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Adenosine 38:58-Cyclic Monophosphate (cAMP)-Inducible Pyrimidine
58-Nucleotidase and Pyrimidine Nucleotide Metabolism of Chick
Embryonic Erythrocytes
By Stefanie Dragon, Rainer Hille, Robert Götz, and Rosemarie Baumann
Terminally differentiating erythrocytes degrade most of their
RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 58-nucleotidase; deficiency of this
enzyme causes hemolytic anemia in humans. Details of the
regulation of its activity during erythroid differentiation are
unknown. The present study arose from the observation that
the immature red blood cells (RBCs) of mid-term chick
embryos contain high concentrations of uridine 58-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days
13 to 14 onward. We analyzed two key enzymes of RBC
pyrimidine nucleotide metabolism: pyrimidine nucleoside
phosphorylase (PNP) and pyrimidine 58-nucleotidase (P-58N), to evaluate if changes of enzyme activity during embry-
onic development are correlated with changes of RBC UTP.
Secondly, we tested if these enzymes are under hormonal
control. The results show that embryonic RBCs contain only
minimal activity of PNP. In contrast, P-58-N increases from
day 13 on, suggesting that the enzyme is a limiting factor in
UTP degradation. Activation of b-adrenergic and A2Aadenosine receptors causes transcription-dependent de novo
synthesis of P-58-N. Because b-adrenergic and adenosine
receptors are also found on adult erythroid cells, P-58-N
might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 38:58-cyclic monophosphate (cAMP).
r 1998 by The American Society of Hematology.
I
uridine 58-triphosphate (UTP),8 which decrease rapidly from
about day 14 onwards. In the second week of incubation,
circulating embryonic RBCs are predominantly (polychromatic/
orthochromatic) erythroblasts that have concluded their terminal division but retained considerable transcriptional and protein synthetic activity.9 For the majority of the RBCs, the
transition to mature definitive erythrocytes and shutdown of
transcriptional activity is only accomplished in the last (third)
week of incubation.9 Thus, the nucleated embryonic chick
RBCs are a good experimental system to study pyrimidine
metabolism in the penultimate stages of erythroid differentiation.
We have recently shown that adenosine 38:58-cyclic monophosphate (cAMP)-dependent processes control major aspects
of the metabolism of embryonic RBCs in the second half of
incubation, including the coordinated activation of 2,3bisphosphoglycerate (2,3BPG) and carbonic anhydrase II (CAII)
synthesis.8,10-12 In late chick embryos, an increase of the RBC
cAMP concentration is initiated by the rapid increase of plasma
norepinephrine (NE), activating RBC adenylyl cyclase via
b-adrenergic receptors.11 The physiologic stimulus for the NE
release is hypoxia.11 These events occur at the time when the
UTP concentration of embryonic RBCs decreases. Besides the
b-adrenergic receptor, we have found an adenosine A2-receptor
coupled to adenylyl cyclase. In vitro adenosine receptor activation induces the same metabolic processes we observed with
b-adrenergic receptor activation.8,12
In addition, we could show that in vitro incubation of
embryonic RBCs from day 11 with b-adrenergic or adenosine
receptor agonists causes transcription-dependent stimulation of
the synthesis of several other RBC proteins besides CAII.
Therefore, we have analyzed the activity of two key enzymes of
pyrimidine metabolism, pyrimidine nucleoside phosphorylase
(PNP) and P-58-N, to find out (1) if changes in the enzyme
activity are correlated with the decrease of the RBC UTP
concentration during terminal differentiation and (2) if the
enzyme activities are under hormonal control by catecholamines and adenosine.
The results show that, during the second week of incubation,
UTP is the second most abundant organic phosphate compound
T IS WELL-KNOWN THAT, during the final steps of
erythroid differentiation, the RNA content of mammalian
and nonmammalian red blood cells (RBCs) is drastically
reduced.1 Little attention has been paid to the metabolic fate of
nucleotides liberated in this process. Studies on human RBCs
have shown the presence of a 58-nucleotidase specific for
pyrimidine mononucleotides but with no affinity for purine
mononucleotides2,3 whose molecular properties have been partially characterized.4 A reduction of pyrimidine 58-nucleotidase
(P-58-N) activity due to genetic defects or lead poisoning causes
hemolytic anemia in humans.2,5 The circulating RBCs show
significant accumulation of pyrimidine nucleotides as well as
incomplete degradation of RNA and ribosomes, which indicate
the important role of the enzyme for RBC maturation, because
the enzymatic step liberates cell-permeable nucleosides. Details
of the regulation of P-58-N activity and its influence on the
nucleotide pattern during erythroid development are unknown.
