Vol. 91, No.4, December
1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
28, 1979
Pages 1250-1257
STIMULATION
OF ERYTHROID
CYCLASE
Jean kstitut
BY SOLUBLE
Piau2,
Siegmund
and Georges
Delaunay
ADENYLATE
FACTORS
Jean-Pierre
Beaumont',
Carole
CELLS
Fischer,
Schapira
&Gculake,CHU cochin,24 rue du Faubourg St Jacques Paris V, U-15 INSJZRM,Laboratoire Associ6 no85 CNRS
de Pathologic
Universit6 Received
October
11,1979
SUMMARY : Soluble factors obtained from human, rat and rabbit erythroid cell lysates are capable to stimulate basal and hormone activated adenylate cyclase of erythroid cell membranes from homologous sources. Extensive dialysis and removal of hemoglobin from the soluble factors do not modify their activity. Human erythrocyte soluble factors stimulate the human reticulocyte enzyme. Nevertheless human erythrocyte adenylate cyclase is not stimulated by either of the soluble factors. The presence of active soluble factors in human erythrocytes where the adenylate cyclase is no longersensitive to these factors, as well as to guanylnucleotides or protaglandins, indicates that the enzyme has been altered during the maturation processes.
In erythrocyte despite that
the these
contrary the
from
presence receptors
are
in erythrocytes
reported
from
In this throid
cell
tivate
basal
also
nucleotides activity
species
not
sensitive
coupled
with
the
adenylate
cyclase
to hormones, the enzyme
other
mammals like
rat,
receptors
remains
throughout
systems,
of AC activity for
is
(l-4).
the
(AC)
indicating On the
coupling
between
the
span of
life
(5-7).
In non-erythroid stimulation
mammalian
no longer
enzyme and catecholamine
the erythrocyte
is
several
of receptors
mamnalian report
erythroid evidence
cytoplasm
from
AC activity
shown that
Similar
is given several
modulate
soluble
activators
to demonstrate
species
and to modulate maintain membranes
from
the
of soluble
the
hormonal
have not
the
action
soluble other
presence
factors
been
factors
to ac-
effecters.
stimulated which
in ery-
capable
of positive
whose AC is not
active
ABBREVIATIONS USED : CAMP : adenosine glycol bis-(aminoethyl) - tetraacetic immidodiphosphate CPP(NH)P : 5'-guanylyl thiothreitol ; AC : adenylate cyclase. IPresent adress : U.49 INSERM, Hgpital 2To whom to adress correspondance.
can modulate
species.
de Pont-Chaillou,
1250
It
by guanyl-
3' 5'-monophosphate ; ECTA : ethylene acfd ; SDS : sodium dodecyl sulfate ; ; NaF : sodium fluoride ; DTT : di-
0006-291X/79/241250-08$01.00/0 Copyright @I 1979 by Academic Press. Inc. AN rights of reproduction in any form reserved.
factors
cells.
human erythrocytes
or hormones in erythroid
cytoplasmic
(8-11).
Rennes.
AC
BIOCHEMICAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHODS INDUCTION OF RETICULOCYTOSIS AND PREPARATION OF MEMBRANES : Reticulocytes were obtained by injecting rats with 0 5 ml phenylhydrazine hydrochloride (15 mg/ml) for 3 consecutive days. After a four day rest blood was collected. Reticulocytes were always higher than 90 X. In rabbits, reticulocytosis was induced by cardiac bleedings (15 ml per day per kg of body weight). On the 5th day, reticulocytes varied from 15 to 35 per cent. After collection of blood on heparine the plasma membranes were prepared according to the procedure of Dodge et al. (12). Aliquots of membrane proteins at concentration of 4-7 mg/ml were stored in liquid nitrogen. or reticulocyte rich blood colPREPARATION OF SOLUBLE FACTORS : Erythrocyte lected on heparine was washed three times in 150 mM NaCl, 5 mMHP042' (pH 7.5), then lysed in two volumes of double distilled water for 30 minutes, and adjusted to a final concentration of 50 mM Tris buffer (pH 7.5), 1 mM DTT by addition of 10 times concentrated solutions. The suspension was then centrifuged at 100,000 g for 30 min. The supernatant was collected and stored in liquid nitrogen, or dialyzed against large volume of 50 mM Tris buffer (pH 7.4), 1 mM DTT, with or without 2 mM MgC12. ASSAY OF ADENYLATE CYCLASE : Adenylate cyclase was assayed according to Salomon et al. (13) 'th the modifications previously described by us (14). 50 to 100 pg of rat :lmbrane proteins were incubated for 10 min., and 150 to 300 pg human or rabbit membrane proteinsfor 30 min. The reaction was linear for at least 45 min. and 90 min., respectively. Protein was assayed by the procedure of Lowry et al. (15) using bovine serum albumin as a standart. Specific activity was expressed in pmol of CAMP formed per mg of protein per 10 min. RESULTS In rat branes
than
times
higher
the basal
in erythrocyte (Table
AC system
from
cytes)
further
is
It
in erythrocyte
involment
of guanylnucleotides
cyte
rat
respectively)
basal
and hormone
reticulocytes (less
than It
is
stimulation stimulated
or erythrocytes 1.5 % of the conceivable
factors
; this
the basal
preparations occured
This
the known
effect
(Table
factors
(RSF-ND
in both
erythro-
factors
of membranes in the
by extensive
I,
were
dialysis
The
from either negligible
presence
agent
I).
col.
of isoproterenol.
soluble
GTP was the stimulatory
1251
GTP) was
30 to 70 % (Table
of the
was investigated
(or
of sti-
reflecting
AC activity
activities
has
the extent
soluble
between
in the absence
sensitive
observation
of hormonal
16
or erythro-
+ Gpp(NH)p
in the presence
AC activities
corresponding that
catecholamine
effectors,probably
and erythrocyte
stimulated
activity
(reticulocytes
(16).
