Taurocholate uptake by adult rat hepatocytes in primary culture

Adult rat hepatocytes were cultured on Petri dishes for 25--30 h prior to measuring their ability to transport taurocholate. A rapid uptake of the bile acid (25 muM) was observed: about 20% was accumulated in the cells within 15 min. The taurocholate transport was saturable with an apparent Km of 28 +/- 10 muM and a maximal velocity V of 0.07 +/- 0.02 nmol/(micrograms DNA x min). Uptake was shown to be energy dependent as it was inhibited about 65% by antimycin A (20 micrograms/ml). The monohydroxylated bile acid taurolithocholate and the dihydroxylated taurochenodeoxycholate inhibited taurocholate transport to about 30 and 40% resp. of the control. The transport process was strongly dependent on sodium ions. It is concluded that the characteristics of taurocholate uptake into adult rat hepatocytes are very similar either in freshly prepared cells or in hepatocytes which are cultured on Petri dishes for 25--30 h.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture

Summary Alterations in peroxisomes and catalase activity and their responsiveness to clofibrate in adult rat hepatocytes in primary culture were investigated. The numbers of peroxisomes with and without crystalloid nucleoids per unit cytoplasmic area were preserved in cultured hepatocytes for 2 d after seeding at a level comparable to that of freshly isolated hepatocytes. At Day 3 in culture, the number of anucleoid peroxisomes was reduced in untreated hepatocytes, accompanied by more significant reduction in the number of nucleoid-containing peroxisomes, which decreased until Day 5. Peroxisome diameters were reduced in untreated hepatocytes at Day 2 and this decrease in the diameter was continued until Day 7. Catalase activity in untreated hepatocytes decreased markedly with culture age. The number of anucleoid peroxisomes was significantly greater in hepatocytes treated with 2mM clofibrate in culture than in freshly isolated hepatocytes for 2 d or in untreated hepatocytes of the same culture age through 7 d. The number of nucleoid-containing peroxisomes in the treated cells began to decrease in 3 d, but was greater than that of untreated cells at Days 3 and 5. Peroxisomes with well-developed nucleoids were observed frequently in the treated cells even at Day 7. Peroxisome diameters were greater in the treated cells than in untreated cells at Days 3, 5, and 7. Catalase activity was always higher in the treated cells than in untreated cells. These results suggest that clofibrate is effective in inducing peroxisome proliferation as well as in maintaining the organelles in cultured hepatocytes. This work was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture, Japan, 448143, 50168, 501069, and 577196, and by a Grant-in-Aid from Hokkaido Geriatrics Research Institute.     

Properties of peroxisomes and their induction by clofibrate in normal adult rat hepatocytes in primary culture

Alterations in peroxisomes and catalase activity and their responsiveness to clofibrate in adult rat hepatocytes in primary culture were investigated. The numbers of peroxisomes with and without crystalloid nucleotids per unit cytoplasmic area were preserved in cultured hepatocytes for 2 d after seeding at a level comparable to that of freshly isolated hepatocytes. At Day 3 in culture, the number of anucleoid peroxisomes was reduced in untreated hepatocytes, accompanied by more significant reduction in the number of nucleoid-containing peroxisomes, which decreased until Day 5. Peroxisome diameters were reduced in untreated hepatocytes at Day 2 and this decrease in the diameter was continued until Day 7. Catalase activity in untreated hepatocytes decreased markedly with culture age. The number of anucleoid peroxisomes was significantly greater in hepatocytes treated with 2 mM clofibrate in culture than in freshly isolated hepatocytes for 2 d or in untreated hepatocytes of the same culture age through 7 d. The number of nucleoid-containing peroxisomes in the treated cells began to decrease in 3 d, but was greater than that of untreated cells at Days 3 and 5. Peroxisomes with well-developed nucleoids were observed frequently in the treated cells even at Day 7. Peroxisome diameters were greater in the treated cells than in untreated cells at Days 3, 5, and 7. Catalase activity was always higher in the treated cells than in untreated cells. These results suggest that clofibrate is effective in inducing peroxisome proliferation as well as in maintaining the organelles in cultured hepatocytes.     

Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8 + cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8 +, but not CD 4 + helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10–20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes. Supported by DFG grants Ra 362/5-2 and SFB 311 A7 (G.R.) and A5 (H.P.D.)     

Cell density dependent morphological changes in adult rat hepatocytes during primary culture

In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (less than about 0.1 X 10(5) nuclei (cm2)-1) and at a high cell density (greater than about 1 X 10(5) nuclei (cm2)-1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numerous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver.     

Isolation and primary culture of adult human hepatocytes

Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.     

The release of hepatic triglyceride lipase from rat monolayered hepatocytes in primary culture

The release of hepatic triglyceride lipase from cultured rat hepatocytes and its hormonal regulation were studied. The activity of lipase released into the medium in the presence of heparin was increasing for 24 hours on the 2nd day of culture. The activity in the absence of heparin was only 10% of that in the presence of heparin. When hepatocytes were cultured with anti-hepatic triglyceride lipase IgG, the lipase activity was suppressed by 92%. The results suggest that the enzyme released into the culture medium is identical to hepatic triglyceride lipase which can be released only in the presence of heparin, the mode of release being similar to that of lipoprotein lipase from adipocytes. The addition of colchicine and monensin to the medium resulted in the inhibition of lipase secretion by 20% and 61%, respectively. Insulin enhanced lipase activity only 20%, whereas dexamethasone suppressed the activity by 44%. These data indicated that hepatic triglyceride lipase is secreted and released from hepatocytes in the presence of heparin and its secretion is regulated by hormones.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

Polygonal networks, "geodomes", of adult rat hepatocytes in primary culture

Polygonal networks, "geodomes", in cultured hepatocytes of adult rats were examined by both light and electron microscopy. On light microscopical examinations of specimens stained with Coomassie blue after the treatment with Triton X-100, the networks were detected 5 days after culture, which consisted of triangles arranged mainly in hexagonal patterns. They surrounded main cell body, looking like a headband, or were occasionally situated over nuclei, looking like a geodesic dome. Scanning electron microscopical observations after Triton treatment revealed that these structures were located underneath surface membrane. Transmission electron microscopical investigations revealed that the connecting fibers of networks consisted of microfilaments which radiated in a compact bundle from electron-dense vertices.     

Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture

Summary Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10⁷ cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 μg/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture

Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [¹⁴C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [¹⁴C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline