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Stimulation of erythropoiesis by cyclic adenosine monophosphate
Life S c i e n c e s Vol. 10, Part II, pp. 895-900, Printed in Great Britain
1971.
Pergamon Press
STIMULATION OF ER~fHROPOIESIS BY CYCLIC ADENOSINE MONOPHOSPHATE* Anthony S. Gidari +, Esmail D. Zanjani and Albert S. Gordon Laboratory of Experimental Hematology, Department of Biology, Graduate School of Arts and Science, New York University, New York City, New York 10003
(Received in final form 2 July 1971) Sun=aary CYCLIC AMP stimulated the incorporation of radioiron by the red blood cells in polycytheraic mice. This effect was potentiated by prior incubation of cyclic AMP with dialyzed rat serum and was partially abolished i__~nvlvo, but not in vitro, by anti-erythropoietin. These experiments suggest both direct and indirect stimulatory effects of cyclic AMP on erythropoiesis. In recent years the role of 3'5'-adenosine monophosphate (cyclic AMP) as an intracellular messenger mediating a variety of hormonal effects has been well established (1-3).
Hormonal stimulation of a membrane-bound adenyl
cyclase enzyme results in increased levels of intracellular cyclic AMP in responsive cells of the target organ.
The intracellular cyclic AMP then
mediates hormonal action by affecting the activities of enzymes, permeability processes, or the synthesis or release of other hormones (1-3).
Recently,
Winkert and Birchette (4) have demonstrated that, i_~nviyo, the dibutyryl derivative of cyclic AMP (db-cyclic AMP) stimulates radioiron incorporation into red blood cells.
In addition, cyclic AMP and db-cyclic AMP have been
reported to enhance delta-aminolevulinic acid (ALA) synthetase activity i__nn vitro (5).
Moreover, db-cyclic AMP has been shown to augment heme synthesis
in human marrow cells in vitro (6).
Our studies were undertaken to determine
the possible relation of cyclic AMP to erythropoietln (ESF), the hormone * Supported by research grant 5 ROI HE03357-14 and training grant 5 TOI HE0564506 from the National Heart and Lung Institute, National Institutes of Health, United States Public Health Service. +Predoctoral NIH Trainee.
895
896
Erythropoiesis and Cyclic AMP
regulating
erythropoiesls
in higher organisms
Vol. 10, No. 15
(7).
Methods Selum from male Long-Evans ethylenediamlne
tetraacetate
rats (250-300 g) was dialyzed
(EDTA) before use (5).
AMP (Sigma Chem. Co.) with saline or EDTA-dialyzed were conducted
All incubations
of cyclic
normal rat serum (EDTA-NRS)
for one hour in a water bath with shaking at 37 ° C in vessels
open to the air. peritoneal
against disodium
The incubation mixtures,
injections,
were assayed
administered
for erythropoietic
had been rendered polycythemic
by hypoxla
was used as the erythropoietic
criterion,
(6).
as single, activity
2 ml intra-
in mice which
Percent RBC-59Fe
incorporation
Five mice were used for each sample
tested. Results Intraperitoneal
administration
of incubation
fluid containing
mg or 6.0 mg of cyclic AMP in saline resulted in a significant incorporation
of radioiron
into the RBC of polycythemic
effect was greater when cyclic AMP was incubated, NRS.
Thus significantly
observed at all dosage those obtained
enhancing
to ESF (anti-ESF,
international
units
activity generated
incorporation,
anti-ESF.
However,
to eliminate
incorporation
the action of cyclic AMP, in
involved
stimulation
of the production
of ESF in vitro.
at a different
by 2 mg of cyclic AMP, previously was only partially
AntI-ESF
(0.5
site but simultaneously
([U) of ESF was completely abolished
results
were
in serum than
Table 2 shows that the erythropoletic
saline and 1 ml of EDTA-NRS,
with EDTA-
kindly supplied by Drs. R.D. Lange and
intraperitoneally
with cyclic AMP or ESF.
