP, and, possibly, also esterase activity against the former. The relationships of function (as lipolytic agents and substrates for cyclic nucleotide phosphodiesterases) to structure among a variety of 3′, 5′-cyclic nucleotides were investigated using adipocytes and partially purified enzymes. An incubation medium consisting of a sodium chloride-phosphate buffer devoid of Mg²⁺, Ca²⁺, and K⁺ permitted entry of all nucleotides into adipocytes. In such permeable cells, all purine and pyrimidine cyclic nucleotides, with the exception of the thymine analogs, stimulated lipolytic activity to various extents; however, no relationships, inverse or otherwise, existed between the potencies of the compounds as activators of lipase activity and their susceptibility to hydrolysis by the cyclic nucleotide phosphodiesterase. Combinations of saturating concentrations of cyclic nucleotides produced rates of lipolysis in cells that were similar to the rate produced by the more potent member of each pair when tested alone; only the lipolytically inactive thymine cyclic nucleotides inhibited the effects of other cyclic nucleotides upon lipolysis. Such results indicated that all active cyclic nucleotides stimulated the same lipolytic mechanism in adipose tissue.     

16, 16-dimethyl prostaglandin E2 modulates aflatoxin B1-induced injury of rat hepatocytes in primary culture: Possible role of cAMP

The hepatocellular cytoprotective effects of 16,16-dimethyl prostaglandin E2 (dmPGE2), an analogue of PGE2, were investigated using primary cultures of rat hepatocytes and aflatoxin B1 as the hepatotoxin. Lactic dehydrogenase (LDH) release by hepatocytes was used as an index of hepatotoxicity. When aflatoxin-treated hepatocytes were co-cultured with 16,16-dmPGE2 (0.01-0.5 micrograms/mL) LDH release was significantly reduced and ultrastructural changes of hepatocellular injury were markedly diminished. The magnitude of the cytoprotective effect was not dependent on the concentration of the prostaglandin over the range tested. A significant cytoprotective effect was also induced when hepatocellular cyclic AMP (cAMP) levels were increased by the addition of dibutyl-cAMP. In contrast to 16,16-dmPGE2, PGF2 alpha Tromethamine, an analogue of PGF2 alpha, which does not stimulate cAMP, induced insignificant changes in cytoprotection. These findings indicate that only a low concentration of 16,16-dmPGE2 (> or = 0.01 micrograms/mL) is necessary to induce a maximal hepatocellular cytoprotective effect and suggest that this effect may be dependent on activation of cAMP.     

Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat

BACKGROUND/AIMS: Alterations in hepatobiliary transporters may render fatty livers more vulnerable against various toxic insults. METHODS: We therefore studied expression and function of key organic anion transporters and their transactivators in 8-week-old obese Zucker rats, an established model for non-alcoholic fatty liver disease. RESULTS: Compared to their heterozygous littermates, obese animals showed a significant reduction in canalicular bile salt secretion, which was paralleled by significantly diminished Oatp2 mRNA and protein levels together with reduced nuclear HNF3beta, while expression of bile salt export pump, organic anion transporter (Oatp) 1 and multidrug resistance-associated protein (Mrp) 4 were unchanged. Impaired bile salt-independent bile flow in obese rats was associated with a 50% reduction of biliary secretion of the Mrp 2 model-substrates glutathione disulfide and S-(2,4-dinitrophenyl)glutathione. In line Mrp2 protein expression was reduced by 50% in obese rats. CONCLUSIONS: Oatp2 and Mrp2 expression is decreased in fatty liver and may impair metabolism and biliary secretion of numerous xenobiotics. Reduction of bile salt secretion and absence of biliary GSH excretion may contribute to impaired bile flow and posthepatic disorders associated with biliary GSH depletion.     

Substrate selection in the isolated working rat heart: effects of reperfusion,afterload, and concentration

A study of substrate selection in the isolated heart was made using¹³C NMR isotopomer analysis, a method that unequivocally identifies relative substrate utilization. This technique has several advantages over conventional approaches used to study this problem. It detects the labeling of metabolic end-products present in tissue, as opposed to more indirect methods such as measurement of respiratory quotient, arteriovenous differences, or specific activity changes in the added substrate. It also has advantages over methods such as¹⁴CO₂ release, which may involve dilution of label with unlabeled pools before CO₂ release. Furthermore, it can measure the relative oxidation of up to four substrates in a single experiment, which other labeling techniques cannot conveniently achieve. Substrates selection was considered in light of its effects on myocardial efficiency and recovery from ischemia. A mixture of four substrates (acetoacetate, glucose, lactate, and a mixture of long chain fatty acids), present at physiological concentration (0.17, 5.5, 1.2, and 0.35 mM, respectively), was examined. This is the first use of such a mixture in the study of substrate selection in an isolated organ preparation. At these concentrations, it was found that fatty acids supplied the majority of the acetyl-CoA (49%), and a substantial contribution was also provided by acetoacetate (23%). This suggests that the ketone bodies are a more important substrate than generally considered. Indeed, normalizing the relative utilizations on the basis of acetyl-CoA equivalents, ketone bodies were by far the preferred substrate. The relative lactate oxidation was only 15%, and glucose oxidation could not be detected. No change in utilization was detected after 15 min of ischemia followed by 40 min of reperfusion. The change in substrate selection with afterload was examined, to mimic the stress-related changes in workload found with ischemia. Only minor changes were found. Substrate selection from the same group of substrates, but employing concentrations observed during starvation, was also assessed. This represents the state during which most clinical treatments and evaluations are performed. In this case, acetoacetate was the most used substrate (78%), with small and equal contributions from fatty acids and endogenous substrates; the oxidation of lactate was suppressed.