|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4314-4320
Ontogeny of Catecholamine and Adenosine Receptor-Mediated cAMP
Signaling of Embryonic Red Blood Cells: Role of
cGMP-Inhibited Phosphodiesterase 3 and Hemoglobin
By
Rosemarie Baumann,
Christian Blass,
Robert Götz, and
Stefanie Dragon
From the Physiologisches Institut, Universität Regensburg,
Regensburg, Germany.
 |
ABSTRACT |
We have previously shown that the cAMP signaling pathway controls
major aspects of embryonic red blood cell (RBC) function in avian
embryos (Glombitza et al, Am J Physiol 271:R973, 1996; and
Dragon et al, Am J Physiol 271:R982, 1996) that
are important for adaptation of the RBC gas transport properties to the
progressive hypercapnia and hypoxia of later stages of avian embryonic
development. Data about the ontogeny of receptor-mediated cAMP
signaling are lacking. We have analyzed the response of primitive and
definitive chick embryo RBC harvested from day 3 to 18 of development
towards forskolin,  -adrenergic, and A2 receptor agonists. The
results show a strong response of immature definitive and primitive RBC to adenosine A2 and -adrenergic receptor agonists, which is
drastically reduced in the last stage of development, coincident with
the appearance of mature, transcriptionally inactive RBC. Modulation of
cGMP-inhibited phosphodiesterase 3 (PDE3) has a controlling influence
on cAMP accumulation in definitive RBC. Under physiological conditions,
PDE3 is inhibited due to activation of soluble guanylyl cyclase (sGC).
Inhibition of sGC with the specific inhibitor ODQ decreases
receptor-mediated stimulation of cAMP production; this effect is
reversed by the PDE3 inhibitor milrinone. sGC is acitivated by nitric
oxide (NO), but we found no evidence for production of NO
by erythrocyte NO-synthase. However, embryonic hemoglobin releases NO
in an oxygen-linked manner that may activate guanylyl cyclase.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HORMONAL SIGNALS THAT control the
function of circulating embryonic erythrocytes have been scarcely
investigated.1,2 In contrast to adult erythropoiesis of
higher vertebrates, in which erythrocytes are released into the
circulation as transcriptionally inactive or anucleate red blood cell
(RBC) with very limited metabolic capacity, embryonic RBC initially
enter the circulation as immature precursor cells with ongoing
translational and transcriptional activity. This may enable embryonic
RBC to respond to changes of developmental conditions with a broader
repertoire for adaptative measures than is possible in the adult.
We have previously shown that, in chick embryos, activation of the cAMP
signaling pathway via A2a or -adrenergic receptors controls major
aspects of embryonic RBC function of older chick embryos,1-3 including oxygen affinity and carbonic
anhydrase II synthesis. These cAMP-induced changes allow rapid
adaptation of RBC gas transport properties to the progressive
hypercapnia and hypoxia characteristic for late embryonic
development.4 Hypoxia causes release of norepinephrine (NE)
into the blood of chick embryos, which then activates the
-adrenergic receptors.2 A second physiological stimulus
linked to hypoxia is adenosine.1 Additional experimental
evidence suggests that cAMP signaling is also involved in the
regulation of cellular pyrimidine nucleotide metabolism during terminal
erythroid differentiation, because the key enzyme pyrimidine
5'-nucleotidase is induced by cAMP.3
Basic data about the ontogeny of receptor-mediated cAMP signaling of
embryonic RBC are lacking. We have analyzed the response of embryonic
erythrocytes from 3- to 18-day-old chick embryos towards stimulation
with forskolin as a direct activator of adenylyl cyclase and adenosine
A2 and -adrenergic receptor agonists. During this period, the
composition of the circulating erythroid population changes from
primitive basophilic erythroblast (day 3) to mature definitive RBC (day
17/18) and covers nearly all stages of late erythroid differentiation.
Definitive RBC enter the circulation from day 6 onwards, initially as
immature polychromatic erythroblasts with substantial translational and
transcriptional activity. Mature definitive RBC appear at the end of
the second week of incubation, coincident with this, one observes a
progressive restriction of transcriptional and translational activity,
which is completed by day 17/18.5 A second aim of the
present study was to assess the role of cAMP degradation by
phosphodiesterase on receptor-mediated cAMP signaling; basic
information on this aspect of cAMP metabolism in immature erythroid
cells is also lacking. In several systems, the cAMP response is
modulated extensively by the activity of regulated phosphodiesterases
such as the cGMP-inhibited phosphodiesterase 3 (PDE3).6
Thus, in mammalian blood platelets, the activity of cGMP-inhibited PDE3
has a major influence on intracellular cAMP levels.7 We
therefore tested if alterations of guanylyl cyclase activity and PDE3
activity influence embryonic RBC cAMP levels.
