/. Embryol. exp. Morph. Vol. 27, 3, pp. 525-532, 1972
525
Printed in Great Britain
Effect of erythropoietin on
haemoglobin synthesis and haem synthesizing
enzymes of mouse foetal liver cells in culture
By R. I. FRESHNEY, 1 JOHN PAUL 1 AND DAVID CONKIE 1
From The Beatson Institute for Cancer Research, Glasgow
SUMMARY
The activities of aminolaevulinate synthetase, aminolaevulinate dehydratase and haem
synthetase have been examined in short-term cultures of embryonic mouse liver.
Although synthesis of haemoglobin was induced by erythropoietin in these cultures no
increase in activity was detected in any of the three enzymes over 24 h in culture. In each case,
however, enzyme activity was higher when erythropoietin was present than in its absence.
The significance of these findings is discussed in relation to control of haemoglobin
synthesis and it is concluded that enzyme activity is not rate limiting during induction of
haemoglobin synthesis in vitro.
INTRODUCTION
It has been shown that three haem synthesizing enzymes - aminolaevulinate
synthetase (ALAS), aminolaevulinate dehydratase (ALAD), and haem synthetase - fluctuate in mouse foetal liver during the hepatic phase of erythropoiesis (Freshney & Paul, 1971). The activities of these enzymes increase
sequentially from the 13th to the 17th days of gestation when the foetal liver is
erythropoietic, and may be related to the rapid increase in the rate of synthesis of
haemoglobin between the 13th and 15th days. Since haemoglobin synthesis in
cell cultures from mouse foetal livers 13 days post-fertilization can be stimulated
by erythropoietin (Cole & Paul, 1966), this provides a suitable system for
investigating the participation of haem synthesizing enzymes in the regulation of
hepatic erythropoiesis.
Levere & Granick (1965) and Wainwright & Wainwright (1970) reported that
ALAS activity might be rate-limiting in the synthesis of haemoglobin by chick
blastoderm. Moreover, Wilt (1968) suggested that RNA synthesis was not ratelimiting at the onset of haemoglobin synthesis in chick blastoderm. Haem
synthesis may also regulate the synthesis of haemoglobin in other erythroid
systems (Levere, Kappas & Granick, 1967; Grayzel, Horchner & London, 1966).
Hence, using the same system as Cole & Paul (1966), haemoglobin synthesis has
1
Authors' address: The Beatson Institute for Cancer Research, 132 Hill Street, Glasgow,
G3 6UD, U.K.
526
R. I. FRESHNEY, J. PAUL AND D. CONKIE
been measured before and after treatment with erythropoietin. The activities
of ALAS, ALAD and haem synthetase have been measured in replicate samples
and fluctuations in their activities compared to that of haemoglobin synthesis.
MATERIALS AND METHODS
Porton white Swiss mice were killed by cervical fracture late on the 13th day of
pregnancy (counting the detection of a vaginal plug as day zero). Livers were
dissected from the embryos under aseptic conditions, disaggregated and cultured
by the method of Cole & Paul (1966). The dispersed cells were grown at about
106/ml in Waymouth's medium MB 752/1 in medical flat bottles, 4 oz or 32 oz
(approx. 0 1 or 11), or in test-tubes, depending on the number of cells required
for each enzyme determination.
To determine the rate of haemoglobin synthesis 59Fe, preincubated for 18 h
with mouse serum (as a source of transferrin), was added to aliquots of the
cultures for 1 h. 59Fe-labelled haem was extracted in butanone from an acidified
Drabkin's lysate of the washed cells and counted on glass-fibre discs in a scintillation counter (after Cole & Paul, 1966).
ALAS activity was measured in a hypotonic lysate of washed cells by the
method of Freshney & Paul (1970, 1971). ALAD activity was measured in a
0 - 1 5 M - K C 1 homogenate by spectrophotometric determination of porphobilinogen produced from aminolaevulinate in anaerobic conditions (Shemin,
1962). Haem synthetase activity was measured in a 0 - 1 5 M - K C 1 homogenate,
containing 0-4 % (v/v) Tween 20, by measuring incorporation of 59Fe into haem
under anaerobic conditions (Freshney & Paul, 1971).
Erythropoietin was prepared from human urine and was a gift from the
National Heart and Lung Institute of the U.S.A. It was procured by the
Department of Physiology, University of the North East, Corrientes, Argentina,
and processed by the Haematology Research Laboratories, Children's Hospital of
Los Angeles, for distribution by the National Heart Institute under Research
Grant HE-1O88O. It was authorized for distribution by the Committee on
Erythropoietin of the National Heart Institute.