in the modulation
membrane
A similar
that
membranes
by isoproterenol
of both
reticulocyte
and reticulocyte
3 and 5).
soluble
addition
mem-
stimulated
2) and we have shown that
membranes
the simple
Non dialyzed
plasma
in reticulocyte
catecholamine
by GTP or Gpp(NH)p column
more than
and ESF-ND,
cell
stimulated I,
and the
higher
has been demonstrated
erythroid
(Table
was 30 times
ones
I).
rat
been confirmed mulation
AC activity
present
of metWanes). in these
of the
soluble
Vol. 91, No. 4, 1979
5IOCHEMICAL
AND BIOPHYSICAL
TABLE RAT ERYTHROID EFFECT OF Gpp(NH)p,
RESEARCH COMMUNICATIONS
I
CELLS PLASMA MEMBRANE ADENYLATE CYCLASE : DIALYZED AND NON-DIALYZED SOLUBLE FACTOR(S) RETICULOCYTE none ------
MEMBRANE RSF:ND
GPP(NH)P
RSF:D
ESF:ND
ESF:D
BASAL I
424
1 080
II III
350 360
377
4 676
5 914
4 482 3 088
4 000
726
479
630
687
:
:
543 640
842 795
6 022
4 642
-
7 905 5 447
6 608 4 569
ESF:D -
RSF:ND -
RSF:D -
ISOPROTERENOL I
6 742 -
ERYTHROCYTE none -
6 122
MEMBRANES ESF:ND -
GPP(NH)P ~
BASAL 51
149
::
22
22
18
26
III
24
58
E
:g
:
:
I ::I
291 425 126
576 311 805
493 912 223
540 790 193
471 -
471 -
ISOPROTERENOL
Adenylate cyclase was measured as described in Material and Methods. Assays were carried out in duplicate on three different preparations for both membranes and soluble factors. 100 ~1 of incubation medium contained : 0.5 mM Mg ATP ;9mMMgC12 : 2.2 mM Theophyllin ; 0.3 mM EGTA ; 20 mM creatine phoskinase phate ; 50 U/ml creatine ; 0.1 % bovine serum albumin ; 20-60 cpm/pmol (d 32P) ATP ; 25-150 pg protein. After a 10 minute incubation, the reaction was stopped by addition of 100 cl1 of a soluble containing : 1.4 mM cyclic AMP ; 5 mM ATP ; 2 % SDS ; 150 000-200 000 cpm/ml crlic (8-3H) AMP Gpp(NH)p and isoproterenol when present were lo-4M and 5.10M respectively. Soluble factors were prepared as described in Material and Methods, diluted to l/10 vol/vol and added in 10 1. ESF, RSF are erythrocyte or reticulocyte soluble factors. D or ND are dia Y yzed or non-dialyzed soluble factor.
factors The
from
reticulocytes
capability
of
isoproterenol
effect
These
suggest
not
results responsible To
Amicon prox.
effect.
10,000
should
mol.
of
wt.)
did
soluble
of
DTT
to
potentiate
colums
4 and
AC and
(Table be
I,
freely
factors
resulted the
in
dialyzable
modify
a PM-10 their
shown).
1252
were
minimal
hemoglobin
through not
presence
and
Mg2+. the 6).
was
probably
stimulation. the
system
the basal
unchanged
which
Concentration
in stimulate
almost
observed
(CM52),
ultrafiltration
to
GTP which
hemoglobin,
methyl-cellulose tory
that the
erythrocytes
factors
remained
for
eliminate
and
soluble
depleted membrane stimulatory
treated
changes
with of
dialyzed
carboxy-
their
stimula-
fraction
(exclusion
limit
activity
(results
by apnot
BlOCHEMlCAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
II
THE EFFECT OF HOMOLOGOUS SOLUBLE FACTORS ON THE AC ACTIVITY OF HUMAN AND RABBIT ERYTHROID MEMBRANES HUMAN ERYTHROCYTE Mb BASAL F-
HUMAN RETICULOCYTE Mb
1.7 40.7 1.7
5.5 318.2 6.5 5.6
2.3
8.9
2.0 .!::bBLE FACTOR(S) PGEl + SOLUBLE FACTOR(S)
RABBIT RETICULOCYTE Mb 11.0 142.3 17 11.0 52
Assays were performed as described in Table I except that incubation time was 30 minutes, (d 3BP) ATP 150-200 cpm, and prote'n concentration lOO4 250 pg.NaF and PGEl when present were lO-lM and lo- M respectively. Results are expressed in pm01 CAMP : mg protein/l0 min. Human erythrocyte membranes, where isolated from normal human blood with reticulocytosis lower than 0.3 per cent. Human reticulocyte membranes were isolated from blood obtained from a patient with hereditary spherocytosis (reticulocyte count 10 %). Rabbit reticulocyte membranes were isolated from blood artificially enriched in reticulocytes by density gradient centrifugation. The original reticulocytosis of 15 % was increased to 68 %. In these conditions basal AC was increased 8 times and NaF stimulated activity 3.5 times compared to the starting whole blood. Soluble factors from either human and rabbit were isolated from normal blood.
and rabbit
Human
compared
to the
rat
by PGEI and not determine
if
activity.
This
of soluble
erythroid
by isoproterenol
the
soluble
the
from
AC activity not
ble
obtained
AC from
soluble
factors,
remarkable than
that
if
erythroid
several from
although
was the
less
erythrocytes
membranes
membrane
under
the
slightly
of
to
basal
the
addition
obtained
AC
from
conditions
same
membranes
interest
on the
nevertheless
species
than
was not
homologous
of stimulation
attained,
level
F- whereas
(results
the
factors.
own soluble not
(Table
shown).
be activated were
III).
stimulated
by its
high
its
could
preparations
was further
with
of human or rat to either
erythrocyte
different
reticulocytes
obtained
upon addition responsive rat
rat
by F- but only
in membranes
whereas
activity
affected
rabbit).
to test factors,
factors
II,
in Table
blood,
specific
had no effect
the PGEr effect rich
low
It was therefore
4, 14).
human and rabbit
shown for
In order
stimulated
by themselves
potentiated reticulocyte
vated
(3,
factors
was the case as shown
factors
gous soluble
AC was of very
It was strongly
one.
human or rabbit (results
cell
F- stimulated Human erythrocyte
factors
incubated
with
Isoproterenol
soluacti-
by human erythrocyte factors. which
What was was even higher
AC remained
unchanged
membranes
or soluble
factors
In the membranes
prepared
1253
by heterolo-
AC was not
prepared from
from
reticu-
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I I I
TABLE
THE EFFECT OF HOMOLOGOUS AND HETEROLOGOUS SOLUBLE FACTORS ON HUMAN AND RAT RETICULOCYTE AC ACTIVITY RAT RETICULOCYTES --RAT :SF
HUMAN+ ESF
5505 440 6685
FBASAL
301.5 4930
5795 590
ISOPROTERENOL
4949
7590
PGEl
Assays
locyte
rich
slight
effect
performed
human blood,
and rabbit which
were
on the
still
as described
both
the
the
present
in
AC is
RAT+ESF
109 6
Ii7
I;6
7
9
10
Table
no longer
I and
rat
AC. These
in the cell
HUMAN+ESF
-
human and the
PGEl stimulated
erythrocytes
are
HUMAN RETICULOCYTES
soluble
results
factors
show that
sensitive
and active
II
Table
elicited in human
to soluble
factors,
on the AC of other
systems.
DISCUSSI(IN In rat,
rabbit
ma membranes thus
the
than
and by guanylnucleotides
(14).
which,
was also
obtained
in reticulocytes,
erythrocytes,
(In
Rat AC was
furthermore, previous
which
the AC was no longer
in plas-
from erythrocytes,
and ourselves
in erythrocytes.
experi-
was barely
seen
was slighisolated from
responsive
to the PGEl
14). now,
the
on AC activity
(8-11)
but
to our
been shown with
presence
of soluble
has been knowledge
soluble
factors
demonstrated
this
factors
is
the first
isolated
capable
to modulate
in some non-erythroid time
from
that
a similar
non-nucleated
hormonal systems effect
has
erythroid
. Several
characteristics
1) This
stimulatory
ultrafiltration peaks
blood
(5-6)
higher
AC from human and rabbit reticulocytes in the plasma membranes by PGE1. On the contrary
activated
Until
than
rich
by others effect
effect
cells
reticulocyte
work
was significantly
experiments).
human or rabbit (l-4,
AC activity
catecholamine
potentiation
in further tly
from
previous
by isoproterenol,
potentiated ments
isolated
confirming
activated
and human,
10,000.
of this activity
on PM 10 filters, Column
of soluble
chromatography
activities
eluted
system
(Table its
merit
I)
apparent
with within
1254
further
is not
molecular
Sephadex the
considerations.
dialyzable. weight
G 150 showed
iclusion
volume
As judged is
by
higher
at least of the
two column
BIOCHEMICAL
Vol. 91, No. 4, 1979
(results tor.
not
at which
dialysis
experiments
protein-bound
have shown that
the
2) In rat by soluble than
It is improbable
shown).
Several
were
similar
to that
are completly
isoproterenol
the from
attainable with
obtained
with
stimulation
In the human reticulocyte than
man membranes
have modified
factors
interact.
throcytes cannot
; both
that is
level
reticulocyte cyclase
shown).
memactivity
On the
contrary,
factors
ten-
soluble
suggest
factors
that
to which
the hu-
soluble
in human and rabbit
factors
of either
factors.
by soluble
NaF. This
evident
t
can be explained
of activation
with
was
of NaF.
of soluble
some of the sites
specially
by addition
effect
in the presence
NaF alone,
(9),
of the enzyme to the soluble
observed
have active
be potentiated
with
the
or lost
This
cells
not
seen by the addition
system,
t PGEI is much lower
(results stimulation
to that
out
was even higher
When rat
the adenylate
responsiveness
concentration
dialyzed
or human erythrocytes
F- alone
fac-
of the catecholamine
fluoride.
an extra
or NaF,compared
might
rat with
F- + Gpp(NH)p,
yielded
as an increased
potentiation
activating
2 mM Mg2+,
was maintained.
the higher
tatively
against
activity
isolated
factors
Therefore
performed
GTP is the
nucleotides
incubated
the soluble
free
guanyl
reticulocytes,
factors
that
stimulatory
the stimulation
branes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
but the effect
homologous
ery-
of PGEI
or heterogous
soluble
of a cytoplasmic
activa-
factors. 3) A protein tor
(CDR) which
of (Cat2 t Mgt2)
to be responsible rase tion
for
the
mimics
Ca2'
dependent
cyclase
activities
It is unlikely
that
such protein
lowing
adenylate
cyclase
(17-18) has been shown
activation
of both
phosphodieste-
might
observed
be responsible
in these
for
the
activa-
for
the
experiments,
fol-
: a) EGTA present b) Additive
found
in rat
luble
factors
such additive NaF t soluble c) High factors,
in our
effect
brain
assay
of soluble
(18).
This
to erythrocyte
effect
was found
plasma
human and rabbit
active
at 10m3M abolishes
factor
plus
membranes in rat
CDR action
(20).
NaF or guanylnucleotides with
human,
rat
not
shown),
(results
reticulocyte
AC, only
was
or rabbit
so-
although
by addition
of
factors. phosphodiesterase
and in their
from both
system
was not observed
added
activity
was found
membranes
(unpublished
ty in these membranes was high. ties were8 times lower than rat shown cells.
properties
(19-20).
and adenylate of basal
the
in human red blood cell
ATPase
factors
were
soluble found
in rat
erythroid
results).
soluble
The AC activi-
On the contrary human and rabbit AC activiones. Phosphodiesterase was almost absent factors in both
1255
and from
their
of them as well
membranes. as in rat
As
erythroid
Vol. 91, No. 4, 1979
Hypothesis der
i)
sal
activity
its
BIOCHEMICAL
explaining
action
The stimulation factors
brane
bound
converts
AC into
luble crease
the
either
conditions It
through
in
they
membranes ions tion,
for
and/or
local
Pre iminary
1the
are
pH resulting now under
experiments
observed
the activated an increase is
the AC is
units
the
in changes response
role(s)
of the
way to isolate
indicate
that
By
from
cytoplasmic intracellular of membrane
is
systems the
stu-
catechola-
and reticuin some erythroid
factors
which
concentration protein
pro-
cells
erythrocytes that
soluble
in the
in erythroid
possible
of the
(22).
of some membrane
in both
by soluble
so-
or in-
modifications
which
therefore
is
; the
protein)
receptor
known that kinase,
formed
in the AC activity.
on the possible
membranes, is
GTP
the effecters is
a GTP binding
of phosphorylation
by modifying the
with
complex
uncoupled
be modulated
alter
together
In two of the membrane
It
that of mem-
generated
ones,
in rat
of ba-
assuming
membranes.
coupled.
example,
and thereby Efforts
for
remain
AC could
activated,
added
protein
rabbit
whereas
It
pattern
the plasma
human and the
min receptor(s), locytes,
systems.
CAMP-dependent
mainly the
factors
is
increase
effect.
(21).
to speculate
the
; the
an hormone-receptor
net effect
in the erythroid
teins,
in the
the phosphorylation
ATP)
add GTP (through
tempting
of CAMP change
died,
to catalyse
of the enzyme for the
has to consi-
can be explained
contains
form
soluble
either
levels present
able
system
presence
affinity
is quite
factors
basal
kinases
might
results
activity
of the
factors
which
factor(s)
of the assay
In their
itself,
soluble
of PGEI or isoproterenol
a more active
The effect
of the
RESEARCH COMMUNICATIONS
potentiation
contain GDP (the
different.
effect
on the enzyme
and ii)
these
the
AND BIOPHYSICAL
might
be
of some
phosphoryla-
cell.
and purify
more than
these
one protein
soluble are
factor(s). responsible
activation. ACKNOWLEDGEMENTS
This work was supported by Grants from the "Institut de la Sante et de la Recherche Medicale" and the "Delegation G&&ale a la Recherche Scientifique et Technique. REFERENCES 1. 2. 3. 4. 5.
J.E. (1975) Biochim. Biophys. Acta Rasmussen, H., Lake, W., and Allen, 411, 63-73. Murti, C.R. (1975) Med. Biol. BX6u, C.R., Azhara, S., and Krishna -53, 148-155. Res. Comm. -64, 1263Hosey, M.M., and TAO, M. (1975) Biochem. Biophys. 1269. Rodan, S-B., Rodan, G.A., and Sha'afi, R.I. (1976) Biochim. Biophys. Acta -428, 509-515. Gauger, D., Kaiser, G., Quiring, K., and Palm, D. (1975) Naunyn-Schmied Arch. Pharmacol. 289, 379-398.
1256
Vol. 91, No. 4, 1979
6. 7.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13.
M.E., BjXnd, D.B., Beckman, B.S. (1976) Life SC. 19, 243-250. J.P., Spiegel, A.M., Brown, E.M., Aurbach, G.B. 7T977) Mol. 13, 775-785. m., Brostrom, C.O., Breckeuridge, B.M., and Wolff, D.J. (1976) J. Biol. Chem. 251, 4750-4755. Pecker, F., and HanouncJ. (1977) J. Biol. Chem. 252, 2784-2786. Sanders, R.B., Thompson, W.J., and Robison, G.A. (T977) Biochim. Biophys. Acta 498, 10-20. Doberska, C.A., and Martin, B.R. (1977) FEBS Letters 82, 273-277. Dodge, J.T., Mitchell, C., and Hanahan, D.J. (1963) AZh. Biochem. Biophys. 100, 119-129. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. g, 541-
14.
Piau,
15.
17.
Lowry,O.M. Rosebrough, R.J. (1951) J. Biol. Chem. 193. 265-275. Bilezikian, J.P., Spiegel, A.M., Gammon, D.E., and Aurbach, G.D. (1977) Mol. Pharmacol. 13, 786-795. Gopinath, R.M., %id Vincenzi, F.F. (1977) Biochem. Biophys. Res. Conn~.
18.
Znnett,
8. 1;: 11. 12.
Charness, Bilezikian, Pharmacol. Brostrom,
548.
16.
J.P., Fischer, S., (1978) Biochim. Biophys.
Delaunay, Acta 544, N.H., Farr,
J.G.,
Tortolero, 482-488. B., and Randall,
M.,
and Schapira,
77, 1203-1209. 19.
H.W.,
and Penniston,
-77, 1210-1209. Cheung,
W.Y.,
Bradham,
L.S.,
J.T.
(1977) Biochem.
Lynch,
T-J.,
Liu,
Y.M.,
Biophys. Tallant,
20.
(1975) Biochem. Biophys. Res. Commun. 66, 1055-1062. Lynch, T.J., Tallant, E.A., and Cheung3.Y. (1977) Arch.
21. 22.
Biophys. 182, 124-133. Pfeuffer,TP. (1977) J. Biol. Chem. 252, 7224-7234. Hanski, E., Rimon, G., and Levittki (T979) Biochem.
1257
Res. E.A. Biochem.
-18, 846-853.
Comm.
G.
