THE ANATOMICAL RECORD 214~1-711986) Plasma Membrane Reregionalization in Cultured Mouse Hepatocytes JUN WATANABE, SHINSUKE KANAMURA, AND KAZUO KANA1 Department of Anatomy, Kansai Medical Universtty, Moriguchi, Osaka 570, Japan ABSTRACT The cytochemical localization of alkaline phophatase (ALPase) activity and the autoradiographic distribution of glucagon receptors were examined in the plasma membrane of cultured mouse hepatocytes. After 24 hours of culture, ALPase activity was exclusively localized on the plasma membrane in areas of cellcell contact, and glucagon receptors were more numerous in the plasma membrane at the periphery of re-formed cell trabeculae. These results indicate that plasma membrane regionalization of hepatocytes, lost by cell isolation, reappeared during culture. The cells maintained this plasma membrane regionalization until 48 hours of culture. By 72 hours of culture, however, ALPase activity was seen on the external surface of all regions of plasma membrane, and the glucagon receptors decreased markedly in number and became scattered in all regions of plasma membrane. Thus, the re-formed plasma membrane regionalization disappeared in the cells by 72 hours of culture. In the intact liver, alkaline phosphatase (ALPase) activity is exclusively localized on the bile canalicular membrane (De Wolf-Peeters et al., 1972; Karasaki, 19751, and glucagon receptors are more numerous in the sinusoidal membrane (Asada-Kubota et al., 1983). Isolated hepatocytes, however, lose the polarity of the plasma membrane, i.e., the plasma membrane regionalization; ALPase activity (Groothuis et al., 1981) and glucagon receptors (Watanabe et al., 1984) are present over the entire cell surface. During monolayer culture, hepatocytes reconstruct cell trabeculae and bile canaliculi (Chapman et al., 1973; Wanson et al., 1977; Miettinen et al., 1978). It is therefore of interest to examine whether hepatocytes also regain the plasma membrane regionalization of ALPase activity and glucagon receptors during culture. Little is known about the localization of ALPase activity in cultured hepatocytes. Jung et al. (1982) reported that the activity was seen on all regions of the plasma membrane of rat hepatocytes until 72 hours of culture. In previous study, we observed high ALPase activity on the plasma membrane a t the periphery of re-formed cell trabeculae after 72 or 96 hours of culture (Watanabe et al., 1983). There have been no reports in the literature on the distribution of glucagon receptors in cultured hepatocytes. In the present study, therefore, we examined the distribution of ALPase activity and glucagon receptors in cultured mouse hepatocytes. The purpose was to determine whether hepatocytes regain the plasma membrane regionalization under culture conditions. MATERIALS AND METHODS Male ddY mice 2-3 months old were used. They had free access to food and water prior to the experiments. 0 1986 ALAN R. LISS, INC. Cell Culture Cells were isolated from livers by perfusion of collagenase solution via the portal vein (Klauning et al., 1981) under Nembutal anesthesia. The parenchymal cells were separated from nonparenchymal cells by four centrifugations at 50g for 1minute at 4°C (Watanabe et al., 1983, 1984) and suspended in Williams’ medium E containing 10% calf serum, 1 nM dexamethasone, and 100 nM insulin. One milliliter of the cell suspension (6 x lo5 cells/ml) was poured onto a 35-mm plastic dish (Falcon, #3001). The cells were cultured as a monolayer at 37°C under 5% C 0 2 and 95% air. The medium was changed after 24 and 48 hours. Cytochemical A LPase Activity After 12, 24, 48, or 72 hours of culture, hepatocytes were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at 4°C for 5 minutes and washed in 0.1 M sodium cacodylate containing 5% sucrose (pH 7.4) a t 4°C for 1 hour. Then, the cells were incubated in the reaction medium for cytochemical demonstration of ALPase activity (20 mM