Participation of hepatic α/ß-adrenoceptors and AT1 receptors in glucose release and portal hypertensive response induced by adrenaline or angiotensin II.

It has been previously demonstrated that the hemodynamic effect induced by angiotensin II (AII) in the liver was completely abolished by losartan while glucose release was partially affected by losartan. Angiotensin II type 1 (AT1) and adrenergic (∝1- and ß-) receptors (AR) belong to the G-proteins superfamily, which signaling promote glycogen breakdown and glucose release. Interactive relationship between AR and AT1-R was shown after blockade of these receptors with specific antagonists. The isolated perfused rat liver was used to study hemodynamic and metabolic responses induced by AII and adrenaline (Adr) in the presence of AT1 (losartan) and ∝1-AR and ß-AR antagonists (prazosin and propranolol). All antagonists diminished the hemodynamic response induced by Adr. Losartan abolished hemodynamic response induced by AII, and AR antagonists had no effect when used alone. When combined, the antagonists caused a decrease in the hemodynamic response. The metabolic response induced by Adr was mainly mediated by ∝1-AR. A significant decrease in the hemodynamic response induced by Adr caused by losartan confirmed the participation of AT1-R. The metabolic response induced by AII was impaired by propranolol, indicating the participation of ß-AR. When both ARs were blocked, the hemodynamic and metabolic responses were impaired in a cumulative effect. These results suggested that both ARs might be responsible for AII effects. This possible cross-talk between ß-AR and AT1-R signaling in the hepatocytes has yet to be investigated and should be considered in the design of specific drugs.

The combination of atorvastatin and ethanol is not more hepatotoxic to rats than the administration of each drug alone.

Animal studies and premarketing clinical trials have revealed hepatotoxicity of statins, primarily minor elevations in serum alanine aminotransferase levels. The combined chronic use of medicines and eventual ethanol abuse are common and may present a synergistic action regarding liver injury. Our objective was to study the effect of the chronic use of atorvastatin associated with acute ethanol administration on the liver in a rat model. One group of rats was treated daily for 5 days a week for 2 months with 0.8 mg/kg atorvastatin by gavage. At the end of the treatment the livers were perfused with 72 mM ethanol for 60 min. Control groups (at least 4 animals in each group) consisted of a group of 2-month-old male Wistar EPM-1 rats exposed to 10% ethanol (v/v) ad libitum replacing water for 2 months, followed by perfusion of the liver with 61 nM atorvastatin for 60 min, and a group of animals without chronic ethanol treatment whose livers were perfused with atorvastatin and/or ethanol. The combination of atorvastatin with ethanol did not increase the release of injury marker enzymes (alanine aminotransferase, aspartate aminotransferase, and lactic dehydrogenase) from the liver and no change in liver function markers (bromosulfophthalein clearance, and oxygen consumption) was observed. Our results suggest that the combination of atorvastatin with ethanol is not more hepatotoxic than the separate use of each substance.

Identification of receptors in the liver that mediate endocytosis of circulating tissue kallikreins.

The liver plays an important role in the clearance, by receptor-mediated endocytosis, of circulating glycoproteins. It has been demonstrated that tissue kallikreins, which are acid glycoproteins, circulate in plasma, where they are poorly inhibited by plasma proteins. We have shown that the liver is the main organ that clears tissue kallikreins from the circulation. We now report the identification of receptors involved in this clearance. Using a perfused rat-liver system, and as models, pig pancreatic (PPK) and horse urinary (HoUK) kallikreins, we have found that: (a) the binding of PPK to the perfused liver was inhibited by 50 mM methyl alpha-D-mannoside and 20 microM mannan, was partially inhibited by 50 mM mannose and was unaffected by 1.5 microM asialofetuin; (b) binding of HoUK to the perfused liver was inhibited by 1.5 microM asialofetuin, 50 mM galactose and 50 mM lactose and was unaffected by 50 mM mannose; (c) the clearance rate of both kallikreins followed the equation y = a.xb; (d) their binding was Ca2+-dependent and their clearance was inhibited by 3 mM chloroquine and 10 mM methylamine. Using isolated liver cells and tritiated HoUK, we calculated that 500,000 receptors/cell were present and the Scatchard plot showed that there were two apparent affinity constants: 0.24.10(9) l/M) (high-affinity) and 0.3.10(8) l/M (low-affinity). These results show that PPK is recognized by a liver mannose receptor and HoUK by the galactose receptor. The liver uptake of native and circulating tissue kallikreins thus emerges as a mechanism by which their levels in plasma are regulated.

Ethanol challenge in non-alcoholic patients with schistosomiasis.