This
in saline.
of ESF in the assay mouse or the generation ml) was administered
before injection,
for RBC radioiron
was used to determine whether
RBC radioiron
increase in the
mice (Table I).
levels of cyclic AMP after incubation
for cyclic AMP incubated
An antibody T.P. McDonald)
greater values
either 3.0
effect of 0.05
by anti-ESF.
The
incubated with I ml of
inhibited
by the injected
listed in Table 3 show that anti-ESF was not able
the erythropoietic
activity of this mixture when added i_~nvitro,
Vol. 10, No. 15
(Group 5).
Erythropoiesis and Cyclic A M P
In these experiments
tion period and allowed globulin
to react
(GARGG; Antibodies,
mixture and allowed
897
anti-ESF was added at the end of the incubafor I0 minutes;
goat anti-rabbit
gam~ma
Inc. Davis, Calif.), was then added to this
to react for 15 minutes at room temperature.
itate was removed by centrifugation
and the supernatant
retained
The precipfor testing
TABLE I Effect of cyclic AMP on RBC-radioiron
incorporation
polycythemic
mice.
Cyclic AMP was dissolved
in a mixture
of 6 ml of saline and 6 ml of EDTA-NRS
for 60 minutes at 37 ° C. fluid containing
values in
in 12 ml of saline or and incubated
Each mouse received 2 ml of incubation
the indicated amount of cyclic AMP.
Each value is
the mean ± i S.E.M.
% RBC-59Fe Saline
cyclic AMP
(mz)
Incorporation EDTA-NRS
1.5
2.91 ± 0.64
9.87 :< 1.83
3.0
7.75 :: 1.64
11.97 • 2.11
6.0
7.37 ± 0.90
15.24 :: 1.79
controls : 0.90% NaCI
0.43 + 0.ii
0.05 IU ESF
4.37 + 2.04
0.20 IU ESF
* ESF-3A,
15.3
Step III sheep plasma erythropoietln
Heart Institute, (Group 5).
used in this way, was able to neutralize
(Group 8).
of anti-ESF was able to abolish experiments
was supplied by the National
United States Public Health Service.
The GARGG,
of 0.2 ml anti-ESF
~L 1.77
The results of group 7 indicate the activity of 0.2 IU ESF.
the activity that 0.2 ml
The above
concerned with both the in vivo and in vitro effects of anti-ESF
were repeated on 3 occasions with essentially
the same results.
898
E r y t h r o p o i e s i s and Cyclic AMP
Vol. 10, No. 15
TABLE 2 The effect of anti-erythropoietln a cyclic AMP-dialyzed Cyclic AMP, previously EDTA-NRS,
on the erythropoietic
rat serum (EDTA-NRS)
activity
of
incubation mixture.
incubated with i ml of saline and I ml of
or ESF was administered
alone to assay mice or together
with 0.5 ml of anti-erythropoietin.
Each value is the mean
± I S.E.M.
Treatment
% RBC-59Fe
2 mg cyclic AMP
Incorporation
12.17 -~ 2.65
2 mg cyclic AMP; 0.5 ml anti-ESF
6.27 ::: 1.50 12.79 ± 0.96
0.05 IU ESF 0.05 IU ESF; 0.5 ml anti-ESF
0.83 * 0.05
0.90% NaCl
0.64 i 0.18
Discussion The data indicate AMP is attributable, mouse.
that the erythropoiesis
in part,
to an increased
The mechanism underlying
stimulating production
effect of cyclic
of ESF in the assay
the enhancing action of serum on cyclic AMP
activity
in vivo is not clear.
Serum may serve as a vehicle
carriage
and release of cyclic AMP to its target
sites.
for more efficient
The results also show
that cyclic AMP does not induce ESF generation when added to serum in vitro. It operates apparently The possibility investigated.
by stimulating
the production
that the kidney mediates The inability
cyclic AMP on erythropoiesis directly enhancing
of anti-ESF suggests
erythropoiesis
this response
is presently
to completely
abolish
in the polycythemic
stimulate ALA synthetase
being
the effects
of
that this substance may also operate by
shown for certain 5~-H steroid metabolites metabolites
of ESF within the animal.
(i0).
mouse.
However,
activity in embryonic
This has been
whereas
these
chick liver cells
Vol. 10, No. 15
E r y t h r o p o i e s i s and Cyclic AMP
(ii), cyclic AMP apparently does not exert this effect (12).
899
On the other
hand, Bottomley, et al. (5) have reported the stimulation of ALA synthetase activity by cyclic AMP and db-cyclic AMP in rabbit marrow cultures. TABLE 3 The effect of anti-erythropoietin on the erythropoietic activity of a cyclic AMP-dialyzed rat serum (EDTA-NRS) incubation mixture. Antl-erythropoietin was added in vitro; incubation procedures are described in the text.
Group
Each value is the mean ± 1 S . E . M .
Treatment
% RBC-59Fe Incorporation
1
2 ml saline
0.98 • 0.18
2
i ml EDTA-NRS + i ml saline
1.39 • 0.29
3
1.5 mg cyclic AMP + 2 ml saline
3.45 =! 0.84
4
1.5 mg cyclic AMP + i ml EDTA-NRS + I ml saline
8.12 ~ 0.66
5
( (1.5 mg cyclic AMP + 0.6 ml saline + I ml EDTA-NRS) + 0.2 ml anti-ESF) + 0.2 ml GARGG*
6.56 • 0.93
6
0.2 IU ESF + 2 ml saline
7
(0.2 IU ESF + 1.6 ml saline + 0.2 ml anti-ESF) + 0.2 ml GARGG
2.80 ~: 0.68
8
(0.2 ml anti-ESF + 0.2 ml GARGG) + 0.2 IU ESF + 1.6 ml saline
14.92 + 1.86
13.25 ± 2.40
Goat anti-rabbit gamma globulin. The erythropoiesis-stlmulating action of cyclic AMP may be a phenomenon attributable to nucleotides in general.
Thus our preliminary experiments have
shown that administration of 5'-AMP and 5'-ATP to polycythemlc mice also increased the incorporation of radloiron into the circulating red cells. may be due to a stabilizing influence on ALA synthetase (5).
This
However, no
stimulating effect on erythropolesis was observed with tetrasodium pyrophosphate
900
Erythropoiesis and Cyclic A M P
Vol. I0, No. 15
References I.
E.W. SUTHERLAND, I. ~YE, R.W. BUTCHER, Recent Prosr. Hormone Res. 21,623 (1965).
2.
E.W. SUT}{ERLAND and G.A. ROBISON, Pharmacol. Rev. 18,145 (1966).
3.
G.A. ROBISON, R.W. BUTCHER, E.W. SUTHERLAND, Ann. Rev. Biochem. 37,149 (1968).
4.
WINKERT, J. and C. BIRCHETTE, Fed. Proc, 29,843 (1970).
5.
BOTTOMLEY, S.S., W.H. WHITCOMB, G.A. SMITHEE, M.Z. MOORE, J. Lab. Clin. Med. 77,793 (1971).
6.
GORSHEIN, D. and F.H. GARDNER, Blood 36,847 (1970).
7.
A.S. GORDON and E.D. ZANJANI, in Regulation of llematopoiesis, A.S. Gordon, Ed. (Appleton-Century-Crofts, New York), p. 413 (1970).
8.
E.D. ZANJANI, J.F. CONTRERA, G.W. COOPER, A.S. GORDON, K.K. WONG, Science 156,1367 (1967).
9.
J.F. CAMISCOLI, A.H. WEINTRAUB, A.S. GORDON, Ann. N.Y. Acad. Sci. 149,40
(1968). i0.
A.S. GORDON, E.D. ZANJANI, R.D. LEVERE, A. KAPPAS, Proc. Nat. Acad. Sci. U.S. 65,919 (1970).
II.
S. GRANICK and A. KAPPAS, Proc. Nat. Acad. Sci. U.S. 57,1463 (1967).
12.