1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
28, 1979
Pages 1250-1257
STIMULATION
OF ERYTHROID
CYCLASE
Jean kstitut
BY SOLUBLE
Piau2,
Siegmund
and Georges
Delaunay
ADENYLATE
FACTORS
Jean-Pierre
Beaumont',
Carole
CELLS
Fischer,
Schapira
&Gculake,CHU cochin,24 rue du Faubourg St Jacques Paris V, U-15 INSJZRM,Laboratoire Associ6 no85 CNRS
de Pathologic
Universit6 Received
October
11,1979
SUMMARY : Soluble factors obtained from human, rat and rabbit erythroid cell lysates are capable to stimulate basal and hormone activated adenylate cyclase of erythroid cell membranes from homologous sources. Extensive dialysis and removal of hemoglobin from the soluble factors do not modify their activity. Human erythrocyte soluble factors stimulate the human reticulocyte enzyme. Nevertheless human erythrocyte adenylate cyclase is not stimulated by either of the soluble factors. The presence of active soluble factors in human erythrocytes where the adenylate cyclase is no longersensitive to these factors, as well as to guanylnucleotides or protaglandins, indicates that the enzyme has been altered during the maturation processes.
In erythrocyte despite that
the these
contrary the
from
presence receptors
are
in erythrocytes
reported
from
In this throid
cell
tivate
basal
also
nucleotides activity
species
not
sensitive
coupled
with
the
adenylate
cyclase
to hormones, the enzyme
other
mammals like
rat,
receptors
remains
throughout
systems,
of AC activity for
is
(l-4).
the
(AC)
indicating On the
coupling
between
the
span of
life
(5-7).
In non-erythroid stimulation
mammalian
no longer
enzyme and catecholamine
the erythrocyte
is
several
of receptors
mamnalian report
erythroid evidence
cytoplasm
from
AC activity
shown that
Similar
is given several
modulate
soluble
activators
to demonstrate
species
and to modulate maintain membranes
from
the
of soluble
the
hormonal
have not
the
action
soluble other
presence
factors
been
factors
to ac-
effecters.
stimulated which
in ery-
capable
of positive
whose AC is not
active
ABBREVIATIONS USED : CAMP : adenosine glycol bis-(aminoethyl) - tetraacetic immidodiphosphate CPP(NH)P : 5'-guanylyl thiothreitol ; AC : adenylate cyclase. IPresent adress : U.49 INSERM, Hgpital 2To whom to adress correspondance.
can modulate
species.
de Pont-Chaillou,
1250
It
by guanyl-
3' 5'-monophosphate ; ECTA : ethylene acfd ; SDS : sodium dodecyl sulfate ; ; NaF : sodium fluoride ; DTT : di-
0006-291X/79/241250-08$01.00/0 Copyright @I 1979 by Academic Press. Inc. AN rights of reproduction in any form reserved.
factors
cells.
human erythrocytes
or hormones in erythroid
cytoplasmic
(8-11).
Rennes.
AC
BIOCHEMICAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHODS INDUCTION OF RETICULOCYTOSIS AND PREPARATION OF MEMBRANES : Reticulocytes were obtained by injecting rats with 0 5 ml phenylhydrazine hydrochloride (15 mg/ml) for 3 consecutive days. After a four day rest blood was collected. Reticulocytes were always higher than 90 X. In rabbits, reticulocytosis was induced by cardiac bleedings (15 ml per day per kg of body weight). On the 5th day, reticulocytes varied from 15 to 35 per cent. After collection of blood on heparine the plasma membranes were prepared according to the procedure of Dodge et al. (12). Aliquots of membrane proteins at concentration of 4-7 mg/ml were stored in liquid nitrogen. or reticulocyte rich blood colPREPARATION OF SOLUBLE FACTORS : Erythrocyte lected on heparine was washed three times in 150 mM NaCl, 5 mMHP042' (pH 7.5), then lysed in two volumes of double distilled water for 30 minutes, and adjusted to a final concentration of 50 mM Tris buffer (pH 7.5), 1 mM DTT by addition of 10 times concentrated solutions. The suspension was then centrifuged at 100,000 g for 30 min. The supernatant was collected and stored in liquid nitrogen, or dialyzed against large volume of 50 mM Tris buffer (pH 7.4), 1 mM DTT, with or without 2 mM MgC12. ASSAY OF ADENYLATE CYCLASE : Adenylate cyclase was assayed according to Salomon et al. (13) 'th the modifications previously described by us (14). 50 to 100 pg of rat :lmbrane proteins were incubated for 10 min., and 150 to 300 pg human or rabbit membrane proteinsfor 30 min. The reaction was linear for at least 45 min. and 90 min., respectively. Protein was assayed by the procedure of Lowry et al. (15) using bovine serum albumin as a standart. Specific activity was expressed in pmol of CAMP formed per mg of protein per 10 min. RESULTS In rat branes
than
times
higher
the basal
in erythrocyte (Table
AC system
from
cytes)
further
is
It
in erythrocyte
involment
of guanylnucleotides
cyte
rat
respectively)
basal
and hormone
reticulocytes (less
than It
is
stimulation stimulated
or erythrocytes 1.5 % of the conceivable
factors
; this
the basal
preparations occured
This
the known
effect
(Table
factors
(RSF-ND
in both
erythro-
factors
of membranes in the
by extensive
I,
were
dialysis
The
from either negligible
presence
agent
I).
col.
of isoproterenol.
soluble
GTP was the stimulatory
1251
GTP) was
30 to 70 % (Table
of the
was investigated
(or
of sti-
reflecting
AC activity
activities
has
the extent
soluble
between
in the absence
sensitive
observation
of hormonal
16
or erythro-
+ Gpp(NH)p
in the presence
AC activities
corresponding that
catecholamine
effectors,probably
and erythrocyte
stimulated
activity
(reticulocytes
(16).
in the modulation
membrane
A similar
that
membranes
by isoproterenol
of both
reticulocyte
and reticulocyte
3 and 5).
soluble
addition
mem-
stimulated
2) and we have shown that
membranes
the simple
Non dialyzed
plasma
in reticulocyte
catecholamine
by GTP or Gpp(NH)p column
more than
and ESF-ND,
cell
stimulated I,
and the
higher
has been demonstrated
erythroid
(Table
was 30 times
ones
I).
rat
been confirmed mulation
AC activity
present
of metWanes). in these
of the
soluble
Vol. 91, No. 4, 1979
5IOCHEMICAL
AND BIOPHYSICAL
TABLE RAT ERYTHROID EFFECT OF Gpp(NH)p,
RESEARCH COMMUNICATIONS
I
CELLS PLASMA MEMBRANE ADENYLATE CYCLASE : DIALYZED AND NON-DIALYZED SOLUBLE FACTOR(S) RETICULOCYTE none ------
MEMBRANE RSF:ND
GPP(NH)P
RSF:D
ESF:ND
ESF:D
BASAL I
424
1 080
II III
350 360
377
4 676
5 914
4 482 3 088
4 000
726
479
630
687
:
:
543 640
842 795
6 022
4 642
-
7 905 5 447
6 608 4 569
ESF:D -
RSF:ND -
RSF:D -
ISOPROTERENOL I
6 742 -
ERYTHROCYTE none -
6 122
MEMBRANES ESF:ND -
GPP(NH)P ~
BASAL 51
149
::
22
22
18
26
III
24
58
E
:g
:
:
I ::I
291 425 126
576 311 805
493 912 223
540 790 193
471 -
471 -
ISOPROTERENOL
Adenylate cyclase was measured as described in Material and Methods. Assays were carried out in duplicate on three different preparations for both membranes and soluble factors. 100 ~1 of incubation medium contained : 0.5 mM Mg ATP ;9mMMgC12 : 2.2 mM Theophyllin ; 0.3 mM EGTA ; 20 mM creatine phoskinase phate ; 50 U/ml creatine ; 0.1 % bovine serum albumin ; 20-60 cpm/pmol (d 32P) ATP ; 25-150 pg protein. After a 10 minute incubation, the reaction was stopped by addition of 100 cl1 of a soluble containing : 1.4 mM cyclic AMP ; 5 mM ATP ; 2 % SDS ; 150 000-200 000 cpm/ml crlic (8-3H) AMP Gpp(NH)p and isoproterenol when present were lo-4M and 5.10M respectively. Soluble factors were prepared as described in Material and Methods, diluted to l/10 vol/vol and added in 10 1. ESF, RSF are erythrocyte or reticulocyte soluble factors. D or ND are dia Y yzed or non-dialyzed soluble factor.
factors The
from
reticulocytes
capability
of
isoproterenol
effect
These
suggest
not
results responsible To
Amicon prox.
effect.
10,000
should
mol.
of
wt.)
did
soluble
of
DTT
to
potentiate
colums
4 and
AC and
(Table be
I,
freely
factors
resulted the
in
dialyzable
modify
a PM-10 their
shown).
1252
were
minimal
hemoglobin
through not
presence
and
Mg2+. the 6).
was
probably
stimulation. the
system
the basal
unchanged
which
Concentration
in stimulate
almost
observed
(CM52),
ultrafiltration
to
GTP which
hemoglobin,
methyl-cellulose tory
that the
erythrocytes
factors
remained
for
eliminate
and
soluble
depleted membrane stimulatory
treated
changes
with of
dialyzed
carboxy-
their
stimula-
fraction
(exclusion
limit
activity
(results
by apnot
BlOCHEMlCAL
Vol. 91, No. 4, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE
II
THE EFFECT OF HOMOLOGOUS SOLUBLE FACTORS ON THE AC ACTIVITY OF HUMAN AND RABBIT ERYTHROID MEMBRANES HUMAN ERYTHROCYTE Mb BASAL F-
HUMAN RETICULOCYTE Mb
1.7 40.7 1.7
5.5 318.2 6.5 5.6
2.3
8.9
2.0 .!::bBLE FACTOR(S) PGEl + SOLUBLE FACTOR(S)
RABBIT RETICULOCYTE Mb 11.0 142.3 17 11.0 52
Assays were performed as described in Table I except that incubation time was 30 minutes, (d 3BP) ATP 150-200 cpm, and prote'n concentration lOO4 250 pg.NaF and PGEl when present were lO-lM and lo- M respectively. Results are expressed in pm01 CAMP : mg protein/l0 min. Human erythrocyte membranes, where isolated from normal human blood with reticulocytosis lower than 0.3 per cent. Human reticulocyte membranes were isolated from blood obtained from a patient with hereditary spherocytosis (reticulocyte count 10 %). Rabbit reticulocyte membranes were isolated from blood artificially enriched in reticulocytes by density gradient centrifugation. The original reticulocytosis of 15 % was increased to 68 %. In these conditions basal AC was increased 8 times and NaF stimulated activity 3.5 times compared to the starting whole blood. Soluble factors from either human and rabbit were isolated from normal blood.
and rabbit
Human
compared
to the
rat
by PGEI and not determine
if
activity.
