Transforming growth factorβ1 increases the number of apoptotic bodies and decreases intracellular pH in isolated periportal and perivenular rat hepatocytes

Transforming Growth Factorzl Increases the Number of Apoptotic Bodies and Decreases Intracellular pH in Isolated Periportal and Perivenular Rat Hepatocytes ANTONIO BENEDETTI, ANTONIO

DI SARIO,GIANLUCA

T r a n s f o r m i n g g r o w t h factor beta 1 (TGF~I) is i n v o l v e d in p r o m o t i n g cell d e a t h by a p o p t o s i s in t h e liver, w h e r e a s t h e a c t i v a t i o n o f Na+/H ÷ e x c h a n g e r h a s b e e n related to cell proliferation. The a i m o f this s t u d y w a s to gain i n f o r m a t i o n o n t h e effects o f TGF~I o n intracellular pH a n d Na÷/I-I+ e x c h a n g e activity in isolated periportal (PP) a n d p e r i v e n u l a r (PV) rat h e p a t o c y t e s u s i n g t h e pH-seusitive dye BCECF in a p e r f u s e d s u b c o n f l u e n t hep a t o c y t e m o n o l a y e r . Steady-state intracellular pH (pHi) in a bicarbonate-free s o l u t i o n (HEPES) w e r e 7.17 _+ 0.031 in P P a n d 7.15 _+ 0.041 in PV cells. T r e a t m e n t w i t h TGF~I (120 pmol/L) for 7 h o u r s i n c r e a s e d t h e n u m b e r o f apoptotic bodies by 25% a n d 38%, a n d d e c r e a s e d steady-state pHi to 7.11 _+ 0.018 (P = .05) a n d to 7.07 + 0.021 (P < .02), respectively, in P P a n d P V h e p a t o c y t e s . In HEPES, cells r e c o v e r e d f r o m an acid load, e x t r u d i n g p r o t o n s at a rate (JH) o f 4.85 -- 1.01 mmol/L/min in P P cells a n d o f 4.91 ___0.99 mmol/L/mJn in P V h e p a t o c y t e s . This r e c o v e r y app e a r e d ~miloride inhibitable (1 retool/L). Culture w i t h TGF~I for 7 h o u r s i n d u c e d (in HEPES) a d e c r e a s e o f pHi r e c o v e r y rate from an acid load m o r e in P V (by 46%) t h a n in P P h e p a t o c y t e s (by 35%, P < .05). Acute administration of e p i d e r m a l g r o w t h factor (EGF) (10 to 100 ng/ mL) i n d u c e d a n i n c r e a s e in Na+/H ÷ e x c h a n g e activity by 32% a n d 27%, respectively, in P P a n d P V cells c o m p a r e d w i t h controls. In contrast, in cells c u l t u r e d for 7 h o u r s w i t h 120 pmol/L T G F ~ , t h e a c u t e a d m i n i s t r a t i o n o f EGF slightly i n c r e a s e d Na+/H ÷ e x c h a n g e activity (by 18%, P < .05) o n l y in P P cells. This study d e m o n s t r a t e s that pHi a n d Na÷/H ÷ e x c h a n g e activity are d e c r e a s e d by TGF~I, w h i c h i n c r e a s e s t h e n u m b e r o f apoptotic bodies in periportal a n d p e r i v e n u l a r rat h e p a t o c y t e p r i m a r y cultures. (HEPATOLOGY 1995;22:1488-1498.)

Abbreviations: EGF, epidermal growth factor; TGF, transforming growth factor; PP, periportal; PV, perivenular; BCECF, 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein; BrdU, bromodeoxyuridine;/~, intrinsic buffering power. From the Clinica di Gastroenterologia and Istitute di Patologia Sperimentale, University of Ancona, School of Medicine, Ancona, Italy. Received March 21, 1995; accepted June 26, 1995. Supported by CNR (Progetto Finalizzato FATMA, 94.00527PF41). Presented in part at the 95th Annual Meeting of the American Gastroenterological Association, New Orleans, LA, May 15-18, 1994, and published in abstract form (Gastroenterology 1994; 106:A865). Address reprint requests to: Antonio Benedetti, MD, Istituto di Patologia Sperimentale, C.P. 538, University of Ancona, 60100 Ancona, Italy. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2205-002353.00/0

SVEGLIATI BARONI, AND A N N E M A R I E J E Z E Q U E L

In recent years, specific factors have been identified to promote or inhibit liver cell proliferation. 1 Among these are hepatocyte growth f a c t o r y epidermal growth factor (EGF), 4 and transforming growth factor-aS which stimulate cell proliferation. Transforming growth factor~l (TGF~I) inhibits hepatocyte DNA synthesis in vitro 6"~° and induces cell death by apoptosis in cultured hepatocytes and in regressing liver. 1~ Cell proliferation is not, in fact, the only determinant of liver mass. Apoptosis is a type of cell death aimed at elimination of excessive or unwanted cells during remodeling of embryonic tissues, organ involution, and regression of tumors, ~2'13 thus representing an important factor in the control of organ volume under a number of different conditions. Apoptosis is under the control of growth regulatory signals, such as hormones, 14 and it is involved in normal and preneoplastic liver cell turnover. ~5 We have recently shown that, in rat and h u m a n liver, apoptosis is more frequently observed in the perivenular zone (or zone 3) of the liver acinus either in normal or in pathological conditions.~6-~9 In contrast, the majority of proliferating hepatocytes are observed in the periportal zone (or zone 1) of the acinus in steady-state conditions. 2° Identification of endogenous factors that could be mutually associated to the control of apoptosis and proliferation is a matter of considerable interest. In hepatocytes, Na+/H ÷ exchange activity is involved in the regulation of intracellular pH, 21'22 cell volume, 23 and bile secretion. In hepatocytes as well as in different mammalian cells, t h i s p u m p appears activated by EGF and other cytokines, 24-27 thus suggesting a role for Na +/ H ÷ exchange in regulating cell turnover. No data are available on the role of TGFz~ on intracellular pH and Na÷/H ÷ exchange activity, especially during different events leading to cell death by apoptosis. In the current study, Na÷/H ÷ exchange activity has been studied in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF and a perfused subconfluent hepatocyte monolayer system cultured up to 7 hours in the presence of TGFzl. MATERIALS A N D M E T H O D S Materials. Hepatocytes were isolated from 175- to 200-g male Sprague-Dawley rats, CD strain (Charles River, Italy).