P-glycerophosphate; 3.9 mM magnesium sulfate; 2.0 mM lead citrate; 250 mM sucrose; 28 mM Tris-HC1 buffer, pH 9.2) (Mayahara et al., 1967) at 20°C for 30 minutes. As controls, the cells were incubated in the reaction medium containing 10 mM levamisole as a n enzyme inhibitor (Van Belle, 1972)or lacking (3-glycerophosphate.For light microscopy, the cells were washed in distilled water, immersed in 1%ammonium sulfide for 10 seconds, washed again in distilled water, and mounted in glycerin-jelly. For electron microscopy, the cells were postfixed in buffered 1%osmium tetroxide Received February 15, 1955; accepted July 11, 1985 J. WATANABE, S. KANAMURA, AND K. KANA1 2 (pH 7.4) at 4°C for 1hour, dehydrated in graded ethanol and embedded in Spurr's resin. Thin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H-700H electron microscope. Biochemical ALPase Activity Cultured hepatocytes were removed from the dishes by a rubber policeman. The cells were homogenized with 10 volumes of 0.25 M sucrose in a Potter-Elvehjem homogenizer at 2,000 rpm for 2 minutes at 4°C and centrifuged a t 3,OOOg for 10 minutes at 4°C. The supernatant was used for measurement of ALPase activity. The activity was assayed by a modified Bodansky's method (Shinowara et al., 1942). Protein was measured by the method of Lowry et al. (1951). The activity was expressed as nanomoles of phosphorous liberated per milligram of protein per minute. Glucagon Receptor Incubation with '25Cglucagon Hepatocytes (6 x lo5 cells/dish) cultured for 12,24,48, or 72 hours were incubated in triplicates with 1 ml of Krebs-Ringer bicarbonate buffer containing 0.2 nM 1251glucagon (specific activity of 94-114 pCi/,ug, NEN, Boston, MA), 0.6 mM bacitracin, and 3% bovine serum albumin a t 37°C for 10 minutes. Nonspecific binding was estimated by adding 100 pg of unlabeled glucagon to the medium before the incubation. Autoradiography After incubation with l"I-glucagon, hepatocytes were fixed in 2%) glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C for 1 hour, postfixed in buffered 1%~ osmium tetroxide (pH 7.4) at 4°C for 1hour, dehydrated, and embedded in Spurr's resin. Semithin and ultrathin sections were cut from the Spurr blocks. For light microscopy, semithin sections (1 pm in thickness) were attached to slide glasses, stained with methylene blue, coated with evaporated carbon (5 nm in thickness), and coated with Sakura NR-HZ emulsion by a dipping method. For electron microscopy, ultrathin sections were placed on collodion-coated copper grids, stained with Fig 1. Phase-contrast micrographs of hepatocytes cultured for a1 24 and b) 72 hours. Note polyhedral hepatocytes forming wide cell trabeculae. ~ 4 0 0 . Fig. 2. Light micrographs of hepatocytes cultured and treated with alkaline phosphataae (ALPase) reaction. a ) Cultured for 12 hours. The staining reaction for ALPase activity is seen on the entire surface of the cells. However, intense reaction is observed in the intercellular space between aggregated cells (arrowhead). ~ 4 0 0 bl . Cultured for 24 hours. The staining reaction is seen in the intercellular space between adjoining cclls. ~ 4 0 0c) . Cultured for 24 hours. Hepatocytes incubated in the reaction medium lacking 8-glycerophosphate. No reaction is seen in the cells except for slight staining in round cells. x400. d) Cultured for 48 hours. The staining reaction is observed i n the intercellular space between adjoining cells. x400. e ) Cultured roo,. 