The results of the present study show a strong response of immature
definitive and primitive RBC to -adrenergic and adenosine receptor
agonists that is lost in the transition from transcriptionally active
reticulocyte to mature nucleate erythrocyte. Furthermore, we can show
that modulation of cGMP-inhibited PDE3 has a controling influence on
RBC cAMP concentration of definitive embryonic RBC. Under physiological
conditions, PDE3 is inhibited due to activation of soluble
NO-dependent guanylyl cyclase. Experimental data suggest that NO is apparently released from embryonic hemoglobin (Hb) and
perhaps other intracellular NO donors, such as S-nitrosoglutathione.
| |
MATERIALS AND METHODS |
Fertilized eggs from white leghorn chicken were incubated for 3 to 18 days in a commercial forced draft incubator with automatic rotation at
37.5°C and 60% relative humidity. Blood was taken from the embryos
by cutting an extraembryonic blood vessel and aspirating the effluent
with a pasteur pipette. Blood was collected into ice-cold buffer of the
following composition: 120 mmol/L NaCl, 4 mmol/L KCl, 5 mmol/L glucose,
1.5 mmol/L CaCl2, 50 mmol/L Tris, pH 8.0. After day 10, heparin was added as anticoagulant. For cAMP measurements, RBC were
suspended for varying periods of time in a buffer of the following
composition: 135 mmol/L NaCl, 4 mmol/L KCl, 5 mmol/L glucose, 1.5 mmol/L MgCl2, 1.5 mmol/L CaCl2, 20 mmol/L
HEPES, pH 7.4, at 37°C. The cytokrit of the suspension was adjusted
to 10%. For experiments determining the response to forskolin,
adenosine A2 receptor and -adrenergic receptor stimulation cells
were preincubated for 15 minutes at 37°C and stimulated with
agonist for 5 or 15 minutes with 100 µmol/L forskolin, 10 µmol/L
NE, or 10 µmol/L 5'-(N-cyclopropyl)-carbamidoadenosine (CPCA)
as adenosine receptor agonist; the concentrations were chosen to
achieve maximum stimulation of -adrenergic and A2 receptors, respectively.1,2 CPCA was used as A2a receptor agonist,
because with embryonic RBC, it is as effective as the A2a-specific
agonist CGS 21680.1 At the end of the incubation, a small
sample was taken for Hb determination. The reaction was stopped by
addition of ice-cold ethanol, and the mixture was left on ice for at
least 5 minutes. This was followed by centrifugation of the sample at 13,000g for 5 minutes at 4°C. The supernatant was
transferred into an Eppendorf test tube and dried overnight at
50°C. The dried samples were stored at  20°C. Before the
measurements, the samples were mixed for 2 minutes with 40 µL
ice-cold perchloric acid, sonicated for 1 minute, and neutralized with
10 µL 2 mol/L KOH. The precipitate was removed by centrifugation and
the clear supernatant used for cAMP measurements with the fluorometric
enzymatic test of Sugiyama and Lurie,8 using a Perkin Elmer
LB 50 fluorescence spectrometer with microplate reader (Perkin Elmer,
Norwalk, CT). All measurements were performed in
triplicate; cAMP concentrations were calculated as picomoles of cAMP
per milligram of Hb. For experiments analyzing the effect of guanylyl
cyclase and PDE3 activity on cyclic AMP production, erythrocytes were
preincubated for 15 minutes with either 0.5 to 30 µmol/L 1 H-[1,2,4] oxydiazolo[4,-a] quinoxaline-1-one (ODQ), a specific
inhibitor of soluble guanylyl cyclase (sGC); 20 µmol/L Ly 83583 (6-Anilino-5,8-quinolinequinone), an inhibitor of soluble and
particulate guanylyl cyclase; or 20 µmol/L milrinone
[1,6-Dihydro-2-methyl-6-oxo-(4'-bipyridine)-5-carbonitrile], a
specific PDE3 inhibitor and were subsequently stimulated for 15 minutes
with NE at the indicated concentration, in the continued presence of
the respective inhibitor. To test the effect of Hb oxygenation on cAMP
signaling, RBC samples suspended in measuring buffer were
preequilibrated for 25 minutes at 37°C in a radiometer micro-tonometer unit with either water vapor saturated air or with
different oxygen/nitrogen mixtures ranging from 1% oxygen to 8%
oxygen, which were provided by a Wösthoff gas mixing pump. After
10 minutes of preequilibration, ODQ (30 µmol/L) was added as
indicated and the samples were incubated for an additional 15 minutes.
The cells were then stimulated for 15 minutes with 10 µmol/L NE.
Samples were processed for cAMP measurements as described above.
Chemicals.
Enzymes for cAMP determination were obtained from Boehringer Manheim
(Manheim, Germany); all other reagents were purchased from
Sigma Chemicals (St Louis, MO).
Statistics.
Mean values were tested for statistical difference using the
t-test for paired or unpaired samples; P < .05 was
considered significant.
| |
RESULTS |
Basal cAMP concentration and response to forskolin for erythrocytes
harvested between 3 to 17 days of incubation.
