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Biomedical subjects

J Mandl

Publications and source records attributed to J Mandl.

At least 73 records · Page 4Linked to original sources
PubMed1988

Cyclic AMP-dependent phosphorylation in the control of biotransformation in the liver.

The possibility of a short-term cAMP-dependent regulation of mixed-function oxidation and of glucuronide formation was investigated in isolated mouse hepatocytes and in mouse liver microsomal membranes. N6, O2-dibutyryl cAMP (in accordance with its increasing effect on gluconeogenesis) decreased aminopyrine oxidation and p-nitrophenol conjugation in isolated hepatocytes, while the phenolphthalein conjugation remained unaltered. Similar to dibutyryl cAMP the Ca2+ ionophore A 23187 also decreased aminopyrine oxidation. In cell-free systems the phosphorylation of isolated microsomal membranes by the exogenous cAMP-dependent protein kinase was inhibitory on aminopyrine oxidation and p-nitrophenol glucuronide formation but aniline oxidation and phenolphthalein glucuronidation were not affected. The correlation between the negative cAMP-dependent control of certain processes of biotransformation and the positive cAMP-dependent regulation of gluconeogenesis is discussed.

Aminopyrine↗
PubMed1988

Interrelationship between drug oxidation ureogenesis and gluconeogenesis in isolated hepatocytes.

1. The effect of increased ureogenesis--provoked by NH4Cl and ornithine--on gluconeogenesis and aminopyrine oxidation was studied in isolated hepatocytes prepared from 24 hr starved mice; lactate or fructose was used as gluconeogenic precursor. 2. Increased ureogenesis caused about 40% inhibition both on aminopyrine oxidation and gluconeogenesis when lactate was added as gluconeogenic substrate. 3. On the other hand, only 10% inhibition of aminopyrine oxidation and about 15% inhibition of gluconeogenesis were observed when fructose was used as gluconeogenic precursor. 4. Aminopyrine has been reported to inhibit gluconeogenesis from fructose by 30% and from lactate by 85%. The inhibitory effect of the combined addition of aminopyrine, NH4Cl and ornithine on gluconeogenesis was also dependent on the applied gluconeogenic precursor. 5. The provoked ureogenesis by ammonia and ornithine was not inhibited by aminopyrine. N6, O2-dibutyryl cAMP known to cause an increase of gluconeogenesis a decrease of aminopyrine oxidation enhanced the inhibitory action of increased ureogenesis on aminopyrine oxidation and on gluconeogenesis further. 6. The role of NADPH in the regulation of drug oxidation and ureogenesis is underlined.

Aminopyrine↗
PubMed1988

cAMP dependent inhibition of thromboxane A2, prostacyclin and PGF2 alpha synthesis in mouse hepatocytes.

The role of cAMP dependent regulation in thromboxane A2, prostacyclin and PGF2 alpha synthesis (measured by radioimmunoassay) was investigated in isolated mouse hepatocytes and in microsomal membranes prepared from these cells. In isolated hepatocytes N6,O2-dibutyryl cAMP inhibited the formation of all the three derivatives, while calcium ionophore A 23187 stimulated their synthesis. Addition of the dissociated catalytic subunit of cAMP dependent protein kinase and ATP to microsomal membranes inhibited the production of TXA2, PGI2 and PGF2 alpha by about 50% and this inhibition was counteracted by the combined addition of heat stable inhibitor protein of cAMP dependent protein kinase. It is concluded that in parenchymal liver cells cAMP dependent phosphorylation is directly involved in the inhibition of prostanoid synthesis.

Animals↗
PubMed1987

Proteolytic activation of protein kinase C in the extracts of cells treated for a short time with phorbol ester.

