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Evidence against dopamine as the mediator of the rise of cyclic AMP in the superior cervical ganglion of the rat
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCE AGAINST DOPAMINE AS THE MEDIATOR OF THE RISE OF CYCLIC AMP IN THE SUPERIOR CERVICAL GANGLION OF THE RAT
Toni Lindl
8 and Hinrich Cramer ~
FachbereichBiologie, University of Konstanz, D-775Konstanz,POB 7733
§
Neurologische Universit~tsklinikmitAbteilungftirNeurophysiologie D'78 Freiburg i.Br., Hansastr.
9
W.-Germany Received May 28,1975 SUMMARY. In isolated intact superior cervical ganglia of the rat dopamine is able to increase the levels of adenosine 3', 5'-monophosphate (cAMP) only if used in concentrations about two orders of magnitude higher than norepinephrine or epinephrine. The methylxanthines papaverine and theophylline have no or liminal effects on cAMP levels in control ganglia or ganglia incubated with dopamine. However, papaverine potentiates the norepinephrine-induced accumulation of cAMP both in ganglia and incubation media. Repeated incubations with norepinephrine in the presence of papaverine show a rapid decrement of the response and a sustained fall of cAMP in ganglia and media. The catecholamine induced rise was blocked by B-, and partially inhibited by ~-adrenergic antagonists. The role of adenosine 3', 5'-monophosphate
(cAMP)
in
the sympathetic peripheral nervous system has been the subject of numerous investigations in the past four years (I-8). It has been shown that electrical stimulation,
ca-
techolamines and histamine induce an accumulation of cAMP in the ganglia. Moreover,
evidence from experiments with
slices of bovine superior cervical ganglia and isolated intact rabbit ganglia has led to the suggestion that cAMP is involved in dopamine-mediated inhibition in the ganglion
(3,6). However, direct evidence that dopamine
alone causes an elevation of cAMP in the intact ganglion is as yet lacking;
in addition it is unclear whether the
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
731
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
catecholamine-induced catecholaminergic
rise
in cAMP
inhibition
is c a u s a l l y
of t r a n s m i s s i o n
related
to
in the intact
ganglion. We p r e s e n t
evidence
that d o p a m i n e
10 -5 to 10 -3 M does not alter ganglia
of the rat,
a potent
increase medium
epinephrine agent,
was
both
another
abolished
causes
in g a n g l i a
potent
(9), is present.
and p a r t i a l l y
in
inhibitor.
in the c A M P - c o n t e n t
inhibitor
of
of t h e o p h y l l i n e ,
we show that n o r e p i n e p h r i n e
w h e n papaverine,
esterase
the c A M P - c o n c e n t r a t i o n
even in the p r e s e n c e
phosphodiesterase
Moreover,
in c o n c e n t r a t i o n s
and in the
cAMP-phosphodi-
The effect
by a B - a d r e n e r g i c
blocked
a large
of nor-
blocking
by an e - a d r e n e r g i c
blocking
agent.
M A T E R I A L S A N D METHODS. M a l e S p r a g u e - D a w l e y rats were k i l l e d by cervical d i s l o c a t i o n and the g a n g l i a w e r e r a p i d l y removed, desheathed, and kept on ice until used. I n c u b a t i o n p r o c e d u r e s w e r e e s s e n t i a l l y the same as d e s c r i b e d p r e v i o u s ly (5, 10). The cAMP c o n t e n t of g a n g l i a was d e t e r m i n e d acc o r d i n g to G i l m a n (11) after p u r i f i c a t i o n of e x t r a c t s on Dowex lX-2 columns (4). The i n c u b a t i o n m e d i a w e r e lyophilized, taken up in 50 m M acetate b u f f e r pH 4.0 and assayed for cAMP by the same method.
RESULTS
AND DISCUSSION.
10-3 M did not affect 10 -2 M c o n c e n t r a t i o n lation
Concentrations cAMP
did there
of the n u c l e o t i d e
(10-4 M)
and e p i n e p h r i n e
were m a x i m a l
(figure
Theophylline cant effect
levels
in ganglia,
appear
(10 -5 M)
up to
only at
a moderate
at a time w h e n
accumu-
norepinephrine
induced
increases
I).