Cyclic AMP levels of unstimulated erythrocytes showed only moderate
changes during development. Basal values ranged from a peak value of 4 pmol/mg Hb at day 4 to 0.2 to 0.4 pmol/mg Hb beween days 12 and 17 (Fig 1). Stimulation with forskolin (100 µmol/L) for 5 minutes increased cAMP approximately 20-fold (Fig 1)
between days 3 and 11. The ability of forskolin to stimulate adenylyl cyclase activity decreases markedly after day 11/12.
View larger version (21K):
[in this window]
[in a new window]
| Fig 1.
Basal and forskolin-stimulated cAMP production of
embryonic RBC at different stages of development. Embryonic RBC were
stimulated for 5 minutes with 100 µmol/L forskolin at pH 7.4 and
37°C. For details, see Materials and Methods. Data are the mean
values and SD derived from at least 5 determinations at each point.
|
|
Effect of -adrenergic and adenosine A2 receptor stimulation.
Figure 2 shows the erythrocyte cAMP level
after 5 minutes of stimulation with 10 µmol/L NE or 10 µmol/L CPCA
with cAMP concentrations related to mg Hb (Fig 2). Between day 3 to
about day 13/14, both NE and CPCA cause a large increase of RBC cAMP.
Basal cAMP levels are increased 50- to 100-fold within 5 minutes (Fig
2). After day 13/14, the response decreases rapidly for NE. In
contrast, the response to CPCA decreases gradually after day 6 and in a marked fashion after day 12. Note that both NE as well as CPCA are far
more effective stimulators of cAMP production than forskolin and that
maximum values for cAMP are at least 5 times higher than what is
observed with forskolin.
View larger version (18K):
[in this window]
[in a new window]
| Fig 2.
Effect of adrenergic effector NE (10 µmol/L) and
adenosine A2 receptor agonist CPCA (10 µmol/L) on cAMP production of
embryonic RBC harvested from 3- to 17-day-old chick embryos; data for
day 11 to 17 are taken from Dragon et al.3 Results are the
mean values and SD of at least 5 determinations.
|
|
Role of sGC and cGMP inhibited PDE3 in the control of
RBC cAMP.
We first analyzed the effect of guanylyl cyclase inhibition on
NE-stimulated cAMP production with RBC from day 11 using the nonspecific inhibitor Ly 83 583. After 5 minutes of stimulation, the
following results (mean values from 3 experiments) were obtained: control, 0.4 pmol cAMP/mg Hb; NE (10 µmol/L), 55.3 pmol cAMP/mg Hb;
NE + 20 µmol/L Ly 83583, 32.4 pmol cAMP/mg Hb; 100 µmol/L dibutyryl-cGMP + NE + Ly 83583, 42.8 pmol cAMP/mg Hb.
Because Ly 83583 is a nonspecific inhibitor of guanylyl cyclase, we
next tested the effect of ODQ, a specific inhibitor of sGC, on cAMP
levels. The dose-response curve for inhibition of NE-induced cAMP
accumulation by ODQ in day-11 RBC is shown in Fig 3. With 30 µmol/L ODQ, cyclic AMP was
reduced by nearly 80%, from 234 pmol cAMP/mgHb/15 minutes (10 µmol/L
NE) to 54 pmol cAMP/mg Hb/15 minutes (10 µmol/L NE + 30 µmol/L
ODQ). In addition, we tested the effect of ODQ on cells stimulated with
physiological concentrations of NE (10 and 30 nmol/L) encountered in
late development.2 With 30 nmol/L NE, cAMP increased to
76.7 pmol/mg Hb/15 minutes (standard deviation [SD], 5.6 pmol/mg Hb)
compared with 45.0 pmol/mg Hb/15 minutes (SD, 7.9 pmol/mg Hb) in cells
treated with 30 nmol/L NE + 30 µmol/L ODQ; when cells were stimulated
with 10 nmol/L NE, we found that ODQ decreased cAMP from 13.3 pmol/mg
Hb to 9.3 pmol/mg Hb (mean values from 3 experiments). On the other
hand, we found no effect of ODQ on basal cAMP levels of unstimulated cells (0.9 pmol/mg Hb). Likewise, the addition of cGMP to unstimulated cells (0.5 mmol/L) had no effect on basal cAMP (control, 0.8 pmol cAMP/mg Hb; +0.5 mmol/L cGMP, 1.1 pmol/mg Hb; mean values from n = 5).
However, cGMP increased cAMP levels of cells stimulated with 10 µmol/L NE by approximately 20% (control with 10 µmol/L NE, 254.1 pmol cAMP/mg Hb [SD, 21.0 pmol/mg Hb; n = 5]; 10 µmol/L NE + 0.5 mmol/L cGMP, 303.4 pmol cAMP/mg Hb [SD, 16.8 pmol/mg Hb; n = 5]). The
results suggest that alterations of cGMP concentration are important
for the regulation of cAMP in embryonic RBC stimulated by NE (or other
agents) activating the cAMP pathway; moreover, the marked effect of ODQ
suggests that sGC is the major cGMP-producing enzyme of the embryonic
RBC.