Experimental evidence suggests that the P-58-N activity of
immature RBCs is substantially increased. In RBCs of human
fetuses from the 17 to 23 weeks of gestation, the activity of
P-58-N was about threefold higher than in the RBCs of adults.6
Likewise, the RBCs of adult rabbits with an increased reticulocyte fraction contained significantly higher activity of the
enzyme.7
The present investigation of the pyrimidine metabolism arose
from the observation that circulating RBCs of midterm chick
embryos (days 10 to 12) contain millimolar concentrations of
From the Physiologisches Institut, Universität Regensburg, Regensburg, Germany.
Submitted September 22, 1997; accepted December 1, 1997.
Supported by DFG Ba691/5.
Address reprint requests to Stefanie Dragon, PhD, Physiologisches
Institut, Universität Regensburg, 93040 Regensburg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9108-0036$3.00/0
3052
Blood, Vol 91, No 8 (April 15), 1998: pp 3052-3058
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cAMP-INDUCIBLE PYRIMIDINE 58-NUCLEOTIDASE
of the embryonic RBCs. The embryonic RBCs contain only
minimal activities of PNP, precluding a use of uridine by
embryonic RBCs. In contrast, the P-58N activity increases
significantly between days 13 and 15 of development. Incubation of embryonic RBCs of day 11 with b-adrenergic or
adenosine receptor agonists or forskolin causes transcriptiondependent de novo synthesis of the enzyme, which in turn
increases the amount of released uridine into the incubation
medium. The results show that P-58N synthesis is partly
controlled by cAMP during terminal erythroid differentiation
and that the enzyme is rate limiting for the release of uridine
during RBC maturation.
MATERIALS AND METHODS
Fertilized eggs of White Leghorn chickens were incubated at 37.5°C
and 60% relative humidity in a commercial forced-draft incubator for
up to 19 days of development.
Blood was sampled after a large extraembryonic vessel was cut. The
effluent blood was aspirated and transferred to cold washing buffer [50
mmol/L tris(hydroxymethyl)aminomethane (Tris), 120 mmol/L NaCl, 4
mmol/L KCl, 5 mmol/L glucose, 1.5 mmol/L CaCl2, pH 7.4]. The RBCs
were washed three times with cold washing buffer before use.
Determination of PNP activity. The PNP activity of embryonic
RBCs was analyzed by the method of Laurensse et al,13 which
determines the degradation of uridine to uracil via analysis by reversedphase high-performance liquid chromatography (HPLC).
For lysis, 50 µL of packed RBCs was diluted with 950 µL hypotonic
Tris-EDTA buffer (50 mmol/L Tris, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), pH 7.4). After 10 minutes on ice, the lysate was
centrifuged for 10 minutes at 14,000g and 4°C to remove debris. Eight
hundred microliters of supernatant was mixed with 280 µL Tris-EDTA
buffer and 60 µL 0.8 mol/L KH2PO4, pH 7.4, and incubated for 10
minutes at 37°C. The reaction was started by the addition of 60 µL
uridine stock solution (20 mmol/L). The reaction was stopped by
heating 200 µL of the sample for 3 minutes at 95°C. After centrifugation, the supernatant was stored at 240°C until HPLC analysis with a
Pharmacia HPLC system (Pharmacia, Uppsala, Sweden) and RP-18
column (LiChroSorb; 250 3 4 mm; 10-µm particle size; Merck,
Darmstadt, Germany) according to Laurensse et al.13 The hemoglobin
(Hb) was converted to cyanmethemoglobin and measured spectrophotometrically. One unit of PNP is defined as the conversion of 1 µmol
uridine to uracil per minute at pH 7.4 and 37°C.
Determination of P-58-N activity. P-58-N activity was analyzed
with the spectrophotometric method of Amici et al,14 which quantitates
the metabolization of uridine 58-monophosphate (UMP) to uridine by
HPLC analysis. Samples for the test were prepared as follows: 100 µL
of packed RBCs was resuspended in 100 µL of washing buffer and lyzed
by two freeze-thaw cycles with liquid nitrogen/ice water. The lysate was
centrifuged at 14,000g for 10 minutes. One hundred microliters of the
supernatant was added to 900 µL of incubation buffer (50 mmol/L Tris,
10 mmol/L MgCl2, 10 mmol/L dithiothreitol, pH 7.5). After 10 minutes
of preincubation at 37°C, the reaction was started by addition of 10 µL
of 100 mmol/L UMP. Between days 4 and 11, RBCs from several
embryos were pooled to obtain the necessary sample size for a single
experiment. In intervals of 10 minutes (0 to 70 minutes), the reaction
was stopped by adding 50 µL ice-cold HClO4 (1.2 mol/L) to a 100-µL
sample. After centrifugation (5 minutes at 14,000g and 4°C), 130 µL of
the supernatant was neutralized with 35 µL of 1 mol/L K2CO3 and the
sample was stored at 240°C until HPLC analysis with a Pharmacia
HPLC system and an RP-18 column (LiChroSorb; 250 3 4 mm; 10 µm;
Merck). One unit of P-58-N is defined as conversion of 1 µmol of UMP
to uridine per minute at pH 7.5 and 37°C.
3053
In vitro incubations. To determine the effect of 10 µmol/L NE, 10
µmol/L epinephrine (E), 10 µmol/L 58-(N-cyclopropyl)-carboxamidoadenosine (CPCA), 100 µmol/L forskolin, and 35 µmol/L actinomycin D
on P-58-N activity, erythrocytes from 11-day-old embryos were incubated for 16 hours at 37°C in a gyratory water bath [cytokrit 4%, Ham’s
medium F10 supplemented with 20 mmol/L N-(2-hydroxyethyl)piperazine-N8-2-ethanesulfonic acid (HEPES), and 10% fetal calf serum
(FCS), pH 7.4] in the absence and presence of the tested substances.
Following the dose-response curves for CPCA, NE, and E determined
previously,11,12 the agonist concentrations were chosen to give maximal
stimulation. Because FCS may contain small quantities of catecholamines, we added the b-adrenergic blocker propranolol during some
control incubations.
We also measured the effect of the b-adrenergic agonists on RBC
uridine release during 16 hours of incubation. Uridine concentration of
the supernatant and RBCs and RBC UTP/uridine 58-diphosphate (UDP)
concentrations were determined at the end of the incubation period.
Nucleotide analysis. RBC nucleotides were analyzed by reversedphase HPLC following the method of Stocchi et al15 with slight
modifications. Fresh whole blood for nucleotide analysis of RBCs from
7-day-old to 17-day-old chick embryos was taken using the following
procedure: a short glass capillary with bevelled tip was inserted into a
2-mL pipette. The 2-mL pipette served as reservoir for the collected
blood and was surrounded by a cooling jacket, which was perfused with
a cold (23°C) water-acetone mixture. The pipette was mounted onto a
Leitz micromanipulator and the capillary inserted into an extraembryonic blood vessel under stereomicroscopic control. In general, collection of blood took less than 2 minutes. The collected blood was
immediately transferred into ice-cold Eppendorf cups and centrifuged
for 5 seconds at 14,000g. After removal of the plasma and buffy coat,
RBCs were washed rapidly (twice for 5 seconds) with ice-cold washing
buffer. Fifty µL of packed RBCs was added to 50 µL HClO4 (1 mol/L)
and centrifuged (14,000g for 10 minutes at 4°C). The supernatant was
neutralized with 10 µL of 5 mol/L K2CO3 and stored at 240°C until
analysis.
cAMP determination. RBC cAMP concentrations were determined
using the fluorometric enzymatic test of Sugiyama and Lurie.16 For the
cAMP determinations, RBCs were first preincubated for 30 minutes at
37°C. One hundred µL of packed RBCs was added to 400 µL of
incubation buffer consisting of 10% FCS, 135 mmol/L NaCl, 4 mmol/L
KCl, 5 mmol/L glucose, 1.5 mmol/L MgCl2, 1.5 mmol/L CaCl2, and 20
mmol/L HEPES, pH 7.4. The incubation was performed in a shaking
water bath. After the preincubation period, the cells were incubated with
agonists for 5 minutes. To stop the reaction, 100 µL of cell suspension
was mixed with 1 mL of ice-cold ethanol. After 5 minutes on ice, the
sample was centrifuged for 5 minutes at 13,000g at 4°C and the
supernatant was transferred to an Eppendorf test tube. To remove the
ethanol, the sample was dried at 50°C and stored at 280°C until
analysis. For the cAMP determination, the sample was dissolved in 40
µL of ice-cold 0.5 mol/L HClO4 by mixing for 2 minutes and sonicating
for 1 minute. After neutralization with 10 µL of 2 mol/L KOH and
centrifugation for 10 minutes (13,000g and 4°C), the supernatant was
used for the cAMP determination.16 Fluorescence measurements were
performed in microtitration plates (Nunc, Wiesbaden, Germany) at an
emission wavelength of 460 nm and excitation wavelength at 360 nm,
using the Perkin-Elmer spectrofluorometer LS 50B with attached
microplate reader (Perkin-Elmer, Norwalk, CT).