A 10 min treatment of human neutrophils with phorbol 12-myristate 13-acetate (PMA) has been reported to induce accumulation of the proteolytically activated Ca2+/phospholipid-independent catalytic fragment of protein kinase C in the cytosol of intact cells [(1986) J. Biol. Chem. 261, 4101-4105]. We investigated the proteolytic conversion of protein kinase C to Ca2+/phospholipid-independent form in the cytosol and membrane fractions of pig neutrophils. The activity of protein kinase C was measured with its specific oligopeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously. In our experiments the short-term treatment of neutrophils with PMA did not induce the accumulation of the proteolytically activated form of protein kinase C in the cytosol of intact cells. However, treatment of cells with PMA enhanced the limited proteolysis of protein kinase C during the preparation of cell extracts.

Animals↗
PubMed1987

Interrelationship between aminopyrine oxidation and gluconeogenesis in hepatocytes prepared from fructose-pretreated mice.

Aminopyrine oxidation was studied in isolated hepatocytes prepared from 24-h-starved mice (i) after induction of the NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase, but not the mixed function oxygenases by fructose, (ii) after induction of both mixed function oxygenases and NADPH-generating malic enzyme and glucose-6-phosphate dehydrogenase by phenobarbital and (iii) without any pretreatment. Phenobarbital pretreatment, as expected, increased the rate of aminopyrine oxidation of isolated hepatocytes. However, fructose pretreatment also enhanced the rate of N-demethylation of aminopyrine by more than 100% supporting the view that the availability of NADPH is rate limiting in drug oxidation under certain conditions. The role of malic enzyme and glucose-6-phosphate dehydrogenase in the NADPH supply for aminopyrine oxidation was investigated by the addition of two groups of gluconeogenic precursors: lactate or alanine and glycerol or fructose with the simultaneous measurement of glucose synthesis and aminopyrine N-demethylation. There was a clear correlation between the increased rate of aminopyrine oxidation and the decreases of glucose production caused by aminopyrine. Gluconeogenesis in the presence of 1 mM aminopyrine was decreased by 70-80% when alanine or lactate were used as precursors, it was decreased by only 35-40% when glucose production was started from glycerol or fructose; in an accordance with the facts that NADPH generation and gluconeogenesis starting from alanine or lactate share two common intermediates--malate and glucose-6 phosphate--, while there is only one common intermediate--glucose-6 phosphate--if fructose or glycerol are used. Similar results were obtained with the addition of the structurally dissimilar hexobarbital. It is concluded that besides malic enzyme, glucose-6-phosphate dehydrogenase also takes part in NADPH supply for drug oxidation in glycogen-depleted hepatocytes.

Aminopyrine↗
PubMed1987

Prostanoid synthesis in isolated parenchymal and nonparenchymal mouse liver cells in the presence of arachidonic acid.

Prostanoid synthesis was investigated in suspensions of isolated mouse hepatocytes and nonparenchymal liver cells. A stable metabolite of thromboxane A2 (TXB2) of prostacyclin (6-keto PGF1 alpha) and one of the prostaglandins (PGF2 alpha) was detected by radio-immuno-assay (RIA). Hepatocytes synthesized mainly TXB2, while smaller amounts of 6-keto PGF1 alpha and PGF2 alpha were detected during 60 min incubation. Homogenization of hepatocytes caused a slight increase of TXB2 production and provoked the synthesis of PGF2 alpha and 6-keto PGF1 alpha. The addition of arachidonate to hepatocytes did not influence prostanoid production at concentrations below 10-5M. Higher concentrations further increased TXB2 production and also increased the synthesis of 6-keto PGF1 alpha and PGF2 alpha. Nonparenchymal cells synthesized all the three types of prostanoids and homogenization of these cells did not result in a marked change. The addition of 10(-7)-10(-5)M arachidonate increased the TXB2, 6-keto PGF1 alpha and PGF2 alpha synthesis in nonparenchymal cells. No further increase was found at higher concentrations.

Animals↗
PubMed1987

The effect of the cAMP-dependent protein kinase on protein phosphorylation and phosphatidylinositol 4-phosphate formation in a hepatocyte membrane preparation.