View larger version (15K):
[in this window]
[in a new window]
| Fig 3.
Inhibition of sGC with ODQ reduces NE-stimulated cAMP
production of RBC from day-11 embryos. The ordinate gives cAMP
production obtained with 10 µmol/L NE. Erythrocytes from day-11
embryos were incubated for 15 minutes at 37°C with 10 µmol/L NE
and varying concentrations of ODQ. Data are the mean values and SD of 3 experiments.
|
|
We next tested if the reduction of the cAMP level by ODQ is due to
activation of PDE3. The results are shown in
Fig 4. Milrinone, a specific inhibitor of
PDE3, reverses the antagonistic effect of ODQ on cAMP formation. On the
other hand, addition of milrinone together with NE did not
significantly increase cAMP (Fig 4) compared with the control with NE;
therefore, the PDE3 of the controls must be largely inhibited. The
results show that changes of PDE3 activity have a very large effect on
RBC cAMP, indicating cGMP-dependent inhibition of PDE3 as a major point
of control. The finding that neither ODQ nor added cGMP had an
influence on basal cAMP levels of unstimulated cells from day 11 is
presumably due to the fact that the basal cAMP concentration (~0.1
µmol/L) is much lower than the reported Km of about 0.5 µmol/L for
PDE3.9
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
PDE3 inhibitor milrinone reverses ODQ-induced decrease of
cAMP. RBC from day 11 were incubated for 15 minutes at 37°C with 10 µmol/L NE (control); 10 µmol/L NE + 30 µmol/L ODQ; 10 µmol/L
NE and 20 µmol/L milrinone (MIL); and 10 µmol/L NE, 30 µmol/L
ODQ, and 20 µmol/L milrinone, respectively. Data are the mean values
and SD from 5 experiments in each case. *Significant difference from
control (P < .01).
|
|
Effect of ODQ and milrinone on RBC cAMP production between days 6 and
18.
Because the composition of the circulating RBC population changes
completely during development, we tested the effect of ODQ and
milrinone on NE-stimulated cAMP formation between days 6 and 18 (Fig 5). At day 6, more than 90% of the
circulating erythrocytes are primitive RBC; at day 18, more than 90%
are mature definitive RBC.5 The data show that, at all
stages, ODQ decreases NE-induced cAMP. However, primitive RBC from day
6 are only moderately affected by ODQ (Fig 5), whereas definitive RBC
reduce cAMP by between 77% (day 11) and greater than 95% (days 15 and
18). At day 15, RBC stimulated with NE have 70 pmol cAMP/mg Hb, and
those incubated with NE and ODQ have only 2.9 pmol/mg Hb. Thus, in
those stages of development when definitive RBC are physiologically
stimulated by NE and adenosine,1,2 inhibition of PDE3 is a
prerequisite for a significant net increase of cellular cAMP. Milrinone
reverses the effect of ODQ at all stages.
View larger version (23K):
[in this window]
[in a new window]
| Fig 5.
Effect of ODQ and milrinone on NE-stimulated cAMP
accumulation of RBC from 6- to 18-day-old chick embryos. RBC were
stimulated for 15 minutes with 10 µmol/L NE in the absence and
presence of ODQ (30 µmol/L) and milrinone (20 µmol/L),
respectively. Data are the mean values and SD from 3 to 5 experiments
in each case.
|
|
Effect of milrinone and ODQ on intracellular cAMP degradation.
To test the effect of ODQ and milrinone on intracellular cAMP
degradation, cells were first incubated for 2 hours with 10 µmol/L
NE, rapidly washed 3 times to remove NE, and resuspended in fresh
buffer to which ODQ and milrinone were added as indicated. Cyclic AMP
concentrations were measured over 60 minutes after the addition of ODQ
or milrinone. The data are presented in Fig 6. After 2 hours of stimulation with NE, the cAMP concentration was
approximately 520 pmol cAMP/mg Hb. In the controls, half of the initial
cAMP is degraded by 25 minutes, whereas in the presence of ODQ, the
half-life for cAMP is reduced to less than 10 minutes and basal cAMP
values of unstimulated cells are reached by 45 minutes. With milrinone,
we still find 230 pmol cAMP/mg Hb at the end of the 1-hour incubation
period, compared with approximately 35 pmol cAMP/mg Hb for the control.
These results support the important role of PDE3 and sGC activity in
the regulation of cAMP signal strength.
