Components of the kallikrein-kinin system in rat urine

Using a direct radioimmunoassay and a kininogenase assay, we determined that 68% of rat urinary kallikrein was enzymatically active while 32% was in an inactive form which was activated by trypsin. Inorganic cations, at concentrations found in rat urine, were inhibitory in an amidase assay but appeared to potentiate kininogenase activity of pure rat urinary kallikrein. In random urines, kinin concentration was 4.2 +/- 0.7 ng/ml. Trypsinization of the urines generated 52.9 +/- 25.8 ng kinin/ml, indicating that kininogen was present. The rate of kinin formation in vivo may depend on the availability of kininogen and the concentration of inorganic cations in urine, as well as on other well-recognized factors, such as the kallikrein activity of the urine.     

Direct measurement of aminopyrine N-demethylase and antipyrine hydroxylase activities in a monolayer rat primary isolated hepatocyte system

This paper describes a simple method for monitoring changes in aminopyrine N-demethylase and antipyrine hydroxylase activities in isolated primary hepatocyte monolayer culture. Aminopyrine N-demethylase activity was determined by monitoring the rate of formation of 14CO2 derived from the N-demethylation of [dimethylamino-14C]aminopyrine (AP). The rate of AP N-demethylation increased linearly with time for 60 min and proportionately with cell concentrations between 4.1 x 10(5) to 1.67 x 10(6) cells/incubation. As expected, non-linear AP N-demethylase kinetics were observed with hepatocytes as well as with microsomal preparations derived from control rats. Hepatocytes prepared from phenobarbital (PB)-pretreated animals exhibited increased AP N-demethylase activity and typical Michaelis-Menten kinetics. In contrast, microsomal preparations from PB-treated animals exhibited non-linear N-demethylase kinetics that differed from the kinetics of preparations derived from control animals. Antipyrine hydroxylase activity was determined by monitoring the rate of formation of non-extractable conjugated 4-hydroxyantipyrine from [N-14C-methyl]antipyrine. Antipyrine hydroxylase activity was found to increase linearly for 120 min and proportionately with cell concentrations. Antipyrine hydroxylation by hepatocytes prepared from control and PB-pretreated animals followed typical Michaelis-Menten kinetics. AP N-demethylase activity immediately after plating was 10 per cent lower than at 4 hr, whereas antipyrine hydroxylase activities were similar. Culturing hepatocytes for 24 hr resulted in a decline to 40 and 60 per cent of control for AP N-demethylase activity and antipyrine hydroxylase activity respectively.     

Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung

Acute hypoxia in the intact animal and in cultured endothelial cells has been shown to be associated with a decrease in conversion of angiotensin I (AI) to angiotensin II (AII). Alterations in capillary surface and in contact time resulting from hemodynamic changes have been shown to influence the rate of pulmonary AI conversion. The dependency of AI conversion on hemodynamics complicates the interpretation of experiments showing changes in AI conversion in intact animals. We studied the effect of acute hypoxia on AI conversion in the isolated rat lung perfused at constant flow without recirculation of perfusate. Three levels of oxygenation were produced by ventilating lungs and equilibrating perfusate with a range of hypoxic gas mixtures. AI (1 μg) was injected into the pulmonary artery, and the effluent was collected for measurement of AI and All. Instead of the expected hypoxic inhibition, percent conversion of AI to All increased slightly but significantly from 69.3 ± 3.1 (mean ± S.E.M.) at normal oxygenation to 74.4 ± 3.0 at moderate hypoxia (P < 0.005, paired t) and to 73.5 ± 3.9 at severe hypoxia (P < 0.01, paired t). Decreasing mean transit time of substrate through the lung (by increasing perfusate flow rate from 5 to 20 ml/min) resulted in a significant decrease in conversion of AI from 88.7 ± 2.9 to 73.4 ± 2.1% (P < 0.001, paired t). These data confirm the effect of contact time on the rate of AI conversion in the lungs. The isolated rat lung preparation does not exhibit the phenomenon of hypoxia-induced inhibition of AI conversion. The authors speculate that hypoxia-induced inhibition of AI conversion in vivo may be secondary to the effects of hypoxia on hemodynamics.     

Conjugation kinetics of acetaminophen by the perfused rat liver preparation

Sulfate conjugation of acetaminophen was studied in the perfused rat liver preparation. Varying input concentrations of unlabeled acetaminophen (0.22 to 6.0 μg/ml or 1.5 to 40 μM) were added in a stepwise fashion once-through the liver preparation at a flow rate of 10 ml/min. For the concentration range tested, acetaminophen sulfate conjugate remained the only detectable metabolite in perfusate plasma. Other metabolites such as the glucuronide and the glutathione conjugates, however, were detected only in bile and, together with the sulfate conjugate and unconjugated acetaminophen, constituted 5 per cent of the total administered dose. Hepatic elimination of acetaminophen in the formation of sulfate conjugate was apparently maximal at input concentrations ? 1.0 μg/ml (6.7 μM) and could be viewed as mediated via a uni-enzyme system. Sulfate conjugation also decreased with preloading of the liver, especially with high concentrations of acetaminophen. A plot of the ratio of the steady-state output concentrations of drug to metabolite versus the reciprocal of drug extraction ratio, which might prove useful in the prediction of metabolite concentrations in the liver, was introduced.     