Chemicals. Analytical grade reagents, nucleotides, nucleosides,
FCS, norepinephrine, epinephrine, and propranolol were purchased
from Sigma Chemicals (Deisenhofen, Germany). CPCA and forskolin
were obtained from RBI Biotrend (Köln, Germany), and Ham’s
F10-medium were obtained from Biochrom KG (Berlin, Germany).
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3054
DRAGON ET AL
RESULTS
Table 1. CTP, CDP, and UDP Concentrations of Erythrocytes
From 11- to 15-Day-Old Chick Embryos
Developmental changes of embryonic RBC ATP and UTP
concentration, PNP, and P-58-N activity. Figure 1 shows the
ATP and UTP concentrations of RBCs between day 7 and day
17 of chicken development. In agreement with previous data,17
we find that early definitive RBCs contain excessively high
concentrations of ATP (13 mmol/L RBCs at day 7), which
decrease from 9 mmol/L at day 13 to 2.5 mmol/L at day 17. The
UTP concentration increases between day 7 and day 10 from
about 3.5 mmol/L to 6 mmol/L and decreases from 5 mmol/L at
day 13 to 0.9 mmol/L at day 17. The UTP concentration profile
suggests that, during the second week of incubation, UMP
released from RNA degradation is to a considerable extent
phosphorylated to yield UTP and retained in the RBCs rather
than metabolized to uridine, indicating a limiting role of P-58-N
and/or PNP. Table 1 contains data for UDP, cytidine
58-diphosphate (CDP), and cytidine 58-triphosphate (CTP)
concentrations measured between days 11 and 15. They show
that the CTP concentration decreases from 2.29 mmol/L RBCs
at day 11 to 0.16 mmol/L RBCs at day 15 and that changes in
UDP and CDP concentration are less conspicuous but follow the
same trend.
We analyzed the developmental profile of PNP and P-58-N
activity to find if the changes in nucleotide pattern correlated
with altered enzyme activities. We found no significant activities of PNP when analyzing RBCs from day 11 to day 15. The
activity was below the limit of detection (,0.42 mU/g Hb).
This finding explains the results of Mathew et al,18 who reported
that extracellular uridine was not a suitable substrate for the
energy metabolism of RBCs from 14-day-old chick embryo. In
contrast to this, the P-58-N activity showed substantial changes
of activity throughout development (Fig 2). Peak activities
(0.47 U/g Hb; 0.013 standard deviation [SD]) were found early
in development (day 4), when the circulating blood consists
predominantly of polychromatic primitive erythroblasts,
whereas, at day 6, when the blood contains primarily mature
primitive RBCs, the activity was significantly reduced (0.17
U/g Hb; 0.053 SD). Between day 6 and day 13, the activity of
P-58-N showed only a small gradual increase to 0.27 U/g Hb by
day 13, but increased rapidly to 0.54 to 0.62 U/g Hb at days 14
to 15, followed by a rapid decrease to 0.1 U/g Hb at day 19. The
rapid decrease of RBC UTP concentration is closely coordinated to the increase of P-58-N activity (Fig 2). The same can be
inferred for CTP (Table 1).
Effect of b-adrenergic and adenosine receptor agonists on
pyrimidine 58-nucleotidase activity. The observed increase of
P-58-N activity is correlated with the increase of NE concentration in the blood of the chick embryo.11 Because we could
previously show that adrenergic agonists stimulate the synthesis
of several, not yet identified, embryonic RBC proteins via
b-adrenergic receptor activation, we tested if they influence
P-58-N activity. To this end, embryonic RBCs from day 11 were
incubated in vitro for 16 hours (for details, see the Materials and
Methods) in the absence and presence of NE or E (both agonists
are equally effective on the b-adrenergic receptor of embryonic
chick RBCs).11 As shown in Fig 3, the addition of 10 µmol/L NE
doubled the P-58-N activity during a 16-hour incubation period.
This increase is completely blocked by the b-adrenergic
antagonist propranolol. Chick embryonic RBCs also possess an
adenosine A2-receptor coupled to adenylyl cyclase.12 The
adenosine receptor agonist CPCA also increases the P-58-N
activity of isolated RBCs of 11-day-old chick embryos (Fig 4).
Direct stimulation of adenylyl cyclase with 100 µmol/L forskolin leads to a less prominent activation of P-58-N than
Fig 1. Changes of RBC UTP and ATP concentration during chick
embryonic development (day 7 to day 17). Data are presented as the
mean and SD of 3 to 14 determinations at each point.
Fig 2. Changes of erythrocyte P-58-N activity during chick embryonic development (day 4 to day 19). Data are the mean values and SD
from three to six experiments at each point. The dotted line gives
parallel changes of RBC UTP concentration (data taken from Fig 1).
Nucleotide
CTP
CDP
UDP
Day 11
Day 14
Day 15
2.29 (0.7)
1.27 (0.44)
0.16 (0.06)
0.37 (0.07)
0.43 (0.14)
0.14 (0.03)
1.15 (0.17)
1.1 (0.28)
0.54 (0.03)
Mean values and SD (in parentheses) for embryonic RBC concentrations of CTP, CDP, and UDP in moles per liter. Data are from three
different experiments.
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cAMP-INDUCIBLE PYRIMIDINE 58-NUCLEOTIDASE
3055
Effect of CPCA and NE on cAMP production of embryonic
RBCs harvested from 11- to 17-day-old chick embryos. RBCs
from day-11 to day-17 embryos were stimulated for 5 minutes
with saturating concentrations of CPCA and NE. cAMP production was assessed and compared with control cells. The results
are presented in Fig 6. The response to both NE and (particularly) CPCA is drastically decreased in RBCs from embryos
older than 15 days.
DISCUSSION
Fig 3. Adrenergic stimulation of RBC P-58-N activity. RBCs (cytokrit 4%) from day 11 were incubated for 16 hours at 37°C in the
absence and presence of 10 mmol/L NE, 10 mmol/L propranolol
(prop), and 35 mmol/L actinomycin D (actD). Data are given as the
mean value and SD from five experiments in each case. (***P F .001,
**P F .01).
b-adrenergic or adenosine receptor activation, corroborating
previous results showing only moderate stimulation of adenylyl
cyclase by forskolin.12
The cAMP-dependent increase of protein synthesis in embryonic RBCs requires transcriptional activation, because actinomycin D abolishes the effect of adenylyl cyclase stimulation by
adrenergic or adenosine A2-receptor agonists.11,12 We therefore
tested the influence of actinomycin D on adrenergic induction of
P-58-N. Actinomycin D not only inhibited the adrenergic
stimulation of P-58-N (Fig 3), but in its presence the P-58-N
activity falls significantly below that of the controls. This
suggests that the enzyme, as well as its RNA, has a considerable
turnover in the embryonic RBCs.
Effect of b-adrenergic stimulation and actinomycin D on
uridine release from embryonic RBCs and UTP concentration
in RBCs. Figure 5A shows the uridine release from embryonic
RBCs from day 11 during 16 hours of incubation with 10
µmol/L epinephrine in the absence and presence of propranolol
(10 µmol/L) and actinomycin D (35 µmol/L). When cells are
stimulated with epinephrine, they increase the uridine release by
about 40% compared with the control with propranolol, whereas,
in the presence of actinomycin D, uridine release is less than
50% of the control value. There are corresponding changes of
the RBC UTP concentration, which is decreased in the presence
of epinephrine and increased in the presence of actinomycin D
(Fig 5B). This suggests that in vivo the embryonic RBC is a
substantial source for provision of pyrimidine nucleosides and
that the activity of P-58-N is the limiting factor for uridine
release.
P-58-N synthesis of embryonic RBCs is stimulated by cAMP.
The developmental profile of the P-58-N activity of chick
embryonic RBCs shows a transient peak between days 13 and
15 of development and a high activity in immature primitive
embryonic RBCs from day 4.
The following results support the conclusion that the increased P-58-N activity of RBCs from day 4 and day 13 to day
15 is due to cAMP-dependent stimulation of P-58-N synthesis.