RESULTS
Haemoglobin synthesis
The stimulation by erythropoietin of 59Fe incorporation into haem was
demonstrated by Cole & Paul (1966) and was confirmed in the present series of
experiments (Figs. 1-3). A similar experiment was performed in which the
acidification of the Drabkin's lysate was omitted. This resulted in a negligible
recovery of 59Fe-labelled haem (Table 1) and, since the acidification is necessary
for the cleavage of haem from globin, this implies that the haem labelled in this
assay is normally protein-bound. There was a decline in the recovery of free
Haem synthesizing enzymes in culture
527
Table 1. Recovery of ™Fe from free haem after culture with [&9Fe]transferrin
5B
Fe recovered
in free haem (p-moles)
Incubation
time (h)
Erythropoietin
(units/ml)
1
24
24
0
0
0-6
Mean
S.D.
0083 ±0015
0028 ± 0006
0034 ±0008
No. samples
(3)
(6)
(6)
Hours incubation
Fig. 1. Suspensions of 13-day embryonic liver cells, prepared as in Materials and
Methods, were inoculated into medical flat bottles. Cultures of 107 cells/10 ml were
used for 59Fe incorporation in 4 oz (approx. 0-1 1) bottles; they were pulsed for 1 h
before sampling with 0-5 ml 59Fe/transferrin, 13/*Ci per ml, 504/tCi per /tmole.
5 x 107 cells in 50 ml were cultured in 32 oz (approx. 11) bottles for ALAS assay.
Duplicate sample bottles were removed at the times indicated and the cells washed
in balanced salt solution and assayed immediately as indicated in Materials and
Methods. The values for 59Fe incorporation per 106 cells were low in this experiment
and may have been due to the low concentration of 59Fe used (one third of the
normal). Broken lines are 59Fe incorporation, solid lines ALAS activities. Open symbols are controls, closed symbols samples cultured in the presence of human urinary
erythropoietin, 0-6 units per ml.
haem between 0 and 24 h, suggesting that there might have been a decrease in
haem synthesis relative to globin synthesis over this period.
Aminolaevulinate synthetase
The activity of ALAS in trypsinized suspensions was 50-70 % lower than in
fresh liver. After culture for 24 h the activity declined a further 75 % relative to
528
R. I. FRESHNEY, J. PAUL AND D. CONKIE
£ 80 -
0
2
4
20
Hours incubation
Fig. 2. One ml of a cell suspension containing 1-37 x 106 cells per ml from 13-day
embryonic liver were inoculated into each of a number of Pyrex test-tubes. Before
collection, duplicate samples were labelled for 30 min by the addition of 015 ml
[59Fe]transferrin, 13/*Ci per ml, 448/*Ci per /imole. At the times indicated, duplicate 59Fe-labelled samples and unlabelled samples were removed, washed twice in
ice-cold Hanks's balanced salt solution, and stored frozen.
Broken lines indicate 59Fe incorporation and solid lines ALAD activity. Open
symbols represent control samples, the closed symbols represent samples cultured
with 0-6 units per ml human urinary erythropoietin.
the initial activity (Fig. 1). In the presence of erythropoietin the decrease was
less (about 60 % of the initial value). In this and in two other similar experiments
there was no indication of any increase of ALAS activity following treatment with
erythropoietin.
Aminolaevulinate dehydratase
ALAD activity was not reduced by overnight trypsinization. The activity of
this enzyme declined during culture though not as rapidly as that of ALAS
(Fig. 2). Induction of haemoglobin synthesis could be produced regularly (five
separate experiments) without induction of ALAD activity. After culture in the
presence of erythropoietin ALAD activity was slightly higher than in the absence
of erythropoietin.
Haem synthetase
The activity of haem synthetase was not reduced by overnight trypsinization.
In the presence of erythropoietin virtually no decline in haem synthetase activity
was detected over 24 h in culture, while in the absence of erythropoietin the
activity fell to less than half of the initial value (Fig. 3). This result was obtained
in two separate experiments.
Haem synthesizing enzymes in culture
529
150 -
100 -
24
Hours incubation
Fig. 3. One ml aliquots of an embryonic liver-cell suspension containing 081 x 106
cells per ml were incubated in test-tubes. 59Fe incorporation was measured in
duplicate tubes by incubation for 1 h prior to sampling with 0-15 ml [59Fe]transferrin,
13 /tCi per ml, 510 /*Ci per/tmole. The samples were then washed in ice-cold balanced
salt solution and stored frozen in 1 ml Drabkin's solution. For haem synthetase
estimations, 10 test-tube cultures were pooled, the cells centrifuged at 1000 g for
5 min, washed twice in balanced salt solution and stored frozen at - 7 0 °C. Broken
lines are 59Fe incorporation into haemoglobin in culture, solid lines haem synthetase
activity of extracts. Solid symbols are samples cultured with 0-6 units per ml human
urinary erythropoietin, open symbols are sample controls.
DISCUSSION
None of the enzymes studied demonstrated any induction comparable to the
extent of the induction of haemoglobin synthesis. This suggests that increased
synthesis of these enzymes is not necessary for the induction of haemoglobin
synthesis by erythropoietin in 13-day-mouse embryo liver cultures. This is in
contrast to the report by Bottomley & Smithee (1969), who showed that ALAS
was induced in marrow cultures stimulated by erythropoietin, although Hrinda
& Goldwasser (1968) were unable to demonstrate ALAS induction with this
material. The regulation of haemoglobin synthesis by ALAS activity (Levere &
Granick, 1965; Wilt, 1968; Wainwright & Wainwright, 1970) may be restricted
to yolk-sac erythropoiesis and may not be important in hepatic erythropoiesis.
However, although enzyme activities were never higher than at the beginning
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R. I. FRESHNEY, J. PAUL AND D. CONKIE
of culture, they were always relatively higher in cells incubated for 24 h in the
presence of erythropoietin. Some possible explanations are as follows:
(1) Whilst most cells demonstrate a reduction of enzyme activity, a small
population sensitive to erythropoietin may exhibit increased activity; induction
in this small population may be masked by the more general pattern of loss of
enzyme activity in the remainder.
(2) Erythropoietin may induce, directly, the synthesis of enzyme at the
translational level or the synthesis of messenger at the transcriptional level but
this may be counteracted by degradation of the enzyme. Bottomley & Smithee
(1969) have suggested that erythropoietin induces ALAS at the transcriptional
level though they did not report any induction of haem synthetase.
(3) The rate of enzyme degradation may be influenced by erythropoietin.
Schimke & Doyle (1970) have stressed the importance of enzyme degradation as
a controlling step in the induction of arginase and tryptophan pyrrolase in rat
liver. The higher enzyme activities in erythropoietin-treated cultures may represent a reduced rate of degradation of enzymes. Since Pitot, Peraino, Lamar &
Kennan (1965) have shown that template stability may be subject to regulatory
changes, and, since an increase in template stability has been proposed during
hepatic erythropoiesis (Djaldetti, Chui, Marks & Rifkind, 1970), the observed
differences in enzyme activity could be due to an increase in the stabilities of the
messenger RNA templates for these enzymes.
(4) Induction of haemoglobin synthesis by erythropoietin may bring about a
reduction in the haem pool. Since haem may be a co-repressor in the transcriptional control of ALAS synthesis (Kappas & Granick, 1968), any reduction in
haem would cause derepression of ALAS synthesis. The same situation may also
hold for the other two enzymes. Moreover, haem may directly inhibit the
activity of these enzymes. In particular there have been reports that haem
inhibits ALAS activity (Scholnick, Hammaker & Marver, 1969), although this
was not confirmed by Kappas & Granick (1963). Hence any fluctuation in the
haem pool produced by elevated haemoglobin synthesis might be reflected in a
change in enzyme activity.
(5) ALAS activity in Rhodopseudomonas spheroides is influenced by the concentration of an intracellular inhibitor (Marriot, Neuberger & Tait, 1969;
Tuboi, Kim & Kikuchi, 1969). If a similar inhibitor existed in erythroid cells
then it might be influenced by erythropoietin.
With the possible exception of the ALAS assay, where the concentration of
the enzyme extract is high, cellular factors are diluted so greatly in the assay
mixtures that it seems unlikely that haem or other inhibitors operate in these
assay conditions.
Since ALAS declines very soon after the 13th day in vivo a decline in activity in
culture of late 13-day liver might be expected. ALAD and haem synthetase,
however, would be expected to rise prior to the induction of haemoglobin
synthesis, if the phenomena observed in vivo were precisely reproduced in culture.
Haem synthesizing enzymes in culture
531
As they are not, the induction of haemoglobin synthesis in vitro by erythropoietin is not strictly comparable to the 13- to 15-day induction of haemoglobin
synthesis in the liver. The in vitro observations are of short duration (24 h);
prolonged hepatic erythropoiesis as seen in vivo may require stimulation of
haem-synthesizing enzymes.
In conclusion, these results imply that the induction of haemoglobin synthesis
by erythropoietin is not a consequence of induction of haem-synthesizing
enzymes, although erythropoietin does have some effect on them. The major
effect of erythropoietin must be presumed to result from another mechanism.
The authors wish to express their gratitude to Mrs Sheila Wilson for expert technical
assistance. This work was supported by grants from the Cancer Research Campaign and the
Medical Research Council. Erythropoietin was procured by the Department of Physiology,
University of the Northeast, Corrientes, Argentina, and processed by the Haematology
Research Laboratories, Children's Hospital of Los Angeles, for distribution by the National
Heart Institute under Research Grant HE-10880. It was authorized for distribution by the
Committee on Erythropoietin of the National Heart Institute.
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SHEMIN,
(Manuscript received 20 December 1971)