(0.5 mM),
which
on the cAMP content,
alone was w i t h o u t was unable
the r e s p o n s e
to dopamine.
tion
by high c o n c e n t r a t i o n s
induced
of d o p a m i n e
The time course
732
signifi-
to p o t e n t i a t e
of the a c c u m u l a -
of d o p a m i n e
(10 -2 M)
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T
.~±~ / /
,-i
S
z
~3
/ / / / / / /
iI
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~4 A
d IO-~M I04M CONTROL
Fi@ure
10-2}
DOPAMIN£
1:
E f f e c t of d o p a m i n e on levels of cyclic A M P in superior cervical g a n g l i a after five m i n u t e s of i n c u b a t i o n w i t h the amine in the m e d i u m at the c o n c e n t r a t i o n s indicated. H a t c h e d bars rep r e s e n t g a n g l i a i n c u b a t e d for 20 m i n in the p r e s e n c e of theop h y l l i n e (0.5 mM) p r i o r to the a d d i t i o n of d o p a m i n e (n = 4).
differed
notably
norepinephrine cAMP
f r o m that d e s c r i b e d
(4), and showed
concentrations
probably
represents
dopamine
rather
sensitive
has
little
(physical,
than
specific
(figure
required,
and indirect
activation
however,
of
2). the effect
actions
of
of a d o p a m i n e -
that e x o g e n o u s
on the c A M P - c o n t e n t
metabolic,
ing the receptors.
increase
and
cyclase.
be ruled out,
effect
30 m i n
concentration
non-specific
adenylate
It c a n n o t
a sustained
for at least
In v i e w of the h i g h
for e p i n e p h r i n e
uptake)
prevent
because dopamine
But e l e c t r o p h y s i o l o g i c a l
733
dopamine
barriers f r o m reach-
experiments
of
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
3 u4
2
I
1'0
6
2'0
3'0
TIME (MIN)
Figure
2:
Time course of a c c u m u l a t i o n of cAMP in f r e s h l y d i s s e c t e d g a n g l i a on i n c u b a t i o n w i t h d o p a m i n e (10 -2 M). (n = 4) * * s i g n i f i c a n t l y d i f f e r e n t f r o m controls, p ~ O . O 5 s i g n i f i c a n t l y d i f f e r e n t from controls, p-=~O.O2
McAfee cause
and G r e e n g a r d
(12)
hyperpolarization
or cervical penetrate
ganglion
of CAMP
Papaverine ganglia
cells
which
in rat superior
with papaverine lation
were
cyclic AMP
of p o s t g a n g l i o n i c of the rabbit
dopamine
neurons
does
of superi-
and hence
does
into the ganglia.
Norepinephrine, cyclase
show that e x o g e n o u s
is a p o t e n t cervical
(10 -4 M) caused in g a n g l i a
alone
ganglia a more
(figure
did not affect
incubated
with
from the tissue
activator (4),
of a d e n y l a t e in c o m b i n a t i o n
than a d d i t i v e
accumu-
3). the c A M P - c o n t e n t
it for 6 minutes.
when
Release
of
into the m e d i u m was also en-
734
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[ i
40
,
I
, J.
O m
o
30
4.!
g, I L
~
Ik
20
a
lO
CON NE
NE
CON N E
NE + PAP
t-
Fi@ure
PAP
3:
C o n c e n t r a t i o n of cAMP in g a n g l i a after the first and the fourth of a series of s t i m u l a t i o n w i t h n o r e p i n e p h r i n e (NE, 10 -4 M) or n o r e p i n e p h r i n e + p a p a v e r i n e (PAP, 10 -4 M). G a n g l i a w e r e i n c u b a t e d for 15 m i n in the p r e s e n c e of the drugs between i n c u b a t i o n p e r i o d s of 20 m i n in d r u g - f r e e m e d i u m (n = 6). ~p
hanced when ganglia epinephrine verine
incubated
or in the p r e s e n c e
(figure
On r e p e a t e d drug-free
were
incubations
fell
after
near
the d e t e c t i o n
second
with alternating
medium,
the cyclic
incubation
limit
fall was only d e t e c t a b l e the c o m b i n a t i o n
of n o r e p i n e p h r i n e
of nor-
and papa-
4).
incubation the
in the p r e s e n c e
after
period
nucleotide
were
norepinephrine/papaverine.
735
with content
and r e a c h e d
the fourth
when ganglia
washes
values
incubation. incubated
The with
No s i g n i f i c a n t
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
W
Z
o z
3.0
~9
÷i
~
20
n
1.o
1
2
3
4
Fi@ure 4: Release of cyclic AMP into the incubation m e d i u m after sequential s t i m u l a t i o n of ganglia w i t h n o r e p i n e p h r i n e J 'J or n o r e p i n e p h r i n e + p a p a v e r i n e 177"]. S t i m u l a t i o n period: 15 min w i t h an a d d i t i o n a l w a s h i n g p e r i o d of 20 min. C o n c e n t r a t i o n of norepinephrine: 10 -4 M; papaverine: 10 -4 M (n = 6 + S.D.). ~ = control. N u m b e r s under the bars r e p r e s e n t the p e r i o d of stimulation.
changes were o b s e r v e d during control incubations with norepinephrine entire i n c u b a t i o n
period,
the cyclic n u c l e o t i d e
content of
alone was still ele-
, w h i l e the cAMP content of ganglia
norepinephrine/papaverine
and during
alone. A t the end of the
the ganglia exposed to n o r e p i n e p h r i n e vated
incubations
incubated with
was b e l o w the control values
(figure 3). A decrease stimulations
in the cAMP c o n c e n t r a t i o n of adenylate
in b r a i n slices
after sequential
cyclase has also been o b s e r v e d
(13). The a p p a r e n t
736
refractoriness
of the
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
adenylate cyclase to the catecholamine may be due to the exhaustion of a limited pool of ATP, the substrate of adenylate cyclase, within the membrane compartment
(14-16).
A limited supply of ATP can also explain the observation that the rate of conversion of radioactive adenine into cAMP in the ganglion during electrical stimulation falls rapidly with increasing frequency, 2400 pulses were given
or when more than
(17).
Pharmacological evidence from previous studies
(4, 6)
led to the conclusion that two types of catecholamine receptors, alpha and beta, can lead to elevations in cAMP in the peripheral nervous system, and that dopamine activates adenylate cyclase by a mechanism involving an ~adrenoceptor. In the rat superior cervical ganglion however,
alpha
adrenergic agents such as dopamine and phenylephrine in reasonable concentrations were without effect on the concentration of cAMP in the ganglion
(2,4). Preincubation
with 10 -5 M propranolol abolished the norepinephrine-induced rise of cyclic AMP, but 10 -5 M phentolamine inhibited the rise by only 50%
(figure 5).
These observations can be interpreted as indicating that both types of receptors may be present in the ganglia but that B-receptors for adenylate cyclase prevail in this species while dopamine does not activate the enzyme. Moreover,
it was described recently that propranolol has an
ID 50 of 2.2 x 10 -7 M suggesting nonspecific effects of 10 -5 M phentolamine
(10).
The present experiments
in rat superior cervical ganglia
737
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Z
O
30
.J
C~
**
z
4-1
20
0
~
~
10 u
CON
NE
PHE
NF +
PHA
Figure 5:
NE -t-
PROP
Effects of norepinephrine (NE, 10 -4 M) and phenylephrine (PHE, 10 -2 M) on cyclic AMP levels in ganglia and effects of phentolamine (PHA, 10 -5 M) and propramolol (PROP, 10 -5 M) on the accumulation of cyclic AMP in ganglia induced by norepinephrine (10 -4 M) (n = 6). ~p~O.O1; ~ p~O.OO1 significance of difference from controls.
fail to support the concept of a "dopamine-sensitive late cyclase"
(8) which supposedly mediates
hibitory postsynaptic
potential
(sIPSP)
From this and other investigations gested that,
adeny-
the slow in-
in the ganglion. (4,7)
it can be sug-
if cyclic AMP is actually linked with slow in-
hibitory postsynaptic
potential
(sIPSP), norepinephrine
is
more likely to be responsible
for this action because of its
marked potency in stimulating
adenylate cyclase and its known
capacity to influence transmission
through the ganglion.
Acknowledgement: This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70 and SFB 138). REFERENCES I.
McAfee, D.A., Schorderet, M., and Greengard, Science 171, 1156-1158 (1971)
738
P.
Vol. 65, No. 2, 1975
2 3 4 5 6 7 8 9 10. II. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cramer, H., Johnson, D.G., Silberstein, S.D., and Kopin, I.J. Pharmacologist 13, 257 (1971) Kebabian, J.W., and Greengard, P. Science 174, 1346-1349 (1971) Cramer, H., Johnson, D.G., Hanbauer, I., Silberstein, S.D., and Kopin, I.J. Brain Res. 53, 97-104 (1973) Lindl, T., and Cramer, H. Biochim. Biophys. Acta 343, 182-191 (1974) Kalix, P., McAfee, D.A., Schorderet, M., and Greengard, P. J.Pharmacol. and Exp.Therap. 188, 676-687 (1974) Otten, U., Mueller, R.A., Oesch, F., and Thoenen, H. Proc. Nat. Acad. Sci. USA 71, 2217-2221 (1974) Greengard, P., and Kebabian, J.W. Fed. Proc. 33, 1059-1067 (1974) Schultz, J., and Daly, J.W. J.Biol.Chem. 248, 853-859 (1973) Lindl, T., Behrendt, H., Heinl-Sawaya, M.C.B., Teufel,E., and Cramer, H. Naun. Schm.Arch.Pharmacol. 286, 283-296 (1974) Gilman, A.G. Proc. Nat. Acad. Sci. USA 67, 305-312 (1970) McAfee, D.A., and Greengard, P. Science 178, 310-312 (1972) Schultz, J., and Daly, J.W. J.Biol.Chem. 248, 860-866 (1973) Reporter, M. Biochem. Biophys. Res. Comm. 48, 598-604 (1972) Shimizu, H., and Okayama, H. J.Neurochem. 20, 1279-1283 (1973) Lindl, T., Heinl-Sawaya, M.C.B., and Cramer, H. Biochem. Pharmacol. 24, 947-950 (1975) Chatzkel, S., Zimmerman, I., and Berg, A. Brain Res. 80, 523-526 (1974)
739
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
EVIDENCE AGAINST DOPAMINE AS THE MEDIATOR OF THE RISE OF CYCLIC AMP IN THE SUPERIOR CERVICAL GANGLION OF THE RAT
Toni Lindl
8 and Hinrich Cramer ~
FachbereichBiologie, University of Konstanz, D-775Konstanz,POB 7733
§
Neurologische Universit~tsklinikmitAbteilungftirNeurophysiologie D'78 Freiburg i.Br., Hansastr.
9
W.-Germany Received May 28,1975 SUMMARY. In isolated intact superior cervical ganglia of the rat dopamine is able to increase the levels of adenosine 3', 5'-monophosphate (cAMP) only if used in concentrations about two orders of magnitude higher than norepinephrine or epinephrine. The methylxanthines papaverine and theophylline have no or liminal effects on cAMP levels in control ganglia or ganglia incubated with dopamine. However, papaverine potentiates the norepinephrine-induced accumulation of cAMP both in ganglia and incubation media. Repeated incubations with norepinephrine in the presence of papaverine show a rapid decrement of the response and a sustained fall of cAMP in ganglia and media. The catecholamine induced rise was blocked by B-, and partially inhibited by ~-adrenergic antagonists. The role of adenosine 3', 5'-monophosphate
(cAMP)
in
the sympathetic peripheral nervous system has been the subject of numerous investigations in the past four years (I-8). It has been shown that electrical stimulation,
ca-
techolamines and histamine induce an accumulation of cAMP in the ganglia. Moreover,
evidence from experiments with
slices of bovine superior cervical ganglia and isolated intact rabbit ganglia has led to the suggestion that cAMP is involved in dopamine-mediated inhibition in the ganglion
(3,6). However, direct evidence that dopamine
alone causes an elevation of cAMP in the intact ganglion is as yet lacking;
in addition it is unclear whether the
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
731
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
catecholamine-induced catecholaminergic
rise
in cAMP
inhibition
is c a u s a l l y
of t r a n s m i s s i o n
related
to
in the intact
ganglion. We p r e s e n t
evidence
that d o p a m i n e
10 -5 to 10 -3 M does not alter ganglia
of the rat,
a potent
increase medium
epinephrine agent,
was
both
another
abolished
causes
in g a n g l i a
potent
(9), is present.
and p a r t i a l l y
in
inhibitor.
in the c A M P - c o n t e n t
inhibitor
of
of t h e o p h y l l i n e ,
we show that n o r e p i n e p h r i n e
w h e n papaverine,
esterase
the c A M P - c o n c e n t r a t i o n
even in the p r e s e n c e
phosphodiesterase
Moreover,
in c o n c e n t r a t i o n s
and in the
cAMP-phosphodi-
The effect
by a B - a d r e n e r g i c
blocked
a large
of nor-
blocking
by an e - a d r e n e r g i c
blocking
agent.
M A T E R I A L S A N D METHODS. M a l e S p r a g u e - D a w l e y rats were k i l l e d by cervical d i s l o c a t i o n and the g a n g l i a w e r e r a p i d l y removed, desheathed, and kept on ice until used. I n c u b a t i o n p r o c e d u r e s w e r e e s s e n t i a l l y the same as d e s c r i b e d p r e v i o u s ly (5, 10). The cAMP c o n t e n t of g a n g l i a was d e t e r m i n e d acc o r d i n g to G i l m a n (11) after p u r i f i c a t i o n of e x t r a c t s on Dowex lX-2 columns (4). The i n c u b a t i o n m e d i a w e r e lyophilized, taken up in 50 m M acetate b u f f e r pH 4.0 and assayed for cAMP by the same method.
RESULTS
AND DISCUSSION.
10-3 M did not affect 10 -2 M c o n c e n t r a t i o n lation
Concentrations cAMP
did there
of the n u c l e o t i d e
(10-4 M)
and e p i n e p h r i n e
were m a x i m a l
(figure
Theophylline cant effect
levels
in ganglia,
appear
(10 -5 M)
up to
only at
a moderate
at a time w h e n
accumu-
norepinephrine
induced
increases
I).
(0.5 mM),
which
on the cAMP content,
alone was w i t h o u t was unable
the r e s p o n s e
to dopamine.
tion
by high c o n c e n t r a t i o n s
induced
of d o p a m i n e
The time course
732
signifi-
to p o t e n t i a t e
of the a c c u m u l a -
of d o p a m i n e
(10 -2 M)
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T
.~±~ / /
,-i
S
z
~3
/ / / / / / /
iI
,.,4 ÷J
//-
,-4 /4 /4 ..4
~4 A
d IO-~M I04M CONTROL
Fi@ure
10-2}
DOPAMIN£
1:
E f f e c t of d o p a m i n e on levels of cyclic A M P in superior cervical g a n g l i a after five m i n u t e s of i n c u b a t i o n w i t h the amine in the m e d i u m at the c o n c e n t r a t i o n s indicated. H a t c h e d bars rep r e s e n t g a n g l i a i n c u b a t e d for 20 m i n in the p r e s e n c e of theop h y l l i n e (0.5 mM) p r i o r to the a d d i t i o n of d o p a m i n e (n = 4).
differed
notably
norepinephrine cAMP
f r o m that d e s c r i b e d
(4), and showed
concentrations
probably
represents
dopamine
rather
sensitive
has
little
(physical,
than
specific
(figure
required,
and indirect
activation
however,
of
2). the effect
actions
of
of a d o p a m i n e -
that e x o g e n o u s
on the c A M P - c o n t e n t
metabolic,
ing the receptors.
increase
and
cyclase.
be ruled out,
effect
30 m i n
concentration
non-specific
adenylate
It c a n n o t
a sustained
for at least
In v i e w of the h i g h
for e p i n e p h r i n e
uptake)
prevent
because dopamine
But e l e c t r o p h y s i o l o g i c a l
733
dopamine
barriers f r o m reach-
experiments
of
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
3 u4
2
I
1'0
6
2'0
3'0
TIME (MIN)
Figure
2:
Time course of a c c u m u l a t i o n of cAMP in f r e s h l y d i s s e c t e d g a n g l i a on i n c u b a t i o n w i t h d o p a m i n e (10 -2 M). (n = 4) * * s i g n i f i c a n t l y d i f f e r e n t f r o m controls, p ~ O . O 5 s i g n i f i c a n t l y d i f f e r e n t from controls, p-=~O.O2
McAfee cause
and G r e e n g a r d
(12)
hyperpolarization
or cervical penetrate
ganglion
of CAMP
Papaverine ganglia
cells
which
in rat superior
with papaverine lation
were
cyclic AMP
of p o s t g a n g l i o n i c of the rabbit
dopamine
neurons
does
of superi-
and hence
does
into the ganglia.
