0022-3565/95/2742-0937$03.00/0
THE Jouiuu
OF PHARMACOLOGY
an
Copyright © 1995 by The American
JPET
274:937-942,
EXPERIMENTAL
Society
for
Vol. 274, No. 2
THERAPEIYFICS
Pharmacology
and
Experimental
Therapeutics
Printed
Coichicine
and Cytochalasin
B Enhance
Accumulation
Via Postreceptor
Actions
JEFFREY
R. JASPER,
Department
of Pediatrics,
Diego,
La Jolla,
Accepted
in U.S.A.
1995
STEVEN
R. POST,
Stanford
California
University,
[S.R.P.
for publication
KAVIN
Stanford
H. DESAI,
[J.R.J.,
K.H.D.,
PAUL
Cyclic
A. INSEL
and
D.B.] and Department
AMP
DANIEL
BERNSTEIN
of Pharmacology,
University
of California
at San
and PA.!.]
April 19, 1995
ABSTRACT
role
The
of cytoskeletal
modulating
cAMP
vestigated
using
microtubules
in S49
generation
the
and
microfilaments
lymphoma
cells was
in
in-
agents
colchicine
and cytochalasin
B,
to disrupt
these structures.
A
with 1 0 pM colchicine
typically
enhanced
maximal
isoproterenol(3-adrenergic
receptor)
stimulated cAMP accumulation
by 100%, whereas
cytochalasin
B
increased
isoproterenol-stimulated
cAMP by 30%. The combirespectively,
which are known
1 -hr pretreatment
of S49 cells
nation
of
hanced
agonist-stimulated
cAMP
A synergistic
increase
served
in cells treated
in cAMP
cholera
otide
colchicine
toxin
(which
regulatory
not ablate
(Ge) protein).
Cytoskeletal
and
For
been
example,
ity ofhormones
variety
of cell
ulation
as
have
multiple
effect
system.
on
lymphoma
components
ofcytoskeletal
with
components
Kennedy,
Received
1
Affiliate
by
model
grants
and
adenylyl
October
from
abil-
cyclase
of this
cytoskeletal
in a
regdrugs
an
20,
the
investigating
the
drugs
549
that
cells,
at the G5 protein
imply
that
regulation
may
and/or
alter
and
of this pathway
contribute
to the action
cytoskeletal
elements.
microtubule
assembly
of
whether
affect
with
Association,
of Health
(P.!. and
California
D.B.).
that
colchicine
and
these
cytoskeletal
gest
microtubules
the
in the
in cellular
alter
and
results
interact
function
cAMP
of these
vinblastine
(Insel
that
ponents
of microfilaments
and
regulation
of
components
microfila.ments
of cAMP
disassembly
facilitates
in
of either
cytoskeletal
interact
than
component
to
The
data
and
microfila-
of postreceptor
formation
of
determine
might
microtubules
interaction
cAMP
to
accumulation.
of both
a greater
involved
is
sugcom-
observed
by
alone.
Methods
lines
cell
and
culture.
Wild-type
cells and 549 clonal variants
were
in the pathway
ofreceptor/G,/adenylyl
tein kinase.
All cells were
grown
heat-inactivated
Heart
3H-
These
can
increases
of drugs
1978)
Kennedy,
Cell
and
disruption
generation.
microfilaments
and that
549
them
(Insel
f3-receptor-stimu-
of isoproterenol-promoted
(a measure
of Gadenylyl
via cAMP
microtubules
interac-
system
on
cydlase coupling).
Thus, colchicine
and cytochalasin
B appear to
enhance
signaling
in the Gjadenylyl
cyclase
pathway
by alterations
of components
distal
to hormone
receptors,
most likely
disassembly
1994.
American
Institutes
for
transduction
cultured
treatment
enhances
expression
binding
in intact
cells
ments
including
signal
that
treatment,
forskolin
inhibitors
function,
effects,
cyclase
in wild-type
(D.B.) and the National
ABBREVIATIONS:
stimulatory
Thus,
1990).
the
of adenylyl
adrenergic
indicated
structures
for publication
Supported
have
a useful
of the
1978).
to influence
toxin
with cytochalasin
B (Insel
and Koachman,
1982) enhances
agonist-stimulated
cAMP
accumulation.
In our study
we assessed
the effects
membrane
et at.,
pertussis
of
lated cAMP accumulation,
indicating
that these cytoskeletal
disrupting
agents do not enhance
responsiveness
in S49 cells
via alterations
in the inhibitory
guanine
nucleotide
regulatory
protein pathway.