AIMS: To evaluate serum gamma glutamyltransferase (GGT) activity in a group of non-alcoholic patients with the hepatointestinal form of schistosomiasis; and the response of both GGT and alkaline phosphatase to an ethanol challenge in two subgroups of patients with different baseline serum concentrations of GGT. METHODS: Seventy six non-alcoholic, non-smoking hepatitis B virus (HBV) negative men with normal body mass index, who denied blood product transfusion or use of medication, were studied (30 healthy volunteers (control group) and 46 patients with the hepatointestinal form of schistosomiasis). GGT activities were determined in all subjects and the ethanol test (measurement of GGT and alkaline phosphatase (ALP) before and 24 hours after the ingestion of 1 g/kg of ethanol) was performed in 14 patients (7 with GGT below 25 IU/l and seven with GGT above 25 IU/l). The ethanol serum concentrations were determined in the samples collected one hour after ingestion of the solution in four patients with schistosomiasis. RESULTS: The mean serum ethanol concentration one hour after the ingestion was 0.7 g/l and all patients were clinically intoxicated. GGT was below 25 IU/l in all 30 volunteers and in 33 of the patients with schistosomiasis. In 13 patients the GGT varied from 28 to 140 IU/l. The two enzymes GGT and ALP determined in the 14 patients submitted to the test were positively correlated in the baseline samples (r = 0.8130) as well as in the samples obtained 24 hours after stimulation (r = 0.7921). Neither the plasma activity of GGT nor the GGT:ALP ratio was affected by the ethanol challenge. CONCLUSIONS: These results suggest that the mechanisms for the increase of GGT serum activity in schistosomiasis and in alcoholism differ. In the latter, microsomal induction increases GGT serum activity, while alterations in the biliary tree may be responsible for the increase observed in patients with schistosomiasis.

Catabolism of vasoactive polypeptides by perfused rat liver.

Exsanguinated rat liver preparations perfused in situ with oxygenated saline solutions inactivated recirculating bradykinin (BK) at rates of 2.3 to 9.1 and isoleucyl5 angiotensin II (AII) at rates of 2.8 to 15.0 nmoles X min-1 X g-1 of liver, depending on the initial concentration of the peptides in the perfusion fluid (3.1 to 18.9 X 10(-6) M for BK and 8.5 to 17.0 X 10(-6) M for AII). On the other hand, at similar concentrations, recirculation of isoleucyl5 Angiotensin I (AI) for 8 min did not lead to decrease of its biological activity when assayed on the isolated rat uterus. Following a single passage through liver, picomole amounts of both BK and AII were inactivated by about 90% as revealed by assays on a superfused rat uterus. The potency ratio AI:AII, assayed on a superfused rat uterus was 1:22 and changed to 1:5 following a single passage of both peptides through liver. This finding and the separation of 4.9% of AII on carboxymethylcellulose columns following recirculation of AI through rat liver indicate a conversion of AI into AII. The dipeptides Phe-Arg, Ser-Pro and Gly-Phe were identified among the hydrolysis products of perfused BK. A peptidyldipeptide hydrolase (EC 3.4.15) may be responsible for both the BK inactivation and AI conversion. The inactivation of AII cannot be attributed to the same enzyme.

Vena cava perfusion in situ: a tool for uptake studies.

While studying the uptake of trypsin and thrombin by the perfused rat liver, we verified that these proteins are internalized neither by hepatocytes nor Kupffer cells. These results raised the possibility that the enzymes might be binding to endothelial cells, either hepatic or vascular. In order to find out if the binding of enzymes to endothelial surface is a liver cell-specific phenomenon, we devised a system to perfuse the rat inferior cava vein in situ. After exsanguination, the vein was perfused with the recirculation of 30 mL of Krebs/BSA solution propellered by a pulsatile flow pump (10 mL/min). The liver was not exsanguinated, but to assure that the organ was indeed excluded from the circuit during the experiment at the end of the perfusion time we added China ink in the perfusion fluid. We verified that trypsin is extracted from the perfusion fluid by the vena cava as efficiently as by the liver, suggesting that the most of the infused trypsin is removed mainly by vascular endothelial cells when the liver perfusion model is used. On the other hand, thrombin is removed mainly by the liver cells since the uptake by the vena cava was insignificant.

Plasma-kallikrein clearance by the liver of acetaminophen-intoxicated rats.