Norepinephrine, cyclase
show that e x o g e n o u s
is a p o t e n t cervical
(10 -4 M) caused in g a n g l i a
alone
ganglia a more
(figure
did not affect
incubated
with
from the tissue
activator (4),
of a d e n y l a t e in c o m b i n a t i o n
than a d d i t i v e
accumu-
3). the c A M P - c o n t e n t
it for 6 minutes.
when
Release
of
into the m e d i u m was also en-
734
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[ i
40
,
I
, J.
O m
o
30
4.!
g, I L
~
Ik
20
a
lO
CON NE
NE
CON N E
NE + PAP
t-
Fi@ure
PAP
3:
C o n c e n t r a t i o n of cAMP in g a n g l i a after the first and the fourth of a series of s t i m u l a t i o n w i t h n o r e p i n e p h r i n e (NE, 10 -4 M) or n o r e p i n e p h r i n e + p a p a v e r i n e (PAP, 10 -4 M). G a n g l i a w e r e i n c u b a t e d for 15 m i n in the p r e s e n c e of the drugs between i n c u b a t i o n p e r i o d s of 20 m i n in d r u g - f r e e m e d i u m (n = 6). ~p
hanced when ganglia epinephrine verine
incubated
or in the p r e s e n c e
(figure
On r e p e a t e d drug-free
were
incubations
fell
after
near
the d e t e c t i o n
second
with alternating
medium,
the cyclic
incubation
limit
fall was only d e t e c t a b l e the c o m b i n a t i o n
of n o r e p i n e p h r i n e
of nor-
and papa-
4).
incubation the
in the p r e s e n c e
after
period
nucleotide
were
norepinephrine/papaverine.
735
with content
and r e a c h e d
the fourth
when ganglia
washes
values
incubation. incubated
The with
No s i g n i f i c a n t
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
W
Z
o z
3.0
~9
÷i
~
20
n
1.o
1
2
3
4
Fi@ure 4: Release of cyclic AMP into the incubation m e d i u m after sequential s t i m u l a t i o n of ganglia w i t h n o r e p i n e p h r i n e J 'J or n o r e p i n e p h r i n e + p a p a v e r i n e 177"]. S t i m u l a t i o n period: 15 min w i t h an a d d i t i o n a l w a s h i n g p e r i o d of 20 min. C o n c e n t r a t i o n of norepinephrine: 10 -4 M; papaverine: 10 -4 M (n = 6 + S.D.). ~ = control. N u m b e r s under the bars r e p r e s e n t the p e r i o d of stimulation.
changes were o b s e r v e d during control incubations with norepinephrine entire i n c u b a t i o n
period,
the cyclic n u c l e o t i d e
content of
alone was still ele-
, w h i l e the cAMP content of ganglia
norepinephrine/papaverine
and during
alone. A t the end of the
the ganglia exposed to n o r e p i n e p h r i n e vated
incubations
incubated with
was b e l o w the control values
(figure 3). A decrease stimulations
in the cAMP c o n c e n t r a t i o n of adenylate
in b r a i n slices
after sequential
cyclase has also been o b s e r v e d
(13). The a p p a r e n t
736
refractoriness
of the
Vol. 65, No. 2, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
adenylate cyclase to the catecholamine may be due to the exhaustion of a limited pool of ATP, the substrate of adenylate cyclase, within the membrane compartment
(14-16).
A limited supply of ATP can also explain the observation that the rate of conversion of radioactive adenine into cAMP in the ganglion during electrical stimulation falls rapidly with increasing frequency, 2400 pulses were given
or when more than
(17).