Plasma membrane preparation obtained from isolated mouse hepatocytes was phosphorylated by exogenous cyclic AMP dependent protein kinase in the presence of 32P-ATP. The phosphorylated proteins were analysed by SDS-polyacrylamide gel-electrophoresis of the membrane and the phosphorylated lipids were analysed by thin layer chromatography of the separated lipid fraction. The protein kinase stimulated the 32P incorporation into phosphatidylinositol 4-phosphate and it catalyzed the phosphorylation of several proteins. The main phosphoprotein bands were found at 51 kDa, 49 kDa, 46 kDa and 34 kDa. A part of the 51 kDa phosphoprotein accompanied the lipids in the course of the separation of the lipid fraction.

Animals↗
PubMed1987

Inhibitory effect of A 23187 on protein synthesis in isolated murine hepatocytes.

The effect of the Ca ionophore A 23187 on amino acid incorporation into protein, on gluconeogenesis and on synthesis of some eicosanoids was investigated in isolated mouse hepatocytes (i) in the presence of 2.5 mM Ca2+, (ii) in the absence of Ca2+ and in the presence of 0.2 mM EGTA, (iii) after calcium depletion. A 23187 inhibited amino acid incorporation into protein both in the presence and in the absence of Ca2+ in a concentration dependent manner, whereas in the absence of Ca2+ the known stimulating effects of A 23187 on eicosanoid synthesis could not be detected. In hepatocytes prepared from 24 h starved mice A 23187 increased the rate of gluconeogenesis and similarly to eicosanoid synthesis this stimulation in the absence of Ca2+ could not be observed. On the other hand, after calcium depletion the inhibition of protein synthesis by A 23187 was markedly moderated. It is concluded that the effect of A 23187 on protein synthesis is also related to its ionophoretic effect but calcium sensitivity in this effect is smaller than in gluconeogenesis or eicosanoid synthesis.

Animals↗
PubMed1985

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes.

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.

Animals↗
PubMed1985

Effect of linear gramicidin on sporulation and intracellular ATP pools of Bacillus brevis.

When Bacillus brevis ATCC 8185 was subjected to nutritional shiftdown from a rich medium to one completely devoid of a nitrogen source, sporulation could be stimulated by the addition of linear gramicidin. Gramicidin-induced sporulation occurred after a considerably longer lag period than the earlier described tyrocidine-induced process (Ristow and Paulus 1982) but involved similar associated biochemical changes, such as extracellular protease production, rapid incorporation of radioactive precursors into RNA, and dipicolinate synthesis. The increased incorporation of [3H]leucine into tyrocidine was a characteristic element in gramicidin-induced sporulation, not being observed when spore formation was accelerated by limited nitrogen supplementation. Nitrogen supplementation (0.02-0.01% nutrient broth) caused a slow and gradual increase in dipicolinate production, in contrast to the sudden, rapid rise of dipicolinate synthesis provoked by the addition of gramicidin or tyrocidine. The induction of sporulation by gramicidin occurred at very low peptide concentrations (0.03 microM), which also brought about an acute depletion of intracellular ATP. In sporulation accelerated by nutrient broth, no depression of ATP level was observed and nonionophoric analogues of gramicidin were unable to substitute for gramicidin in inducing sporulation.

Adenosine Diphosphate↗
PubMed1984

Uptake of arachidonic acid, arachidic acid, oleic acid and their incorporation into phospholipids and triacylglycerols of isolated murine hepatocytes. Effect of thrombin-antithrombin III complex.

Uptake and metabolism of arachidonic acid, arachidic acid and oleic acid were investigated in isolated hepatocytes prepared from mouse liver with the collagenase perfusion method. The rate of uptake of arachidonic acid was time- and concentration- dependent. 94-98% of the arachidonic acid was incorporated into the phospholipid and triacylglycerol fractions following a 60 min incubation period at 37 degrees C. In the presence of thrombin-anti-thrombin III complex a change in the distribution of arachidonic acid incorporated into lipid fractions was found, i.e. increased incorporation into phosphatidyl-serine and phosphatidylethanolamine, whereas the uptake was not altered. There was no change in the uptake and incorporation of arachidic acid and oleic acid.