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
Effect of ODQ and milrinone on cAMP degradation. RBC from
day-11 chick embryos were incubated for 2 hours at 37°C with 10 µmol/L NE, washed, and resuspended in fresh buffer (control) or
buffer with 30 µmol/L ODQ or 20 µmol/L milrinone. Cyclic AMP
measurements started with addition of ODQ or milrinone and were
performed over 60 minutes. Data are the mean values and SD from 3 experiments.
|
|
Effect of NO synthase inhibitor L-NMMA on cAMP production.
The embryonic RBC must produce sufficient cGMP during short-term
incubation (30 minutes at 37°C) to keep PDE3 activity fairly low,
because milrinone per se does not increase cAMP formation in the
presence of NE. Because sGC is activated by NO, we tested if embryonic
erythrocytes contain NO-synthase (NOS) activity. Erythrocytes from day
11 were preincubated for 15 or 120 minutes with the NOS inhibitor
L-NMMA (0.5 mmol/L) and subsequently stimulated for 15 minutes with 10 µmol/L NE in the continued presence of the NOS inhibitor. We found no
effect of NOS inhibition on NE-stimulated cAMP production (control with
10 µmol/L NE, 247 pmol cAMP/mg; 15 minutes of preincubation with
L-NNMA, 245.0 pmol cAMP/mg Hb; 120 minutes of preincubation with
L-NNMA, 237.1 pmol cAMP/mg Hb; mean values from n = 5 in each case).
The results show that guanylyl cyclase activity was unchanged in the
presence of L-NNMA.
Effect of Hb oxygenation and sodium nitroprusside (SNP) on cAMP
formation.
Because we have no evidence for endogenous NO production by NO
synthase, other intracellular sources should provide NO. Recent work
has shown that Hb is an NO donor under certain conditions: NO binds not
only to the heme group of Hb, but upon oxygenation it can be
transferred from the heme group to cysteine -93, forming S-nitrosohemoglobin.10,11 The structural transition from R to T conformation accompanying deoxygenation leads to release of NO
from the S-nitroso group.10,11 To test if Hb of embryonic RBC releases NO in an oxygenation-dependent manner, we investigated the
effect of partial deoxygenation on ODQ-induced inhibition of cAMP
formation. Because ODQ binds competetively to the heme of guanylyl
cyclase,12 increased occupancy of the heme of guanylyl cyclase by NO (due to NO release during deoxygenation) should decrease
the inhibitory effect of ODQ. Erythrocytes from day-11 embryos were
preequilibrated for 25 minutes before the addition of NE either with
1%, 3%, or 8% oxygen in nitrogen or with air. After 10 minutes of
the preequilibration period, ODQ was added. The results are shown in
Fig 7. Control samples incubated only with
NE were unaffected by deoxygenation. In the presence of ODQ, deoxygenation markedly affected cAMP production (Fig 7). In blood samples equilibrated with 1% oxygen, ODQ decreased NE-stimulated cAMP
formation only by 28%, whereas in air-equilibrated samples, ODQ
decreased cAMP formation by 76% (with 8% oxygen, we observed a 60%
reduction; with 3% oxygen, we observed a 52% reduction). Similar results were obtained with RBC from day 15 (data not shown).
View larger version (19K):
[in this window]
[in a new window]
| Fig 7.
Influence of Hb oxygenation on ODQ-induced decrease of
cAMP level of RBC from day 11. RBC from day-11 embryos were
preequilibrated for 25 minutes before addition of NE (10 µmol/L for
15 minutes) with either air, 1% oxygen, 3% oxygen, or 8% oxygen.
After 10 minutes, 30 µmol/L ODQ was added to 1 group of samples. Data
are the mean values and SD from 3 to 7 experiments.
|
|
We then tested if the addition of SNP (100 µmol/L) as an external NO
donor alters the effect of oxygenation and deoxygenation on cAMP
formation. As shown in Fig 8, the addition
of SNP at the start of the 25-minute prequlibration period increased
the formation of cAMP in air-incubated erythrocytes in the presence of
ODQ by approximately 70 pmol/mg Hb (from 72.6 to 141 pmol/mg Hb).The effect was significantly lower for partially deoxygenated RBC (1%
oxygen equilibration). With SNP, cAMP increased by approximately 35 pmol/mg Hb compared with the controls with ODQ (from 176.5 to 211 pmol/mg Hb). SNP had no effect on the cAMP formation in the absence of
ODQ. These results suggest that embryonic Hb is a substantial store for
NO and that the NO release is dependent on Hb oxygen saturation and
most effective when oxygen pressure falls to oxygen pressure of 20 mm
Hg or less, values that are commonly found in the intraembryonic
circulation.13 However, even in the oxygenated state,
sufficient NO must be liberated (from Hb or other compounds such as
S-nitrosoglutathione) to account for the inhibition of PDE3 during
short-term incubation.
View larger version (33K):
[in this window]
[in a new window]
| Fig 8.