Pharmacological evidence from previous studies
(4, 6)
led to the conclusion that two types of catecholamine receptors, alpha and beta, can lead to elevations in cAMP in the peripheral nervous system, and that dopamine activates adenylate cyclase by a mechanism involving an ~adrenoceptor. In the rat superior cervical ganglion however,
alpha
adrenergic agents such as dopamine and phenylephrine in reasonable concentrations were without effect on the concentration of cAMP in the ganglion
(2,4). Preincubation
with 10 -5 M propranolol abolished the norepinephrine-induced rise of cyclic AMP, but 10 -5 M phentolamine inhibited the rise by only 50%
(figure 5).
These observations can be interpreted as indicating that both types of receptors may be present in the ganglia but that B-receptors for adenylate cyclase prevail in this species while dopamine does not activate the enzyme. Moreover,
it was described recently that propranolol has an
ID 50 of 2.2 x 10 -7 M suggesting nonspecific effects of 10 -5 M phentolamine
(10).
The present experiments
in rat superior cervical ganglia
737
Vol. 65, No. 2, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Z
O
30
.J
C~
**
z
4-1
20
0
~
~
10 u
CON
NE
PHE
NF +
PHA
Figure 5:
NE -t-
PROP
Effects of norepinephrine (NE, 10 -4 M) and phenylephrine (PHE, 10 -2 M) on cyclic AMP levels in ganglia and effects of phentolamine (PHA, 10 -5 M) and propramolol (PROP, 10 -5 M) on the accumulation of cyclic AMP in ganglia induced by norepinephrine (10 -4 M) (n = 6). ~p~O.O1; ~ p~O.OO1 significance of difference from controls.
fail to support the concept of a "dopamine-sensitive late cyclase"
(8) which supposedly mediates
hibitory postsynaptic
potential
(sIPSP)
From this and other investigations gested that,
adeny-
the slow in-
in the ganglion. (4,7)
it can be sug-
if cyclic AMP is actually linked with slow in-
hibitory postsynaptic
potential
(sIPSP), norepinephrine
is
more likely to be responsible
for this action because of its
marked potency in stimulating
adenylate cyclase and its known
capacity to influence transmission
through the ganglion.
Acknowledgement: This work was supported by the Deutsche Forschungsgemeinschaft (SFB 70 and SFB 138). REFERENCES I.
McAfee, D.A., Schorderet, M., and Greengard, Science 171, 1156-1158 (1971)
738
P.
Vol. 65, No. 2, 1975
2 3 4 5 6 7 8 9 10. II. 12. 13. 14. 15. 16. 17.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cramer, H., Johnson, D.G., Silberstein, S.D., and Kopin, I.J. Pharmacologist 13, 257 (1971) Kebabian, J.W., and Greengard, P. Science 174, 1346-1349 (1971) Cramer, H., Johnson, D.G., Hanbauer, I., Silberstein, S.D., and Kopin, I.J. Brain Res. 53, 97-104 (1973) Lindl, T., and Cramer, H. Biochim. Biophys. Acta 343, 182-191 (1974) Kalix, P., McAfee, D.A., Schorderet, M., and Greengard, P. J.Pharmacol. and Exp.Therap. 188, 676-687 (1974) Otten, U., Mueller, R.A., Oesch, F., and Thoenen, H. Proc. Nat. Acad. Sci. USA 71, 2217-2221 (1974) Greengard, P., and Kebabian, J.W. Fed. Proc. 33, 1059-1067 (1974) Schultz, J., and Daly, J.W. J.Biol.Chem. 248, 853-859 (1973) Lindl, T., Behrendt, H., Heinl-Sawaya, M.C.B., Teufel,E., and Cramer, H. Naun. Schm.Arch.Pharmacol. 286, 283-296 (1974) Gilman, A.G. Proc. Nat. Acad. Sci. USA 67, 305-312 (1970) McAfee, D.A., and Greengard, P. Science 178, 310-312 (1972) Schultz, J., and Daly, J.W. J.Biol.Chem. 248, 860-866 (1973) Reporter, M. Biochem. Biophys. Res. Comm. 48, 598-604 (1972) Shimizu, H., and Okayama, H. J.Neurochem. 20, 1279-1283 (1973) Lindl, T., Heinl-Sawaya, M.C.B., and Cramer, H. Biochem. Pharmacol. 24, 947-950 (1975) Chatzkel, S., Zimmerman, I., and Berg, A. Brain Res. 80, 523-526 (1974)
739