(1) Under in vitro conditions, forskolin, adenosine receptor
agonist CPCA, and NE as well as E stimulate P-58-N in
embryonic chicken RBCs from day 11. (2) RBCs from day 11 to
day 15 respond to CPCA and adrenergic agonists with a large
increase of cAMP production. (3) During normal development,
plasma catecholamine (NE) levels increase significantly after
day 12.11 (4) Primitive RBCs from day 4 of incubation have an
intrinsic cAMP level that is much higher than that of RBCs from
day 6 (Dragon et al, manuscript in preparation).
From these data one can infer that, during normal develop-
Fig 4. Effect of adenosine A2-receptor activation and direct stimulation of adenylyl cyclase with forskolin on P-58-N activity. RBC cells
from day-11 chick embryos were incubated for 16 hours with either
10 mmol/L CPCA or 100 mmol/L forskolin, and P-58-N activity was
determined for control and stimulated samples at the end of the
incubation period. Data are the mean values and SD from five
experiments. The asterisk indicates statistically significant difference
tested by the Student’s t-test for paired samples (***P F .001).
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3056
DRAGON ET AL
Fig 6. Basal and stimulated cAMP-production of RBCs from 11- to
17-day-old chick embryos. RBCs were stimulated for 5 minutes at
37°C (see the Materials and Methods) with either 10 mmol/L CPCA or
10 mmol/L NE. Data are the mean values and SD from at least five
determinations in each case.
Fig 5. (A) Uridine release from RBCs of day-11 chick embryos
during 16 hours with 10 mmol/L E, 10 mmol/L propranolol (prop), and
35 mmol/L actinomycin D (actD). Incubation conditions were the
same as in Fig 3. Data are given as the mean and SD from five
experiments in each case (**P F .01; ***P F .001). (B) Changes of
RBC UTP concentration during in vitro incubation with E, propranolol
(prop), and actD. Agonist concentrations and incubation conditions
as in (A). Data are the mean values and SD from three experiments in
each case (*P F .05; n.s., not significant; P 5 .055).
ment, the increase of P-58-N activity at the end of the second
week of incubation is largely due to the increased level of
plasma catecholamines and subsequent activation of badrenergic receptors. In addition, external adenosine might also
contribute to activation of P-58-N synthesis by binding to
A2a-receptors.
We have previously demonstrated that the synthesis of CAII
of embryonic RBCs is also controlled by cAMP.12 However, in
contrast to CAII, the P-58-N of immature definitive RBCs seems
to be submitted to a rapid turnover. Thus, in vitro incubation
with actinomycin D lowered the P-58-N activity to less than
50% of the initial value after 16 hours of incubation. This
observation also explains partly the rapid decrease of P-58-N
after day 15, because the transcriptional activity of circulating
RBCs is shut down during this period.9 A second factor that
might contribute to the decrease of P-58N activity is the
observation that, in circulating RBCs from embryos older than
15 days, cAMP production by catecholamines and adenosine
receptor agonist is greatly diminished.
The peak activities for P-58-N reported in the present study
(0.5 to 0.63 U/g Hb) are close to the activities reported for fetal
human RBCs from 17 to 23 weeks of gestation with 0.48
U/g Hb.6
Both b-adrenergic and adenosine receptors have been described for immature adult mammalian RBCs (see Rapoport1),
although the physiologic function of the receptors in RBC
development is not known. Given the homology of major
aspects of erythroid development in avian and mammalian
species, our data suggest that activation of adenylyl cyclase via
coupled receptors might partially control P-58-N synthesis in
differentiating mammalian RBCs during fetal as well as adult
development and thus be of importance for the final steps of
RNA metabolization (Fig 7).
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cAMP-INDUCIBLE PYRIMIDINE 58-NUCLEOTIDASE
Fig 7. Role of cAMP-inducible P-58-N in the metabolism of terminally differentiating erythrocytes. NT, nucleoside transporter; AC,
adenylyl cyclase; b-R, b-adrenergic receptor; A, adenosine; A2R,
adenosine receptor.