Moreover,
colchicine,
but not cytochalasin
B
and
response
to sympathomimetic
may
be mediated
by colchicine’s
of the
studies
are
plasma
pharmacologic
micro-
of surface
Rasenick
influence
B did
and
the mechanism
1983). Antiinflammatory
(Zor,
which
ob-
E1 or
nude-
or the stimula-
regulation
the
1985;
activation
of the
clinical
enhancement
tions
of
although
additional
cells
also
cytochalasin
microtubules
appears
to regulate
Previous
and
in the
cytoskeleton
colchicine,
enhancement
agents.
This
as
et at.,
systems,
defined
is poorly
such
such
Marsh
the
values.
was
of somatostatin
components
1987;
en-
over control
accumulation
implicated
other
et at.,
B synergistically
to 225%
Colchicine
effects
structures,
have
proteins
cytochalasin
with the agonist
prostaglandin
activates
the stimulatory
guanine
the inhibitory
filaments,
(Eide
and
effect
tory
(Mahan
growth
horse
and
conditions
Insel,
serum
1986).
(0.3-1.2
in
(24.3.2)
S49 lymphoma
used,
the variants
having
lesions
cyclase/cAMP-dependent
proat 37#{176}C
in DME containing
10%
a 10% C02;
90% air environment
Cells
were
maintained
in
x 106 cells/ml)
and
viability
logarithmic
was
>95%.
a-receptor,
f32-adrenergic
receptor;
CT, cholera toxin; DMEM, Dulbecco’s
modified
Eagle’s medium;
G,, a-subunit
of the
binding regulatory
protein; Gja, a-subunit
of the inhibitory
guanine-nucleotide
binding regulatory
protein; HEPES,
-piperazine-ethanesulfonic
acid; [1251]-ICYP, [1251]-iodocyanopindolol;
IBMX, isobutylmethylxanthine;
PGE1, prostaglandin
E1.
guanine-nucleotide
4-(2-hydroxyethyl)-1
937
938
Cell
number
Jasper
et al.
and
viability
Vol. 274
were
determined
using
a Coulter
ZBI
cell
counter/Channelizer.
Stimulation
accumulation
in whole
cells. Wild-type
were pretreated
with either
10 p.M colchicine,
10
B [concentrations
of these
agents
found
prey-
ofcAMP
549 lymphoma
cells
p.g/ml cytochalasin
ously
to
be
Kennedy,
optimal
1978;
cytochalasin
for
Insel
enhanced
and
B together
cAMP
Koachman,
for
1 hr
responsiveness
1982)1
at 37#{176}C.In
(Insel
or both
some
and
colchicine
and
experiments,
cells
were treated
with 10 p.M vinblastine,
another
inhibitor
of microtubules. Cells were then stimulated
with 1 M (-)-isoproterenol
or 10
.tM PGE1 for 15 mm (unless
otherwise
noted)
in the presence
of a
phosphodiesterase
inhibitor
(100 iM Ro 20-1724
and/or
100 .tM
isobutylmethyl.xanthine).
Superoxide
dismutase
and
catalase
(10
j.g/ml each) were added
to prevent
oxidation
of drugs
(Mahan
and
Insel,
1984). The reaction
was terminated
by centrifugation
for 20
sec,
aspiration
buffer”
of reaction
xanthine,
described
(Darfier
media,
addition
of 150
ml
of ice
cold
mM
previously
sodium
(Brunton
and
Mayer,
of approximately
in a dry
further
as
1979).
5 x i0
ice-ethanol
disrupted
centrifuged
at
bath,
in
cells/mi.
The
to
fracture
thawed
a Dounce
cells
homogenizer.
x g for 4 sec to remove
12000
were
the
then
cells
frozen
and
then
Disrupted
cells
were
nuclei
and unbroken
cells.
fi-Adrenergic
receptor
binding
assay. After treatment
of S49
cells with colchicine
or cytochalasin
B, intact
cells were centrifuged
at 900 x g for 5 mm at room temperature
and resuspended
in DMEM
containing
20 mM
HEPES
(pH 7.4) and 1 mg/mi
of bovine
serum
albumin.
Replicate
tubes (three
or four) containing
0.6 to 0.9 x 106
cells
were
incubated
with
the
radioligand
[‘251]-ICYP
in the
presence
(nonspecific
binding)
or absence
(total
binding)
of 1 M
(-)-propranolol
in a final volume
ofSOO l for 60 mm at 37#{176}C.
For saturation
binding,
the [1251]-ICYP
concentration
ranged
from 5 to 150 pM.
Binding
reactions
were terminated
by dilution
in 10 ml ice cold wash
buffer (5 mM potassium
phosphate
and 1 mM MgSO4,
pH 7.4) and
then rapidly
filtered
and washed
with an additional
10 ml wash
buffer
over Whatman
GF/C filters
on a Brandel
cell harvester.