Animals↗
PubMed1984

Prostaglandin and thromboxane synthesizing activity in isolated murine hepatocytes and nonparenchymal liver cells.

Prostanoid synthesis from 3H-arachidonic acid was compared in isolated parenchymal and nonparenchymal murine liver cells. The cells incorporated arachidonic acid into phospholipids but no prostanoid synthesis could be measured during 30 min incubation. Conditions necessary for prostanoid synthesis were different in parenchymal and nonparenchymal cells and the products were also different. Prostanoid synthesis could be induced by in vitro partial hepatectomy: parenchymal cells synthesized thromboxane A2 whereas nonparenchymal cells produced prostaglandin E2 and F2 alpha. Prostaglandin E2 and F2 alpha synthesis could be provoked also by homogenization of the nonparenchymal cells prepared from normal liver, while the homogenates of parenchymal cells prepared from normal liver did not synthesize thromboxane. Imidazole and indomethacin inhibited the production of thromboxane and prostaglandins, respectively. Our results suggest that the various cell types of the liver respond by the synthesis of different and specific prostanoids after the same injury.

Animals↗
PubMed1983

Ethanol potentiates the inhibitory effect of D-galactosamine on protein synthesis in isolated hepatocytes.

The effect of the combined addition of D-galactosamine and ethanol on hepatic protein synthesis was studied in isolated mouse hepatocytes. 2.5 mM D-galactosamine or 40 mM ethanol alone caused slight or no inhibition of amino acid incorporation into proteins. However, a profound inhibition (about 80%) was observed if D-galactosamine of the same dose was added after a preincubation of the cells with 40 mM ethanol and vice versa. It shows that there is a strong mutual potentiation between D-galactosamine and ethanol in the inhibition of protein synthesis.

Animals↗
PubMed1982

Specific binding of thrombin-antithrombin III complex to hepatocytes.

Thrombin-antithrombin III complex binds selectively to isolated hepatocytes, whereas antithrombin III alone does not. The binding is time and concentration dependent at 37 degrees C: the apparent Km value is 0.8/microM. The rate of binding is approximately 1.6 X 10(5) molecules h-1 cell-1 at this concentration. At 4 degrees C there is no measurable interaction between the complex and the hepatocytes. The binding is also prevented by pretreatment of cells with trypsin. On the other hand, about 80% of the thrombin-antithrombin III complex bound to hepatocytes is releasable by trypsin digestion. NaF or carboxyatractyloside does not inhibit the process. The interaction of thrombin-antithrombin III complex with hepatocytes seems to be specific, since the complexes of antithrombin III with other proteinases, like trypsin or plasmin, are not bound at the concentrations used. Based on these data, a mechanism for the binding of the inactive complexed form of thrombin to hepatocytes is suggested.

Animals↗
PubMed1982

Reversible inhibition of RNA synthesis and irreversible inhibition of protein synthesis by D-galactosamine in isolated mouse hepatocytes.

The inhibition of RNA synthesis of isolated mouse liver parenchymal cells caused by 10 mM D-galactosamine was reversible, while the inhibition of protein synthesis remained unaltered after the removal of galactosamine. 10(-5)M epinephrine and 10(07)M glucagon have been shown to decrease aminoglycogen formation and thus to reduce the inhibitory effect of galactosamine on protein synthesis (II). However, these hormones did not decrease the inhibition of RNA synthesis. 10 mM D-galactosamine did not effect the nucleoside and amino acid incorporation of isolated non-parenchymal mouse liver cells. The predominant role of aminoglycogen in the inhibition of protein synthesis in galactosamine induced liver injury is discussed.

Animals↗