572
BIOCHEMICAL SOCIETY TRANSACTIONS
Expression of glutamine synthetase and carbonic anhydrase in isolated periportal and perivenous
hepatocytes
NICHOLAS CARTER,* STEPHEN JEFFERY,* PEKKA
NlEMlNEN f and KAI LlNDROSf
*Department of Child Health, St George’s Hospital Medical
School, London SWI 7ORE, U.K. and i A l k o Ltd., Research
Laboratories, I’OB 305, Helsinki 10, Finland
lmmunohistochemical studies first demonstrated that, both
in human and rat liver, glutamine synthetase is expressed
only in a narrow zone surrounding the terminal hepatic
venule (Gebhardt & Mecke, 1983). This peculiar heterogeneous distribution of the enzyme protein has recently been
confirmed by direct analysis of glutamine synthetase activity
in periportal and perivenous cell lysates (Quistorff &
Grunnet, 1987)or hepatocytes (Ugele et al., 1987; K. Lindros
unpublished work) isolated by digitonin-collagenase perfusion (Lindros & Penttila, 1985). The so-called glutamine
cycle encompassing periportal ammonia fixation and glutamine utilization for urea synthesis and perivenous glutamine
synthesis has been established (Haussinger, 1983) and has
been considered to have an important role in hepatic and
systemic pH regulation. The factors governing the local
expression of glutamine synthetase are, however, not known.
Recently, a heterogeneous hepatic expression of another
enzyme, carbonic anhydrase (CA), resembling that of glutamine synthetase, was described (Carter et al., 1987). The
high- and low-activity cytoplasmic forms CAI1 and CAIII,
identified by specific radioimmunoassay, were shown to
exhibit growth hormone-mediated sexual dimorphism in rat
liver. While there was 3-4 times more CAI1 in female than in
male liver, CAI11 was up to 30 times more abundant in male
liver. Immunohistochemistry revealed that although weak
staining was observed all over the lobule, both CAI1 and
CAI11 stained intensively around the central veins. However,
while livers from males exhibited much higher CAI11 activities than females in the perivenous zones, CAI1 activities
were much higher in livers from females. The mitochondria1
CA isoenzyme, CAIV serves the production of urea
(Dodgson et al., 1983). This process is more active in the
periportal zone (Haussinger, 1983; Poso et al., 1986).
Since glutamine synthetase and carbonic anhydrase are
both intimately involved in regulation of pH and metabolism,
and since both are differentiation markers in embryonic
neural retina (Vardimon et al., 1986), their possible coexpression in rat liver was investigated.
Glutamine synthetase was measured radioisotopically, by
separating [ 14C]glutamate and [ 14C]glutamine by ionexchange (Pishak & Phillips, 1979). Total CA activity was
measured by a pH indicator method and CAI1 and CAIII
isoenzymes by specific radioimmunoassay. Periportal and
perivenous hepatocytes were isolated by the digitonincollagenase perfusion technique (Lindros & Penttila, 1985;
Quistorff, 1985). Hepatocytes were inoculated on culture
dishes coated with rat tail collagen at a density of 6.3 x lo4/
cm2. A 22 mwbicarbonate/l5 mM-Hepes buffered 1: 1
mixture of Waymouth MB 752/1 and Ham F-12 was ordinarily supplemented with a 5% (v/v) fetal calf serum, 5%
(v/v) newborn calf serum (Gibco), insulin (10 units/l),
dexamethasone ( 1 p ~ )gentamycin
,
(10 mg/l) and nystatin ( 1
mg/l). Cultures were always supplemented with serum for the
first 4 h after plating.
Analysis of freshly isolated cells revealed a striking and
consistent difference both in total COz hydrase activity and
in CAI1 and CAI11 concentrations between periportal and
Abbreviation used: CA, carbonic anhydrase.
perivenous hepatocytes. The activity of CAI1 in perivenous
hepatocytes was 2 9 4 k 109 (SD, n = 11) and in periportal
cells 66 30 ng/mg of protein ( n = 13). Similar differences
were found for total CA activity. The CAIII isoenzyme
showed a less striking difference (about 2:l) between perivenous and periportal hepatocytes. The reason for the relative difference in the heterogeneous expression between
CAI1 and CAIII in the periportal and perivenous samples
from the male donors, as compared with the immunohistochemical data, is not resolved at the moment.
The activity of glutamine synthetase, in freshly isolated
perivenous hepatocytes, as assayed with a quite specific and
sensitive radioisotopic assay, ranged between 60 and 140
nmol/min per mg of protein. Periportal cells exhibited
extremely little activity: 0.2-0.6 nmol/min per mg of protein.