Effect of SNP on ODQ-dependent decrease of cAMP level of
oxygenated and deoxygenated RBC from day 11. RBC suspensions were
preequilibrated in air or 1% oxygen for 25 minutes before the addition
of NE (10 µmol/L for 15 minutes) in the absence or presence of 100 µmol/L SNP; ODQ (30 µmol/L) was added after 10 minutes of
preequilibration. Data are the mean values and SD from 4 experiments.
|
|
| |
DISCUSSION |
Developmental changes of cAMP signaling in primitive and definitive
RBC.
The results of the present study demonstrate that, for most of
embryonic life, RBC adenylyl cyclase from chick embryo is functionally coupled to -adrenergic receptors and adenosine receptors (A2). Basophilic primitive erythroblasts from day 3 embryos show, within 5 minutes, at least 50-fold stimulation of their basal cAMP production in
the presence of NE or CPCA, and similar or even higher degrees of
activation (up to 100-fold increase over basal cAMP) are observed until
day 12/13, despite the fact that the composition of the RBC population
in the circulation has changed profoundly. Such high degrees of
stimulation by -adrenergic or other receptors linked to adenylyl
cyclase (measured in the absence of added PDE inhibitor) have so far
not been reported for erythroid cells. In primitive RBC, the effect of
adenosine receptor stimulation is slightly larger than -adrenergic
activation. On the other hand, after day 7, when the majority of the
circulating RBC are definitive erythrocytes, adrenergic stimulation
of cAMP production is stronger and the effect of CPCA gradually
decreases. This suggests some reorganization of the signaling system
upon transition from primitive to definitive erythrocytes. The
definitive RBC present in the embryonic circulation during the second
week of development are markedly different from adult RBC. Although
postmitotic, they are still transcriptionally active and have an
approximately 5- to 6-fold higher respiratory rate than RBC from adult
chick.14 Mature definitive RBC first appear
around day 12 to 14, and by day 18, the amount of reticulocytes in the
circulation has decreased to less than 10%.5 The rapid
decrease in cAMP production at this stage (receptor stimulated or
forskolin stimulated) closely correlates with the declining population
of transcriptionally active reticulocytes in the embryonic circulation.
In comparison to NE or CPCA, forskolin is a weak effector.
Nevertheless, the response of the cells is remarkably constant between
day 3 until about day 12, followed by a significant reduction of
forskolin-stimulated adenylyl cyclase activity, which parallels the
decrease of NE- or CPCA-mediated stimulation of adenylyl cyclase.
Although we have previously clarified the physiological role of cAMP
signaling in immature definitive RBC of the chick
embryo,1-3 one can only speculate about the function of
adenosine and -adrenergic receptors in circulating primitive RBC.
The yolk of the freshly laid egg contains substantial amounts of
catecholamine, which are taken up by the chick embryo once the
embryonic circulation is established, ie, after approximately 48 to 55 hours of incubation.15 Between days 2 and 4/5 of chick
embryonic development, uptake of extraembryonic catecholamine is
probably the major source of catecholamine supply for the
embryo.15,16 During this period, primitive embryonic
erythroblasts will be exposed to catecholamine in the yolk sac and the
vitelline circulation. This could account for our previous results
demonstrating that primitive RBC from day 4 had significantly increased
CAII17 and pyrimidine-5'-nucleotidase activity3 when compared with definitive RBC of days 8 to
12,3,17 because both proteins are upregulated by
cAMP.1,3
Transient stimulation of immature primitive RBC by catecholamine may
therefore help to increase the speed of terminal differentiation of the
circulating primitive RBC. NE has also been implicated as indispensible
for early mammalian development; lack of NE caused intrauterine death
that was attributed to cardiovascular failure.18 Our
results suggest that the maturation and function of primitive erythrocytes could also be affected. Taken together, the data indicate
that, in the avian embryo, the main targets of receptor-mediated cAMP
signaling are immature primitive or definitive RBC (basophilic erythroblast to reticulocytes). The rapid decrease of cAMP production in the last week of development correlates with the appearance of
mature definitive erythrocytes.
It is difficult to compare our results with those of other studies that
have investigated cAMP signaling in immature erythroid cells. There are
no comparable data for mamalian embryonic RBC. Most work analyzing cAMP
signaling in intact mammalian erythroid cells has been performed with
transformed erythroleukemia cell lines, which appear less restricted in
differentiation potential than normal erythroid progenitors (see Porzig
et al19).
Porzig et al19 analyzed the response of normal human adult
erythroid progenitors (eg, burst-forming units-erythroid [BFUe] to
colony-forming units-erythroid [CFUe]) derived from
peripheral stem cells towards a variety of hormonal agonists. The
progenitor cells responded to adenosine, PGE, and less so to
isoproterenol with increased cAMP production. Even in the presence of
PDE inhibitor adenosine, as the most potent effector, gave only
moderate (8-fold) stimulation of cAMP production. In late precursor
cells (normocytes and reticulocytes), cAMP signaling was also analyzed
in a few cases, but only very weak responses to receptor agonists were observed in these cells.19
Role of PDE3 and sGC in the control of cAMP production.