The liver synthesizes prokallikrein and is the main organ to clear the active enzyme (plasma-kallikrein) from circulation. This clearance, a receptor-mediated endocytosis, is calcium-independent and not affected by the blockade of Kupffer cells. The effects of endothelial cells blockade and of acetaminophen intoxication on the clearance of 10 nM rat plasma-kallikrein (RPK) by the isolated, exsanguinated and perfused rat liver are now reported. Endothelial cells blockade obtained by the addition of large excess (30 uM) of formaldehyde-treated serum albumin to the perfusion fluid does not affect the hepatic clearance of RPK (the half-lives of hepatic uptake were 15.5 +/- 1.0 min in the absence versus 16.5 +/- 1.4 min in the presence of the treated protein, p > 0.05). Some livers were perfused 24 hours after acetaminophen intoxication: 6.6 mmol/kg given i.p. after a 42-hour period of fast. Hepatocyte injury suggested by elevated aminotransferase activity (ALT 10 times control value, AST 30 times control value), acute phase inflammatory response (serum alpha 2-macroglobulin increase) and reduced synthetic function (serum albumin decrease), was confirmed histologically and only zone 3 hepatocytes were necrotic. A 66-hour period of fast does not affect by itself the hepatic clearance of RPK (16.9 +/- 1.3 min of half-life of hepatic uptake) when compared with the control group (15.5 +/- 1.0 min, p > 0.05). On the other hand the RPK clearance by the livers of rats previously intoxicated with acetaminophen was markedly deficient (the half-life of hepatic uptake was 39.2 +/- 3.2 min). These findings suggest that RPK is internalized by hepatocytes, preferentially by those of the perivenular zone of the hepatic acinus.

Thimet oligopeptidase EC 3.4.24.15 is a major liver kininase.

Bradykinin (BK) is a potent hepato-portal hypertensive agent although it is efficiently inactivated by the liver. The organ converts angiotensin I to AII, but at a much slower rate than it inactivates BK. We had previously identified EC 3.4.24.15 as an hepatic bradykinin inactivating endopeptidase that hydrolyzes BK at the F5-F6 bond. The aim of this study was to determine the relative importance of BIE, as compared to other kininases, in normal, cirrhotic or inflamed rat livers, as well as in samples of human liver. Using specific substrates and inhibitors we showed that: 1) purified BIE preparation hydrolyzed BK and a BK analogue (BK-Q) with similar efficacy; BK-Q was functionally active since it caused an increase in hepato-portal pressure, as did BK itself. 2) BK degradation in rat serum was performed by ACE since BIE and prolylendopeptidase (PEP) activities were negligible. 3) normal rat liver homogenate contained a large amount of BIE activity which was eliminated by a specific EC 3.4.24.15 inhibitor; ACE and PEP activities were negligible. 4) There was no difference (p>0.05) in BIE activity in the liver homogenates from rats with normal, inflamed or cirrhotic livers. 5) BIE activity was efficiently removed from livers (normal, inflamed or cirrhotic) that were perfused with TritonX-100.6) Human liver had an similar enzymatic pattern although ACE activity was detected. We concluded that in normal, inflamed or cirrhotic rat livers, as well as in the human liver, the bradykinin inactivating endopeptidase (EC 3.4.24.15), and not ACE, is the major hepatic kininase.

Plasma kallikrein and thrombin are cleared through unrelated hepatic pathways.

Plasma kallikrein (PK) and thrombin (TH), serine proteinases formed from inactive precursors, participate in important body defence mechanisms. The isolated hepatocyte recognizes TH, and the liver clears PK by calcium-independent receptors through mechanisms that are not yet clearly understood. It is known that heparin impairs the binding of TH to isolated liver cells through the inhibition of high affinity receptors. Using an isolated, exsanguinated and perfused rat liver preparation we confirmed that the TH hepatic clearance is calcium-independent and affected by heparin; PK clearance rates both in the presence (t1/2 10 +/- 2 min) or the absence (t1/2 10 +/- 1 min) of heparin were similar; the presence of beta-galactosides does not impair the TH clearance but adversely affects the PK clearance and a large excess of TH does not impair the PK clearance rate (t1/2 6 +/- 1 min). These results indicate that PK and TH are cleared by calcium-independent but otherwise unrelated hepatic pathways and suggest that TH may indeed facilitate the PK clearance by the liver.

The enteroinsular axis and endocrine pancreatic function in chronic alcohol consumers: evidence for early beta-cell hypofunction.

Chronic alcohol consumers may have, as judged by functional criteria, exocrine as well as endocrine pancreatic dysfunction, the latter represented by a decreased insulin response to an oral glucose load. To investigate whether this decreased insulin response was due to an ethanol-induced beta-cell dysfunction or to an ethanol-induced dysfunction of the enteroinsular axis, we determined glucose, insulin, and C-peptide plasma concentrations following an oral and an intravenous glucose load in 16 healthy volunteer nonalcohol consumers and in 10 chronic alcohol consumers. In each group, total integrated response for glucose did not significantly change whether glucose was given orally or intravenously, indicating isoglycemic glucose loads. The total integrated response values for insulin in the alcoholic group following both glucose loads as well as C-peptide plasma concentrations were significantly lower than in the control group. Moreover, in both groups the insulin TIR values following the oral glucose load were significantly greater than the values obtained following the intravenous glucose load, indicating an incretin effect. These results indicate that the decreased insulin response observed in alcoholics was not caused by a dysfunction of the enteroinsular axis because it also occurred following an intravenous glucose load, but by an ethanol-induced beta-cell dysfunction because C-peptide and insulin were proportionally decreased in this group.

Kininogen and kininogenase synthesis by the liver of normal and injured rats.

Isolated livers from normal rats or from others at 2 days after subcutaneous injection of turpentine have been perfused with a simplified medium. Estimation of kininogen, kininogenase and 2 plasma proteins in the perfusates thus obtained indicate that both kininogen and kininogenase are synthesized in the liver. Furthermore, because twice as much kininogen and kininogenase was synthesized by the livers from the rats which had been injected with turpentine as by those from the normal rats, these two proteins must be considered to be members of the group of plasma proteins known as acute phase reactants.

Plasma kallikrein clearance by the liver: a review.

1. The liver is the main organ clearing both plasma and tissue kallikreins from the circulation. Hepatocytes are responsible for the internalization of rat plasma kallikrein (RPK) and the clearance of plasma kallikrein by the liver is Ca(2+)-independent. The binding site of RPK to the liver cell is located on its heavy chain which is not exposed on prokallikrein. An S-type lectin accounts for the receptor-mediated endocytosis of RPK. 2. These properties of the liver are affected by pathological situations, particularly the acute-phase response to inflammation, in which the kallikrein-kinin system plays a major role. The hepatic clearance of the alpha 2-macroglobulin-plasma kallikrein complex is less efficient than the clearance of the free enzyme.

Chronic administration of ethanol does not alter plasma kallikrein hepatic clearance.

1. The clearance of plasma kallikrein by the isolated and perfused liver of rats chronically intoxicated with ethanol was studied. Alcohol was added to the diet as 36% of total calories, and the animals were kept on this diet for 5-7 weeks. 2. The hepatic clearance of plasma kallikrein by these rats (uptake half-life, 15 +/- 2 min; N = 3) was similar to that observed in the control groups (normal diet, uptake half-life, 14 +/- 2 min; N = 5, or normal diet with sucrose added as 36% of total calories, uptake half-life, 16 +/- 3 min; N = 4). 3. These results provided indirect evidence that the endocytosis mechanism of plasma kallikrein by the liver differs from that described for glycoproteins which use the galactosyl receptor, since liver endocytosis via this latter system is reduced by chronic alcohol intoxication.

Native plasma kallikrein is cleared at similar rates by mammalian and avian livers.

Rat plasma kallikrein (RPK) is a serine protease that circulates as an inactive precursor, prokallikrein, and once activated is efficiently cleared by the liver by a carbohydrate-dependent, Ca(2+)-independent mechanism. Seven hepatic lectin systems have been described for mammals but not all of these animal lectins are expressed in the avian liver. Using a liver perfusion system we compared the plasma kallikrein clearance of rats (N = 10) and pigeons (N = 4). Our results show that the lectin responsible for the hepatic clearance of plasma kallikrein is also present in pigeon liver and that this organ clears the enzyme with an efficiency (11.4 +/- 1.3 pmol/g, 20 min) similar to that of the rat liver (10.0 +/- 0.7 pmol/g, 20 min).

Clearance of the alpha 2 macroglobulin-trypsin complex and uptake of trypsin by perfused liver.