Role of red cell membrane lipid peroxidation in hemolysis due to phenylhydrazine

Although red cell membrane lipid peroxidation has been identified as a consequence of certain oxidizing hemolytic drugs, the relative contribution of lipid peroxidation to red cell damage leading to hemolysis is unclear. This has been evaluated by studying the response to phenylhydrazine of vitamin E-deficient rats as compared to vitamin E-supplemented rats. Following repetitive phenylhydrazinc injections, a lower hematocrit was observed in the vitamin E-deficient group which was associated with higher levels of lipid peroxidation, as indicated by the fluorescence of lipid-containing red cell extracts. However, no significant difference in the initial extent of hemolysis following phenyl-hydrazine injection was observed. Evidence was also obtained suggesting that malonaldehyde, a decomposition product of polyunsaturated fatty acids, is capable of cross-linking hemoglobin to the red cell membrane. These findings suggest that red cell membrane lipid peroxidation is of relatively minor consequence in the acute response to phenylhydrazine but may be of importance in chronic hemolysis due to this oxidizing drug.     

Effect of selenium on glutathione metabolism. Induction of gamma-glutamylcysteine synthetase and glutathione reductase in the rat liver

The effect of sodium selenite (Na₂SeO₃, Se) on cellular glutathione metabolism was examined, particularly with respect to its ability to alter the activities of γ-glutamylcysteine synthetase and glutathione disulfide (GSSG) reductase. The treatment of rats with Se (5, 10 and 20 μmoles/kg) caused time- and dose-dependent increases in the activities of the synthetase and the reductase in the liver. The activity of γ-glutamylcysteine synthetase, the rate-limiting enzyme of the glutathione (GSH) biosynthesis, was particularly susceptible to Se treatment. The Se-mediated increases in the activities of the above enzymes were inhibited by puromycin and the increases could not be elicited in vitro. Selenium treatment caused time-dependent perturbations in the levels and ratio of GSSG and GSH in the liver. When compared to the control animals, rats treated for 3 hr with 10 and 20 μmoles Se/kg showed increased cellular levels of GSSG; in contrast, 24 hr after Se treatment the concentration of GSH was increased significantly. The activity of γ-glutamyl transpeptidase, which catalyzes the initial reaction in GSH breakdown, was unaltered by Se treatment. Repeated administration of low doses of Se (7.0 μmoles/kg, three times) also increased the activities of the reductase and the synthetase as well as the cellular levels of hepatic GSH and GSSG. It is suggested that the Se-mediated increases in the activities of γ-glutamylcysteine synthetase and GSSG-reductase represent cellular responses to Se-mediated perturbations in the levels and ratio of GSH and GSSG.     

Correction of drug binding defects in uremia in vitro by anion exchange resin treatment

Serum protein binding of weakly acidic drugs is impaired in uremia, but that of basic drugs tends to be normal. Treatment of uremic serum with anion exchange resin (Amberlite CG-400, acetate form) corrected binding defects for three acidic drugs (nafcillin, salicylate and sulfamethoxazole) but did not affect the binding of two basic drugs (trimethoprim and quinidine). Resin treatment of normal human serum did not alter the binding of these five drugs. Extraction of the acetate buffer eluate from resin exposed to uremic serum with n-butyl chloride at acidic pH (3.0) resulted in a fraction that could induce similar binding defects in normal human serum. The factor(s) responsible for binding defects in uremia appears to be lipid soluble, weakly acidic, and dialyzable. It is believed to be tightly bound to albumin at physiologic pH, but dissociates from it at acidic pH. These findings further support the previously proposed hypothesis that drug-binding defects in uremia are due to accumulation of certain endogenous metabolic product(s).     

