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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 3052-3058
Adenosine 3 :5-Cyclic Monophosphate (cAMP)-Inducible
Pyrimidine 5-Nucleotidase and Pyrimidine Nucleotide Metabolism
of Chick Embryonic Erythrocytes
By
Stefanie Dragon,
Rainer Hille,
Robert Götz, and
Rosemarie Baumann
From the Physiologisches Institut, Universität Regensburg,
Regensburg, Germany.
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ABSTRACT |
Terminally differentiating erythrocytes degrade most of their RNA
with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5 -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 5-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 5-nucleotidase (P-5-N), to evaluate if changes of enzyme
activity during embryonic 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-5-N increases from day 13 on,
suggesting that the enzyme is a limiting factor in UTP degradation.
Activation of  -adrenergic and A2A-adenosine receptors
causes transcription-dependent de novo synthesis of P-5-N.
Because -adrenergic and adenosine receptors are also found on adult
erythroid cells, P-5-N might be an enzyme of differentiating
RBCs whose expression is in part controlled by adenosine
3:5-cyclic monophosphate (cAMP).
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INTRODUCTION |
IT 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 5-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
5-nucleotidase (P-5-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-5-N activity and its influence on
the nucleotide pattern during erythroid development are unknown.
Experimental evidence suggests that the P-5-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-5-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 uridine 5-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 3:5-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 2,3-bisphosphoglycerate
(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 -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 -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 -adrenergic receptor
activation.8,12
In addition, we could show that in vitro incubation of embryonic RBCs
from day 11 with -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-5-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 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-5N activity
increases significantly between days 13 and 15 of development.
Incubation of embryonic RBCs of day 11 with -adrenergic or adenosine
receptor agonists or forskolin causes transcription-dependent de novo
synthesis of the enzyme, which in turn increases the amount of released
uridine into the incubation medium. The results show that P-5N
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.
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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 reversed-phase 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  40°C until HPLC analysis with a
Pharmacia HPLC system (Pharmacia, Uppsala, Sweden) and
RP-18 column (LiChroSorb; 250 × 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-5-N activity.
P-5-N activity was analyzed with the spectrophotometric method
of Amici et al,14 which quantitates the metabolization of uridine 5-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
40°C until HPLC analysis with a Pharmacia HPLC system and an
RP-18 column (LiChroSorb; 250 × 4 mm; 10 µm; Merck). One unit
of P-5-N is defined as conversion of 1 µmol of UMP to uridine
per minute at pH 7.5 and 37°C.
In vitro incubations.
To determine the effect of 10 µmol/L NE, 10 µmol/L epinephrine (E),
10 µmol/L 5-(N-cyclopropyl)-carboxamidoadenosine (CPCA), 100 µmol/L forskolin, and 35 µmol/L actinomycin D on P-5-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-N-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 -adrenergic
blocker propranolol during some control incubations.
We also measured the effect of the -adrenergic agonists on RBC
uridine release during 16 hours of incubation. Uridine concentration of
the supernatant and RBCs and RBC UTP/uridine 5-diphosphate (UDP)
concentrations were determined at the end of the incubation period.
Nucleotide analysis.
RBC nucleotides were analyzed by reversed-phase 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
(3°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 40°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 80°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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RESULTS |
Developmental changes of embryonic RBC ATP and UTP concentration, PNP,
and P-5-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-5-N and/or PNP. Table
1 contains data for UDP, cytidine 5-diphosphate (CDP),
and cytidine 5-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.
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| 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.
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We analyzed the developmental profile of PNP and P-5-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-5-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-5-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-5-N activity (Fig 2). The same can be inferred
for CTP (Table 1).
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| Fig 2.
Changes of erythrocyte P-5-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).
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Effect of -adrenergic and adenosine receptor agonists
on pyrimidine 5-nucleotidase activity.
The observed increase of P-5-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 -adrenergic receptor activation, we
tested if they influence P-5-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 -adrenergic receptor of
embryonic chick RBCs).11 As shown in
Fig 3, the addition of 10 µmol/L NE
doubled the P-5-N activity during a 16-hour incubation period. This increase is completely blocked by the -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-5-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-5-N
than -adrenergic or adenosine receptor activation, corroborating previous results showing only moderate stimulation of
adenylyl cyclase by forskolin.12
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| Fig 3.
Adrenergic stimulation of RBC P-5-N activity. RBCs
(cytokrit 4%) from day 11 were incubated for 16 hours at 37°C in
the absence and presence of 10 µmol/L NE, 10 µmol/L propranolol
(prop), and 35 µmol/L actinomycin D (actD). Data are given as the
mean value and SD from five experiments in each case. (***P < .001, **P < .01).
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| Fig 4.
Effect of adenosine A2-receptor activation
and direct stimulation of adenylyl cyclase with forskolin on
P-5-N activity. RBC cells from day-11 chick embryos were
incubated for 16 hours with either 10 µmol/L CPCA or 100 µmol/L
forskolin, and P-5-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 < .001).
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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-5-N. Actinomycin D not only inhibited the adrenergic stimulation of P-5-N (Fig 3), but in its presence the P-5-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 -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-5-N is the limiting factor for uridine release.
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| Fig 5.
(A) Uridine release from RBCs of day-11 chick embryos
during 16 hours with 10 µmol/L E, 10 µmol/L propranolol (prop), and 35 µmol/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 < .01; ***P < .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 < .05; n.s., not significant; P = .055).
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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.
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| 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 µmol/L CPCA
or 10 µmol/L NE. Data are the mean values and SD from at least five determinations in each case.
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DISCUSSION |
P-5-N synthesis of embryonic RBCs is stimulated by cAMP.
The developmental profile of the P-5-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-5-N activity of RBCs from day 4 and day 13 to day 15 is due to
cAMP-dependent stimulation of P-5-N synthesis. (1) Under in
vitro conditions, forskolin, adenosine receptor agonist CPCA, and NE as
well as E stimulate P-5-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 development, the
increase of P-5-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 -adrenergic receptors. In addition, external adenosine might also contribute to activation of
P-5-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-5-N of immature definitive RBCs seems to be
submitted to a rapid turnover. Thus, in vitro incubation with
actinomycin D lowered the P-5-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-5-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-5N 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-5-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 -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-5-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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| Fig 7.
Role of cAMP-inducible P-5-N in the metabolism of
terminally differentiating erythrocytes. NT, nucleoside transporter;
AC, adenylyl cyclase; -R, -adrenergic receptor; A, adenosine;
A2R, adenosine receptor.
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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 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-5-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
-alanine and serve as precursor for synthesis of carnosine in
skeletal muscle; indeed, analysis of embryonic myoblasts shows
increased uptake of -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-5-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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FOOTNOTES |
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.
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REFERENCES |
1. Rapoport SM: The Reticulocyte. Boca Raton, FL, CRC,
1986
2.
Valentine WN,
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Abstract
Introduction
Abstract