J.A. SIMONS, Science 167,1378 (1970).
1971.
Pergamon Press
STIMULATION OF ER~fHROPOIESIS BY CYCLIC ADENOSINE MONOPHOSPHATE* Anthony S. Gidari +, Esmail D. Zanjani and Albert S. Gordon Laboratory of Experimental Hematology, Department of Biology, Graduate School of Arts and Science, New York University, New York City, New York 10003
(Received in final form 2 July 1971) Sun=aary CYCLIC AMP stimulated the incorporation of radioiron by the red blood cells in polycytheraic mice. This effect was potentiated by prior incubation of cyclic AMP with dialyzed rat serum and was partially abolished i__~nvlvo, but not in vitro, by anti-erythropoietin. These experiments suggest both direct and indirect stimulatory effects of cyclic AMP on erythropoiesis. In recent years the role of 3'5'-adenosine monophosphate (cyclic AMP) as an intracellular messenger mediating a variety of hormonal effects has been well established (1-3).
Hormonal stimulation of a membrane-bound adenyl
cyclase enzyme results in increased levels of intracellular cyclic AMP in responsive cells of the target organ.
The intracellular cyclic AMP then
mediates hormonal action by affecting the activities of enzymes, permeability processes, or the synthesis or release of other hormones (1-3).
Recently,
Winkert and Birchette (4) have demonstrated that, i_~nviyo, the dibutyryl derivative of cyclic AMP (db-cyclic AMP) stimulates radioiron incorporation into red blood cells.
In addition, cyclic AMP and db-cyclic AMP have been
reported to enhance delta-aminolevulinic acid (ALA) synthetase activity i__nn vitro (5).
Moreover, db-cyclic AMP has been shown to augment heme synthesis
in human marrow cells in vitro (6).
Our studies were undertaken to determine
the possible relation of cyclic AMP to erythropoietln (ESF), the hormone * Supported by research grant 5 ROI HE03357-14 and training grant 5 TOI HE0564506 from the National Heart and Lung Institute, National Institutes of Health, United States Public Health Service. +Predoctoral NIH Trainee.
895
896
Erythropoiesis and Cyclic AMP
regulating
erythropoiesls
in higher organisms
Vol. 10, No. 15
(7).
Methods Selum from male Long-Evans ethylenediamlne
tetraacetate
rats (250-300 g) was dialyzed
(EDTA) before use (5).
AMP (Sigma Chem. Co.) with saline or EDTA-dialyzed were conducted
All incubations
of cyclic
normal rat serum (EDTA-NRS)
for one hour in a water bath with shaking at 37 ° C in vessels
open to the air. peritoneal
against disodium
The incubation mixtures,
injections,
were assayed
administered
for erythropoietic
had been rendered polycythemic
by hypoxla
was used as the erythropoietic
criterion,
(6).
as single, activity
2 ml intra-
in mice which
Percent RBC-59Fe
incorporation
Five mice were used for each sample
tested. Results Intraperitoneal
administration
of incubation
fluid containing
mg or 6.0 mg of cyclic AMP in saline resulted in a significant incorporation
of radioiron
into the RBC of polycythemic
effect was greater when cyclic AMP was incubated, NRS.
Thus significantly
observed at all dosage those obtained
enhancing
to ESF (anti-ESF,
international
units
activity generated
incorporation,
anti-ESF.
However,
to eliminate
incorporation
the action of cyclic AMP, in
involved
stimulation
of the production
of ESF in vitro.
at a different
by 2 mg of cyclic AMP, previously was only partially
AntI-ESF
(0.5
site but simultaneously
([U) of ESF was completely abolished
results
were
in serum than
Table 2 shows that the erythropoletic
saline and 1 ml of EDTA-NRS,
with EDTA-
kindly supplied by Drs. R.D. Lange and
intraperitoneally
with cyclic AMP or ESF.
This
in saline.
of ESF in the assay mouse or the generation ml) was administered
before injection,
for RBC radioiron
was used to determine whether
RBC radioiron
increase in the
mice (Table I).
levels of cyclic AMP after incubation
for cyclic AMP incubated
An antibody T.P. McDonald)
greater values
either 3.0
effect of 0.05
by anti-ESF.
The
incubated with I ml of
inhibited
by the injected
listed in Table 3 show that anti-ESF was not able
the erythropoietic
activity of this mixture when added i_~nvitro,
Vol. 10, No. 15
(Group 5).
Erythropoiesis and Cyclic A M P
In these experiments
tion period and allowed globulin
to react
(GARGG; Antibodies,
mixture and allowed
897
anti-ESF was added at the end of the incubafor I0 minutes;
goat anti-rabbit
gam~ma
Inc. Davis, Calif.), was then added to this
to react for 15 minutes at room temperature.
itate was removed by centrifugation
and the supernatant
retained
The precipfor testing
TABLE I Effect of cyclic AMP on RBC-radioiron
incorporation
polycythemic
mice.
Cyclic AMP was dissolved
in a mixture
of 6 ml of saline and 6 ml of EDTA-NRS
for 60 minutes at 37 ° C. fluid containing
values in
in 12 ml of saline or and incubated
Each mouse received 2 ml of incubation
the indicated amount of cyclic AMP.
Each value is
the mean ± i S.E.M.
% RBC-59Fe Saline
cyclic AMP
(mz)
Incorporation EDTA-NRS
1.5
2.91 ± 0.64
9.87 :< 1.83
3.0
7.75 :: 1.64
11.97 • 2.11
6.0
7.37 ± 0.90
15.24 :: 1.79
controls : 0.90% NaCI
0.43 + 0.ii
0.05 IU ESF
4.37 + 2.04
0.20 IU ESF
* ESF-3A,
15.3
Step III sheep plasma erythropoietln
Heart Institute, (Group 5).
used in this way, was able to neutralize
(Group 8).
of anti-ESF was able to abolish experiments
was supplied by the National
United States Public Health Service.
The GARGG,
of 0.2 ml anti-ESF
~L 1.77
The results of group 7 indicate the activity of 0.2 IU ESF.
the activity that 0.2 ml
The above
concerned with both the in vivo and in vitro effects of anti-ESF
were repeated on 3 occasions with essentially
the same results.
898
E r y t h r o p o i e s i s and Cyclic AMP
Vol. 10, No. 15
TABLE 2 The effect of anti-erythropoietln a cyclic AMP-dialyzed Cyclic AMP, previously EDTA-NRS,
on the erythropoietic
rat serum (EDTA-NRS)
activity
of
incubation mixture.
incubated with i ml of saline and I ml of
or ESF was administered
alone to assay mice or together
with 0.5 ml of anti-erythropoietin.
Each value is the mean
± I S.E.M.
Treatment
% RBC-59Fe
2 mg cyclic AMP
Incorporation
12.17 -~ 2.65
2 mg cyclic AMP; 0.5 ml anti-ESF
6.27 ::: 1.50 12.79 ± 0.96
0.05 IU ESF 0.05 IU ESF; 0.5 ml anti-ESF
0.83 * 0.05
0.90% NaCl
0.64 i 0.18
Discussion The data indicate AMP is attributable, mouse.
that the erythropoiesis
in part,
to an increased
The mechanism underlying
stimulating production
effect of cyclic
of ESF in the assay
the enhancing action of serum on cyclic AMP
activity
in vivo is not clear.
Serum may serve as a vehicle
carriage
and release of cyclic AMP to its target
sites.
for more efficient
The results also show
that cyclic AMP does not induce ESF generation when added to serum in vitro. It operates apparently The possibility investigated.
by stimulating
the production
that the kidney mediates The inability
cyclic AMP on erythropoiesis directly enhancing
of anti-ESF suggests
erythropoiesis
this response
is presently
to completely
abolish
in the polycythemic
stimulate ALA synthetase
being
the effects
of
that this substance may also operate by
shown for certain 5~-H steroid metabolites metabolites
of ESF within the animal.
(i0).
mouse.
However,
activity in embryonic
This has been
whereas
these
chick liver cells
Vol. 10, No. 15
E r y t h r o p o i e s i s and Cyclic AMP
(ii), cyclic AMP apparently does not exert this effect (12).
899
On the other
hand, Bottomley, et al. (5) have reported the stimulation of ALA synthetase activity by cyclic AMP and db-cyclic AMP in rabbit marrow cultures. TABLE 3 The effect of anti-erythropoietin on the erythropoietic activity of a cyclic AMP-dialyzed rat serum (EDTA-NRS) incubation mixture. Antl-erythropoietin was added in vitro; incubation procedures are described in the text.
Group
Each value is the mean ± 1 S . E . M .
Treatment
% RBC-59Fe Incorporation
1
2 ml saline
0.98 • 0.18
2
i ml EDTA-NRS + i ml saline
1.39 • 0.29
3
1.5 mg cyclic AMP + 2 ml saline
3.45 =! 0.84
4
1.5 mg cyclic AMP + i ml EDTA-NRS + I ml saline
8.12 ~ 0.66
5
( (1.5 mg cyclic AMP + 0.6 ml saline + I ml EDTA-NRS) + 0.2 ml anti-ESF) + 0.2 ml GARGG*
6.56 • 0.93
6
0.2 IU ESF + 2 ml saline
7
(0.2 IU ESF + 1.6 ml saline + 0.2 ml anti-ESF) + 0.2 ml GARGG
2.80 ~: 0.68
8
(0.2 ml anti-ESF + 0.2 ml GARGG) + 0.2 IU ESF + 1.6 ml saline
14.92 + 1.86
13.25 ± 2.40
Goat anti-rabbit gamma globulin. The erythropoiesis-stlmulating action of cyclic AMP may be a phenomenon attributable to nucleotides in general.
Thus our preliminary experiments have
shown that administration of 5'-AMP and 5'-ATP to polycythemlc mice also increased the incorporation of radloiron into the circulating red cells. may be due to a stabilizing influence on ALA synthetase (5).
This
However, no
stimulating effect on erythropolesis was observed with tetrasodium pyrophosphate
900
Erythropoiesis and Cyclic A M P
Vol. I0, No. 15
References I.
E.W. SUTHERLAND, I. ~YE, R.W. BUTCHER, Recent Prosr. Hormone Res. 21,623 (1965).
2.
E.W. SUT}{ERLAND and G.A. ROBISON, Pharmacol. Rev. 18,145 (1966).
3.
G.A. ROBISON, R.W. BUTCHER, E.W. SUTHERLAND, Ann. Rev. Biochem. 37,149 (1968).
4.
WINKERT, J. and C. BIRCHETTE, Fed. Proc, 29,843 (1970).
5.
BOTTOMLEY, S.S., W.H. WHITCOMB, G.A. SMITHEE, M.Z. MOORE, J. Lab. Clin. Med. 77,793 (1971).
6.
GORSHEIN, D. and F.H. GARDNER, Blood 36,847 (1970).
7.
A.S. GORDON and E.D. ZANJANI, in Regulation of llematopoiesis, A.S. Gordon, Ed. (Appleton-Century-Crofts, New York), p. 413 (1970).
8.
E.D. ZANJANI, J.F. CONTRERA, G.W. COOPER, A.S. GORDON, K.K. WONG, Science 156,1367 (1967).
9.
J.F. CAMISCOLI, A.H. WEINTRAUB, A.S. GORDON, Ann. N.Y. Acad. Sci. 149,40
(1968). i0.
A.S. GORDON, E.D. ZANJANI, R.D. LEVERE, A. KAPPAS, Proc. Nat. Acad. Sci. U.S. 65,919 (1970).
II.
S. GRANICK and A. KAPPAS, Proc. Nat. Acad. Sci. U.S. 57,1463 (1967).
12.
J.A. SIMONS, Science 167,1378 (1970).