Incubation Of Rat Hepatocytes Research Articles

Extracellular calcium effects on cell viability and thiol homeostasis.

Studies of chemically induced cell injury and death, which have used as model systems freshly isolated rat hepatocytes and hepatocytes in culture, are discussed. An important model uses the omission of Ca2+ from the medium during rat hepatocyte incubations. Ca2+ omission induces an intense oxidative stress within hepatocytes incubated in a 95% O2 + 5% CO2 atmosphere. The relationship of calcium homeostasis to the parameters of oxidative stress is important to understanding the progression from reversible to irreversible injury. In the Ca2+ omission model, the vitamin E (Vit. E) content of hepatocytes is important for the prevention of cell injury. Recent studies with rat hepatocytes show that ruthenium red (RR) and La3+, which block Ca2+ translocation through the mitochondrial uniport, can prevent malondialdehyde (MDA) formation, reduced glutathione (GSH), and protein-SH loss, Vit. E loss, and LDH leakage induced by Ca2+ omission from the incubation medium. Ca2+ omission promoted a marked loss of mitochondrial transmembrane potential (delta phi) that was prevented by RR, EGTA, Vit. E, and desferrioxamine. The absence of extracellular Ca2+ may cause mitochondrial Ca2+ cycling that contributes to the observed oxidative stress, resultant loss of cell viability, and protein thiol homeostasis. Chemical agents including a glutathione-depleting agent, ethacrynic acid, and a redox cycling agent, adriamycin, increase the loss of cell viability caused by a Ca2(+)-free medium, but they have some additional effects on cellular processes. The demise of cell viability by the agent is also preventable by Vit. E supplementation. Ca2+ has a role in cell injury that appears to uniquely involve mitochondrial homeostasis.

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A 2

The effect of phospholipase A 2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membranebound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A 2 (0.9 units /dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 ± 0.15 nmol/min per mg compared to 3.9 ± 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units /dish of phospholipase A 2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 ± 0.10 nmol/min per mg to 2.6 ± 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 ± 0.16 nmol/min per mg to 1.8 ± 0.20 nmol/min per mg). The effect of phospholipase A 2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepaotcytes in the presence of phospholipase A 2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 ± 0.02· 10 −5 dpm/dish to 3.1 ± 0.1 · 10 −5 dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 ± 0.05 · 10 −5 to 1.4 ± 0.03 · 10 −5 dpm) and phosphocholine fractions (from 8.5 ± 0.07 · 10 −5 to 6.9 ± 0.05 · 10 −5 dpm). We conclude that phospholipase A 2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.

Identification of a cholesterol‐regulated 180‐kDA microsomal protein in rat hepatocytes

The immunoprecipitation by antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of extracts of [35S]methionine-pulse-labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97-kDa HMG-CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2-mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180-kDa/97-kDa proteins. These facts suggest that the 180-kDa protein is not an aggregated form of HMG-CoA reductase. A different batch of antibodies obtained from a newly purified HMG-CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180-kDa protein, showing that there is no cross-reactivity between these proteins. The 180-kDa polypeptide is a glycoprotein of N-linked high-mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa. The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180-kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate-free 164-kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180-kDa protein is not a modified form of 97-kDa reductase, but probably a different protein related to cholesterol metabolism, and also that the N-linked, high-mannose chains, which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.

Phosphorylation of Native 97-kDa 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase from Rat Liver: Impact on Activity and Degradation of the Enzyme

Abstract Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the linker segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.

The metabolism ofN, N-Dimethylaniline by Isolated Rat Hepatocytes: Identification of a NovelN-Conjugate

1. Incubation of N,N-dimethylaniline (DMA) with isolated rat hepatocytes resulted in the production of N-methylaniline, aniline, N,N-dimethylaniline N-oxide (DMA N-Oxide) and a highly water-soluble metabolic tentatively identified as N-methylaniline N-glucuronide. 2. After the removal of aniline, N-methylaniline and DMA, treatment of the media with either strong acid or beta-glucuronidase, resulted in the release of N-methylaniline, identified by chromatography and mass spectrometry. 3. Pre-incubation of rat hepatocytes with 2 mM D-galactosamine, which decreased 7-hydroxycoumarin conjugate formation by 40%, selectively decreased the formation of this highly water-soluble metabolite from DMA by 70%. DMA N-demethylase and N-oxidase activities remained unchanged. 4. Incubation of rat hepatocytes with N-methylaniline resulted in the production of the novel metabolite, the formation of which was proportional to cell number, incubation time, and N-methylaniline (substrate) concentration. 5. The N-glucuronidation of the secondary N-alkylarylamine, N-methylaniline, by rat hepatocytes represents a quantitatively important and previously uncharacterized route of metabolism in these cells. Further studies are, however, required to identify this metabolite unequivocally as the N-glucuronide of N-methylaniline.

The effect of glucagon on the synthesis and degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Incubation of rat hepatocytes with glucagon results in a time- and dose-dependent decrease in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We demonstrate, using immunoprecipitation of radiolabeled enzyme, that 10 nM glucagon inhibits the synthesis of the enzyme by approximately 50%, but that the apparent rate of degradation of the enzyme is not affected by the hormone. We also demonstrate that the intact reductase polypeptide contained phosphoserine. We conclude that glucagon inhibits the activity of the reductase by inhibition of enzyme synthesis.

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 μM Gpp[NH]p, 30 μM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component N s and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase a activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.

Metabolism of cysteine in rat hepatocytes. Evidence for cysteinesulphinate-independent pathways.

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mM- or 25 mM-cysteinesulphinate. In similar incubations with 1 mM- or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14CO2 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production of ammonia plus urea N; cysteinesulphinate catabolism by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.

Metabolism of Cysteine and Cysteinesulfinate in Rat and Cat Hepatocytes

The metabolism of cysteine and cysteinesulfinate was studied in freshly isolated hepatocytes from fed rats and cats. In incubations of rat hepatocytes with cysteinesulfinate, the rate of hypotaurine plus taurine production was approximately the same as the rate of conversion of the 1-carbon of cysteinesulfinate to CO2. In contrast, no significant production of hypotaurine plus taurine occurred in incubations of cat hepatocytes with cysteinesulfinate. These data are consistent with the species difference in the activity of hepatic cysteinesulfinate decarboxylase, which converts cysteinesulfinate to hypotaurine. In incubations of either rat or cat hepatocytes with cysteine, no hypotaurine plus taurine production was detected. However, the 1-carbon of cysteine was converted to CO2 and the production of urea plus ammonia nitrogen was significantly increased over the rates observed in incubations of cells without substrate. Our results suggest that most cysteine oxidation by hepatocytes occurs by pathways that do not involve formation of cysteinesulfinate.

High Performance Liquid Chromatography Determination of Chlorpropham and Its Metabolites in Isolated Rat Hepatocyte Incubations

Abstract Free chlorpropham (CIPC) and its metabolites (4-hydroxy CIPC, 3-chloroani-line and 3-chloroacetanilide) were separated by reverse phase high performance liquid chromatography (H.P.L.C.) using an isocratic procedure. The separation was used to quantify the metabolism of CIPC in rat hepatocyte incubations. The free metabolites were diethylether-extracted at pH 7.4. The sulfate and glucuronide conjugates of 4(OH)CIPC were enzymatically split and the aglycone determined by HPLC. Beside the unmetabolized CIPC which accounts for 33% of the dose, 4(OH)CIPC as the sulfoconjugate and glucuroconjugate represents 35% and 20% respectively, while free 4(OH)CIPC accounts for only 3%. About 10% of the CIPC is hydrolytically split yielding 3-chloroaniline (2%) which further transformed into 3-chloroacetanilide (5%). In spite of the great sensitivity of the proposed method (0.2 μg/ml incubation medium) no other metabolites were recorded. This extraction and separation procedure could be applicable to future metab...

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1,2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca 2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase α and cytosolic Ca 2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase α or between the changes in cytosolic Ca 2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca 2+ and activation of glycogen phosphorylase. Addition of an α 1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca 2+ and phosphorylase α, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca 2+-mobilizing hormones regulate phosphorylase α through a Ca 2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.

Enhanced formation of ethane and n-pentane by rat hepatocytes in the presence of dimethyl sulfoxide

Incubation of rat hepatocytes with 14 mM dimethyl sulfoxide (DMSO) produced an increase in the formation of ethane, measured by capillary column gas chromatography, to 18.0 pmoles/hr/10 7 cells from 11.2 pmoles/hr/10 7 cells in control hepatocytes, and an increase in the formation of n-pentane to 17.5 pmoles/hru/10 7 cells from 5.6 pmoles/hr/10 7 cells in control hepatocytes. This was about one-third the stimulation of ethane and n-pentane formation produced by incubation of hepatocytes with 13 mM carbon tetrachloride. DMSO-stimulated ethane and n-pentane formation was inhibited up to 63% by 0.1 μM α-tocopherol and up to 89% by N 2. Formation of dimethylsulfide from DMSO by hepatocytes was the same in air and N 2. DMSO increased methane production by hepatocytes to 31.3 pmoles/hr/10 7 cells from 6.9 pmoles/hru/10 7 cells in control hepatocytes. Although DMSO apparently stimulated lipid peroxidation by hepatocytes, as measured by ethane and n-pentane formation, there was no increase in the formation of thiobarbituric acid reactive material. DMSO was not toxic to hepatocytes, measured by release of cytosolic lactate dehydrogenase, over a 2-hr incubation. Possible mechanisms for the increase in alkane formation by DMSO are discussed.

Phosphorylation of HMG-CoA reductase induced by mevalonate accelerates its rate of degradation in isolated rat hepatocytes

Incubation of rat hepatocytes with 10 mM mevalonate produces a decrease in HMG-CoA reductase activity and in the rate of synthesis of both monomeric and dimeric HMG-CoA reductase, and an increase in the rate of degradation of the monomeric form without significant change in that of the dimeric form. Since mevalonate promotes a short-term phosphorylation of the monomeric form without affecting the dimeric form, it is suggested that the mechanism of degradation of reductase is controlled by its phosphorylation state.

Phosphorylation and inactivation of rat hepatocyte glycogen synthase by phorbol esters and mezerein

incubation of rat hepatocytes with active phorbol esters and mezerein provoked a decrease in glycogen synthase activity. After the incubation of [ 32P] phosphate-labeled cells with these tumor promoters, an increase in the amount of 32P bound to the immunoprecipitated enzyme was observed. The decrease in activity highly correlated with the phosphorylation in the smaller CNBr fragment (CB-1) and only at high concentration of the phorbol ester the increase in the phosphorylation of the larger CNBr fragment (CB-2) became significative. Tryptic degradation of CB-1 showed two phosphopeptides after isoelectro focusing analysis (pI 3.9 and pI 3.4) and only one of them (pI 3.9) increased its phosphorylation state after treatment of the cells. These results indicate that the decrease in activity of glycogen synthase by phorbol esters and mezerein is a result of the phosphorylation of the enzyme and that a single site located in CB-1 is preferentially phosphorylated by these agents.

Rat liver HMG CoA reductase, a glycoprotein of the endoplasmic reticulum, is in equilibrium between monomeric and dimeric forms

Incubation of rat hepatocytes with [ 35S]methionine in pulse and pulsechase experiments followed by immunoprecipitation of the HMG CoA reductase and SDS-PAGE results in two labelled polypeptides of 104 and 180 Kdaltons. These two polypeptides have half lives of 80 and 46 minutes respectively. When hepatocytes are incubated with mevalonolactone, and a pulse of [ 35S]methionine is given, the rate of synthesis of both the 180 and 104 Kd peptides, is strongly diminished. After treatment of the [ 35S] labelled immunoprecipitates with endoglycosidase H, the 180 Kd reductase splits into two labelled peptides of 110 and 97 Kd. We suggest that in addition to the 104 Kd reductase, the endoplasmic reticulum contains the dimer of two reductases linked by a carbohydrate chain. The equilibrium monomer-dimer probably regulates the rate of degradation of reductase.

Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions.

Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium. This was not leakage from dead cells, as 90-95% of the hepatocytes had retained their integrity at the end of the incubation. Release of carbonyl products from cells stimulated to undergo lipid peroxidation may be a mechanism for spreading an initial intracellular disturbance to affect critical targets outside the parent cell.

A novel approach to the analysis of mass spectrally assayed stable isotope-labeling experiments.

A novel approach to the analysis of mass spectrally assayed stable isotope-labeling experiments for studies of biosynthetic pathways is reported. This method determines in a mixture of product molecules, the relative number of product molecules synthesized from the stable labeled precursor pathway and those that were either present prior to the labeling period or were produced by an alternate pathway during the course of an experiment. In addition, the isotopic enrichment of the labeled atoms in the product molecules produced from the stable labeled precursor is determined. These isotopic enrichments represent the isotopic enrichment in the immediate precursors which form the product molecules and would reflect any cellular compartmentation of precursor pools. The feasibility of the method using 15NH4Cl and L-[5-15N]glutamine as precursors to study the de novo pyrimidine biosynthetic pathway in isolated rat hepatocytes is demonstrated. The results of these studies show that after incubation of rat hepatocytes with either precursor it is possible to determine the fraction of the uracil nucleotide pool that is formed by the de novo pathway during the period of exposure. The pattern of 15N labeling in the N1 and N3 positions in the uracil moiety is different for the two precursors; however, in most cases the 15N enrichment of each position remained relatively constant for each precursor with either time (15-120 min) or precursor concentration (1 to 10 mM). This method will allow the actual quantitation and isotopic enrichment of product formed by a specific biosynthetic pathway during the course of an experiment and, as such is an improvement over existing labeling techniques.

High density lipoproteins and lecithin dispersions increase the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase by increasing the rate of synthesis and decreasing the rate of degradation of the enzyme.

Incubation of rat hepatocytes, prepared from rats fed a normal diet, with either human high density lipoproteins or lecithin dispersions resulted in a dose-and time-dependent increase in the specific activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Incubation of cells with both high density lipoprotein and lecithin dispersions resulted in an additive stimulation of enzymic activity. We used pulse and pulse-chase experiments, followed by immunoprecipitation of the radiolabeled reductase, to demonstrate that high density lipoproteins and lecithin dispersions resulted in increased rates of synthesis and decreased rates of degradation of the reductase. We also demonstrate, by the use of immunoblots, that the increased enzymic activity was paralleled by an equivalent increase in the mass of the reductase. Addition of human low density lipoprotein to rat hepatocytes resulted in a small decrease in both enzymic activity and enzyme synthesis.

Regulation of Ca2+-Mg2+-ATPase activity in hepatocyte plasma membranes by vasopressin and phenylephrine.

A high affinity Ca2+-stimulated, Mg2+-dependent ATPase (Ca2+-Mg2+-ATPase) was identified in microsomes and plasma membrane vesicles isolated from rat hepatocytes. The distribution of this enzyme was similar to that of the plasma membrane marker enzymes alkaline phosphodiesterase and 5'-nucleotidase. The Ca2+-Mg2+-ATPase had an apparent half-saturation constant of approximately 75 nM for Ca2+. After incubation of rat hepatocytes with 25 nM vasopressin for 3 min, the activity of Ca2+-Mg2+-ATPase was decreased 15-30%. The effect of vasopressin on the activity of this enzyme was near maximal after incubating hepatocytes with vasopressin for only 15 sec. The concentration of vasopressin needed for half-maximal inhibition of this enzyme in hepatocytes was approximately 6 nM. Treatment of the hepatocytes with 10 microM phenylephrine caused about a 10% decrease in ATPase activity while 10 nM glucagon or 200 microU/ml insulin did not affect the enzyme. These findings suggest that inhibition of the Ca2+-Mg2+-ATPase activity may be part of the mechanism by which vasopressin and alpha-adrenergic agonists elevate cytosolic Ca2+ in hepatocytes.

Trifluoperazine and other anaesthetics inhibit rat liver CTP: phosphocholine cytidylyltransferase

Chlorpromazine (25 μM) and trifluoperazine (25 μM) inhibited by 5-fold the activity of CTP: phosphocholine cytidylyltransferase, the rate-limiting enzyme for phosphatidylcholine biosynthesis, in rat liver cytosol. Addition of saturating amounts of rat liver phospholipid to the enzyme assay rapidly reversed the drug-mediated inhibition. Three-fold or greater concentrations of these drugs were required to produce a 50% inhibition of the microsomal cytidylyltransferase. Incubation of rat hepatocytes with 20 μM trifluoperazine or chlorpromazine did not inhibit phosphatidylcholine biosynthesis. These results provide additional evidence for the hypothesis that the active form of cytidylyltransferase is on the endoplasmic reticulum and the enzyme in cytosol appears to be latent.