Immunocytochemical Identification of Phenylalanine Hydroxylase and Albumin

Rhodamine-conjugated antibodies specific for phenylalanine hydroxylase and serum albumin were employed as cytochemical probes to identify these two proteins in H4 hepatoma cells and in isolated rat hepatocytes . Each fluorescent antibody stained the cells specifically and in a distinctive manner . In both cell types, albumin staining was discretely localized in cytoplasmic "bundles," whereas, phenylalanine hydroxylase staining was diffuse and cytoplasmic and in H4 cultures varied somewhat from cell to cell . Evidence from cultures of REB15 cells, a strain derived by cloning H4 cells in tyrosine-free medium, suggested that the staining variability of H4 cells could reflect a variability in phenylalanine hydroxylase content. Hydrocortisone-treated H4 and REB15 cultures contain increased amounts of phenylalanine hydroxylase ; and all cells in the culture appear to be induced by the hormone . Evidence was presented to show that the albumin visualized within the isolated hepatocytes had been synthesized by these cells, and, furthermore, that quantitatively nearly all intracellular albumin in the isolated rat hepatocytes appeared to be entrained in the secretion pathway (analogous data already exist for H4 cells [Baker, R . E ., and R . Shiman . 1979 . J . Biol . Chem. 254: 9633-9639]) . By scoring specific fluorescence, 86 and 98% of the H4 cells and 89 and 98% of the isolated hepatocytes were found to contain phenylalanine hydroxylase and albumin, respectively . Therefore, almost all cells in each population appeared to synthesize both proteins . An implication of these findings is that in rat virtually all liver parenchymal cells must synthesize both phenylalanine hydroxylase and albumin . Serum albumin synthesis and phenylalanine hydroxylase expression are differentiated functions of normal mammalian liver . Both functions are also expressed by the Reuber hepatoma (rat) cell line H4 (1) . Recently, we developed a new method for measuring protein turnover and used it to determine the half-life ofphenylalanine hydroxylase in H4 cultures (2). Because the method, which we hoped also to apply in studies with perfused rat liver, involved the use of albumin as a reference protein, the question immediately arose as to whether or not albumin and phenylalanine hydroxylase were simultaneously synthesized within the same cells. Several studies (3-7) indicate that only a minority of liver parenchymal cells contain and, by implication, synthesize albumin. To our knowledge, there is no existing information regarding the distribution of phenylalanine hydroxylase among hepatic cells either in vivo or in culture. To answer the cosynthesis question, THE JOURNAL Of CELL BIOLOGY " VOLUME 90 JULY 1981 145-152 ©The Rockefeller University Press " 0021-9525/81/07/0145/OB $1 .00 we employed immunocytochemical methods to identify albumin and phenylalanine hydroxylase in H4 cultures and also in freshly isolated rat hepatocytes. Our results, reported here, are of quite general interest, because they supply information at the cellular level relating to the expression of specialized functions by differentiated tissue and by a permanent cell line derived from that tissue . MATERIALS AND METHODS Cell Culture and Hepatocyte Isolation We used the Reuber hepatoma cell line H4 (1) and REBIS, a strain of H4 derived in this laboratory . H4 cells were grown in monolayer culture with a modification (8) ofMedium S-77 containing two timesglutamne, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4, and 10% fetal calf serum, but containing no antibiotics . Detailed cell culture conditions have been described (9). 145 on S etem er 1, 2017 jcb.rress.org D ow nladed fom REB15 was derived as follows : H4 cells were selected for three passages in culture medium lacking tyrosine. (Fetal calf serum used in the preparation of tyrosine-free S-77 was exhaustively dialyzed against P04-buffered saline' [PBS]) . Surviving cells were plated sparsely in tyrosine-free medium and, after 2 wk, colonies were picked using small stainless steel cylinders (l0). One such clone, designated REB 15, was propagated in the manner described for H4 but using tyrosine-free S-77. Isolated hepatocytes were prepared from livers of normal, fed rats by a modification of the collagenase perfusion method of Berry and Friend (11) . Livers were initially perfused for 15 min in a flow-through mode with Ca"-free Krebs-Henseleit bicarbonate buffer (12), pH 7 .4, containing 27 mM glucose, 5 mM monosodium glutamate, 5 mM sodium pyruvate, and 10% (vol/vol) washed bovine erythrocytes. The perfusion system was then switched to a recirculating mode and 35 mg of collagenase (CLS 11, lot 4713275, Worthington Biochemicals, Freehold, N. J .) was added to the remaining 100 ml of buffer . After 30 min of digestion the liver was gently passed through a stainless-steel sieve (E-C Cellector, 10-mesh; E-C Apparatus Corp., Cambridge, Mass .) and any large pieces of undigested tissue were removed by filtration through a single layer of surgical gauze. Hepatocytes were collected and washed by three cycles of low-speed centrifugation and resuspended as previously described (13) with the same buffer that was used in the perfusion, but lacking erythrocytes, and supplemented to contain 2.4 mM Ca", 3% bovine serum albumin (Fraction V; Miles Laboratories, Miles Research Products, Elkhart, Ind.), and amino acids at 7.5 times the normal rat plasma level (14). After the final wash, cells were resuspended in 20 vol of buffer and either processed immediately for immunofluorescent staining or incubated in 250-ml polycarbonate Erlenmeyer flasks (40 ml suspension per flask) at 37°C in a shaking water bath (rotary motion, 150 rpm) . All perfusion, wash, and incubation buffers were maintained at 37°C and equilibrated with an oxygen-carbon dioxide mixture (95 :5%) .