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BENEDETTI ET AL 1489

TABLE 1. C o m p o s i t i o n o f B u f f e r S o l u t i o n s U s e d for Measurement of Intracellular pH Solution

Component

Na ÷ K÷ NH~ Mg2+ Ca2+ Choline TMA C1-

H2P04 SO~HCO~ HEPES Glucose CO2 (%) O2 (%) pH

1 HEPES

2 HEPES NH~

3 HEPES 0 Na +

4 (Standards)

141.0 5.9 0 1.0 1.25 0 0 142.2 1.2 1.0 0 10.0 5.0 0 100 7.4

121.0 5.9 20.0 1.0 1.25 0 0 142.2 1.2 1.0 0 10.0 5.0 0 100 7.4

0 5.9 0* 1.0 2.5 130.0 6.0t 137.2 1.2 1.0 0 10.0 0 0 100 7.4

0 121.2 0 1.0 0 10.0 05 130.0 1.2 1.0 0 20.0 0 0 100 6.8-7.2-7.6

NOTE. All concentrations are given in mmol/L. Abbreviation: TMA, tetramethylammonium. * In experiments to determine/~i, different [choline-C1-] was replaced with different [NH4C1]. t pH was titrated to 7.4 with ~6 mmol/L TMA-OH. $ pH was titrated to different pH (6.8, 7.2, 7.6) with TMA-OH.

All animals used in this study received humane care in compliance with the institution's guidelines. Collagenase A and digitonin were obtained from Sigma Chemical Co. (St. Louis, MO). The fluorescent dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was purchased from Molecular Probes (Eugene, OR). BCECF/AM was made up as a 1.0 mmol/L stock in dimethylsulfoxide. Matrigel, TGF~I, and EGF were from Collaborative Research (Lexington, MA), and culture media were from GIBCO (Grand Island, NY). Bisbenzimide H33258 was from Fluka Chemie (Buchs, Switzerland). All other reagents were from Sigma Chemical Co. (St. Louis, MO). Solutions. The composition of different solutions used for studies of pHi regulation is shown in Table 1. Isolation and Culture of Periportal and Perivenular Hepe~ tocyte.Enriched Fractions. The isolation of periportal (PP) or perivenular (PV) hepatocyte-enriched fractions was performed as previously described. 2s After anesthesia with sodium thiopental (Farmotal, 100 mg/kg intraperitoneally) the portal vein was cannulated with a 16-gauge cannula and perfused in situ for 7 minutes at 37°C. The papilliform lobe was then removed and the vena cava superior was cannulated. A 7 mmol/L digitonin solution was then infused at a rate of 10 mL/min for 18 to 25 seconds (37°C) through the portal vein to destroy PP hepatocytes and preferentially isolate PV cells, or through the supradiaphragmatic part of the inferior cava vein to destroy PV hepatocytes and preferentially obtain PP cells. The infusion was stopped when the pattern of the liver surface characteristic of selective destruction was clearly delineated. Perfusion with Ca +÷, Mg++-free solution (pH 7.5) was initiated through the opposite cannula after stopping the infusion of digitonin, and continued for 10 minutes. The liver was then perfused from the direction opposite to the digitonin

pulse with Ca ++, Mg++-containing L-15 media supplemented with collagenase A (360 U/L), HEPES (25 mmol/L), bovine serum albumin (1 g/L) (pH 7.4) for 10 minutes at 37°C. When the capsule began to detach, the perfusion was stopped, and the liver was immersed in ice-cold modified L-15. The cells were released by gentle combing into 50 mL of L-15 culture media on a Petri dish, filtered, and pelleted twice as previously described. The initial cell viability determined by trypan blue exclusion varied considerably but was commonly above 70%. After a partial purification obtained as previously described, 29 the viability generally exceeded 83% to 88%. Aliquots of 1.5 mL of the cell pellet were resuspended in 30 mL of HCO3/CO2-containing medium (alfa-MEM medium). This medium was supplemented or not with 10% fetal calf serum, pH 7.40, and 100/~g/mL bovine serum albumin, as required by the experimental condition. Cells (10 mL/dish) were then layered on glass coverslips (3 x 0.5 cm) precoated with an extracellular biomatrix (Matrigel) diluted 1:1.5 with L-15-HEPES medium. Hepatocytes were incubated at 37°C up to 7 hours, during which time they formed a subconfluent monolayer on the glass coverslips with a density o f - 10z cells/ mm 2. The medium was not renewed for all the culture period (up to 7 hours). Enzymatic Determinations. Four marker enzymes were analyzed, as markers, in the purified cell fractions to evaluate the good reproducibility of the separation technique. All assays were carried out in duplicate at 37°C. Proteins were determined according to the method of Lowry et alJ ° Glutamine synthetase activity w a s carried out by measuring the rate of gamma-glutamyl hydroxamate formation in the transferase reaction catalyzed by the enzymeJ 1 Lactate dehydrogenase activity was assayed by the method of Bergmeyer et alJ 2 Pyruvate kinase and alanine transaminase were measured spectrophotometrically as described by Boyer3~ and Wroblewski and La Due, 84 respectively. The periportal dominance of alanine aminotransferase and lactate dehydrogenase and the perivenous dominance of glutamine synthetase and pyruvate kinase have been previously observed in several studies. EVALUATION OF A P O P T O T I C C E L L S Apoptotic bodies in the culture system have been characterized as recently described, s~ After 7 hours of incubation, the medium was gently removed, and cultures (n -- 36, 18 PP and 18 PV cultures) were fixed for 5 minutes with 3% paraformaldehyde in phosphate-buffered saline (pH 7.4) at 4°C, washed with distilled water, and dried at room temperature. Cells were stained with H33258 (8 #g/mL) for 5 minutes, washed, and mounted in Moviol. Fluorescent H33258-stained nuclei were scored and classified according to the condensation and staining characteristics of chromatin as follows: (1) normal nucleus, uncondensed chromatin dispersed over the whole nucleus; (2) nucleus with condensed chromatin, masses of condensed chromatin located at the nuclear membrane; (3) fragmented nucleus, groups of isolated pieces of condensed chromatin after nuclear fragmentation. 35 An example is shown in Fig. 1. Fluorescence was observed using an AHBT3 photomicrographic microscope system (Vanox, Olympus, Tokyo, Japan) equipped with a 40x objective. Either nuclei with condensed chromatin or fragmented nuclei were considered as apoptotic bodies and were counted as percent of total nuclei per field. At least 10 nonoverlapping fields per dish were counted in 12 PP and PV hepatocyte monolayers treated up to 7 hours with or without TGFzl (120 pmol/L).

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i

FIG. 1. Morphologyof hepatocytes treated with TGF~I. (A-D) Fluorescence microscopy of H33258 staining. (Original magnification ×200.) (A) Control with normal nuclei. (B-D) Hepatocyte cultures treated with TGFzl (120 pmol/L) for 7 hours. Arrows indicate condensation of the nuclear chromatin and aggregation at the nuclear membrane (B), and fragmented nuclei in PP (C) and PV (D) hepatocyte cultures.

EVALUATION OF P R O L I F E R A T I N G HEPATOCYTES To measure the percentage of proliferating PP and PV hepatocytes in culture, cell cultures (n = 36, 18 PP and 18 PV cultures) grown on coverslips for 7 hours were exposed to bromodeoxyuridine (BrdU, 10 #mol/L) for 30 minutes at 37°C, then fLxed in paraformaldehyde 4% in phosphate-buffered saline overnight. The cells were incubated in formamide 95% in Na÷-citrate at 70°C for 45 minutes and exposed to mouse anti-BrdU (Dako, M 744, 1:25) in Tris-buffered saline for 30 minutes followed by horseradish peroxidase-labeled rabbit anti-mouse (Dako, P 161, 1:50) for 30 minutes and horseradish peroxidase-labeled swine anti-rabbit (Dako, P 217, 1:100) for 30 minutes at room temperature. The immunoreactive sites were detected by incubation with 0.6 mg/mL diaminobenzidine in Tris-buffered saline containing 0.01% H202 for 10 minutes and brief staining with hematoxylin. The positive nuclei appeared brown against light blue background. 2° Ten fields per dish were counted using an AHBT3 photomicrographic microscope system (Vanox, Olympus) equipped with a 40× objective. The average number of BrdU-positive nuclei per field was calculated in PP and PV subconfluent monolayers cultured in different conditions (see Results) and expressed as percentage of total nuclei.

M E A S U R E M E N T OF pH, pHi was measured using the fluorescent indicator BCECF given as the acetoxymethyl-ester (BCECF/AM) with a Perkin-Elmer LS-50 spectrofluorimeter as described in detail elsewhere. 36 Background fluorescence intensity was measured, and thereafter cells were loaded with BCECF (10 #mol/L) for about 10 minutes and subsequently washed by perfusion with dye-free solution for an additional 10 minutes. All solutions used to superfuse the cells were prewarmed. At the beginning of the experiment, values for fluorescence intensity at 450 nm were 7.98 _+ 4.31 (SD) (range, 5.4 to 18.3)-fold higher than background values. pHi was measured during continuous superfusion (10 mL/ min) as the fluorescent intensity ratio between the pHi-sensitive wavelength (Fisoo) and the isosbestic point (Fi45o), thus correcting for changes in intracellular dye concentration as described for other cell systems. pHi was calculated correlating the measured Fisoo/Fi45oratio with an intracellular calibration curve obtained at the end of each experiment by perfusing with the H+-K÷ ionophore nigericin (12.5 #mol/L) in high-K + medium buffered at different pHo (6.8, 7.2, 7.6; Table 1) as previously described. 36 After completing each set of experiments, the viability of

HEPATOLOGYVol. 22, No. 5, 1995 the preparation was reassessed by trypan blue exclusion and routinely ranged between 68% and 78%, including the cells exposed to NH~, amiloride, TGF~I, or EGF. Protocols for lntracellular Acid Loading. Cells were acid loaded by pulse exposure to 20 mmol/L NH4CI.36 Recovery of pHi from an acid load was assessed under control conditions in a HCO~-free solution (HEPES solution) (Table 1) as well as during perfusion with 1 mmol/L amiloride. TGFzl (120 pmol/L) was added to the culture medium, and then the cells were cultured for 7 hours or it was acutely added to the perfusate 20 minutes before the calculation of pHi and then was present throughout the experiment. Media were supported with 100 #g/mL bovine serum albumin to avoid unspecific binding of TGFzl. 10 to 100 ng/mL EGF was acutely added to the perfusate 5 minutes before the NH4Cl-withdrawal maneuver. Each experiment was assessed relative to a paired control, performed in varying order in three to five separate cell preparations on different days. The pHi recovery to baseline was quantitated as (1) maxim u m pHi recovery rate over a 2-minute period after the acid load ((~pH/(~tmax).The ~pH/~tm~ was measured by the tangent from the experimental plots; (2) maximum H ÷ rate (Jn), calculated by multiplying 5pH/Stmax by intracellular buffering power (see below). Statistical comparisons were performed using the paired or unpaired Student's t-test as appropriate. Data are presented as means _+ SD. Determination of Intracellular Intrinsic Buffering Power (~,). The intrinsic buffering power (f~i) (in the absence of the open buffering system HCOJCO2) of isolated periportal (n = 5) or perivenular (n = 7) hepatocytes was determined at different pHi as described. 36 RESULTS

BENEDETTI ET AL 1491 2.0 A ® ~ o "4 ~

1.6 1.2 0.8

~, 0.4

= C

0.0

PP PV controls

PV

+ TGFbeta1

2.0 1.6 ~¢" 2O ~ ~5 1.2 oo "~ ~ 0.8 "~ gE~ 0.4 o.o

I'll

T

PP PV controls

PP PV + TGFbeto 1

PP PV controls

PP PV + TGFbeta I

2.o C

Isolation of Periportal and Perivenular Hepatocyte-

1.6 ._~ o ~ ~ 1"2'

in n u c l e a r m o r p h o l o g y s h o w n b y H 3 3 2 5 8 s t a i n i n g allowed to q u a n t i f y a p o p t o s i s in vitro. 3~ F o r t h i s p u r p o s e we d i s t i n g u i s h e d t h r e e k i n d s of nuclei: n o r m a l nuclei, nuclei w i t h c o n d e n s e d c h r o m a t i n , a n d f r a g m e n t e d nuclei (Figs. I a n d 2). A f t e r 7 h o u r s of culture, t h e subconf l u e n t m o n o l a y e r still c o n t a i n e d 95% of t h e cells p r e s e n t a f t e r plating, e i t h e r in control c u l t u r e s or in c u l t u r e s t r e a t e d w i t h TGFB~ (20 to 120 pmol/L). T r e a t m e n t w i t h TGFz~ (120 pmoUL) for 7 h o u r s induced a significant i n c r e a s e in t h e t o t a l n u m b e r of apoptotic bodies (% of nuclei w i t h c o n d e n s e d c h r o m a t i n + f r a g m e n t e d nuclei) w i t h r e s p e c t to controls e i t h e r in P P or in P V h e p a t o c y t e s (0.60 _+ 0.10% vs. 0.40 +_ 0.10%, P < .05 a n d 0.75 _+ 0.15% vs. 0.50 _ 0.10%, P < 0.05, in P P a n d PV cells, respectively) (Fig. 2A). T h i s inc r e a s e w a s p a r t i c u l a r l y e v i d e n t for nuclei w i t h con-

PP

B

E n r i c h e d Fractions. P e r i p o r t a l a n d p e r i v e n u l a r h e p a t o c y t e - e n r i c h e d f r a c t i o n s w e r e c h a r a c t e r i z e d according to t h e d i s t r i b u t i o n p a t t e r n of four m a r k e r e n z y m e s . In agreement with previous data, the animals showed h i g h e r activities for l a c t a t e d e h y d r o g e n a s e a n d for ala-

nine transaminase in the periportal zone, with a PP/ PV ratio of 1.9 and of 1.3, respectively. The activity of glutamine synthetase was exclusively present in PV hepatocyte preparations. Pyruvate kinase activity was slightly higher in hepatocytes isolated from the perivenular zone, showing a PP/PV ratio of 0.8. Morphometric Analysis of Apoptotic Bodies. Changes

i lll T

E

® o ~

0.8

0.4.

0.0

FIG. 2. Morphometrical evaluation of apoptosis in PP and PV hepatocyte cultures treated with TGF~I (120 pmol/L) for 7 hours. (A) Total number of apoptotic bodies (nuclei with condensed chromatin + fragmented nuclei) expressed as percentage of total nuclei. (B) Nuclei with condensed chromatin (% of total nuclei). (C) Fragmented nuclei (% of total nuclei). (A) 7 hours' culture with TGF~I (120 pmol/ L) significantly increases total number of apoptotic bodies with respect to controls either in PP or in PV hepatocytes (*P < .05 vs. corresponding controls). (B) The increase was significant for nuclei with condensed chromatin, particularly in PV cell cultures (*P < .05 vs. corresponding PP controls, and **P < .02 vs. corresponding PV controls). (C) No significant differences have been observed for percentage of fragmented nuclei in cell cultures treated for 7 hours with TGFzl with respect to corresponding PP or PV controls.

1492

BENEDETTI ET AL

HEPATOLOGY November 1995

50.0

~

¢o.o

o.o

- -

20.0

ioo

0.0

I PP PV controls

PP

PV

+ EGF

PP

PV

+ EGF & TGFbeto I

FIG. 3. Morphometrical evaluation of proliferating PP and PV hepatocytes. (Blank box) Controls (# = P < .05 vs. PV control cells). (Narrow left diagonal) PP and PV hepatocyte cultures treated for 7 hours with EGF (100 ng/mL) (* = P < .001 vs. corresponding controls). (Narrow crosshatch) PP and PV hepatocyte cultures treated for 7 hours with EGF (100 ng/mL) and TGFzl (120 pmol/L) (** = P < .005 vs. PV cells treated with EGF alone).

densed chromatin in cultures of PV hepatocytes (0.60 + 0.05% in TGFzl-treated cells vs. 0.35 + 0.05% in controls; P < .02), whereas, in P P hepatocyte cultures, the difference in percentage of nuclei with condensed chromatin was less but still significantly higher (0.45 _+ 0.10% in TGFz~-treated cultures vs. 0.30 + 0.05% in controls, P < .05) (Fig. 2B). In contrast, no significant differences have been observed for percent of fragmented nuclei either in PP or in PV hepatocyte monolayers after 7 hours of incubation with TGFzl (120 pmol/L) (Fig. 2C). When the cells were treated up to 7 hours with 20 pmol/L TGFzl, no significant differences were observed for the total number of apoptotic bodies (percent of nuclei with condensed chromatin + fragmented nuclei) either in PP or in PV cell cultures (data not shown), suggesting that the induction of apoptosis by TGF~I is dose dependent.

Morphometric Analysis of Proliferating Hepatocytes. Incubation with EGF for 7 hours significantly increased the number of proliferating hepatocytes (percent of total) with respect to control cultures (21.5 ± 3.5% vs. 6.5 ± 0.15%, P < .001 and 15.5 _ 1.5% vs. 4.9 ± 0.05%, P < .001, in PP and PV cells, respectively) (Fig. 3) as detected by BrdU-anti-BrdU immunohistochemistry. Moreover, the increase in percentage of proliferating hepatocytes was significantly higher in PP than in PV cultures (P < .05). Because no significant differences in cell proliferation were observed in hepatocytes exposed to 10 or 100 ng/mL EGF either in PP or PV fractions, the two doses were considered as a single group of treatment. The simultaneous addition to the culture medium of TGFB~ (120 pmol/L) together with EGF (100 ng/mL) significantly reduced the increase in number of proliferating hepatocytes in PV fraction (8.5 ± 2.5% in PV cells cultured with EGF + T G F ~ vs. 15.5 ± 1.5% in PV cells cultured with EGF

alone, P < .005), but there was only a slight decrease in PP fractions (17.0 _+ 4.0% in PP cells cultured with EGF + TGFzl vs. 21.5 _+ 3.5% in PP cells cultured with EGF alone, P = NS) (Fig. 3). Intrinsic B u f f e r i n g Power. Intrinsic buffering power (Bi) appears strongly dependent on pHi, with fli increasing at low pHi either in periportal or in perivenular hepatocytes. 3s fli values were grouped into 0.2-pH unit buffering domains and plotted _+ 1 SD versus midpoint of buffering domain (average fli). Only between phi 6.55 and 6.75, fli averaged points were significantly higher in isolated periportal (66.20 _+ 0.99 mmol/L/pHu at phi 6.55) than in perivenular hepatocytes (59.18 + 2.46 mmol/L/pHu at pHi 6.55, P < .05) either in the presence of or in the absence of 20 to 120 pmol/L TGFzl in the culture medium. When measured at different pHi (in range of pHi between 6.8 and 7.7),/?i did not significantly differ in both isolated cell fractions in any culture condition, measuring 20.18 _+ 1.63 mmol/L/pHu in PP cells and 21.00 _+ 1.26 in PV hepatocytes at pHi 7.35. Baseline phi. I n a nominally bicarbonate-free HEPES-buffered medium, the difference in baseline pHi between isolated P P and PV cells was not significant (7.17 + 0.031, n = 9, vs. 7.15 + 0.041, n = 8, respectively) (Table 2, series 1 and 8). Culture with TGFzl (120 pmol/L) for 7 hours significantly reduced baseline pHi either in PP or PV hepatocytes with respect to corresponding controls (7.11 _+ 0.018, n = 6, vs. 7.17 _+ 0.031, n = 9, in PP hepatocytes, respectively treated or not with TGFB1 , P = .05; and 7.07 _+ 0.021, n = 6, vs. 7.15 _+ 0.041, n = 8, in PV cells, respectively treated or not with TGFB1 , P < .02) (Table 2, series 3 and 10). However, the difference in baseline pHi observed between PP and PV hepatocytes treated for 7 hours with TGFz~ did not reach statistical significance. One hundred twenty picomolar TGFzl slightly and reversibly decreased baseline pHi ( - 0 . 0 4 ± 0.02 pH~i~, n = 6, P = .05 vs. controls) when acutely administered (Fig. 4). Recovery of pHi F r o m a n A c u t e A c i d Load. To study the activity of sinusoidal Na+PH + exchange, which mediates acid extrusion when the cells are perfused in a nominally bicarbonate-free medium, recovery of pHi from an acute acid load has been evaluated by applying and then withdrawing 20 mmol/L NH4CI from a H E P E S solution buffered to pH 7.4. The recovery of pHi after an acute acid load was similar in control PP (JH = 4.85 + 1.01 mmol/L/min, n = 9) and PV hepatocytes (JH = 4.91 --+ 0.99 mmol/L/ min, n = 8) (Table 2, series 1 and 8). The superfusion with 1 mmol/L amiloride inhibited pHi recovery from an acid load by 80% either in PP (JH = 1.30 _+ 0.33 mmol/L/min, n = 5, P < .001 vs. corresponding controls) or in PV cells (JH = 1.39 + 0.29 mmol/L/min, n = 5, P < .001 vs. corresponding controls) (Table 2, series 2 and 9). Inhibition was reversible, and phi recovered to baseline after withdrawal of amiloride.

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BENEDETTI ET AL 1493

TABLE 2. Parameters o f pHi; Recovery From an Acute Acid Load U n d e r Various E x p e r i m e n t a l Conditions Condition PP hepatocytes 1. HEPES alone 2. HEPES, amiloride 3. HEPES, TGF~I (120 pM, 7 hr) 4. HEPES, TGF~I (120 pM, 20 min) 5. HEPES, EGF (100 ng/ml) 6. HEPES, EGF, amiloride 7. HEPES, EGF, TGFsl (7 hr) PV hepatocytes 8. HEPES alone 9. HEPES, amiloride 10. HEPES, TGF~I (120 pM, 7 hr) 11. HEPES, TGF~I (120 pM, 20 min) 12. HEPES, EGF (100 ng/ml) 13. HEPES, EGF, amiloride 14. HEPES, EGF, TGF~I (7 hr)

N

Baseline pHi

Nadir pHi

9 5 6

7.17 _+ 0.031 7.16 _+ 0.039 7.11 _+ 0.018

6.80 _+ 0.049 6.58 _+ 0.018 6.72 _+ 0.029

4.85 + 1.010 1.30 _+ 0.330* 3.16 _+ 0.290

7 8 8 8

7.16 7.19 7.16 7.14

6.76 6.82 6.75 6.78

4.80 6.39 1.44 3.72

8 5 6

7.15 _+ 0.041 7.14 _+ 0.023 7.07 _+ 0.021

6.76 _+ 0.052 6.56 + 0.021 6.70 _+ 0.021

4.91 _+ 0.990 1.39 _+ 0.290* 2.66 _+ 0.180¶

7 8 8 8

7.13 7.17 7.18 7.13

6.71 6.80 6.75 6.80

3,48 6.23 1.44 2.66

+_ 0.038 _+ 0.031 _+ 0.041 + 0.021

_+ 0.048 _+ 0.042 _+ 0.031 _+ 0.025

_+ 0.052 _+ 0.063 _+ 0.041 _+ 0.039

_+ 0.031 _+ 0.059 +_ 0.039 _+ 0.030

H + e f f i u x (JK) ( r e t o o l / L - l / r a i n - l )

+_ 0.660 _+ 0.910~ _+ 0.6905 _+ 0.310§]]

_+ 0.480# _+ 0.590t +_ 0.7305 _+ 0.450

NOTE. Values are means _+ 1 SD; N = number of experiments. * P < .001 vs. corresponding controls. t P < .01 vs. corresponding controls. $ P < .001 vs. corresponding samples treated only with EGF. § P < .05 vs. samples incubated in absence of TGF~I. ]]P < .01 vs. PV cells. ¶ P < .05 vs. corresponding PP cells. # P < .05 vs. corresponding PP cells.

C u l t u r e w i t h TGFz~ (120 pmol/L) for 7 h o u r s signific a n t l y d e c r e a s e d pHi recovery from a n acid load b o t h in P P cells (by 35%, P < .01) a n d in P V h e p a t o c y t e s (by 46%, P < .01) w i t h respect to controls. H o w e v e r , the r e d u c t i o n of pHi recovery from a n acid load was significantly h i g h e r in PV- t h a n in P P - t r e a t e d cells (JH = 2.66 +_ 0.18 mmol/L/min, n = 6, vs. 3.16 _+ 0.29 m m o l / L/min, n = 6, respectively, P < .05) (Fig. 5 a n d Table 2, series 10 a n d 3).

9.0 ¸

1'

HEPES

_. I_HEPEs

TGFbete 1

I

8.5 ¸

8.0 ~F f~

7.5

7.0

6.5

6.0

I

I

I

I

I

200

400

600

800

1000

1200

time (sec)

FIG. 4. Effect of TGF~I(120 pmol/L) on baseline pHi in PV hepatocytes (7 hours culture). When acutely administered, TGF~I slightly and reversibly decreased baseline pHi, similarly in PP (data not shown) and PV cell cultures. The recording is representative of nine similar experiments in PP and PV hepatocytes.

As shown in Fig. 6, w h e n t h e r a t e s of pHi recovery from a n acid load w e r e plotted a g a i n s t t h e n a d i r pHi, t h e i n t e r c e p t of t h e curve was lower for b o t h P P a n d P V h e p a t o c y t e s c u l t u r e d for 7 h o u r s in the p r e s e n c e of TGF~I w i t h respect to c o r r e s p o n d i n g controls. This indicates t h a t , for a n y given pHi, Na+/H + exchange activity was lower w h e n e i t h e r P P or PV cells w e r e cult u r e d w i t h TGF~I. In addition, t h e absence of significant changes in slope b e t w e e n the curves suggests t h a t t h e e x p o s u r e to this cytokine m o s t likely r e s u l t s in a decrease in t h e n u m b e r of m e m b r a n e u n i t s available for t r a n s p o r t u n d e r t h e s e conditions. TGF~I (120 pmol/L) added to the culture m e d i u m j u s t 20 minutes before starting pHi m e a s u r e m e n t s (time 0) significantly decreased pHi recovery from a n acid load only in PV cells with respect to controls (JH = 3.48 +_ 0.48 mmol/L/min, n = 7, vs. 4.91 _+ 0.99 mmol/L/min, n = 8, respectively, P < .05) (Table 2, series 11). In contrast, in the same experimental condition, no significant difference was observed in P P cells t r e a t e d with 120 pmol/L TGFzl with respect to controls (Table 2, series 4). Acute a d m i n i s t r a t i o n of E G F (100 ng/mL) 5 m i n u t e s before NH~Cl-withdrawal significantly i n c r e a s e d pHi recovery from a n acid load in P P (by 32%, n = 8, P < .01) as well as in PV cells (by 27%, n = 8, P < .01) w i t h r e s p e c t to controls (Fig. 7 a n d Table 2, series 5 a n d 12). i mmol/L amiloride r e v e r s i b l y i n h i b i t e d the increase in pHi r e c o v e r y i n d u c e d by acute a d m i n i s t r a t i o n of E G F (JH = 1.44 _+ 0.69 mmol/L/min, n = 8, a n d 1.44 _+ 0.73, n = 8, in P P a n d P V cells, respectively) (Table 2, series 6 a n d 13).

1494 BENEDETTI ET AL

HEPATOLOGYNovember 1995

PERIPORTAL (PP) HEPATOCYTES

PERIVENULAR ( I N ) HEPATOCYTES

8.0-

8.0-

HEPES I

N_H_4CI

HEPES

I

H~ES I

I

NH~C,

'I

HEPES

I

7.5-

7.5. -I-

"r a.

Q.

7.0.

7.0'

6.5 0

I 200

I 400

I 600

I 800

I 1000

6.5 1200

0

I

I

I

I

I

200

400

600

800

1000

1200

time Csec)

time (sec)

FIG. 5. Effect of 7 hours culture with TGFzl (120 pmol/L) on pHi recovery from an acute acid load in the nominal absence of H C O J CO2. Seven hours' culture with TGFBI (120 pmol/L) decreased pHi-recovery from an acute acid load more in PV than in PP cells with respect to corresponding controls. The recordings are representative of six similar experiments, respectively, in PP and PV hepatocytes.

The acute administration of EGF (100 ng/mL) slightly increased pHi recovery from an acid load (by 18%, n = 8) in PP but not in PV cells incubated with 120 pmol/L TGFzl for 7 hours with respect to hepatocytes treated with EGF alone (100 ng/mL) (P = .05) (Fig. 8 and Table 2, series 7 and 14). DISCUSSION

The results of the current study show that modifications of pHi and Na÷/H ÷ exchange activity are evident 8 ¸

6¸

E "1"

2

0 6.3

6.5

6.7

6.9

7.1

7.3

PHi FIG. 6. Relationship between maximal H ÷ effiux rates (JH) and nadir pHi after the NH4C1 pulse in control PP (e) and PV (O) hepatocytes or in P P (A) and PV (A) cells treated for 7 hours with TGFzl (120 pmol/L). The regression equation in control cells was y = 353.2 - 48.6x (r = .991, P < .0001) and y = 355.9 - 48.2x (r = .990, P < .0001), respectively, in P P and PV hepatocytes. In contrast, in cells treated for 7 hours with TGFzl (120 pmol/L), the regression equation was y = 381.1 - 47.5x (r = .968, P < .001) and y = 392.5 - 47.7x (r = .973, P < .001), respectively, in PP and PV hepatocytes. The corresponding set points (intercept with the x axis) averaged 7.15 _+ 0.01 and 7.13 --- 0.02, respectively, in control PP and PV cells. In cells treated for 7 hours with TGF~I (120 pmol/L), the corresponding set points averaged 6.98 _+ 0.01 and 6.91 _+ 0.02, respectively, in P P and PV hepatocytes.

in hepatocytes that underwent stimuli promoting cell death by apoptosis. A lobular gradient appears evident in different kinds of response of parenchymal cells to TGFzl. Our data demonstrate that TGFzl, known to induce apoptosis of liver parenchymal cells, has a significant inhibitory effect on Na÷/H ÷ exchange activity in shortterm cultured rat hepatocytes, especially in cells isolated from the PV zone, where apoptosis has been more frequently observed in vivo. 1~ A number of conclusions can be drawn from our findings (Fig. 9): (1) in the nominal absence of bicarbonate, pHi recovery from an acid load is similar in PP and PV control hepatocytes, pHi recovery from an acid load is amiloride dependent in both cell fractions, i.e., mediated by Na÷/H ÷ exchange as previously demonstrated36; (2) incubation with TGFzl for 7 hours increases the number of apoptotic bodies, especially in PV hepatocyte cultures, and decreases pHi recovery from an acid load in PV more than in P P cells. When TGFz~ is acutely added to the perfusion system, phi recovery from an acid load significantly decreases only in PV hepatocytes; (3) EGF stimulates the rate of proliferation of isolated hepatocytes and particularly of cells isolated from PP zone. TGFz~ reduces this stimulation induced by EGF, especially in PV cells; (4) acute administration of EGF stimulates pHi recovery from an acid load in both hepatocyte fractions. This stimulation is amiloride dependent and is still partly present only in PP cell fractions when hepatocytes are cultured for 7 hours in the presence of TGFzl. Cell death has traditionally been considered as a passive degenerative phenomenon consequent to toxic injury. However, a nontoxic type of cell death is known to occur in the embryo or during metamorphosis and is strictly related to the developmental program. This type of cell death has been termed "programmed cell death. ''~7'8s According to this concept, apoptosis is the complement of mitosis, and the maintenance, growth,

HEPATOLOGYVol. 22, No. 5, 1995

BENEDETTI ET AL 1495 PERNENULAR(PV) HEPATOCYTES

PERIPORTAL(PP) HEPATOCY't~ 8.0

8.0

HEPES I I

....

J .....

_.

HE~I

.....

NH4Cl

I

HEPES

I !

EGF

7.5 ¸

7,5

°-"TO.

I

EGF

1

°

:E D.

7.0

7.0-

6.5

I

0

6.5

200

400

600

800

1000

1200

0

I

I

I

I

I

200

400

600

800

1000

(Hc)

tlme

time

1200

(sec)

FIG. 7. Effect of acute administration of EGF (100 ng/mL) on pHi recovery from an acute acid load in PP and PV hepatocytes. Acute administration of EGF (100 ng/mL) 5 minutes before NH4C1withdrawal significantly increased pHi recovery from an acute acid load either in PP or PV cells with respect to corresponding controls. The recordings are representative of eight similar experiments, respectively, in PP and PV hepatocytes.

or involution of tissues are dependent on the equilibrium between these two phenomenons. The current study confirms an increase of apoptotic bodies in shortterm cultures in the presence of TGF~I. These are characterized by DNA condensation and formation of oligosomal fragments. 35 Previous data 3~ have shown that TGFz~-induced apoptosis of cultured hepatocytes after longer exposure compared with the current study, but to maintain the culture for several days, many hormones and growth factors had to be added. In contrast, when cells are cultured for a short time, the system maintains good functional and morphological features for 6 to 8 hours. 89 At that time, the number of apoptotic bodies

in control PP and PV cells was very low, but increased by 25% and 38%, respectively, in PP and PV hepatocytes treated with 120 pmol/L--not with 20 pmol/L TGFzl, providing evidence of a dose-dependent effect in the induction of apoptosis by this cytokine. The increased number of apoptotic bodies includes a significant increase in nuclei showing condensed chromatin but not in fragmented nuclei (Fig. 3). The whole process of apoptosis in vivo takes approximately 3 hours. 4° In contrast, the first visible stage of apoptosis, i.e., chromatin condensation, has a duration of a few minutes in vivo but requires 90 minutes in vitro. 85 This suggests that nuclei with condensed chromatin persist longer in vitro than in vivo before fragmentation of the nucleus

PERIVENULAR (PC) HEPATOCYI"ES

PERIPORTAL (PP) HEPATOCY'rEs 8.0

l I HEPES I NHACI I I'IEPIES . . . . . I____I__I . . . . . . . . . . . . .

J i INHJCI I HEPIES I____%____I . . . . . . . . . . . . . . .

8°0 '

, I

._'2__'__',

=

I NI.I .

I

L_'-':__t i

I

I_ . . . . . . . . . . . . . . . . . . . .

I

:

7.5- ~

'

0

, I I

7.5

~7.0-

6.5

I [~F

7.0

t

I

I

t

400

800

1200

1600

time

(sec)

6.5 2000

I

I

I

I

400

800

1200

1600

time

2000

(sec)

FIG. 8. Effect of EGF (100 ng/mL) on pHi recovery from an acute acid load in PP and PV hepatocytes cultured for 7 hours in the presence of TGFzz (120 pmol/L). In cells cultured for 7 hours in the presence of TGF~z (120 pmol/L), the acute administration of EGF (100 ng/mL) slightly increased pHi recovery from an acute acid load only in PP cells. The recordings are representative of eight similar experiments, respectively, in PP and PV hepatocytes.

1496

BENEDETTI ET AL 150-

[::]

HEPATOLOGY November 1995

PP cells PV cells

<

o, 100.

o=OO ~

s0-

K

bJ > 0 (J bJ nr

,,'~o

I

.-

u-

•

FIG. 9. Summary of experiments on pHi recovery from an acute acid load. Percent of recovery from an acute acid load in P P and PV isolated hepatocytes in HEPES-buffered solutions.

and may account for the increased configuration of apoptotic bodies showing condensed chromatin versus fragmented nuclei. We have recently shown that, in rat and human liver, apoptosis is more frequently present in the PV zone of the liver acinus in either physiological or pathological conditions. ~8'~9Current data show that the lobular gradient of apoptosis is maintained in isolated hepatocytes in primary short-term culture also in the presence of TGF~, suggesting that, after isolation, the cells maintain features related to aging as proposed by the theory of the "streaming liver. "4~ Our findings also confirm a higher proliferative rate of PP with respect to PV hepatocytes when cells are exposed to EGF (Fig. 3), in agreement with previous data 42 and different from results obtained after partial hepatectomyY However, the higher proliferative capacity of PP hepatocytes is provided by the observation that in hepatocytes simultaneously cultured in the presence of EGF and TGFzl, the EGF-increased rate of cell proliferation is significantly reduced only in PV cells (Fig. 3). Identification of endogenous factors mutually associated with the control of apoptosis and cell proliferation remains a matter of considerable interest. It has been demonstrated that Na÷/H ÷ exchange is activated by EGF 2426 and that the inhibition of this pump abolishes growth-factor-stimulated DNA synthesisY Although these observations suggest a role for Na+/H ÷ exchange in the activation of hepatocellular proliferation, nothing is known about a possible modification of Na÷/H ÷ exchange activity in cells receiving stimuli known to induce cell death by apoptosis. Na+/H ÷ exchange in the liver is important in bile secretion, intracellular pH, and volume regulation. 21-23 To study modifications of Na÷/H ÷ exchange activity in different steps of the cell cycle, we have paid special attention to the role of cytokines that stimulate cell

proliferation or death. This has been performed in cells at short intervals stimulated by different cytokines, to obtain data concerning early steps of cell death or proliferation. Experiments performed in HEPES perfusate confirm our recent data 21'22 demonstrating that Na+/H ÷ exchange activity is not significantly different in control PP and PV hepatocytes. 36 TGFB1 induces (1) a very slight acidification of baseline pHi similar in PP and PV hepatocytes when acutely administered, but higher in PV with respect to PP cells after 7 hours' culture; (2) a decrease of Na÷/H ÷ exchange activity only in PV cells when acutely administered; (3) a higher decrease ofNa+/H ÷ exchanger activity in PV than in PP hepatocytes after 7 hours' culture; (4) no modification ofintracellular buffering power (fli). All these data suggest an inhibitory effect of TGFB1 on Na÷/H ÷ exchange activity similar to specific inhibitors. In contrast with amiloride, TGFz~ is slower in inihibiting Na÷/H + exchange activity. This is also confirmed by the effect of acute administration of TGF~I on steady-state pHi, inducing a very slight decrease of pHi. This phenomenon remains to be explained but is likely attributable to an insufficient length of incubation or to a low activity of Na÷/H ÷ exchange at the pHivalue considered (7.15 to 7 . 2 0 ) . 21'24 In contrast, the acidifying effect of TGFzl cannot be explained by a modification of the intracellular buffering power, because TGFzl does not induce alterations of/~i. When cells were incubated for a longer time (7 hours) with TGF~, a decrease of Na÷/H + exchange activity was significantly higher in PV than in PP cells. This partially accounts for the reduced baseline pHi observed in these conditions, especially in PV cells. Moreover, the observed decreased Na+/H ÷ exchange activity cannot be firstly explained by an acute effect of TGF~I at a modifier site of the exchanger. Rather, as for any giver pHi, the activity ofNa÷/H ÷ exchanger was lower if cells had been chronically (7 hours) treated with TGF~, whereas the slope of the curve did not appear significantly different. These data (Fig. 7) suggest a reduced number of exchanger units available for transport under these conditions. This seems to be indicated also by the necessity of 7 hours' incubation to observe these effects. However, more and different studies are needed to clarify this aspect. In fact, a direct inhibiting effect of TGF~ at a modifier site of the exchanger cannot be completely excluded, as indicated by the slight and partial decrease of Na÷/I-I+ exchange activity observed after the acute treatment with TGF~, especially in PV cells. These physiological data are of importance because they occur in a cell population where TGF~I has been demonstrated to induce cell death by apoptosis. The effect of TGF~I on intracellular pHi in vivo could be different when HCO3/CO2 system is physiologically present. In fact, it is known that an intracellular acidification higher than that observed in this study (by approximately 0.3 to 0.4 pH~its), seems to protect hepatocytes toward cell damage rather than to induce cell

HEPATOLOGYVol. 22, No. 5, 1995 death. However, TGFz~ i n d u c e s apoptosis of h e p a t o cytes e i t h e r in vitro or in vivo w h e r e bicarbonate/CO2 is p r e s e n t ~5 a n d Na+/H ÷ e x c h a n g e is similarly involved in h e p a t o c y t e proliferation in b o t h in vivo and in vitro models. T h e s e o b s e r v a t i o n s could t h e r e f o r e s u g g e s t t h a t inhibition of Na+/H + e x c h a n g e b y different cytokines r a t h e r t h a n modifications of pHi could r e p r e s e n t t h e k e y point in e a r l y steps of cell death. E G F exerts a proliferative action m o r e on P P t h a n on PV cells in culture, a n d this effect is c o u n t e r a c t e d by T G F z l , especially in P V cells, w h i c h are k n o w n to show a low r a t e of proliferation either in vivo or in

BENEDETTI ET AL 1497

9. 10. 11. 12. 13.

vitro. A s t i m u l a t o r y effect of E G F on Na+/H + e x c h a n g e activity h a s been p r e v i o u s l y s h o w n in h e p a t o c y t e s , 2428 h e p a t o m a cell lines, 44 or different m a m m a l i a n cells. The i n h i b i t o r y effect of Na+/H ÷ e x c h a n g e inhibitors on D N A s y n t h e s i s a n d cell proliferation h a s been also d e m o n s t r a t e d . ~ I n a g r e e m e n t w i t h previous studies, 24-2s a clear s t i m u l a t i o n o f N a + / H ÷ e x c h a n g e activity b y E G F also r e s u l t s from t h e c u r r e n t data, w i t h a p r e v a l e n t effect on P P cells. This s t i m u l a t i o n is amiloride depend e n t a n d is completely i n h i b i t e d in P V b u t n o t in P P cells by i n c u b a t i o n w i t h TGFzl for 7 hours. T h e s e e x p e r i m e n t s confirm the h i g h e r proliferative c a p a c i t y of P P cells w i t h r e s p e c t to PV h e p a t o c y t e s , which, in contrast, a p p e a r m o r e exposed to d e a t h b y apoptosis. I n conclusion, a lobular g r a d i e n t a n d a m o d u l a t i o n of Na÷/H ÷ e x c h a n g e activity in liver p a r e n c h y m a l cells u n d e r g o i n g e i t h e r proliferation or cell d e a t h by apoptosis could likely r e p r e s e n t a m e c h a n i s m by w h i c h v a r i o u s steps of t h e cell cycle are r e g u l a t e d .

Acknowledgment:

We would like to express our m o s t sincere t h a n k s to L u c i a n o Trozzi a n d A n t o n e l l a F a v a for t h e i r excellent technical assistance. REFERENCES

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