7 2 hours. Moderate to weak reaction for ALPase activity is seen in the intercellular space between adjoining cells and on the upper free surface of the cells. However, intense reaction is occasionally seen a t the periphery of cell traheculae (arrowheads). x400. PLASMA MEMBRANE IN CULTURED HEPATOCYTES Fig. 3. Electron micrographs of portions of hepatocytes cultured and treated with ALPase reaction. a) Cultured for 24 hours. Horizontal section. The reaction product is localized in the narrow intercellular space between adjoining cells (arrowheads). There is very little or no reaction product on the plasma membrane at the periphery of cell trabeculae (P). x 16,600. b,c) Cultured for 24 hours. b) Horizontal and c) vertical sections. The reaction product is observed in the narrow intercellular space between adjoining cells (C), but very little or no 3 reaction product is present on the bile canalicular membrane (BC)and the plasma membrane adjoining the dish (D). b, ~ 9 , 0 0 0c,; x 12,000. d,e) Cultured for 48 hours. d) Horizontal and e) vertical sections. The reaction product is localized in the narrow intercellular space between adjoining cells (arrowheads) and bile canalicular membrane (BC,, hut very few or no reaction product is present on upper free surface of the cells (FSI. The reaction product is occasionally seen also on the plasma membrane adjoining the dish (D). d, x 8,600; e, x 7,000. 4 J. WATANABE, S. KANAMURA, AND K. KANA1 uranyl acetate, coated with evaporated carbon, and coated with the emulsion by a wire-loop method (Caro et al., 1962). Then these specimens were exposed at 4°C for 6 weeks and developed with Konidol X. Grain counting Four independent cultures were used per each time point. Eight Spurr blocks (2 blocks/culture) were chosen at random. Sixteen horizontal sections cut from eight blocks (2 sectionsiblock) were prepared for light microscopic autoradiography as described above. Four photographs taken at ~ 5 0 0from each autoradiographic section were enlarged to a final magnification x3,400. An area in each photograph covered about 3,360 pm2. Then, two of four photographs were chosen at random. The length of plasma membrane was measured by the point-counting method (Weibel, 1979). The number of grains associated with plasma membrane (within 0.5 pm from the plasma membrane) was counted in the same photograph. The values were expressed as number of grains per unit length (1pm) of plasma membrane in each region. Assay of '251-glucagonbound to the cells Cultured hepatocytes were incubated with 0.2 nM '"1glucagon as described above. The cells were washed four times with 0.25 M sucrose, removed from the dishes by a rubber policeman, and suspended in 1 ml of 0.25 M sucrose. Then radioactivity of the cells was assayed by an auto gamma counter (Packard, 5330). RESULTS About 99%.of the cells used for culture were parenchymal cells as determined by phase-contrast microscopy. On the basis of trypan blue exclusion, about 90% of the cells were viable. Twelve hours after plating, hepatocytes adhered to the bottom of dish, and some of the cells formed short cell trabeculae. After 24,48, or 72 hours of culture, hepatocytes formed wide and flattened cell trabeculae (Fig. la,b). A few round cells were seen on the upper free surface of the flattened polyhedral cells. For the facility of describing the results, the plasma membrane of the cultured cells was divided into four regions; 1)upper free surface, 2) periphery of trabeculae, 3) portion in cell to cell contact and bile canaliculus, and 4)portion adjoining to the dish. Cytochemical ALPase Activity Light microscopy After 12 hours of culture, the staining reaction for ALPase activity was generally observed on the entire surface of hepatocytes (Fig. 2a). However, the reaction was not seen on the upper free surface, but was present in the intercellular space between some of the cells forming short cell trabeculae. After 24 or 48 hours of culture, the reaction became exclusively observed in the intercellular space between adjoining cells (Fig. 2b,d). After 72 hours of culture, however, both the intercellular space and upper free surface showed moderate to weak reaction (Fig. 2e). On the other hand, moderate to intense reaction was sometimes seen at the periphery of cell trabeculae, as reported in our previous paper (Watanabe et al, 1983). In the cells incubated in the reaction medium containing levamisole or lacking 