Filters
were
counted
number
ofbinding
ICYP
saturation
Koachman,
1982).
Adenylyl
cyclase
in
a
gamma
sites per
isotherms
counter
at
cell and Kd were
as described
assay.
Determination
-80%
efficiency.
The
determined
previously
from
(Insel
of adenylyl
cyclase
[125J}
and
ac-
in plasma
membrane
fractions
used the method
of Salomon
et
al. (1974).
Briefly,
membranes
(20 mg protein/20
ml membrane
buffer)
were stimulated
for 15 mm with various
agents
at 30#{176}C
in a
buffer
containing
50 mM HEPES,
pH 8.0, 5 mM MgC12,
5 mM
tivity
f3-mercaptoethanol,
20-1724
(to inhibit
of creatine
HC1, 10 mM MgCl2,
Tris
100
mg/ml
bovine
phosphodiesterases),
phophokinase,
serum
10 mM
1 mM cAMP,
albumin,
100
M
buffer.
Cytochalasin
B and D and colchicine
were purchased
from either
Aldrich
or Sigma
Chemical
Co. (St. Louis, MO); prostaglandin
E1, isoproterenol,
isobutylmethyixanthine
and vinblastine
from Sigma;
[3H]-CAMP
from Amersham
Corp. (Arlington
Heights,
Reagents.
IL); and cholera
bell,
and pertussis
toxins
from List Biochemicals
(Camp-
CA);
Ho 20-2724
was a gift from Hoffmann-La
Roche (Nutley,
NJ).
All other
chemicals
were purchased
from standard
sources.
Data analysis
and presentation.
Data shown are either representative
or average
of at least three
separate
experiments
(unless
otherwise
noted)
and include
± 1 S.E.M.
Results
Isoproterenol-stimulated
cAMP
accumulation
in intact 849 cells.
Cytochalasin
B (10 .tg/ml) or cytochalasin
D
(10 .tWml) pretreatment
of whole 549 cells enhanced
isoproterenol-mediated
cAMP
accumulation
by 22 ± 8% and 32 ±
8%, respectively
(fig.
1). Colchicine
(10 .tM) pretreatment
resulted
in a 2-fold increase
in cAMP
levels (98 ± 26%). The
combination
of either
cytochalasin
B or D and colchicine
synergistically
increased
isoproterenol-stimulated
cAMP
accumulation
more
than
3-fold (218 ± 28% and 239 ± 33%,
respectively)
over control
values.
Inhibition
of microtubule
assembly
by 10 M vinblastine
instead
of colchicine
yielded
similar
results
(data not shown).
Although
disruption
of the
cytoskeleton
increased
the amount
of cAMP
produced,
it did
not alter
the length
of time
required
to elicit
a maximal
response
(fig.
2, top).
Cells stimulated
with PGE1,
which
activates
adenylyl
cyclase
via
a different
G-protein-coupled
receptor,
demonstrated
a similar
increase
in responsiveness
with either
colchicine
or cytochalasin
B alone,
and a similar
synergistic
enhancement
of cAMP
stimulation
(fig. 2, bottom).
This in-
X0
..
L
4-,
0
.1
-a)
L
E0
4-,
OC
CO
w4
Ro
..---
creatine
phosphate,
400 pM ATP, 1 to 2
10 U/ml
x 106 cpm of [a-32P}ATP.
Incubations
were
performed
in the presence of 100 M GTP,
100 M Gpp(NH)p,
10 mM NaF, or 10 p.M
isoproterenol
+ 100 p.M GTP.
[3H]-Forskolin
binding
in whole S49 cells. [3H]-forskolin
binding was determined
as described
by Alousi
et al. (Alousi et al. , 1991).
549 cells were treated
with 10 p.M colchicine,
10 p.g/ml cytochalasin
B, colchicine
and cytoshalasin
B or ethanol vehicle for 1 hr at 37#{176}C
in
growth
medium.
The cells were sedimented
by centrifugation
and
resuspended
in DMEM
containing
20 mM
HEPES,
pH 7.4, and
stimulated
for 10 mm with 10 p.M isoproterenol
in the absence
(total
binding)
or presence
(nonspecific
binding)
of 10 p.M unlabeled
fors-
at 22#{176}C.
The cells were
rapidly
with 5 ml ice-cold wash buffer (50
pH 7.4) and washed
with an additional
20 ml cold
acetate
Preparation
of cell membranes.
Isolated cellular membranes
from 549 lymphoma
cells were prepared
as described
previously
(Insel and Kennedy,
1978). 549 cells were pretreated
with drugs
or
untreated
and washed
with Dulbecco’s
phosphate-buffered
saline.