This
of soluble
erythroid
by isoproterenol
the
soluble
the
from
AC activity not
ble
obtained
AC from
soluble
factors,
remarkable than
that
if
erythroid
several from
although
was the
less
erythrocytes
membranes
membrane
under
the
slightly
of
to
basal
the
addition
obtained
AC
from
conditions
same
membranes
interest
on the
nevertheless
species
than
was not
homologous
of stimulation
attained,
level
F- whereas
(results
the
factors.
own soluble not
(Table
shown).
be activated were
III).
stimulated
by its
high
its
could
preparations
was further
with
of human or rat to either
erythrocyte
different
reticulocytes
obtained
upon addition responsive rat
rat
by F- but only
in membranes
whereas
activity
affected
rabbit).
to test factors,
factors
II,
in Table
blood,
specific
had no effect
the PGEr effect rich
low
It was therefore
4, 14).
human and rabbit
shown for
In order
stimulated
by themselves
potentiated reticulocyte
vated
(3,
factors
was the case as shown
factors
gous soluble
AC was of very
It was strongly
one.
human or rabbit (results
cell
F- stimulated Human erythrocyte
factors
incubated
with
Isoproterenol
soluacti-
by human erythrocyte factors. which
What was was even higher
AC remained
unchanged
membranes
or soluble
factors
In the membranes
prepared
1253
by heterolo-
AC was not
prepared from
from
reticu-
Vol. 91, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I I I
TABLE
THE EFFECT OF HOMOLOGOUS AND HETEROLOGOUS SOLUBLE FACTORS ON HUMAN AND RAT RETICULOCYTE AC ACTIVITY RAT RETICULOCYTES --RAT :SF
HUMAN+ ESF
5505 440 6685
FBASAL
301.5 4930
5795 590
ISOPROTERENOL
4949
7590
PGEl
Assays
locyte
rich
slight
effect
performed
human blood,
and rabbit which
were
on the
still
as described
both
the
the
present
in
AC is
RAT+ESF
109 6
Ii7
I;6
7
9
10
Table
no longer
I and
rat
AC. These
in the cell
HUMAN+ESF
-
human and the
PGEl stimulated
erythrocytes
are
HUMAN RETICULOCYTES
soluble
results
factors
show that
sensitive
and active
II
Table
elicited in human
to soluble
factors,
on the AC of other
systems.
DISCUSSI(IN In rat,
rabbit
ma membranes thus
the
than
and by guanylnucleotides
(14).
which,
was also
obtained
in reticulocytes,
erythrocytes,
(In
Rat AC was
furthermore, previous
which
the AC was no longer
in plas-
from erythrocytes,
and ourselves
in erythrocytes.
experi-
was barely
seen
was slighisolated from
responsive
to the PGEl
14). now,
the
on AC activity
(8-11)
but
to our
been shown with
presence
of soluble
has been knowledge
soluble
factors
demonstrated
this
factors
is
the first
isolated
capable
to modulate
in some non-erythroid time
from
that
a similar
non-nucleated
hormonal systems effect
has
erythroid
. Several
characteristics
1) This
stimulatory
ultrafiltration peaks
blood
(5-6)
higher
AC from human and rabbit reticulocytes in the plasma membranes by PGE1. On the contrary
activated
Until
than
rich
by others effect
effect
cells
reticulocyte
work
was significantly
experiments).
human or rabbit (l-4,
AC activity
catecholamine
potentiation
in further tly
from
previous
by isoproterenol,
potentiated ments
isolated
confirming
activated
and human,
10,000.
of this activity
on PM 10 filters, Column
of soluble
chromatography
activities
eluted
system
(Table its
merit
I)
apparent
with within
1254
further
is not
molecular
Sephadex the
considerations.
dialyzable. weight
G 150 showed
iclusion
volume
As judged is
by
higher
at least of the
two column
BIOCHEMICAL
Vol. 91, No. 4, 1979
(results tor.
not
at which
dialysis
experiments
protein-bound
have shown that
the
2) In rat by soluble than
It is improbable
shown).
Several
were
similar
to that
are completly
isoproterenol
the from
attainable with
obtained
with
stimulation
In the human reticulocyte than
man membranes
have modified
factors
interact.
throcytes cannot
; both
that is
level
reticulocyte cyclase
shown).
memactivity
On the
contrary,
factors
ten-
soluble
suggest
factors
that
to which
the hu-
soluble
in human and rabbit
factors
of either
factors.
by soluble
NaF. This
evident
t
can be explained
of activation
with
was
of NaF.
of soluble
some of the sites
specially
by addition
effect
in the presence
NaF alone,
(9),
of the enzyme to the soluble
observed
have active
be potentiated
with
the
or lost
This
cells
not
seen by the addition
system,
t PGEI is much lower
(results stimulation
to that
out
was even higher
When rat
the adenylate
responsiveness
concentration
dialyzed
or human erythrocytes
F- alone
fac-
of the catecholamine
fluoride.
an extra
or NaF,compared
might
rat with
F- + Gpp(NH)p,
yielded
as an increased
potentiation
activating
2 mM Mg2+,
was maintained.
the higher
tatively
against
activity
isolated
factors
Therefore
performed
GTP is the
nucleotides
incubated
the soluble
free
guanyl
reticulocytes,
factors
that
stimulatory
the stimulation
branes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
but the effect
homologous
ery-
of PGEI
or heterogous
soluble
of a cytoplasmic
activa-
factors. 3) A protein tor
(CDR) which
of (Cat2 t Mgt2)
to be responsible rase tion
for
the
mimics
Ca2'
dependent
cyclase
activities
It is unlikely
that
such protein
lowing
adenylate
cyclase
(17-18) has been shown
activation
of both
phosphodieste-
might
observed
be responsible
in these
for
the
activa-
for
the
experiments,
fol-
: a) EGTA present b) Additive
found
in rat
luble
factors
such additive NaF t soluble c) High factors,
in our
effect
brain
assay
of soluble
(18).
This
to erythrocyte
effect
was found
plasma
human and rabbit
active
at 10m3M abolishes
factor
plus
membranes in rat
CDR action
(20).