0-glycerophosphate, no stain- ing reaction was observed in the cells except for round cells lying on the upper free surface of the main sheet of cells (Fig. 2c). Electron microscopy The reaction product for ALPase activity was mainly localized on the external surface of all regions of the plasma membrane except that adjoining the dish after 12 hours of culture. However, abundant reaction product was occasionally seen in the narrow intercellular space between aggregated cells, i.e., on the plasma membrane in cell-cell contact. After 24 or 48 hours of culture, the reaction product was almost exclusively localized on the plasma membrane in cell-cell contact (Fig. 3a-e). Reformed bile canalicular membranes showed irregular reaction; some displayed an intense reaction and others showed weak or no reaction (Fig. 3b-e). After 72 hours of culture, the reaction product was observed on the external surface of all regions of the plasma membrane. The plasma membrane adjoining the dish was devoid of the reaction product after 12 or 24 hours of culture (Fig. 3c), and displayed only slight reaction product after 48 or 72 hours of culture (Fig. 3e). Biochemical ALPase Activity ALPase activity in hepatocytes cultured for 12, 24, or 48 hours was similar to that in freshly isolated hepatocytes (Fig. 4). The activity, however, increased significantly in the cells cultured for 72 hours. Protein contents of the cells were constant until 72 hours of culture (mg per d i s h means k S.D. for five experiments: 1.08 & 0.12, isolated cells; 1.01 k 0.10, 12 hours; 0.98 t 0.13,24 hours; 1.04 k 0.15, 48 hours; 0.94 0.18, 72 hours). T o 12 24 48 TIME IN CULTURE 72 (HOURS) F g 4.Changes in ALPase activity duringculture Vnlucs a r e means i S.U. for f i v e experiments. :'.P < .05: significantly different from value of freshly isolated hepatocytes tStudent's t-test).Pi. phosphorous. PLASMA MEMBRANE IN CULTURED HEPATOCYTES 5 Flg. 5 Light microscopic (a-di a n d electron microscopic ie.11 autoradiohvaphs of cultui-ed mouse hepatocytes incubated with 0.2 nM '"1~ glucagon a t 37°C for 10 minutes. a,bi Cultured tor ai 12 and hi 24 hours. Plasma-membrane-associated hvains are numerous 'IL t h c periphery of cell trabeculae. x 1,000. ci Cultured for 48 hours. M a n y g a i n s are localized a t the peripherv of cell trabeculae, but some grains a r c present in the intercellular space hetween adjoining cells. x 1,000. di Cultured lor 72 hours. The grains decrease markedly In number. ~ 1 , 0 0 0ei . Ciiltiired for 24 hours. The gyains are numerous in the plasma membrane a t the periphery ofcell trabeculae (Pi. A fen p a i n s are seen in the narrow interccllular space between aghyegated cells iC). ~ 6 , 3 0 011 . Cultured for 48 hours. Some grains become seen also i n t h e plasma membrane in cell-cell contact tCi. x 7,000. Glucagon Receptor Grain counting The values are summarized in Table 1.The number of plasma-membrane-associated grains was significantly greater in region I (the plasma membrane a t the periphery of cell trabeculae and upper free surface) than in region I1 (the plasma membrane in cell-cell contact and bile canalicular membrane) in the cells cultured for 12, 24, or 48 hours. After 72 hours of culture, however, no difference was seen between region I and region 11. Autoradiography About 30% of the cell-associated labels were internalized. However, we paid attention to the plasma-membrane-associated grains. After 12 or 24 hours of culture, developed grains associated to the plasma membrane were mainly seen at the periphery of cell trabeculae and upper free surfaces (Fig. 5a,b,e). Very few grains were seen a t the plasma membrane in cell-cell contact or at bile canaliculi. After 48 hours of culture, although many grains were still seen at the periphery of cell trabeculae, more grains became present in the plasma membrane in cell-cell contact (Fig. 5c,f). After 72 hours of culture, the grains decreased markedly in number, and