Factors influencing halothane hepatotoxicity in the rat hypoxic model

Halothane, a widely used inhalation anesthetic, was shown to be hepatotoxic to male, phenobarbital-pretreated rats, only when administered under hypoxic conditions (fraction of inspired oxygen = 0.14). The degree of hepatotoxicity as determined from morphological alterations and serum glutamic-pyruvic transaminase (SGPT) activities, correlated well with concentrations of hepatic cytochrome P-450 and concentration of inspired halothane. Maximal lesion intensity developed within 12 to 24 hr after exposure to 1% halothane for as little as 30 min. By 4 days after exposure, the liver had repaired, since no morphological alterations were apparent and SGPT activities had returned to normal values. Female rats, when pretreated with phenobarbital and exposed to 1% halothane under hypoxic conditions did not develop liver injury. SKF-525A and metyrapone reduced the severity of liver injury when administered preanesthesia and 4 hr postanesthesia. The free sulhydryl-containing compounds, cysteine, cystamine, and N-acetylcysteine afforded protection when administered at 4 or 8 hr (cystamine) after ending anesthesia. These results support the hypothesis that reductive or noxoxygen-dependent biotransformation of halothane results in toxic intermediates that can initiate halothane-induced liver injury.     

Impaired drug metabolism in experimental cirrhosis in the rat

Cirrhosis was produced in the rat by chronic administration of phenobarbital and carbon tetrachloride for 10 weeks. The metabolism of three substrates, aminopyrine. hexobarbital and propranolol, has been investigated in a 9000 g supernatant fraction of liver homogenate and in intact hepatocytes in cirrhotic livers and compared to appropriate phenobarbital-treated controls. In the 9000 g supernatant preparation, the maximal velocity of metabolism for each substrate and the cytochrome P450 concentration were reduced significantly in cirrhotic livers, while total protein and DNA concentration remained unchanged. In intact hepatocytes, the V_maxfor each substrate was reduced, while the apparent K_m remained unchanged. In both in vitro systems, the metabolism of propranolol and of hexobarbital was influenced by cirrhosis to a greater extent than that of aminopyrine. It is concluded that the carbon tetrachloride-phenobarbital model of cirrhosis in the rat is a suitable model in which to study the effects of chronic liver disease on drug disposition.     

Potentiation of the hepatotoxicity of carbon tetrachloride following preexposure to chlordecone (kepone) in the male rat.

The effect of carbon tetrachloride (CCl₄) challenge on several parameters of hepatic injury was determined in chlordecone (CD)-pretreated male rats. Following a 15-day feeding of 0 or 10 ppm CD, rats received a single intraperitoneal challenge of 0, 25, 50, 100, or 200 μl CCl₄/kg. Twenty-four hours after CCl₄, biliary excretion of phenolphthalein glucuronide (PG), bile secretion, and serum transaminase (SGPT, SGOT) activities were examined as functional indices of hepatic injury. Feeding of 10 ppm CD alone resulted in decreased biliary excretion of PG (59% of control) but had no effect on bile flow or on serum transaminases. Twenty-four hours after CCl₄ alone, the two high doses caused decreased biliary excretion of PG in the absence of any effect on bile flow and doubled serum transaminases at the highest dose (200 μl/kg). A single challenge of CCl₄ to CD-fed rats resulted in a dose-dependent impairment of PG excretion at a dose of 50 μl/kg and higher. Bile secretory function was severely impaired in the CD-fed animals receiving CCl₄ at the doses of 100 and 200 μl/kg. Decreased bile flow was not seen in any other groups. Greatly potentiated hepatotoxicity was reflected in the form of elevated SGPT and SGOT activities which increased in excess of 30- and 10-fold, respectively, in CD-fed rats challenged with CCl₄ at 100 and 200 μl/kg. Parenchymal liposis (cytoplasmic sudanophilic droplets) developed in all CD-fed rats receiving CCl₄, while necrosis occurred after CCl₄ at 50 μl/kg and increased in a dosedependent manner. CCl₄ controls exhibited parenchymal liposis and limited centrilobular necrosis only at the two highest doses of CCl₄. These data indicate a great potential for production of severe liver damage resulting from interactions of CCl₄ and CD exposure at levels which may be independently nontoxic.     

Effect of cimetidine on δ-aminolevulinic acid synthase and microsomal heme oxygenase in rat liver

Cimetidine is a well known inhibitor of the heme-containing enzyme cytochrome P-450. We have found that it also inhibits δ-aminolevulinic acid synthase (ALA-S) and microsomal heme oxygenase, the rate-limiting enzymes for heme synthesis and heme degradation respectively. Cytochrome P-450 content was decreased but microsomal heme concentration remained unaltered for a period of 30 min after in vivo cimetidine administration to rats. In vitro incubation of cimetidine with each of the above enzymes revealed no direct effect of cimetidine on ALA-S but about 50% inhibition of heme oxygenase and 20% reduction in cytochrome P-450 content. This suggests that a metabolite of cimetidine inhibits ALA-S activity in vivo, while the drug itself or a metabolite inhibits heme oxygenase both in vivo and in vitro. A rise in ALA-S activity seen after its early inhibition and its return to approximate control values after 60 min suggest a reversible inhibition of ALA-S by a metabolite of cimetidine and may correspond to its clearance from the animal. An elevation in microsomal heme content paralleled the rise in ALA-S activity while microsomal heme oxygenase activity returned to only 65% of control value 60 min after cimetidine treatment. Cytochrome P-450 content did not change after its initial decrease, suggesting that irreversible alteration had occured.     

Studies on the mechanism of phospholipid storage induced by amantadine and chloroquine in Madin Darby canine kidney cells

Previous studies suggested that hepatic lipidosis caused by cationic amphiphilic drugs in rats is related to the capacity of these drugs to concentrate in liver lysosomes. These drugs inhibit lysosomal phospholipases, causing phospholipid accumulation. Amantadine, an inhibitor of influenza A virus replication, is a cationic amphiphilic drug which concentrates in the lysosomes of the Madin Darby canine kidney (MDCK) cell. In the present study, amantadine and chloroquine were shown to inhibit soluble lysosomal phospholipases isolated from MDCK cell in. vitro. Both amantadine and chloroquine concentrated in MDCK cell lysosomes. These drugs caused phospholipid storage in cultured MDCK cells, and the amounts of the respective agents required to cause phospholipid storage correlated with the capacities of the agents to inhibit lysosomal phospholipases. The mechanisms involved in this phenomenon are discussed, and a three-step hypothesis is presented predicting which agents will cause phospholipidosis.     

α1-acid glycoprotein involvement in high affinity binding of tricyclic antidepressants to human plasma

The binding of the tricyclic antidepressants imipramine (IMI) and desmethylimipramine (DMI) to human plasma and individual proteins was studied by equilibrium dialysis. Both drugs bound extensively to plasma, albumin, and α₁-acid glycoprotein, while there was very little binding to the γglobulin fraction. The binding of both IMI and DMI to α₁-acid glycoprotein was high affinity (association constant K, 9.2 × 10⁴/M and 4.7 × 10⁴/M respectively) and low capacity (number of binding sites, n = 1 for both IMI and DMI), whereas the binding to albumin was low affinity (K for IMI, 2.3 × 107²/M and for DMI, 3 × 10²/M) and high capacity (n = 7). The binding of IMI to a mixture of human serum albumin and α₁-acid glycoprotein revealed two sets of binding sites; a high affinity binding site corresponding to α₁-acid glycoprotein and a low affinity binding site corresponding to albumin. The binding affinity and/or number of binding sites for IMI binding to albumin decreased with increasing albumin concentrations. The free fraction in plasma of nineteen normal, male controls was significantly correlated with the concentration of α-acid glycoprotein (r = 0.601, P < 0.01), although there was no correlation with albumin or free fatty acid concentrations in plasma.     

Differential effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism in the rat

The effects of chronic ethanol feeding on cytochrome P-448- and P-450-mediated drug metabolism have been studied both in vivo and in vitro in the rat, using caffeine, phenacetin, antipyrine and aminopyrine as test substrates. N-Demethylation of aminopyrine (P-450 mediated) was increased both in vivo and in vitro in rats after chronic ethanol feeding (P < 0.05) whereas in vivoN-demethylation of caffeine and O-dealkylation of phenacetin (P-448 mediated) were unchanged in the same animals. N-rmDemethylation of antipyrine was increased by both phenobarbital and 3-methylcholanthrene pretreatment and by chronic ethanol feeding (P < 0.05), possibly due to cytochrome P-450 induction. Furthermore, the Michaelis affinity constants. K_m, for hepatic microsomal aminopyrine N-demethylase and antipyrine N-demethylase were lower in chronic ethanol-fed animals (P < 0.05), suggesting a qualitative change in the enzymes resulting in greater substrate affinity. These findings suggest a differential effect of chronic ethanol feeding on the induction of cytochrome P-450- and cytochrome P-448-mediated drug metabolism, with a greater effect on the former microsomal system.     

Toxicity of heparin in isolated rat hepatocytes

The effect of heparin on isolated rat hepatocytes in monolayer culture was assessed to investigate the observed increase in serum aminotransferase activity in patients treated with heparin for thromboembolic disorders. Cells were treated with porcine intestinal mucosal heparin or beef lung heparin in concentrations ranging from 0.01 to 100 units/ml. Toxicity was evaluated based on cell damage or death measured by LDH release into the culture media as a fraction of total system LDH (LDH index). Toxicity appeared at concentrations between 1 and 10 units/ml (P less than 0.05). The uptake and binding of heparin by the hepatocyte were evaluated by addition of tritium-labeled heparin to the cultures. Sucrose gradient centrifugation with isolation of the liver plasma membranes (LPM) showed little membrane binding of heparin. The majority of intracellular heparin was located in the cytosol fraction. Heparin gains access to hepatocytes and causes a dose-related toxic effect resulting in cell damage and death. This investigation indicates that the increased serum aminotransferase concentrations seen with heparin treatment may be due to a direct hepatotoxic effect of heparin.