The uptake and degradation of the alpha 2 macroglobulin-trypsin (alpha 2 m-trypsin) complex have been studied using isolated liver cells but not in the liver as a whole. We report the clearance of the complex by the isolated and exsanguinated liver of Wistar male rats, weighing 150- 280 g, and compare it with that of the free enzyme. The hepatic clearance of the alpha 2m-trypsin complex follows a pattern with a distribution phase followed by an elimination phase, which contrasts with that of trypsin where only the distribution phase is observed. The extraction of trypsin from the perfusate is Ca(2+)-independent (156 +/- 14 pmol/g liver in the presence of 2.5 mM Ca2+, N = 9, versus 140 +/- 8 pmol/g liver in its absence, N = 7) and is not affected by 100 mM NH4Cl (152 +/- 7 pmol/g liver, N = 6), 100 U/ml heparin (164 +/- 14 pmol/g liver, N = 5), 30 microliters/ml carbon particle suspension (150 +/- 13 pmol/g liver, N = 7) or an acute-phase situation induced by turpentine (125 +/- 10 pmol/g liver, N = 6) (P > 0.05, ANOVA). The hepatic clearance of the alpha 2m-trypsin complex is Ca(2+)-dependent (1.8 +/- 0.2 ml/min in the presence of Ca2+, N = 8, versus 0.6 +/- 0.03 ml/min in its absence, N = 4), affected by NH4Cl (< 0.1 ml/min, N = 7), heparin (1.1 +/- 0.2 ml/min, N = 6) and the acute-phase (0.6 +/- 0.1 ml/min, N = 6) but not by the carbon particle suspension (1.8 +/- 0.2 ml/min, N = 7). These results show that trypsin is not internalized by hepatocytes (no NH4Cl effect) or Kupffer cells (no carbon particle effect) and that the alpha 2m-trypsin complex is internalized in a Ca(2+)-dependent process by hepatocytes, but not by Kupffer cells, and is affected by an acute-phase reaction.

Protein C deficiency in the compensated form of hepatosplenic schistosomiasis.

Plasma levels of protein C (enzyme immunoassay), albumin (electrophoresis), and transthyretin (radial immunodiffusion) were measured in 15 patients with the compensated hepatosplenic form of schistosomiasis and in 10 healthy volunteers. Plasma levels of protein C were below normal in 47% of the schistosomiasis patients; this deficiency could be explained by diminished hepatic synthesis since it occurred in conjunction with low plasma levels of albumin and/or transthyretin. In 33% of the schistosomiasis patients, plasma levels of protein C were below 0.5 U/ml, a value which has been associated with thrombotic disease. Protein C deficiency may explain the unexpectedly low incidence of hemorrhagic episodes, as well as the occurrence of portal vein thrombosis that is not infrequent in these patients.

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus.

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 +/- 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 +/- 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 +/- 1.3 microg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22. 5 +/- 0.7 microg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 +/- 2.4 microg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

Lysosome injury by perfusion of the isolated rat liver with ethanol or a molasses distillate (cachaça).

An isolated rat liver perfusion model was used to study the effects of acute exposure of the organ to either ethanol or a molasses distillate (cachaça). When ethanol (72 mM) or a molasses distillate (68 mM ethanol) was added to the perfusion fluid, lysosomal injury was indicated by the increased release of tartrate-inhibited acid phosphatase activity at the end of a 3 h period of perfusion. Other cellular compartments were not significantly damaged in these acute experiments, as judged by the release of aspartate and alanine aminotransferases, lactate dehydrogenase and alkaline phosphatase. The behavior of both ethanol itself and the alcoholic beverage was similar as far as enzyme release is concerned but only the molasses distillate caused significant acidosis (a decrease in perfusate pH) at the end of a 3 h period of perfusion. These data may be of importance for a better understanding of the hepatic damage caused by alcohol abuse and useful for laboratory investigation of alcohol intoxication.

Plasma kallikrein clearance by the liver in food-restricted rats.

We measured the clearance rate of plasma kallikrein by the liver in three groups of rats: one recently weaned, and two seven weeks old (control and food-restricted groups). The clearance rates were similar in the three groups when expressed as units/g liver. The livers of the recently weaned and food-restricted rats were, however, smaller than those of the controls and consequently their livers cleared plasma kallikrein less efficiently.

Rat plasma kallikrein clearance by perfused rat liver.

Previous studies have shown that perfused rat liver in situ is able to clear recirculating rat plasma kallikrein (RPK) in two phases: an initial clearance lasting a few minutes, followed by a slow exponential phase. Using purified RPK preparations we now show that: RPK is a glycoprotein; clearance was inhibited by human serum against blood group B and 0.1 M melibiose but was not affected by human serum against blood group A, 0.1 M lactose, 0.1 M mannose, 0.05 M N-acetyl galactosamine, 0.05 M galactose or 15 microM asialofetuin. Prolonged incubation of RPK with alpha-galactosidase reduced RPK clearance. Oligosaccharide structures in RPK may have terminal galactose units since treatment of RPK with neuraminidase did not affect the clearance rate; RPK clearance occurs in the absence of added Ca2+, with either EDTA or EGTA in the perfusion fluid; the exponential phase is reversibly inhibited by the addition of NH4Cl or chloroquine to the perfusion fluid. This observation, along with experiments using liver homogenates, suggests that RPK catabolism is carried out by lysosomal enzymes, probably cathepsin B of possible hepatocyte origin.