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Fig. 1. Effects of dexarnethasone on giutamine sytithetuse
activity in cultured periportal and perivenous hepatocytes
Hepatocytes isolated from the periportal (PP) or the perivenous (PV) region by digitonin-collagenase perfusion were
cultured in presence or absence of serum and 1 p ~ dexamethasone as described in the text. Mean values from
two PV (upper part of Figure) and two PP (lower part) cell
isolations are given. Note the discontinuous scale. 0 , serum
and dexamethasone; 0 , serum but no dexamethasone; A , no
serum, no dexamethasone.
I OX8
573
625th MEETING, LONDON
This remarkable activity difference demonstrates that periportal and perivenous hepatocytes can be virtually completely separated by the digitonin-collagenase perfusion
met hod.
During cultures of perivenous hepatocytes CA activities
diminished. Glutamine synthetase activities also were
reduced (Fig. 1, upper part). This decrease was less steep if
dexamethasone or both serum and dexamethasone were
omitted from the culture medium. By contrast, a gradual
expression of glutamine synthetase activity was seen during
culture of initially quiescent periportal cells (Fig. 1, lower
part). Furthermore, the expression of glutamine synthetase
was much more dramatic if dexamethasone or both serum
and dexamethasone were omitted from the culture medium.
These data suggest that corticosteroids repress the expression of glutamine synthetase. In addition, in a preliminary
experiment we observed that when periportal cells were cocultured with endothelial-cells from rat heart, glutamine synthetase activity was increased, supporting the suggestion
(Gebhardt & Mecke, 1983; Bennett et al., 1987) that close
contact with non-parenchymal cells from the terminal
hepatic venule region are necessary for the expression of
glutamine synthetase. We are currently investigating whether
similar signals are required for expression also of CAI1 and
CAIII. A correlation between CAI1 and glutamine synthetase activities in samples from the periportal region
( r = 0.722; P < 0.01) suggests the possibility of common regu-
latory mechanisms. The perivenous location of glutamine
synthetase makes it potentially useful as a clinical marker for
;ierivenous liver damage.
We acknowledge the help of Gary Brooke, Sean Carter and
Gunilla Ronnholm.
Bennett, A. L., Paulson, K. E., Miller, R. E. & Darnell, J. E., Jr
(1987)1. Cell. Biol. 105, 1073-1085
Carter, N., Jeffery, S., Legg, R., Wistrand, P. & Lonnerholm, G.
(1987) Biochem. SOC. Trans. 15,667-668
Dodgson, S. J., Forster, R. E., Schwed, D. A. & Storey, B. T. (1983)
J. Biol. Chem. 258,7696-7701
Gebhardt, R. & Mecke, D. (1983) EMBO J. 2,567-570
Haussinger, D. (1983) Eur. J. Biochem. J. 133,269-275
Lindros, K. 0.& Penttila, K. E. (1985) Biochem. J. 228,757-760
Pishak, M. R. & Phillips, A. T. (1979)Anal. Biochem. 9 4 , 8 2 4 8
Poso, A. R., Penttila, K. E., Suolinna, E. -M. & Lindros, K. 0.(1 986)
Biochem. J. 239,263-267
Quistorff, B. (1985) Biochem. J . 229,221-226
Quistorff, B. & Grunnet, N. ( 1 987) Biochem. J. 243,87-95
Ugele, B., Locher, M., Burger, H.-J. & Gebhardt, R. (1987) in Bile
Acids and the Liver (Baumgartner, G., Stiehl, A. & Gerok, W.,
eds.), pp. 153-160, MTP Press, Lancaster
Vardimon, L., Fox, L. E. & Moscona, A. A. (1986) froc. Natl. Acad.
Sci. U.S.A. 83, 9060-9064
Received 25 November 1987
Ethanol inhibits endocytosis of insulin in perfused rat liver
JANET FAWCETT, GEOFFREY D. SMITH and
TIMOTHY J. PETERS
Division of Clinical Cell Biology, M.R.C. Clinical Research
Centre, Harrow, Middlesex HA1 3UJ, U.K.
Chronic alcohol abuse can cause hyperinsulinaemia and
insulin resistance resulting in abnormalities in glucose
homoeostasis (Shankar et al., 1986). The cause of these
changes is not clearly understood. Proietto et al. (1984) sug-
105
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Fig. 1. Effect of ethanol on the subcellular distribution of "'I-labelled insulin
Frequency-density distributions of '?'I-labelled insulin from ( a ) control perfusions
( n= 3) and ( h )perfusions in the presence of 50 mM-ethanol ( n= 4). The lower panel
shows representative distributions of (c)latent NADH pyrophosphatase (EC 3.6.1.9)
(endosomes) and ( d )5'-nucleotidase (EC 3.1.3.5) (plasma membranes).
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