In the present investigation, we have tested to which extent the cAMP
signal is modulated by cAMP degradation and have concentrated on the
impact of cGMP regulated PDE3. Our data show that embryonic erythroid
cells possess a cGMP-inhibitable PDE3. Using the specific inhibitors
ODQ (for sGC) and milrinone (for PDE3), we established the presence of
PDE3 and sGC in primitive as well as definitive RBC. Primitive and
definitive RBC differ in their response to sGC inhibition by ODQ. In
primitive RBC, we observed a 25% reduction of NE-stimulated cAMP
formation in the presence of ODQ, whereas in definitive RBC, the signal
is reduced by 77% (day 11) to approximately 95% (day 15).
Activation of sGC in embryonic RBC.
Our data suggest that PDE3 is at least partially inhibited under our
experimental conditions: during short-term incubation, milrinone was
not able to increase NE-dependent cAMP formation in the absence of ODQ.
Inhibition of PDE3 requires continuous activity of sGC, which is
activated by NO. We found no evidence for NO synthase activity. L-NMMA,
which inhibits all known NOS isozymes, had no effect on cAMP levels.
Therefore, other intracellular sources must provide NO. NO can form
S-nitrosocompounds with suitable receptor molecules such as
glutathione,10,11 and there is evidence for the existence
of oxylabile S-nitrosogroups formed at cys -93,10,11 an
invariant residue in all mammalian and avian Hbs.
Our own data give some indirect evidence that the erythrocyte contains
NO donors that activate sGC. The addition of SNP as an NO donor had no
effect on the cAMP accumulation in oxygenated or deoxygenated RBC
stimulated with 10 µmol/L NE. This result is compatible with the
assumption that sGC is continously activated by intracellular NO donors
so that additional NO from an external source will have little effect.
On the other hand, SNP increased cAMP production of cells that were
stimulated with 10 µmol/L NE in the presence of ODQ. Because ODQ
binds competitively to the heme of sGC, increased free NO due to
addition of SNP will reduce the inhibitory effect of ODQ. Our results
also give some evidence that NO is released in in an oxygen-linked
manner from embryonic Hb: increasing free NO on deoxygenation should
diminish the antagonistic effect of ODQ. The results are in agreement
with this prediction: at the lowest oxygen concentration, ODQ inhibited
cAMP formation only by 28%, compared with 76% in the air-incubated
controls. Likewise, SNP stimulated cAMP accumulation to a larger extent in oxygenated RBC treated with ODQ than in deoxygenated RBC. Although further work is needed to test the hypothesis mentioned above, namely
that RBC sGC is activated by intracellular NO donors such as
Nitrosoglutathione or S-nitrosohemoglobin, it is clear that continued
activation of sGC provides a key element for cAMP signaling of
embryonic RBC under physiological conditions. The release of NE and
adenosine under hypoxic conditions and subsequent stimulation of RBC
adenylyl cyclase via adenosine and -adrenergic receptors promote
complex changes of RBC metabolism that rely on transcriptional activation and de novo protein synthesis subsequent to stimulation of
cAMP formation.1,2 This requires that intracellular cAMP levels must be elevated over hours rather than minutes.1,2 A long-lasting cAMP signal is only possible when PDE3 is inhibited. This creates a direct link between the NO-, cGMP-, and cAMP-signaling pathways. In conclusion, we have shown that cAMP signaling of immature
nucleate erythrocytes from chick embryo is embedded in a complex
network of stimulatory and inhibitory controls, which makes the
embryonic erythrocyte susceptible to influences from other embryonic
tissues. This feedback may be necessary to adjust the RBC properties to
changing developmental and/or environmental conditions.
| |
FOOTNOTES |
Submitted May 7, 1999; accepted August 16, 1999.
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.
Address reprint requests to Rosemarie Baumann, MD,
Physiologisches Institut, Universität Regensburg, 93047 Regensburg, Germany.
| |
REFERENCES |
1.
Glombitza S, Dragon S, Berghammer M, Pannermayer M, Baumann R:
Adenosine causes cAMP dependent activation of red cell carbonic anhydrase and 2,3BPG synthesis.
Am J Physiol
271:R973, 1996
[Abstract/Free Full Text]
2.
Dragon S, Glombitza S, Götz R, Baumann R:
Norepinephrine-mediated hypoxic stimulation of red cell embryonic carbonic anhydrase and 2,3BPG synthesis.
Am J Physiol
271:R982, 1996
[Abstract/Free Full Text]
3.
Dragon S, Hille R, Götz R, Baumann R:
Adenosine 3'-5'-cyclic monophosphate (cAMP)-inducible pyrimidine 5'-nucleotidase and pyrimidine nucleotide metabolism of chick embryonic erythrocytes.
Blood
91:3052, 1998
[Abstract/Free Full Text]
4.
Tazawa H:
Oxygen and CO2 exchange and acid-base regulation in the avian embryo.
Am Zool
20:395, 1980
5.
Bruns GA, Ingram VM:
The erythroid cells and haemoglobin of the chick embryo.
Phil Trans R Soc Lond B
266:225, 1973
[Medline]
[Order article via Infotrieve]
6.
Beavo JA, Conti M, Heaslip RJ:
Multiple cyclic nucleotide phosphodiesterases.
Mol Pharmacol
46:399, 1994
[Abstract]
7.
Maurice DW, Haslam RJ:
Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: Inhibition of cAMP breakdown by cGMP.
Mol Pharm
37:671, 1990
[Abstract]
8.
Sugiyama A, Lurie KG:
An enzymatic fluorometric assay for adenosine 3':5'-monophosphate.
Anal Biochem
218:20, 1994
[Medline]
[Order article via Infotrieve]
9.
Tang KM, Jang EK, Haslam RJ:
Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets.
Biochem J
323:217, 1997
10.
Jia L, Bonaventura C, Bonaventura J, Stamler JS:
S-nitrosohemoglobin: A dynamic activity of blood involved in vascular control.
Nature
380:221, 1996
[Medline]
[Order article via Infotrieve]
11.
Gow A, Stamler JS:
Reactions between nitric oxide and haemoglobin under physiological conditions.
Nature
391:169, 1998
[Medline]
[Order article via Infotrieve]
12.
Schrammel A, Behrends S, Schmidt K, Koesling D, Mayer B:
Characterization of 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin as a heme site inhibitor of nitric oxide sensitive guanylyl cyclase.
Mol Pharmacol
50:1, 1996
[Abstract]
13.
Baumann R, Meuer HJ:
Blood oxygen transport in the early avian embryo.
Physiol Rev
72:941, 1992
[Free Full Text]
14.
Grima M, Girard H:
Oxygen consumption by chick blood cells during embryonic and post-hatch growth.
Comp Biochem Physiol
69A:437, 1981
15.
Ignarro LJ, Shideman FE:
Appearance and concentrations of catecholamines and their biosynthesis in the embryonic and developing chick.
J Pharmacol Exp Therap
159:38, 1968
[Abstract/Free Full Text]
16.
Ignarro LJ, Shidemann FE:
Norepinephrine and epinephrine in the embryo and embryonic heart of the chick: Uptake and subcellular distribution.
J Pharmacol Exp Therap
159:49, 1968
[Abstract/Free Full Text]
17.
Million D, Zillner P, Baumann R:
Oxygen pressure dependent control of carbonic anhydrase synthesis in chick embryonic red cells.
Am J Physiol
261:R1188, 1991
[Abstract/Free Full Text]
18.
Thomas SA, Matsumoto AM, Palmiter RD:
Noradrenaline is essential for mouse fetal development.
Nature
374:643, 1995
[Medline]
[Order article via Infotrieve]
19.
Porzig H, Gutknecht R, Kostova G, Thalmeier K:
G protein coupled receptors in normal human erythroid progenitor cells.
Naunyn-Schmiedeberg's Arch Pharmacol
353:11, 1995
[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. S. Hanson, A. H. Stephenson, E. A. Bowles, M. Sridharan, S. Adderley, and R. S. Sprague
Phosphodiesterase 3 is present in rabbit and human erythrocytes and its inhibition potentiates iloprost-induced increases in cAMP
Am J Physiol Heart Circ Physiol,
August 1, 2008;
295(2):
H786 - H793.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. P. Cokic, S. A. Andric, S. S. Stojilkovic, C. T. Noguchi, and A. N. Schechter
Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells
Blood,
February 1, 2008;
111(3):
1117 - 1123.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Zennadi, P. C. Hines, L. M. De Castro, J.-P. Cartron, L. V. Parise, and M. J. Telen
Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-{alpha}v{beta}3 interactions
Blood,
December 1, 2004;
104(12):
3774 - 3781.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mass, E. Simo, and S. Dragon
Erythroid pyrimidine 5'-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia
Blood,
December 1, 2003;
102(12):
4198 - 4205.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Dragon and R. Baumann
Hypoxia, Hormones, and Red Blood Cell Function in Chick Embryos
Physiology,
April 1, 2003;
18(2):
77 - 82.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Dragon and R. Baumann
Erythroid carbonic anhydrase and hsp70 expression in chick embryonic development: role of cAMP and hypoxia
Am J Physiol Regulatory Integrative Comp Physiol,
March 1, 2001;
280(3):
R870 - R878.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Dragon, N. Offenhauser, and R. Baumann
cAMP and in vivo hypoxia induce tob, ifr1, and fos expression in erythroid cells of the chick embryo
Am J Physiol Regulatory Integrative Comp Physiol,
April 1, 2002;
282(4):
R1219 - R1226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION