Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10(-5) M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10(-5) M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide.     

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes

Summary Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10⁻⁵ M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10⁻⁵ M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide. This study was supported by NIH Grant CA30241-01.     

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N′-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.

Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.

No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca²⁺ concentration.

We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.     

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N'-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.

Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.

No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca²⁺ concentration.

We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.     

Expression of alcohol dehydrogenase in primary monolayer cultures of rat hepatocytes

With the use of an extensively modified Leibovitz-15 medium, the alcohol dehydrogenase activity of hepatocytes prepared from male rats was successfully maintained in primary culture at the level observed in freshly isolated hepatocytes. Enzyme activity was higher in freshly isolated cells from female rats than from male rats, but it fell to the level characteristic of the male animals after four days in culture. The levels of activity of the cells in culture from both sexes were unaffected by treatment with estrogens or androgens. The results suggest that the sex-determined differences in alcohol dehydrogenase activity in rats do not arise from direct effects of gonadal steroids on the liver.     

Precocious induction of arginase in primary cultures of fetal rat hepatocytes

Summary Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis. This work was supported by the Institut National Scientifique et de la Recherche Médicale through Grant 85.80.117.     

cAMP-mediated upregulation of gelatinases in primary cultures of isolated rat hepatocytes

Matrix metalloproteinases (MMPs) play a major role in tissue remodelling and repair in pathophysiological conditions, such as liver fibrosis and regeneration. Regulation of the MMPs produced by liver cells is important in maintaining cell-matrix ratio in liver, which is a major target site for hormones that mediate their intracellular effects through cAMP. The possibility of cAMP affecting the activity of MMPs and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) was studied using isolated rat hepatocytes in culture. Zymographic analysis showed that treatment with hormones like epinephrine, thyroxine and dexamethasone and Bt2 cAMP increased 92 kDa MMP-9 activity. Bt2 cAMP caused upregulation of MMP-9 in a dose-dependent manner. The effect of hormones was less on MMP-2. ELISA using specific antibodies showed increase in levels of MMP-9 and TIMP-1 protein. Kinetic analysis of production of MMPs and TIMPs showed that the response to Bt2 cAMP was a delayed one, indicating its effect on de novo protein synthesis. These results suggest the possibility of cAMP dependent regulation of MMP-9 in the hepatocytes.     

Tyrosine O-sulfation promotes proteolytic processing of progastrin.

Tyrosine O-sulfation is a common post-translational modification of secretory and membrane proteins. The biological function of sulfation is known in only a few proteins, where it appears to enhance protein-protein interactions. Based on known sequences around sulfated tyrosines, a consensus sequence for prediction of target tyrosines has been proposed. However, some proteins are tyrosine sulfated at sites that deviate from the proposed consensus. Among these is progastrin. It is possible that the deviation explains the incomplete sulfation characteristic for bioactive gastrin peptides. In order to test this hypothesis, we have performed site-directed mutagenesis of the gastrin gene followed by heterologous expression in an endocrine cell line. The results show that substitution of the alanyl residue immediately N-terminal to the sulfated tyrosine with an acidic amino acid promotes the sulfation of gastrin peptides. Hence, the study supports the proposed consensus sequence for tyrosine sulfation. Importantly, however, the results also reveal that complete sulfation increases the endoproteolytic maturation of progastrin. Thus, our study suggests an additional function for tyrosine sulfation of possible general significance.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1μM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethasone supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture. This study was supported by an Alexander Ralston Peacock Memorial Grant for Cancer Research (No. BC-133A) from the American Cancer Society.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did not prolong cell survival. This study was supported by grant no. BC 133 from the American Cancer Society.     

The rat fibrinogen genes. Linkage of the A alpha and gamma chain genes

We have utilized cDNA probes for the alpha, beta, and gamma chains of rat fibrinogen to isolate the corresponding genes from two rat genomic libraries constructed in bacteriophage Charon 4A. There is a single copy of each gene. Mapping of greater than 92 kilobase pairs of rat genomic DNA has shown that the gamma and alpha chain genes are directly linked in a 5'-3' direction in vivo.     

Agonist-induced down-regulation of the angiotensin II receptor in primary cultures of rat hepatocytes

The ability of angiotensin II to down-regulate its receptor was tested on rat hepatocytes in primary culture for 4 h. Angiotensin II treatment decreased [3H]angiotensin II specific binding in a concentration- and time-dependent manner. The effect was maximum with 1 microM angiotensin II and after 2 h. There was a decrease in the maximum number of binding sites (56% of control) with no significant effect on the apparent dissociation constant. The down-regulation was blocked by the angiotensin II antagonist [Val4,Ile7]angiotensin III and was not induced by other hormones (e.g. vasopressin, norepinephrine, or glucagon) or by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate or A23187 ionophore. The decrease in angiotensin II receptors resulted in correlated decreases in the potency of angiotensin II to activate phosphorylase or lower glucagon-induced cAMP accumulation. However, high concentrations of the agonist were still able to elicit maximal responses in both parameters. Down-regulation of the receptor was not dependent upon active Gi, since it was still observed after ADP-ribosylation and inactivation of Gi by pertussis toxin. The above results indicate that the down-regulation of the hepatic angiotensin II receptor induced by its agonist is homologous and does not involve Gi, Ca2+, or protein kinase C. The correlation of receptor loss with decreases in the potency of angiotensin to activate phosphorylase and inhibit glucagon-induced cAMP accumulation is consistent with the idea that a single receptor population regulates two different messengers, i.e. calcium and cAMP.     

Regulation of secretory component by glucocorticoids in primary cultures of rat hepatocytes

The present study examined the effects of steroid hormones on the production of secretory component (SC) by rat hepatocytes in cell culture. When hepatocytes were incubated in the presence of cortisol (10(-6) M), the levels of SC in media increased significantly after 2 days of incubation. This response was dose-dependent and specific for glucocorticoids because progesterone, dihydrotestosterone, and estradiol had no effect. When estradiol was added to the incubation media along with dexamethasone, a known potent synthetic glucocorticoid, it diminished the glucocorticoid response. The addition of cycloheximide to incubation media significantly decreased the effect of dexamethasone on SC accumulation. These findings suggest that glucocorticoid regulation of hepatocyte SC most likely involves stimulation of its synthesis. In addition, our results suggest that endogenous glucocorticoids may play a role in enhancing the clearance of IgA from blood into bile in the intact animal.     

Carbohydrate-dependent induction of fatty acid synthase in primary cultures of rat hepatocytes

The glucose and insulin-dependent long-term regulation of fatty acid synthase was studied in primary cultures of hepatocytes from adult female rats. Under basic culture conditions, i.e. 5.5 mM glucose and 0.5 nM insulin, the enzyme activity was continuously decreased over 6 days. In the presence of 100 nM insulin this decrease was reduced but it was not prevented. Enhancement of glucose to 20 mM was followed by an increase of the enzyme activity; after 5 days of treatment the activity was three times higher than under basic culture conditions. The simultaneous presence of 20 mM glucose and of 100 nM insulin resulted in a much more pronounced increase of the activity; after 5 days of treatment the activity was eight times higher than under basic culture conditions. The enhancement was prevented by inhibition of glycolysis. This may indicate that the increase of fatty acid synthase was mediated by a metabolite of glucose rather than by glucose itself. The coordinate regulation of fatty acid synthase and of other lipogenic enzymes was specific as demonstrated by comparison with the activity of lactate dehydrogenase and with synthesis and degradation of cytosolic proteins. The enhancement of the enzyme protein, demonstrated by rocket immunoelectrophoresis, was due to an increase in the rate of enzyme synthesis by 600% as well as to a prolongation of the apparent half-life of the enzyme by 50% (45 h).     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Induction of S-adenosylmethionine synthetase isozymes in primary cultures of adult rat hepatocytes

The activities of S-adenosylmethionine synthetase isozymes were studied using adult rat hepatocytes in primary culture. Hepatocytes from adult rats were isolated and cultured for several days. The activities of the synthetase isozymes did not change during primary culture. The activity of the alpha-form increased with increasing ethionine plus adenine or methionine in the medium, and reached about 5 fold after 2 days. However, the increased activity of the beta-form showed less than twice.