NaF or guanylnucleotides with
human,
rat
not
shown),
(results
reticulocyte
AC, only
was
or rabbit
so-
although
by addition
of
factors. phosphodiesterase
and in their
from both
system
was not observed
added
activity
was found
membranes
(unpublished
ty in these membranes was high. ties were8 times lower than rat shown cells.
properties
(19-20).
and adenylate of basal
the
in human red blood cell
ATPase
factors
were
soluble found
in rat
erythroid
results).
soluble
The AC activi-
On the contrary human and rabbit AC activiones. Phosphodiesterase was almost absent factors in both
1255
and from
their
of them as well
membranes. as in rat
As
erythroid
Vol. 91, No. 4, 1979
Hypothesis der
i)
sal
activity
its
BIOCHEMICAL
explaining
action
The stimulation factors
brane
bound
converts
AC into
luble crease
the
either
conditions It
through
in
they
membranes ions tion,
for
and/or
local
Pre iminary
1the
are
pH resulting now under
experiments
observed
the activated an increase is
the AC is
units
the
in changes response
role(s)
of the
way to isolate
indicate
that
By
from
cytoplasmic intracellular of membrane
is
systems the
stu-
catechola-
and reticuin some erythroid
factors
which
concentration protein
pro-
cells
erythrocytes that
soluble
in the
in erythroid
possible
of the
(22).
of some membrane
in both
by soluble
so-
or in-
modifications
which
therefore
is
; the
protein)
receptor
known that kinase,
formed
in the AC activity.
on the possible
membranes, is
GTP
the effecters is
a GTP binding
of phosphorylation
by modifying the
with
complex
uncoupled
be modulated
alter
together
In two of the membrane
It
that of mem-
generated
ones,
in rat
of ba-
assuming
membranes.
coupled.
example,
and thereby Efforts
for
remain
AC could
activated,
added
protein
rabbit
whereas
It
pattern
the plasma
human and the
min receptor(s), locytes,
systems.
CAMP-dependent
mainly the
factors
is
increase
effect.
(21).
to speculate
the
; the
an hormone-receptor
net effect
in the erythroid
teins,
in the
the phosphorylation
ATP)
add GTP (through
tempting
of CAMP change
died,
to catalyse
of the enzyme for the
has to consi-
can be explained
contains
form
soluble
either
levels present
able
system
presence
affinity
is quite
factors
basal
kinases
might
results
activity
of the
factors
which
factor(s)
of the assay
In their
itself,
soluble
of PGEI or isoproterenol
a more active
The effect
of the
RESEARCH COMMUNICATIONS
potentiation
contain GDP (the
different.
effect
on the enzyme
and ii)
these
the
AND BIOPHYSICAL
might
be
of some
phosphoryla-
cell.
and purify
more than
these
one protein
soluble are
factor(s). responsible
activation. ACKNOWLEDGEMENTS
This work was supported by Grants from the "Institut de la Sante et de la Recherche Medicale" and the "Delegation G&&ale a la Recherche Scientifique et Technique. REFERENCES 1. 2. 3. 4. 5.
J.E. (1975) Biochim. Biophys. Acta Rasmussen, H., Lake, W., and Allen, 411, 63-73. Murti, C.R. (1975) Med. Biol. BX6u, C.R., Azhara, S., and Krishna -53, 148-155. Res. Comm. -64, 1263Hosey, M.M., and TAO, M. (1975) Biochem. Biophys. 1269. Rodan, S-B., Rodan, G.A., and Sha'afi, R.I. (1976) Biochim. Biophys. Acta -428, 509-515. Gauger, D., Kaiser, G., Quiring, K., and Palm, D. (1975) Naunyn-Schmied Arch. Pharmacol. 289, 379-398.
1256
Vol. 91, No. 4, 1979
6. 7.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13.
M.E., BjXnd, D.B., Beckman, B.S. (1976) Life SC. 19, 243-250. J.P., Spiegel, A.M., Brown, E.M., Aurbach, G.B. 7T977) Mol. 13, 775-785. m., Brostrom, C.O., Breckeuridge, B.M., and Wolff, D.J. (1976) J. Biol. Chem. 251, 4750-4755. Pecker, F., and HanouncJ. (1977) J. Biol. Chem. 252, 2784-2786. Sanders, R.B., Thompson, W.J., and Robison, G.A. (T977) Biochim. Biophys. Acta 498, 10-20. Doberska, C.A., and Martin, B.R. (1977) FEBS Letters 82, 273-277. Dodge, J.T., Mitchell, C., and Hanahan, D.J. (1963) AZh. Biochem. Biophys. 100, 119-129. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. g, 541-
14.
Piau,
15.
17.
Lowry,O.M. Rosebrough, R.J. (1951) J. Biol. Chem. 193. 265-275. Bilezikian, J.P., Spiegel, A.M., Gammon, D.E., and Aurbach, G.D. (1977) Mol. Pharmacol. 13, 786-795. Gopinath, R.M., %id Vincenzi, F.F. (1977) Biochem. Biophys. Res. Conn~.
18.
Znnett,
8. 1;: 11. 12.
Charness, Bilezikian, Pharmacol. Brostrom,
548.
16.
J.P., Fischer, S., (1978) Biochim. Biophys.
Delaunay, Acta 544, N.H., Farr,
J.G.,
Tortolero, 482-488. B., and Randall,
M.,
and Schapira,
77, 1203-1209. 19.
H.W.,
and Penniston,
-77, 1210-1209. Cheung,
W.Y.,
Bradham,
L.S.,
J.T.
(1977) Biochem.
Lynch,
T-J.,
Liu,
Y.M.,
Biophys. Tallant,
20.
(1975) Biochem. Biophys. Res. Commun. 66, 1055-1062. Lynch, T.J., Tallant, E.A., and Cheung3.Y. (1977) Arch.
21. 22.
Biophys. 182, 124-133. Pfeuffer,TP. (1977) J. Biol. Chem. 252, 7224-7234. Hanski, E., Rimon, G., and Levittki (T979) Biochem.
1257
Res. E.A. Biochem.
-18, 846-853.
Comm.
G.