Immature embryonic RBCs accumulate large amounts of
pyrimidine trinucleotides. The developmental pattern for the
embryonic chick RBC UTP/CTP concentration shows that, in
the second week of incubation, part of the UMP/CMP liberated
from RNA degradation (cellular RNA decreases by more than
50% between days 6 and 10; Dragon et al, unpublished
observation) is retained in the cell and phosphorylated to give
UTP/CTP. In consequence, embryonic RBCs (from day 7 to day
12) contain about 8 to 9 mmol/L RBC pyrimidine trinucleotide
in addition to about 10 to 13 mmol/L ATP. To our knowledge,
the pyrimidine trinucleotide concentration is the highest reported for a cell so far.19
Obviously, RNA degradation can also contribute to the ATP
pool of the erythrocyte and influence the developmental profile
of the ATP concentration in embryonic RBCs. However,
analysis of the ATP metabolism is complicated by the fact that
embryonic RBCs have the capacity to convert significant
amounts of extracellular adenosine to ATP by subsequent
phosphorylation.8,20 That this latter process may be involved in
the physiologic regulation of the ATP concentration is also
indicated by the finding that, under in vitro conditions, early
embryonic RBCs can maintain their high ATP concentration
only in the presence of an extracellular source for adenosine.8
Thus, we are presently unable to evaluate the importance of
adenosine salvage and RNA degradation at a quantitative level
for establishing the high ATP concentration found early in
development. We also do not know to which extent a decreasing
activity of either metabolic pathway contributes to the rapid
decrease of the ATP concentration after day 13. Further insight
will be provided by investigating the developmental profile of
various enzymes involved in the adenosine salvage and purine
nucleotide degradation.
The fact that pyrimidine nucleotides are present in concentrations equimolar or above those of Hb raises the question of
whether they can act as an allosteric effector of Hb, in
competition with or in addition to ATP. This possibility is
currently under investigation.
In addition, the embryonic RBC is a potential source for UTP
release to the extracellular space of the embryo. This is of
interest because a nucleotide receptor specific for UTP has
3057
recently been identified on cardiac endothelial cells21 and in the
human placenta22 and very little is known about potential
cellular sources for extracellular UTP, which in most adult
blood cells amounts to much less than 0.5 mmol/L.19
Our data also show that the circulating embryonic RBC is a
source for pyrimidine nucleosides. Because the embryonic
RBCs cannot use pyrimidine nucleosides for their energy
metabolism, due to absence of PNP, all of the pyrimidine
nucleoside produced by the action of P-58-N is released from
the embryonic RBCs to the extracellular space. It follows that,
at least in the avian embryo, circulating embryonic RBCs are a
substantial source for provision of pyrimidine nucleoside to
other embryonic tissues, particularly in the last part of development, when RBC UTP (CTP) decreases rapidly, thereby reducing the energy requirement for de novo synthesis of nucleoside
at a time when the chick embryo faces increasing limitations for
oxygen uptake.23 The same could be true for purine nucleosides,
because embryonic RBCs contain only a small activity of
purine nucleoside phosphorylase.8
For uridine, there are three metabolic sinks in the embryo. (1)
It can enter de novo RNA synthesis in proliferating tissue24 and
serve as precursor for thymidine nucleotides. (2) It can be
degraded to b-alanine and serve as precursor for synthesis of
carnosine in skeletal muscle; indeed, analysis of embryonic
myoblasts shows increased uptake of b-alanine in the last stages
of development.25,26 (3) It can enter the UDP-glucose pool
required for glycogen synthesis in liver and yolk sac.27 Taken
together, the data indicate that the nucleated embryonic RBCs
offer a variety of services to the embryo that are not connected
to its respiratory function. This conclusion has also been made
in a recent investigation of nucleated human embryonic RBCs,
in which the investigators showed substantial enzymatic capacity for detoxification of endogenous and xenobiotic compounds.28
Our results can be applied to erythropoiesis in adults. In the
adult organism, immature erythroid cells are segregated to the
bone marrow. Upregulation of P-58-N during the late phases of
erythroid differentiation associated with RNA degradation and
consequent release of pyrimidine nucleoside in erythroid foci of
the bone marrow would provide an effective and energy-saving
way to transfer pyrimidine nucleotide precursors to proliferating erythroid cells at earlier stages of differentiation.
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1998 91: 3052-3058
Adenosine 3′:5′-Cyclic Monophosphate (cAMP)-Inducible Pyrimidine 5′
-Nucleotidase and Pyrimidine Nucleotide Metabolism of Chick Embryonic
Erythrocytes
Stefanie Dragon, Rainer Hille, Robert Götz and Rosemarie Baumann
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