This study was directed to use the genetically developed isoprenaline-sensitive (S), isoprenaline-resistant (R) and spontaneous hypertensive rats (SHR) as standard diseased animal models for in vitro liver function evaluation of drug biotransformation. Hepatic hexobarbital hydroxylase and glutathione transferase (GST) were evaluated by using hexobarbital and l-chloro-2,4-dinitrobenzene (CDNB) as substrates, at concentrations of 0.21 mmol/l and 1 mmol/l, respectively. The assay was conducted by using isolated hepatocytes in suspension and hepatocytes in a bioreactor configuration. The data demonstrate that there are certain cellular pharmacokinetic differénces in hexobarbital hydroxylase and GST activities in hepatocytes obtained from Wistar, SHR, R and S strains which can be better demonstrated, when using the model of perfused and immobilized hepatocytes.
The method of cellular immobilization and perfusion was applied to adipocytes. The lipolytic effect of isoprénaline, whose action is produced as a result of receptor-drug interaction, was followed. An agarose solution kept at at 37 °C was mixed 1:1 with the cell suspension. Thereafter, adipocytes were immobilized in the agarose threads. The lipolytic effect of 0.1 ml of isoprénaline (1x10~4 mol/1), that was rapidly introduced to the cell perfusion inlet in a non-recirculating system, was monitored by assessing glycerol production. The immobilized and perfused adipocytes exhibited significant lipolytic activity. After reaching the maximum effect, 0.1 ml of propranol (lxl 0-3 mol/1) that was applied to the bioreactor inlet, abolished the isoprénaline effect. The present data demonstrate the potential applicability of immobilized perfused adipocytes for various kinds of studies.
This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase 1 and phase II of biotransformation reactions. To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/1) and hexobarbital (0.2 mmol/1) were selected. For phase II reactions, glutathione transferase activity was evaluated by using l-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/1). Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time. The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension. At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension. The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension.
The content of phospholipids and their fatty acid composition were followed in the hearts of two inbred strains of rats: IR, resistant against the development of isoprenaline-induced myocardial lesions and IS, sensitive to their development. In the hearts of rats of the resistant strain, a lower content of phosphatidylcholine and its plasmalogen fraction was found compared to IS rats. The total amount of phospholipids was only insignificantly lower in IR rats. Greater differences were found in individual fatty acids. The most important finding concerned lower arachidonic acid and higher linoleic acid content in heart phospholipids of IR rats. These differences were exactly opposite to changes reported in the literature in animals known to have a higher resistance against myocardial damage due to various interventions. Our results do not support the hypothesis claiming the importance of changes in phospholipids and their FA composition for the resistance of the heart against the development of necrotic lesions.
The effects of the selective alpha2-adrenergic agonist p-aminoclonidine, the nonselective adrenergic agonist epinephrine, the selective beta2-adrenergic agonist fenoterol and the adenosine Ai agonist R-PLA on intraocular pressure were studied in control and pertussis toxin-pretreated rabbits. Pretreatment of rabbits with pertussis toxin decreased the ocular hypotensive effects of p-aminoclonidine and epinephrine, did not influence the same effects of fenoterol or R-PIA and markedly potentiated the initial ocular hypertensive effects of epinephrine and R-PIA. As far as the action on adenylyl cyclase in ciliary processes is concerned, isoproterenol stimulated its activity in control rabbits and epinephrine exerted dual, i.e. stimulatory and inhibitory effects on the activity of this enzyme. The data obtained with epinephrine and p-aminoclonidine confirm the view that their ocular hypotensive effects are associated with their inhibitory action on adenylyl cyclase and contradict the opinion that the hypotensive action of adrenergic drugs depends on adenylyl cyclase activation.
At present, the physiological role of NO * synthesis in the liver is ambiguous. Studies directed to reveal the role of NO * in relation to liver function were primarily initiated by an interest in the hepatic response to infections and the consequent modulation of liver function. The purpose of the present investigation was to use perfused rat hepatocytes to test the ability of the latter to produce NO * and to delineate the relationship between exogenously delivered NO* and any alteration in the degree of injury as produced by anoxia/reoxygenation (AR) or D-galactosamine (GalN, 5 mM) intoxication. NO * production in rats was stimulated by a single dose of lipopolysaccharide (LPS, 20 mg/kg i.p.) from which hepatocytes were isolated and perfused. Exogenous NO * was delivered to the perfusate of hepatocytes that were isolated from untreated rats, by the addition of sodium nitroprusside (SNP, 2 mM and 0.2 mM). AR and GalN hepatocyte injury was followed after the addition of SNP. Rat hepatocytes were immobilized in low-gelling agarose and perfused with Williams E medium. Endogenous synthesis of NO ’ and exogenous NO * as produced by SNP was evaluated by estimating the end products of NO * (N02" + N03“) in the perfusion medium. The functional and structural integrity of hepatocytes was evaluated from lactate dehydrogenase (LD) leakage and urea synthesis in the perfusion medium. Normal, AR- and GalN-injured hepatocytes did not exhibit measurable NO * while LPS-treated hepatocytes produced NO * (80 //M N02_ + N03_). SNP-produced NO * significantly increased or decreased LD leakage in AR at 2 mM or 0.2 mM, respectively, and also reduced or increased the rate of urea synthesis, respectively. 0.2 mM SNP increased trypan blue exclusion by hepatocytes. On the other hand, GalN toxicity was not significantly altered by SNP as demonstrated by LD leakage and the rate of urea synthesis was increased by SNP addition. The present data suggest both deleterious and beneficial role of NO * in AR liver injury model depending on the level of NO * generated.
After long-lasting administration of estradiol (4—6 weeks) in the presence or absence of pertussis toxin treatment we followed up the changes in body weight and adenohypophyseal weight in rats subjected to this treatment. The most striking effect was the potentiating effect of pertussis toxin on the estradiol-induced adenohypophyseal growth reaction. Adenylyl cyclase activity in the adenohypophysis was significantly increased in the estradiol- treated group and the addition of pertussis toxin did not further increase this enzyme activity. The lipolytic activity in adipose tissue exhibited a similar response as adenohypophyseal growth. Adrenergic lipolysis stimulated by pertussis toxin was highly significantly increased in tissues of rats treated with pertussis toxin. Our results show that the estrogen-induced adenohypophyseal growth reaction is highly potentiated by the treatment of rats with pertussis toxin and that this effect is in many aspects similar to that observed in adrenergic lipolysis. It thus seems that both processes might be mediated via a pertussis toxin-sensitive G protein which is involved in inhibitory regulation of adenylyl cyclase.
In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion. The rat hepatocytes are isolated from the liver. A 1.8 % low-gelling agarose solution is prepared in warm Krebs-Henseleit solution. The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing. Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix. This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver. Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals.
The present study was designed to investigate the ameliorative effect of cyclosporine A (CsA) pretreatment on an anoxia/reoxygenation injury model by using immobilized perfused hepatocytes. Rats received an i.p. injection of two successive doses of CsA (5 mg/kg/day). Twenty-four hours later hepatocytes were isolated from CsA-treated and control rats. After hepatocyte isolation, immobilization, perfusion, induction of anoxia/reoxygenation, the structural and functional integrity of the hepatocytes was followed in a perfusion medium by measuring the leakage of lactate dehydrogenase (LD) and the time course of urea biosynthesis. CsA pretreatment reduced the initial rate of urea synthesis during normoxia but reduced the drop in the relative percentage rate of urea synthesis during the period of anoxia. LD leakage was increased threefold by anoxia and sevenfold by reoxygenation in cells of untreated animals. After CsA pretreatment in vivo, hepatocytes showed no increase in LD leakage into the medium. These findings demonstrate that the perfused immobilized hepatocytes can be used as a cellular model to assess the effects of liver insults such as anoxia/reoxygenation injury and that CsA modulates the injury. The mechanisms of CsA beneficial effects at the experimental level remain to be elucidated.