were scattered a t the periphery of cell trabecuale and at regions of cell-cell contact (Fig. 5d). There were a few grains after 12,24, and 48 hours of culture and no grains after 72 hours of culture in the plasma membrane adjoining the dish. Radioactivity Associated With the Cells The amounts of l"I-glucagon bound t o the cells after 12, 24, or 48 hours of culture were similar to that of freshly isolated hepatocytes (Fig. 6). After 72 hours of culture, however, the binding decreased markedly. DISCUSSION In the intact liver, hepatocytes maintain plasma membrane regionalization (sinusoidal, lateral, and bile can- 6 J. WATANABE, S. KANAMURA, AND K. KANA1 TABLE 1. Distribution of cell-surface grains in cultured mouse hepatocytes' Time in culture (hours) 12 E C No. of surface bp-ains per cell circumference (1 um) No. of surface grains per photograph (3,356 pm2) Region I Region I1 Total Region I Region I1 75.0 (4.1) 11.8 (7.1) 0.170 (0.023) 0.072 (0.031) 9.8 (2.2) 5.4 (1.3) 0.029 (0.006) 0.021 (0.004) 91.5 (11.0) '3 0.195 (0.053) (0.008) Region I Specific Region I1 Significance 0.141 (0.019) 0.050 (0.028) P < .05 0.159 (0.041) 0.047 (0.005) P < .01 0.122 (0.032) 0.078 (0.014) P < .05 0.038 (0.008) 0.031 (0.010) NS 24 E -) .2 0.062 1.9) 0.033 (0.009) 0.015 (0.004) 88.0 (8.6) 32.3 (4.1) 0.147 (0.038) 0.085 (0.017) 9.5 (3.1) 6.9 (2.4) 0.015 (0.009) 0.008 (0.004) E 29.3 (2.5) 16.7 (4.4) 0.049 (0.012) 0.036 (0.O 13) C 9.2 (2.3) 4.5 (2.0) 0.011 (0.005) 0.006 (0.002) C 10.4 (2.4) 48 E n CI 72 'Hepatocytes were incubated with 1251-glucagon(El or '"I-glucagon plus excess amounts of unlabeled glucagon (0. No. of grains in region I (the plasma membrane a t the periphery of cell trabeculae and a t the upper free surface) and region I1 (the plasma membrane in cell-cell contact and bile canaiicular membrane) were counted as described in t h e text. Values a r e means (?S.D.) for four independent cultures. Student's ttest was used for comparison of values between region I and region 11. NS, not significant. 0 12 24 48 TIME I N CULTURE 72 (HOURS) alicular sites) (De Wolf-Peeters et al., 1972; Karasaki, 1975; Wisher and Evans, 1977; Groothuis et al., 1981; Asada-Kubota et al., 1983). However, the plasma membrane regionalization i s lost in freshly isolated hepatocytes (Groothuis et al., 1981; Wantanabe et al., 1984). After 24 hours of culture, as revealed in the present study, ALPase activity becomes exclusively localized in the plasma membrane in cell-cell contact, and glucagon receptors are more numerous in the plasma membrane a t the periphery of cell trabeculae. Thus, hepatocytes regain plasma membrane regionalization of ALPase and glucagon receptor during 24 hours of culture, although the plasma membrane in cell-cell contact may be not equivalent to bile canalicular and lateral membrane in hepatocytes of the intact liver. The plasma membrane regionalization of ALPase and glucagon receptor was maintained in the cells until 48 hours of culture, but disappeared after 72 hours of culture. Thus, the plasma membrane regionalization reestablished a t early culture time was lost after longer culture. Ichihara et al. (1980) found that protein synthesis, insulin receptors, and 5'-nucleotidase activity in hepatocytes, damaged by cell isolation, recovered during culture for a few davs but disawweared after longer culture. The recovery-and disappearance of the plasma membrane regionalization during culture, observed in the present might be to the results of Ichihara et al. (1980). I Fig. 6. Changes of '""I-glucagon binding to hepatocytes during c u l ~ ture. The cells were incubated with 0.2 nM '"I-glucagon a t 37°C for 10 minutes. Nonspecific binding (7-16%) is subtracted. Values are means S.D. for five experiments. * P < .05: significantly different from value of freshly isolated hepatocytes (Student's t-test). I U~~ ~ ~ PLASMA MEMBRANE IN CULTURED HEPATOCYTES 7 hepatocytes of intact mouse liver. An autoradiographlc study. Exp. There have been few studies on cytochemical ALPase Pathol.. 23:95-101. activity in cultured rat hepatocytes. Jung et al. (1982) M.N., and D.S. Friend (19691High-yield preparation of isolated L activity ~ localized ~ ~ in all ~ regions of the Berry, reported A ra t liver parenchymal cells. A biochemical and fine structural plasma membrane of hepatocytes cultured for 24 or 72 study, J. cell Biol., 43,.506-52~, hours. In the present results, ALPase activity was exclu- Caro, L.G., R.P. Van Tubberghen, and J.A. Roll (196'1 High resolution autoradiogaphy. 1. Methods. J. Cell Biol., 15.173-188. sively localized in the plasma membrane in cell-cell conG.S., A.L. Jones, U.A. Meyer, a nd D.M. Bisscl(1973l Parentact after 24 or 48 hours of culture. ~h~ for the Chapman, chymal cells from adult r a t liver in nonproliferating monolayer discrepancy between Our finding and that Of Jung et al. culture. 11. Ultrastructural studies, J.Cell Biol., 59735-747. is unknown. .DeWolf-Peeters, C., R. De Vos, and V. Desmet (1972)Electron microscopy a nd histochemistry of canalicular differentiation in fetal and Berry and Friend (1969), Drochmans et al, (1975) and neonatal rat liver. Tissue Cell, 4r379-388. Penasse et (1979) reported that hepatocytes Drochmans, P., J.C. Wanson, and R. Mosselmans (19751Isolated and lost the former polarity Of the plasma membrane; the subfractionation on ficoll gradients of adult rat hepatocytes. Size, cells were covered uniformly with short microvilh. Durmorphology, and biochemical characteristics of cell fractions. J. Cell Blol., 66:1-22. ing primary monolayer culture for a few days, the cells Groothuis, G.M., C.E. Hulstaert, D. K a llc hxa n, and M.J. Hardonk attached to each other and formed cell trabeculare 11981)Plasma membrane specialization and intracellular polarity (Chapman et al., 1973; Wanson e t al., 1977; Miettinen et offl-eshiy isolated rat hepatocytes. ELir. J. Cell B i d , 26.43-51. al., 1978). Wanson et al. (19771, Miettinen et al. (19781, Ichihara, A., T. Nakamura, K. Takada, Y Tomita, K. Anoyama, S. Kato, a nd H. Shinno (19801 Biochemical functions of adult r a t and Jung e t ale (1982) suggested that cell to cell attachhepatocyles in primary culture. Ann. N.Y. Acad Sci., 34!1:77-84. merit was important for the maintenance Of Jung,W., R. Gebhardt, a nd D. Mecke (19821Alteration in activity and functions Of the plasma membrane and Of the plasma ultrastructural localization of several phosphatases on the surfacc of adult r a t hepatocytes in primary monolayer culture. Eui- J . Cell membrane regionahzation in hepatocytes. Also in the Blol., 27t230-241. present study, the plasma membrane regionalization of s. 11975) Cell proliferation and subcellular localization of A L and ~glucagon ~ receptors ~ ~ occurred in parallel with Karasaki, alkaline phosphatase activity in rat liver parenchyma during azo the reestablishment of cell to cell attachment during dve carcinotrenesis, cancer R ~ ~~ 5, : ,~ 8 3 - 4 9 1 , Klau"ning, J.E.:'P.J. Goldblatt, D.E. Hilton, M.M. Lipsky, J . Chacku, culture. and B.F. Trump (1981, Mouse liver cell culture. 1. Hepatocyte Wanson et al. (1977), Miettinen et al. (1978,, and Robisolation. In Vitro, 17:913-925. enek et al. (1982) reported reestablishment of focal tight O H . N.J. Rosebrough, A.L. Far!., and R.J. Randall i 1951) junctions a t the plasma membrane in cell-cell contact Lowry, Protein measurement with the Folin phcnol reagent. J. B i d . Chem., during monolayer culture of rat hepatocytes. In the re193365-275. sults of their experiments, hepatocytes cultured as a Mavahara, H., H. Hirano, T. Saito, a nd K. Ogawa (1967, 'The nvw lead citrate method for the ultracytochemlcal dcmonstration if activity monolayer could not reestablish complete sealing of bile of non-specific alkaline phosphatase rortho-phosphoric monoester canaliculi by tight junctions. We previously found that phosphohydrolase,. Histochenne, 11:88-96. hepatocytes began to be separated from neighboring Micttinen, A,, I. Virtanen, a nd E. Lindncr (19781Cellular actin and junction formation during rcaggregatlon of adult rat hepatocytes cells and culture substratum after 96 hours of culture into epithelial cell sheets J Cell Sci., 33:341-353. (Watanabe et al., 1983).Thus, tight junctions appear to Penasse, W., D. Bernaert, K. Mosselmans, J:C. Wanson, and P. Drochbreak down after longer culture. It may be therefore mans i 19791 Scanning electron microscopy of adult rat hrpatocytes possible that early in culture newly formed tight juncin situ, after isolation of pure fractions by elutriation and after tions exclude glucagon receptors from the plasma memculture. Biol. Cell., 34:175-186. brane in cell-cell contact, but later the barriers break Robenek, H.. W. J ung, and R. Gebhardt 11982)The topography of filipin-cholesterol complexes in the plasma membrane of cultured down and the receptors float into the plasma membrane hepatocytes a nd their relation to cell junction (iirmation. J . Cltrain cell-cell contact. Similarly, i t may be that ALPase struct. Re-:, 78:95-106. activity is initially localized to the plasma membrane in Shinowara, C.Y., L.M. Jones. and H.L. Reinhart I 19421The estimation of serum inorganic phosphate and "acid" and "alkalinc~" phosphacell-cell contact and becomes free to move to other memtase activity. J. Biol. Chem., 142921-933. brane areas with time of culture. K., M. Sato, Y. Toniitii. a nd A. Ichiliala 19781i'hochemical Tanaka et al. (1978) reported that protein synthesis Tanaka, studies on liver functions in primary culturcd hcpatocytes of' adult increased in hepatocytes until 5 days of culture. In the rats. 1 Hormonal effects on cell viabillty and protein syntheslh. J. Biocheni., 84:937-946. present results, however, protein content per dish did not change significantly until 72 hours of culture. There- Van Belle, H. (1972) Kinetics and inhihition of alkaline pho3phatase from canine tissues. Biochim Biophys. Acta, 289:158-168. fore, the present results suggest that the increase in the Wanson. J.-C., P. Drochmans, R. Mosselmans, and M.F. Konveaux biochemical ALPase activity is not due to loss of protein (1977)Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics oC intercellular contacts and cell memrelative to a n unchanged ALPase amount. However, it brane differentiations. J . Cell Biol., 745358-877. is unknown whether the increasing ALPase activity is J.. S. Kanamura, M. Asada-Kubota, K. Kanai, and M. Oka due to de novo synthesis of ALPase molecules. On the Watanabe, (1983) Observation of cytochemical alkaline phosphatase activity other hand, we previously found that very few or no on the plasma membrane of culturcd rat hepatocytes by hackscattered electron imaging. Anat. Rec., 207r635-542. glucagon receptors were synthesized in rat hepatocytes cultured under the same conditions (Watanabe et al., Watanabe, J., S. Kanamura, M. Asada-Kubota, K. Kanai, a nd M. Oka (1984)Receptor-mediated endocytosis of glucagon in isolated mouse 1985). This may be the reason why bound glucagon hepatocptes. Anat. Rec., 210:557-567. decreased in number in hepatocytes cultured for 72 Watanabe, J., S. K a na mura , K. Kanai, M. Asada-Kubota, and M. Oka hours. Therefore, the changes in glucagon receptor dis11985) Inhibitory effect of colchicine a nd vinblastine on transport of glucagon receptors to the plasma membrane in cultured rat tribution during culture is probably due to the reshufhepatocytes. J. Endrocrinol., 106t125-131. fling of the receptors that were present a t the time of Weibel, E.R. 11979) Stereologicul Methods 1. Academic Prcss;,London, culture initiation. nn 76-27 rr. _ .' 'I LITERATURE CITED Asada-Kubota, M., J. Watanabe, S. Kanamura, K. Kdnai, and M. Yoshikawa (1983)Binding and internalization of '".-'I-glucagon in Wisher, M.H., and W.H. Evans (1975)Functional polarity of rat hepatocyte surface membrane. Biochem. J., 146:375-388.