The cells were resuspended
in 40 mM HEPES,
pH 7.4, 2 mM MgC12,
1 mM -mercaptoethanol,
10 mg/ml
bovine
serum
albumin
to a
density
mM
“stop
containing
100 iM isobutylmethylpH 4.0) and boiling for 5 mm. cAMP
was
assayed
by the method
of Gilman
(1970) with minor modifications
et al. , 1982). Extrusion
of cAMP
was measured
as described
(50
kolin
and 40 nM [3H]-forskolin
filtered
over Whatman
GF/C filters
,
(;
-
.
#{149}0
f’
\“)
c;
\?
(
c#{176} c,#{176}
‘P
#{231}O0
O#{149}”
Fig. 1. Effect of colchicine,
cytochalasin
B and cytochalasin
D on
isoproterenol-stimulated
cAMP accumulation
in S49 cells. Cells were
pretreated
for 1 hr with either 1 0 p.M colchicine,
10 p.g/ml cytochalasin
B or 1 0 p.g/ml
cytochalasin
D to disrupt the cytoskeleton
and the cells
were then stimulated
for 1 5 mm with the 13-adrenergic
receptor agonist
isoproterenol
and intracellular
cAMP levels measured. Shown is the
average of four independent experiments
(± SEM); isoproterenol-stimulated
cAMP accumulation
control was 260 pmol cAMP/107
cells.
Treatment
with colchicine, cytochalasin
B or D, colchicine or combinations thereof all yielded significantly
(P < .01) elevated levels of cAMP.
Cytoskeletal
1995
939
and cAMP
Disruption
#{149}
CB+COL
1IIIIII
750
C
so
500
00
250
‘C
0
log
EC50=-7.24
I
log
EC50-7.23
0
15
30
Ti..
60
wfth Iso.ncl
45
75
105
90
-8
120
(mn)
-7
-6
-5
-4
(N)
Log [Isoproterenol]
Fig. 3. Effect of colchicine
and cytochalasin
B on the isoproterenolstimulated
cAMP dose-response
relationship.
Cells were pretreated
with 1 0 p.M colchicine and 10 p.g/ml cytochalasin
B (#{149})
or vehicle (0) for
1 hr and stimulated
with varying concentrations
of isoproterenol
for 15
mm. Shown is the representative
of two experiments.
TABLE 1
Effect of cytoskeletal
S49 cell membranes
inhibitors
on adenylyl
cyclase
of
activity
Membrane Adenylyl Cyclase Activity
(pmol cAMP/15 mm/mg Protein)
30
Fig.
2. Effect
of colchicine
45
60
75
Ti..
with
PGE
and
cytochalasin
90
105
(n
120
5)
=
Pretreated
(.)
cells (n
Control
B on the kinetics of
hormone-stimulated
cAMP accumulation in wild-type S49 cells. Cells
were pretreated with either 10 p.M colchicine (A), 1 0 p.g/ml cytochalasin
B (#{149}),
cytochalasin
B + colchicine
(S) or ethanol
vehicle (0) for 1 hr and
stimulated
with
1 p.M isoproterenol
(top) or 1 0 p.M PGE1 (bottom) for
varying times, and cAMP accumulation
was measured.
Shown is the
representative
of three independent
experiments.
dicates
that these
cytoskeletal
inhibitor-mediated
increases
in cAMP
accumulation
are not receptor-specific.
3-adrenergic
receptor
density
and
affinity
and
receptor
activation.
Specific
binding
of the antagonist
radioligand
[‘2511-ICYP
to whole
cells demonstrated
that
treatment with coichicine
and cytochalasin
B did not alter either
receptor
number
(control:
2755 ± 172 sites/cell;
treated:
2569
± 239 sites/cell,
P = NS) or affinity
of the
fl-receptor
for
l25ICyp
(control
KD: 22 ± 1 pM; treated:
21 ± 1 pM, n = 2,
P = NS). Moreover,
the increased
responsiveness
to isoproterenol
in cells treated
with colchicine
and cytochalasin
B
occurred
without
a change
in the EC50 for isoproterenolstimulated
cAMP accumulation
(fig. 3).
Adenylyl
cyclase
activity
of S49 cell membranes.
The
data presented
thus far involved
intact
cells. To eliminate
the
possibility
that cytoskeletal
inhibitors
act directly
on plasma
membrane
components
to enhance
cAMP
accumulation,
we
GTP
ISO + GTP
PGE1 + GTP
Gpp(NH)p
Right
Col
116±20
4)
=
+
Treated membranes
CB
Control
123±37
186 ± 48
166 ± 25
193
110±13
269
229
±
47
217
±
±
42
166
±
49
31
236
±
47
181
±
44
Col
CB
106±7
206 ± 49
175 ± 18
196 ± 92
109
±
+
columns:
cells were pretreated
for 1 h with either 10 p.M colchicine
or 10
p.g/ml cytochalasin
B after which crude membrane
fractions
were prepared.
Adenylyl cyclase activity was measured in response
to various agents [1 00 pM
GTP, 100 p.M Gpp(NH)p, or 10 p.M isoproterenol
+
100 pM GIP]. Left columns:
Membranes were prepared from S49 cells and colchicine
and cytochalasin
B
were added to the membrane
preparations.
Adenylyl
cyclase
activity
was measured in response to above stimulatory
agents.
required
for expression
colchicine
and
metabolism.
cAMP
or
ofthe
cytochalasin
deplete
cAMP
accumulation
in
enhanced
response
produced
Inhibition
of processes
that
extrude
could
explain
the enhancement
S49 cells
treated
with
cytoskeletal
To examine
of decay of intracellular
cAMP
levels
in cells stimulated
the (3-agonist
isoproterenol,
followed
by the f3-antago( )-propranolol.
The decay rates
of cAMP
levels
over
we measured
the
a
-
15-mm
cAMP
mechanism,
cAMP
inhibi-
rate
with
nist
treated
potential
in
tors.
mm)
this
by
B.
period
and
were
colchicine
cells
was
(fig.
not
+
identical
cytochalasin
4). In addition,
different
in
cells
in
B-
(tv2
we found
incubated
control
5.4
(t#{189}=
5.3 mm, n
that extrusion
=
with
=
2)
of
or without
which
cytoskeletal
components
are
mechanically
disrupted.
When
colchicine
and cytochalasin
B
were added
to intact
549 cells and then a crude
membrane
fraction
prepared,
agonist-dependent
and independent
adenylyl
cyclase
activity
was not enhanced
(table
1, left col-
B (“0.7%/mm,
data not shown).
These
results
are strong
evidence
that processes
that extrude
and degrade
cAMP
are
not responsible
for the enhancement
in hormone-stimulated
cAMP
accumulation
observed
with
colchicine
and cytochalasin
B.
Cholera
toxin-stimulated
cAMP
accumulation
in
wild-type
and
mutant
549
cells.
The
bacterial
exotoxin
from
Vibrio
cholera
activates
the G8a subunit
independent
of
umns).
hormone
assayed
arations,
added
again
(table
adenylyl
a
cyclase
system
Similarly,
activity
in isolated
membrane
prep-
in
when
after membranes
failed to enhance
1, right columns).
colchicine
and
cythchalasin
B were
were prepared
from S49 cells, they
stimulated
adenylyl
cyclase
activity
Thus,
the milieu
of the intact
cell is
and
virtually
colchicine
receptors
(Cassel
and
postreceptor
found
cytochalasin
by
inhibiting
Selinger,
1977).
We
site of action
of the
a synergistic
enhancement
the
GTPase
thus
used
cytoskeletal
activity
of G8
CT to test
inhibitors.
for a
We
of CT-stimulated
cAMP
940
Jasper
Vol. 274
et al.
activation
OfGsa.
Treatment
with colchicine
and cytochalasin
B did not restore
responsiveness
of UNC
cells to isoproterenol (data not shown).
However,
UNC
cells did show enhanced
CT-mediated
cAMP
accumulation
after
addition
of either
colchicine
or cytochalasin
B and a synergistic
response
to
C1
.2.,
‘.o
.
2o
simultaneous
0
5
10
15
20
Time with Isoproterenol
Fig. 4. Rate of decay of intracellular
blockade
of isoproterenol-stimulated
treated with 10 p.M colchicine
and
25
(mm)
30
cAMP levels after propranololadenylyl cyclase. Cells were pre10 p.g/ml cytochalasin
B (#{149})
or
and stimulated with isoproterenol.
After 15 mm, 10
were added to block isoproterenol-stimulation
of
a-receptors
and the decay of intracellular
cAMP measured. Shown is
the representative
of two experiments;
the mean half-time values for
the decay of cAMP in the two experiments
were:
control
=
5.4 ± 0.45
mm., colchicine
+ cytochalasin
B = 5.25 ± 1 .7 mm.
vehicle
(0)
for
1 hr
p.M (-)-propranolol
accumulation
in S49 cells by colchicine
and cytochalasin
B
(fig. 5a). Moreover,
the cytoskeletal
inhibitors
did not shorten
the lag time required
for CT to increase
cellular
cAMP
levels.
The mutant
549 cell line UNC
has a point
mutation
in G8
at g372
that uncouples
G8 from hormone
receptors
but does
not interfere
with normal
coupling
of activated
G8a to adenylyl cyclase
as
(Sullivan
et at.
accumulation
cAMP
treatment
with
both
of these
agents
(fig.
5b),
to that
found
in wild-type
S49 cells. These
results
further
demonstrate
that the synergistic
effects
of colchicine
and cytochalasin
B on cAMP
accumulation
are at sites
distal
to hormone
receptors.
Both the kin
S49 cell mutant,
which
lacks cAMP-dependent protein
kinase,
and the cyc
S49 cell mutant,
which
lacks G5a protein,
were also pretreated
with colchicine
and
cytochalasin
B. In kin
cells,
isoproterenol-stimulated
cAMP
accumulation
was enhanced
similar
to the enhancement
demonstrated
in wild-type
549 cells (fig. 6). Importantly,
colchicine
and cytochalasin
B pretreatment
of the cyc
549
cell mutant
did not alter forskolin-stimulated
cAMP accumulation
(data
not shown).
Taken
together,
studies
in mutant
549 cells indicate
that the effects
of colchicine
and cytochalasin
B are at sites
distal
to receptor,
do not require
cAMPdependent
protein
kinase,
but do require
a functional
G8,,
protein.
G1-mediated
inhibition
ofcAMP
accumulation
in S49
similar
CC
isoproterenol
,
1987).
in response
or
PGE1,
These
cells
to hormonal
but
do
respond
do not
increase
agonists,
such
to CT-mediated
Cu,
NO
J
00.
U
0.
<C
U
cells.
Somatostatin
cells,
acting
inhibits
adenylyl
cyclase
activity
in 549
via the inhibitory
guanine
nucleotide
binding
regulatory
protein,
G1 (Jakobs
et al. , 1983).
Inasmuch
as our
data
implicate
a postreceptor
site in the observed
synergistic
effect of colchicine
and cytochalasin
B, we next determined
whether
these
agents
could block
activation
of G1, thereby
enhancing
f3-receptor-stimulated
cAMP
accumulation.
Coincubation
of wild-type
549 cells with isoproterenol
and somatostatin
attenuates
isoproterenol
stimulation
of cAMP;
pretreatment
ofcells
with colchicine
and cytochalasin
B failed to
block this attenuation
(fig. 7). Furthermore,
pertussis
toxin
enhancement
of isoproterenol-stimulate
cAMP accumulation
(Jasper
et at. , 1990) was not affected
by pretreatment
with
colchicine
or cytochalasin
B (data not shown).
Thus,
cytochalasin
B and colchicine
do not enhance
agonist-stimulated
cAMP
accumulation
by blocking
G1-mediated
inhibition
of
adenylyl
cyclase.
Isoproterenol-stimulated
549
cells.
[3H]-forskolin
Binding
of [3H]-forskolin
binding
to intact
549
in
cells
is a
Cu)
. N
C
0
00
so
U
0
I-
lie
<.5
‘CE
U
‘C
0
30
60
Time
90
with
120
cholera
150
toxin
180
(mm)
Fig. 5. Effect of colchicine
and cytochalasin
B on cholera
toxin-stimulated cAMP accumulation
in 549 cells. Wild-type (top) or UNC (bottom)
549 cells were pretreated with either 10 p.M colchicine
(A), 1 0 p.g/ml
cytochalasin
B (#{149}),
cytochalasin
B + colchicine
(#{149})
or ethanol vehicle
(0) for 1 hr and stimulated
with 1 00 ng/ml cholera
toxin and intracellular
cAMP measured at varying times. Shown is the representative
of two
independent
experiments.
Time
Fig.
6.
Effect
hormone-stimulated
of
colchicine
cAMP
45
with
and
60
75
Isoproterenol
cytochalasin
accumulation
90
105
120
(mm)
B
on
the
kinetics
in 549 cells lacking
of
cAMP-
protein
kinase
activity
(kin).
Cells were treated with colchicine and cytochalasin
B and stimulated
with isoproterenol
for varying
times. Shown
is the representative
of two independent
experiments.
dependent
1995
Cytoskeletal
mone
1250
receptors,
the
or G1, or the
oa
At the
1000
receptor
altered
5,.’.
E0
ity
750
‘-4
for
PGE1
I
500
0E
a-receptor
and
response
variant
0.
0
c__
and
were
had
B
In
both
a synergistic
of colchicine
ated inhibition of cAMP
and
cytochalasin
B on
accumulation
somatostatin-medi-
in 549
cells. Cells were pretreated with 10 p.M colchicine
and 10 p.g/ml cytochalasin
B (right bars)
or dimethylsulfoxide
vehicle (left bars) for 1 hr and stimulated
with 1 p.M
isoproterenol
for 15 mm in the presence
(cross-hatched
bars) or ab-
sence (open bars) of 1 p.M somatostatin.
three
independent
Shown is the representative
of
experiments.
as CT
on GTPase
the
inhibitors
distal
proximal
not
measure
of
the
interaction
of
activated
(Alousi
et at. , 1991; Barber,
[3HJ-forskolin
binding
as a means
toskeletal
inhibitors
might
enhance
ment
interaction
of 549
cells
nol-stimulated
cells
kolin
10 p.M
,
assessed
effect
by
and
B decreased
binding
on
compared
G8-adenylyl
by
to
cyclase
B have
a
interactions,
as
binding.
study,
regulation
S49 lymphoma
of plasma
cyclase
system
microfilaments.
were
used
components
to examine
of the
of cytoskeletal
the
adenylyl
microtubules
time
of
G,,/adenylyl
colchicine
and
cytochalasin
B fail
for
inhibitor
activity,
cytoskeletal
cytochalasin
indicating
components
D
exhibit
similar
these
effects
are
that
and
not
nonspecific.
In
gard, the inactive
isomer
of colchicine,
p-lumicolchicine,
not enhance
agonist-mediated
cAMP
accumulation
and Kennedy,
1978; Leiber
et at., 1993).
When
cells
cytochalasin
are
either
may
be
simultaneously
B, hormone
accumulation
with
treated
and
alone.
regulating
adenylyl
cyclase
whether
this
filament
inhibitors
increased
This
suggests
different,
but
pathway.
synergistic
We
action
is related
the
interactive,
have
site
cyclase
activity
by
appear
to
to colchicine
with
our
and
results:
ef-
in the
and
and
have
phosphodiesterase
of transducin.
life-
disruption
G5. A brief
(1992)
Bownds
a
does
of G8 and perhaps
We propose
three
interaction,
molecule
7), nor
assessed
enhancement
and
Simto be
binding,
compatible
the
GTPase
(fig.
of S49 cells
of receptors
follows.
effector
appear
interaction,
of regulation
inhibitors?
be
do
of cAMP.
not
inhibitor
Arshavsky
the
does
must
6) and
of
discus-
demonacts
Similarly,
to
Ross
and colleagues
have
found
that
the effector
phospholipase
C
acts
as a GTPase
activating
(GAP)
protein
for its cognate
G-protein,
Gq (Berstein
et at. , 1992).
We speculate
that one
C2
U)
Ct-
‘no
L. ‘.,.
rLiO
:EC
1’)
‘..
re-
does
COL
CB
COL
CB
and
cAMP
that
two
found
inhibitors
steps
attempted
of microtubules
to their
this
colchicine
above
that
cAMP
specific
(Insel
nonhormone-stimulated
is synergistically
agent
with
metabolism
hydrolysis/exchange,
cellular
“compartments”
sion of each
of these
that
be
(fig.
or
to enhance
adenylyl
cyclase
activity;
thus,
their
effect
on adenylyl
cyclase requires
the presence
of an intact
cytoskeletal
architecture.
The microtubule
inhibitor
vinblastine
and the microfilament
enhancing
would
effects
of
at
and
The addition
of colchicine
or cytochalasin
B
to intact
549 cells produces
a dose-dependent
enhancement
in both
hormone
(isoproterenol,
PGE1)
and nonhormone(CT) stimulated
cAMP
accumulation.
However,
in broken
cell
preparations,
that
GTP
effect
of action
is
kinase
G.,
cyclase
be the sites
by cytoskeletal
agonists.
site
these
protein
response
agents
of cAMP
inhibitory
one
[3H1-forskolin
might
cyclase
these
microfilaments
cytoskeletal
synergistic
B.
in
in the UNC
ofG8 to the
receptor
its
that
and
G8-adenylyl
on
enhance
cells
membrane
by inhibitors
the
Recently,
Discussion
In our
of
fects
strated
suggest
However,
isoproterenol-stimulated
possibilities
Leiber
,
of either
facilitation
What
adenylyl
[3H]-forsof cells with
(c.f.
of action
explain
via
G protein,
colchi-
accumulation
cells,
with
data
by
potenti-
stimulation
seen
in cellular
inhibitory
cytochalasin
control
our
alterations
the
UNC
G8 directly
of microtubules
involve
site
isoprotere-
instead
was stimulated
colchicine
and cytochalasin
[3HJ-forskolin
adenylyl
accumulation
cyclase.
Pretreat-
In contrast,
isoproterenol-stimulated
was not inhibited
by pretreatment
colchicine,
but
1993). Therefore,
differential
cAMP
adenylyl
cytochalasin
[3H1-forskolin
(fig. 8).
binding
et at.
of G8a
with
and
1988). We thus measured
to test whether
the cy-
cyclase
increasing
G8a
that
the
G8 protein.
to cAMP-dependent
to
ilarly,
to
activates
activity,
their
Moreover,
on CT-mediated
similar
Inasmuch
enhanced
that
cAMP
and
affin-
Furthermore,
in both
wild-type
and
is defective
in coupling
wild-type
effect
accumulation
Fig. 7. Effect
toxin
(which
B
their
was
specific.
enhanced
G8
cytochalasin
nor
[‘25I1-ICYP.
a-receptor
B
to cholera
of S49 cells
nor
B, indicating
not
proteins
receptors,
accumulation
cytochalasin
941
cyclase.
colchicine
antagonist
cytochalasin
f3-receptor).
cytochalasin
neither
of cAMP
and
effects
cine
250
<0
the
binding
of adenylyl
of -adrenergic
stimulation
ation
0.0
I-
number
nucleotide
component
level,
the
colchicine
guanine
catalytic
and cAMP
Disruption
to
and
of action
in
the
discern
micro-
on hor-
Fig. 8. Effect of cytochalasin
B on isoproterenol-stimulated
rHJ-forskolin binding in 549 cells. 549 cells were pretreated
for 1 hr with 1 0 p.M
colchicine,
1 0 p.g/ml cytochalasin
B, colchicine
plus cytochalasin
B or
vehicle.
lsoproterenol(10 p.M) stimulated
rH]-forskolin
binding
was
determined
after a 10-mm incubation
at 22#{176}C
in the absence (total
binding) or presence
(nonspecific
binding) of 10 p.M forskolin.
Data are
presented
as isoproterenol-stimulated
rH]-forskolmn
binding over isoproterenol
+ unlabeled
forskolin-stimulated
rH]-forskolin
binding averaged
from five separate
experiments
performed
in quadruplicate.
942
et al.
Jasper
site
of action
of cytoskeletal
is via
system
Vol. 274
functioning
the
as GAP-like
exchange.
Thus,
filaments
intrinsic
not
activity
of GTP/GDP
of the
or microfilaments
decrease
the GTPase
stimulation
necessarily
G8a
activity
of adenylyl
be reflected
subunit,
by colchicine
or
ofGsa
and
cyclase,
in enhanced
for
a period
mechanism
of
to block
time
after
activation;
stimulation
ofthe
a
active
G-protein
a-subunits
(Bourne
and Stayer,
1992). If cytoskeletal proteins
were to play a role in holding
inactive
a8 and
adenylyl
cyclase
together,
then disruption
ofthe
cytoskeleton
would
decrease
the time
that
the inactive
a8 is bound
to
adenylyl
cyclase
and thereby
increase
the activation
state of
adenylyl
does
cyclase.
not
cyclase
Because,
enhance
to the
this
rather
possibility
concept
colchicine,
[3H]-forskolin
interaction),
microtubules
A third
unlike
binding
mechanism
cytochalasin
(a
might
of G8-
measure
be
operative
for
action
of components
in the
and adenylyl
cyclase
“sharing”
of proteins
would
increase
the
cell, but it would also
mains
in the
precision
from
another
system
(e.g. , PGE1).
between
different
signaling
dototal
amount
of cAMP
synthesized
of responsiveness
Although
no
data
destroy
the
to external
are
available
normal
the
three
mately
possibilities
that we have proposed,
each should
ultibe amenable
to experimental
testing.
We have
at-
tempted
study
preliminary
studies
G5 and G in 549 cells
itors.
However,
such
studies.
high
levels
However,
using
treated
immunofluorescence
with
cytoskeletal
of autofluorescence
the
current
have
findings
lead
to
inhibimpeded
us
to hy-
pothesize
that
microtubules
and microfilaments
act at distinct but interactive
sites along the pathway
of cAMP
generation.
It is possible
that colchicine
and cytochalasin
B act to
alter
this pathway
via
lin and actin,
respectively,
the
present
microtubules
previous
components
other
than
the most
likely
explanation
data
are
is
direct regulatory
of the adenylyl
cyclase
have
components
effects
and
cellular
but
perturbed
by
colchicine
tubufor
that
microfflaments
and
effects
on the membrane
system,
and that these
and
F.,
DARFLER,
cytochalasin
B.
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and
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in the cell in spatially
distinct
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and Brunton,
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these
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to interact
with
one another:
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of one type
(e.g. , -adrenergic
receptors)
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and
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appears
to localize
to microtubules
(Lefkowitz
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with our data relates
to the suggestion
that GDP-liganded
Ge-subunits
remain
bound
to their effectors
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Send
reprint
requests
to: Dr. Daniel Bernstein,
Stanford
University,
750 Welch Road, Suite 305,
Department
Palo
Alto,
of Pediatrics,
CA 94304.
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