Japan. J. Plrarmacol. 26, 201-207 (1976)
FURTHER ON
STUDIES
THE
OF
ELECTRICAL OF
201
THE
ACTION
AND
INTESTINAL
CYCLIC
MECHANICAL SMOOTH
Hiromichi
OF
AMP
ACTIVITIES
MUSCLE
OHKAWA
Department of Physiology, Yamaguchi University School of Medicine, Ube 755, Japan Accepted December 16, 1975
Abstract-Effects of externally applied cyclic AMP and other adrenergic stimulants on the electrical and mechanical activities of the cat small intestine were observed by using pressure electrodes. The electrical and mechanical activities were suppressed by cyclic AMP and beta-stimulants. Those inhibitory actions of cyclic AMP and beta-stimulants were potentiated under the treatment with caffeine, theophylline and papaverine which inhibits the phosphodiesterase activity. On the other hand, the inhibitory action of cyclic AMP and beta-stimulants was decreased in imidazole, an agent that increases phosphodiesterase activity. Exogenous applied concanavalin A, an agent that inhibits the adenyl cyclase activity, showed no observable changes in both activities but the effects of beta-stimulants were decreased after treatment with con canavalin A. No obvious changes on both activities were obtained in cyclic GMP and dibutyryl cyclic GMP. These findings tentatively support the hypothesis that cyclic AMP is a second messenger in the inhibitory responses to beta-stimulants on the intestinal smooth muscle. However, it is also concluded that the inhibition of me chanical activity caused by cyclic AMP is partially due to suppression of the membrane activity. In relation
to the relaxation
have emphasized
two subjects,
beta-adrenergic
stimulants
monophosphate
(cyclic AMP).
AMP and dibutyryl
muscle.
and the increase In previous inhibited
smooth muscle.
of intracellular papers
muscle under
the spontaneous
on the enzyme
MATERIALS was removed
For the purpose
of recording
electrical
that cyclic
and mechanical
ac
activity
in the smooth
that the phosphodiesterase (3), papaverine
activity
(6, 7) and imidazole
A (8). of intestinal
activity.
AND METHODS
from
adult
electrical
to 3.5 cm long, was slipped onto a rectangular solution
by
These actions of cyclic AMP and related compounds
is affected by concanavalin
factors
(1, 2, 3)
level of cyclic 3',5'-adenosine
with the action of cyclic AMP on both activities
affecting
The small intestine
authors
(4, 5), it has been reported
applied caffeine (3), theophylline
This report is concerned
in the normal
several
of adenyl cyclase in cell membrane
it has been acknowledged
(3) and that adenyl cyclase activity
isor_ole.
muscles,
relate with adenyl cyclase and phosphodiesterase
On the other hand,
is affected by externally
smooth
of smooth
that is, the activation
cyclic AMP
tivities of the intestinal may thus possibly
mechanism
cats which
and mechanical Lucite holder
at 36-37`-'C in a 200 nil Lucite chamber.
had been anesthetized activities,
a segment,
with 3 cm
and the holder was mounted One end of the segment was
202
H. OHKA WA
fixed on the holder and the other which was made by cuttings along the longitudinal direction of segment, 2 cm in length and 3 mm in width, was connected to the force-transducer. tension development was measured longitudinally.
The
Electrical recordings were made by
using pressure electrodes (3M KCI-agar, 0.5-1 mm capillaries) which were handled with a micromanipulator.
Time constant of the electrical recording system was 2.0 sec. In many
experiments, both activities were recorded simultaneously.
The solution was gassed with
95 % 02 and 5 % CO,. The composition of modified Krebs solution was the same as described in the previous paper (5). The following drugs were used: cyclic 3',5'-adenosine monophos phate (cyclic AMP), adrenaline hydrochloride, noradrenaline hydrochloride, isoprenaline hydrochloride, caffeine sodium benzoate, theophylline, papaverine hydrochloride, imidazole hydrochloride, cyclic 3',5'-guanosine monophosphate
(cyclic GMP), dibutyryl cyclic 3',5'
guanosine monophosphate (dibutyryl cyclic GMP), insulin and concanavalin A (Boehringer Mannheim GmbH). RESULTS Effects of catecholamines and cyclic AMP When a pressure electrode was attached to the serosal surface of the segment, slow waves and spikes were recorded.
Usually, the spontaneous discharges appeared as repetitive spike
discharges superimposed on a slow wave depolarization.
General pattern of the slow waves
and spikes was similar to that described in previous papers (5, 9). Adrenaline (10-9-10-6 g/ml) inhibited the spontaneous activities of the segment (Fig. 1). At a concentration
of 10-9 g/ml, spike generation was partially suppressed.
Higher
concentrations of adrenaline (10-a-10-7 g/ml) strongly blocked the spike generation but the slow waves were continued.
Spike activity was completely abolished at a concentration of
10-6 g/ml. Mechanical activity was also inhibited by raising the concentrations of adrenaline. At a concentration
of 10-6 g/ml, phasic contraction could not be recorded.
Isoprenaline
(10-6g/ml) and noradrenaline (10-6 g/ml) also exhibited a strong inhibition on the spike and
Fin. 1. Effect of adrenaline on the electrical (upper) and mechanical (lower) activities of the cat small intestine. A: Control. B: Adrenaline 10 ~'g/ml. C: Adrenaline 10-1 g!ml. D: Adrenaline 10-7 g/ml and E: Adrenaline 10" g/ml. Lines in A-E in the mechanical activity indicate a standard level in tension. The meaning of lines in the following figures is the same. Calibration; I mV, I g and 30 sec.
CYCLIC
AMP
AND
INTESTINAL
SMOOTH
MUSCLE
203
Fin. 2. Effect of cyclic AMP with and without caffeine on the electrical (upper) and mechanical (lower) activities. A: Control to B. B: Cyclic AMP 10-' g,1ml. C: Control to D and E. D: Caffeine 1C-3 g, nil and E: Cyclic AMP 10 g/ml after caffeine. The results of B and E were recorded after about a 15 min application of cyclic AMP. The following results on the effects of cyclic AMP and adrenaline were recorded in the same manner. Calibration; I mV, I g and 30 sec. mechanical
activities.
After treatment frequency
with cyclic AMP (10-s g/ml), the spike generation
of slow wave was not altered.
and the tone level was also decreased activities
of the intestinal
smooth
The magnitude
was reduced
of phasic contraction
slightly as shown in Fig. 2B. These changes
muscle were similar to those in a previous
Effects of cyclic AMP and adrenaline
under treatment
but the
was decreased in both
paper (5).
with phosphodiesterase
inhibitors
and
activator Caffeine,
theophylline
Cyclic AMP (10 various
and
were
used
however,
inhibitors.
Caffeine
(I0
slow waves were observed
g/ml) caused
continuously.
activity by cyclic AMP which was added during
the treatment
Namely
the frequency
of spike generation
was decreased
without
the treatment
with caffeine
2, C-E).
(Fig.
(10-1 g/ml), spike activity was decreased slightly. ment with theophylline caused stronger inhibition of spikes
were abolished
theophylline
treatment
Papaverine phasic contraction papaverine. normal
solution
was decreased
inhibitors.
was used.
Stronger
abolished
treatment
was also decreased.
than that in normal
that by cyclic AMP with theophylline
application
and was similar activities
This inhibition
solution
of phasic contraction.
continued
completely
action
of spike
Cyclic AMP (10 g/ml) during the treat on slow wave and spike activities. Most
activity
activity
inhibition
with
of spike
with caffeine was observed.
more than
During
Spike and mechanical
The inhibitory
weak inhibition
and the tone level was also reduced
the spike
tit ont ly ss ith :u~ additional
in Fig. 3F, spikes sere to be sporadic.
and mechanical
was more considerable
(10-5 g/ml) caused the inhibition
However,
were suppressed
as phosphodiesterase
g/ml) was added to the organ bath after 15 min during the treatment
phosphodiesterase
generation,
papaverine
in
(Fig. 3, A-C).
The magnitude after treatment
of with
to that seen when
in the papaverinc
of cyclic A M P (10 5g/ml).
and small phasic contractions
treatment As shown
wcrc observed
of cyclic AMP in those phosphodiesterase
inhibitors
204
H.
O HKA WA
Fu.. 3. Effect of cyclic AMP combined with theophylline and papaverine on the electrical (upper) and mechanical (lower) activities. A: Control to B and C. B: Theophylline 10_1 g/ml. C: Cyclic AMP 10-6 g~ml after theophylline. D: Control to E and F. E: Papaverine 10 g;ml and F: Cyclic AMP l0-' g'ml after pa paverine. Calibration: I mV, 1 g and 30 sec. was most effective in papaverine. The effect of adrenaline inhibitors. inhibitory action
After treatment action
(10-s-10
activity
after treatment
with theophylline
than that seen in normal
of adrenaline
chanical
was examined
(10-4 g/ml), adrenaline solution.
(10-7 g/ml) on both activities
was completely
with various
blocked.
As shown
exhibited
a stronger
in Fig. 4C, the inhibitory
was considerable,
However,
phosphodiesterase
in particular
the inhibitory
the me
effect of adrenaline
g//1111)in caffeine (10-4 g/ml) was much the same as that seen in normal
solution
(Fig. 4, D-E). The activatory (3). The action amined.
action
on the phosphodiesterase
of cyclic AMP and adrenaline
When imidazole
Fu,. 4.
of imidazole
(10
Eficet of adrenaline
after treatment
activity
with imidazole
is well known was also ex
g/ml) was added to the organ bath, electrical and mechanical
combined
witli theophylline
and caffeine
on the electrical
(upper) and mechanical (lower) activities. A: Theophylline 10 '' glml. B: Adrenaline 10-', g%ml after theophylline. C: Adrenaline 10 g9nil after theophylline D: Caffeine 10-.ti g;'ml. E: Adrenaline 10 1 glml after caffeine and F: Adrenaline 10
g,/nil after caffeine.
Calibration;
I mV, I g and 30 sec.
CYCLIC
AMP
AND
INTESTINAL
SMOOTH
MUSCLE
205
FIG. 5. Effect of cyclic AMP combined with imidazole on the electrical (upper) and mechanical (lower) activities. A: Control to B and C. B: Imidazole 10-5 g/ml. C: Cyclic AMP 10-5 g/ml after imidazole. D: Control to E and F. E: Imidazole 10-5 g/ml and F: Adrenaline 10-7 g/ml after imidazole. Calibration; I mV, I g and 30 sec. activities
were not
altered
as shown
in Fig. 5B and
cyclic AMP (10-s g/ml) or adrenaline g/tnl) was added. activities
E.
After
15 min of imidazole
treatment,
(10-7
Spike and mechanical
were
not
inhibited
by cyclic
AMP (Fig. 5, C) and inhibitory adrenaline
on
mechanical
activity
spike
action
generation was
not
of and
observed
(Fig. 5F). Effects
of catecholainines
under treatment
with adenyl cyclase inhibitor In the dynamic activation brane
theory,
by beta-adrenoceptive
was emphasized been
receptor
reported
hibited various
the
of adenyl cyclase in cell mem
(1, 2, 3). that
periment,
in the
present
A on the electrical
activities
preparations centrations
activity
In the
was
also A in
the effect of externally
concanavalin chanical
(8).
It has
concanavalin
the adenyl cyclase tissues
stimulants
ex
applied and me
examined.
The
were exposed to various con of concanavalin
A (10-7-10-4
g/ml) for 15 min or more. The spike and mechanical activities were, however, not changed.
Neither
of the activities
was
Fiu. 6. Effect of noradrenaline combined with concanavalin A on the electrical (upper) and mechanical (lower) activities. A: Control. 13: Concanavalin A 2.5 10-1 glml. C: Noradrenaline I0 7 g/ml after concanavalin A and D: Noradrenaline 10 5 g/ml after concanavalin A. Cali bration: I mV, I g and 30 sec.
206
H. OHKA WA
altered
by the externally
applied
insulin
(4>,'l0---,-4',,' 10-3 U/nil).
(10-7-10_6 g/ml) was added to the organ bath after treatment g/ml),
the action
generation
of noradrenaline
of the spikes
disappeared
on both activities
was still observed
at the same concentration
When noradrenaline
with concanavalin
was decreased
with noradrenaline
of noradrenaline
A (2.5
(Fig. 6).
10 '6 -/ml
in normal
10-s
That
is, the
while the spikes
solution.
Effects of cyclic GMP awl clibut_r'rvl c.r•clic G.-1IP Cyclic GM P (7:
10.6-7.
10-5 g;'ml) and dibutyryl
no striking action on the electrical activity caused
by cyclic GMP at a concentration
was no considerable (10-8-10
and mechanical
change in the mechanical
g//1111)in dibutyryl
activities.
A slight inhibition
of the spike
of 7
g ml vvas recorded
'pile
activity.
cyclic GMP
cyclic G M P (10 E; 10-1 g 1111)had
10
The inhibitory
(10-5 g,'ml) vas similar
there
action of noradrenaline to that seen in normal
solution. DISCUSSION Cyclic AMP the intestinal increases
and beta-stimulants
smooth
muscle.
inhibited
According
the adenyl cyclase
activity
the electrical
to the dynamic
and mechanical
receptor
in cell membrane
hypothesis,
and consequently
level of cyclic AMP increases.
This increase of cyclic AMP content
relaxation
muscle.
of intestinal
smooth
by cyclic AMP which indicates smooth
muscle in a manner
AMP may be partially
As shown in results,
due to such inhibition
spike activity
activity.
to the
was suppressed
activity
Relaxation
of the membrane
of
the intracellular
relates positively
that cyclic AMP affects the membrane
similar to that of beta-stimulants.
activities beta-stimulant
of intestinal
caused
by cyclic
In previous
papers
(4, 5), the effect of dibutyryl cyclic AMP on the membrane activity was reported. The effects of cyclic AMP and its dibutyryl derivative on the membrane activity of intestinal smooth muscle were found
to be similar.
The phosphodiesterase theophylline
involved
on the phosphodiesterase data
suggest
treatment
that
activity
the actions
action
is considered
Under
treatment
herein.
Results
However,
In fact, results
to have an activatory
as mentioned
the interaction
AMP or beta-stimulants hibitors and activators,
stimulants inhibited
after treatment
(I, 2, 3).
above appear
between
with caffeine,
to support
in themselves,
modified
of intestinal
activity
receptor
inhibitors,
activators
he excluded
smooth
(3).
was decreased
the dynamic
cannot
under showed
and papaverine.
and adrenaline
hypothesis. and cyclic
because
these in
caused
by beta
the electrical activity.
role in the relaxation
It had been reported A, however,
activity
activity
theophylline
by
(6). These
are potentiated
on the phosphodiesterase
these phosphodiesterase
on the membrane
inhibited
of papaverine
in the present experiment
the effect of cyclic AMP
plays an important
by concanavalin
and cyclic AMP obtained
action
action
than that of theophylline
of cyclic AMP on the mechanical
with Imidazole,
Adenyl cyclasc
greater
of beta-stimulants
muscle was indeed potentiated
is reportedly
(6, 7) and the inhibitory
is apparently
with those inhibitors.
that the inhibitory
Imidazole
in cyclic AMP breakdown
(3), caffeine (3) and papaverine
that in various
the inhibitory
mechanism
tissues this enzyme activity
action
on adenyl cyclase activity
vas by
CYCLIC A:AIP AND INTESTINAL SMOOTH
MUSCLE
207
this agent was limited to within a certain range (8). In the present work, concentrations of 10-7-10-1 g/ml were used. Electrical and mechanical activities were not affected at various concentrations of concanavalin A. of noradrenaline
However, after concanavalin A, the inhibitory action
was decreased suggesting that the inhibition of adenyl cyclase activity
may be caused by externally applied concanavalin A. It had been reported that the intracellular level of cyclic AMP is reciprocally related to the intracellular content of cyclic GMP (10). Though the excitatory action of dibutyryl cyclic GMP on the smooth muscle strip of guinea-pig ileum and rat stomach has been reported (Il,
12), cyclic GMP and dibutyryl cyclic GMP showed no obvious effects on
either activity in the present experiment.
The relationship of cyclic GMP to cholinergic
transmission (12) and Ca ` supply to the contractile element during dibutyryl cyclic GMP treatment (11) was suggested for the mechanism of action of cyclic GMP. The discrepancy between the obtained results and the former data on the effect of cyclic GMP was not eluci dated and further study concerning the action of cyclic GMP on the activity of smooth muscle is now underway in our laboratory. REFERENCES 1) BELLEAU, B.: Pharmacol. Rcr., 21, 131 (1966) 2) ROBINSON, G.A., BUTCHER, R.W. ANDSUTHERLAND, E.W.: Ann. N. Y. Acad. Sci., 139, 703 (1967) 3) BUTCHER, R.W. ANDSUTHERLAND, E.W.: J. biol. Chem., 237, 1244 (1962) 4) TAKAGI, K. et al.: Japan. J. Pharinacol., 21, 477 (1971) 5) OHKAWA, H.: Bull. YamaguchiAled. Sch., 22, 185 (1975) 6) TRINER,L., VULLIEMOZ, Y., SCH\VATL, 1.ANDNAHAS,G.G.: Iioche,n. biophvs.Res. Common., 40, 64 (1970) 7) PocH, G. ANDKUKOVETZ, W.R.: Life Sci., 10, 133 (1971) 8) CUATRECASAS, P. ANDTELL,G.P.E.: Proc. natn. Acad. Sci U.S.A., 70, 485 (1973) 9) OHKAWA, H. ANDWATANABE, M.: TohokuJ. exp. Med., 117, 69 (1975) 10) ILLIANO, G., TELL,G.P.E., SIEGEL,M.I. ANDCUATRECASAS, P.: Proc. natn. Acad. Sci. U.S.A., 70, 2443 (1973) 11) TAKAYANAGI, 1. ANDTAKAGI,K.: Japan. J. Pharmacol., 23, 573 (1973) 12) PUGLISI, L., BERTI,F. ANDPAOLETTI, R.: Experientia,27, 1187 (1971)
FURTHER ON
STUDIES
THE
OF
ELECTRICAL OF
201
THE
ACTION
AND
INTESTINAL
CYCLIC
MECHANICAL SMOOTH
Hiromichi
OF
AMP
ACTIVITIES
MUSCLE
OHKAWA
Department of Physiology, Yamaguchi University School of Medicine, Ube 755, Japan Accepted December 16, 1975
Abstract-Effects of externally applied cyclic AMP and other adrenergic stimulants on the electrical and mechanical activities of the cat small intestine were observed by using pressure electrodes. The electrical and mechanical activities were suppressed by cyclic AMP and beta-stimulants. Those inhibitory actions of cyclic AMP and beta-stimulants were potentiated under the treatment with caffeine, theophylline and papaverine which inhibits the phosphodiesterase activity. On the other hand, the inhibitory action of cyclic AMP and beta-stimulants was decreased in imidazole, an agent that increases phosphodiesterase activity. Exogenous applied concanavalin A, an agent that inhibits the adenyl cyclase activity, showed no observable changes in both activities but the effects of beta-stimulants were decreased after treatment with con canavalin A. No obvious changes on both activities were obtained in cyclic GMP and dibutyryl cyclic GMP. These findings tentatively support the hypothesis that cyclic AMP is a second messenger in the inhibitory responses to beta-stimulants on the intestinal smooth muscle. However, it is also concluded that the inhibition of me chanical activity caused by cyclic AMP is partially due to suppression of the membrane activity. In relation
to the relaxation
have emphasized
two subjects,
beta-adrenergic
stimulants
monophosphate
(cyclic AMP).
AMP and dibutyryl
muscle.
and the increase In previous inhibited
smooth muscle.
of intracellular papers
muscle under
the spontaneous
on the enzyme
MATERIALS was removed
For the purpose
of recording
electrical
that cyclic
and mechanical
ac
activity
in the smooth
that the phosphodiesterase (3), papaverine
activity
(6, 7) and imidazole
A (8). of intestinal
activity.
AND METHODS
from
adult
electrical
to 3.5 cm long, was slipped onto a rectangular solution
by
These actions of cyclic AMP and related compounds
is affected by concanavalin
factors
(1, 2, 3)
level of cyclic 3',5'-adenosine
with the action of cyclic AMP on both activities
affecting
The small intestine
authors
(4, 5), it has been reported
applied caffeine (3), theophylline
This report is concerned
in the normal
several
of adenyl cyclase in cell membrane
it has been acknowledged
(3) and that adenyl cyclase activity
isor_ole.
muscles,
relate with adenyl cyclase and phosphodiesterase
On the other hand,
is affected by externally
smooth
of smooth
that is, the activation
cyclic AMP
tivities of the intestinal may thus possibly
mechanism
cats which
and mechanical Lucite holder
at 36-37`-'C in a 200 nil Lucite chamber.
had been anesthetized activities,
a segment,
with 3 cm
and the holder was mounted One end of the segment was
202
H. OHKA WA
fixed on the holder and the other which was made by cuttings along the longitudinal direction of segment, 2 cm in length and 3 mm in width, was connected to the force-transducer. tension development was measured longitudinally.
The
Electrical recordings were made by
using pressure electrodes (3M KCI-agar, 0.5-1 mm capillaries) which were handled with a micromanipulator.
Time constant of the electrical recording system was 2.0 sec. In many
experiments, both activities were recorded simultaneously.
The solution was gassed with
95 % 02 and 5 % CO,. The composition of modified Krebs solution was the same as described in the previous paper (5). The following drugs were used: cyclic 3',5'-adenosine monophos phate (cyclic AMP), adrenaline hydrochloride, noradrenaline hydrochloride, isoprenaline hydrochloride, caffeine sodium benzoate, theophylline, papaverine hydrochloride, imidazole hydrochloride, cyclic 3',5'-guanosine monophosphate
(cyclic GMP), dibutyryl cyclic 3',5'
guanosine monophosphate (dibutyryl cyclic GMP), insulin and concanavalin A (Boehringer Mannheim GmbH). RESULTS Effects of catecholamines and cyclic AMP When a pressure electrode was attached to the serosal surface of the segment, slow waves and spikes were recorded.
Usually, the spontaneous discharges appeared as repetitive spike
discharges superimposed on a slow wave depolarization.
General pattern of the slow waves
and spikes was similar to that described in previous papers (5, 9). Adrenaline (10-9-10-6 g/ml) inhibited the spontaneous activities of the segment (Fig. 1). At a concentration
of 10-9 g/ml, spike generation was partially suppressed.
Higher
concentrations of adrenaline (10-a-10-7 g/ml) strongly blocked the spike generation but the slow waves were continued.
Spike activity was completely abolished at a concentration of
10-6 g/ml. Mechanical activity was also inhibited by raising the concentrations of adrenaline. At a concentration
of 10-6 g/ml, phasic contraction could not be recorded.
Isoprenaline
(10-6g/ml) and noradrenaline (10-6 g/ml) also exhibited a strong inhibition on the spike and
Fin. 1. Effect of adrenaline on the electrical (upper) and mechanical (lower) activities of the cat small intestine. A: Control. B: Adrenaline 10 ~'g/ml. C: Adrenaline 10-1 g!ml. D: Adrenaline 10-7 g/ml and E: Adrenaline 10" g/ml. Lines in A-E in the mechanical activity indicate a standard level in tension. The meaning of lines in the following figures is the same. Calibration; I mV, I g and 30 sec.
CYCLIC
AMP
AND
INTESTINAL
SMOOTH
MUSCLE
203
Fin. 2. Effect of cyclic AMP with and without caffeine on the electrical (upper) and mechanical (lower) activities. A: Control to B. B: Cyclic AMP 10-' g,1ml. C: Control to D and E. D: Caffeine 1C-3 g, nil and E: Cyclic AMP 10 g/ml after caffeine. The results of B and E were recorded after about a 15 min application of cyclic AMP. The following results on the effects of cyclic AMP and adrenaline were recorded in the same manner. Calibration; I mV, I g and 30 sec. mechanical
activities.
After treatment frequency
with cyclic AMP (10-s g/ml), the spike generation
of slow wave was not altered.
and the tone level was also decreased activities
of the intestinal
smooth
The magnitude
was reduced
of phasic contraction
slightly as shown in Fig. 2B. These changes
muscle were similar to those in a previous
Effects of cyclic AMP and adrenaline
under treatment
but the
was decreased in both
paper (5).
with phosphodiesterase
inhibitors
and
activator Caffeine,
theophylline
Cyclic AMP (10 various
and
were
used
however,
inhibitors.
Caffeine
(I0
slow waves were observed
g/ml) caused
continuously.
activity by cyclic AMP which was added during
the treatment
Namely
the frequency
of spike generation
was decreased
without
the treatment
with caffeine
2, C-E).
(Fig.
(10-1 g/ml), spike activity was decreased slightly. ment with theophylline caused stronger inhibition of spikes
were abolished
theophylline
treatment
Papaverine phasic contraction papaverine. normal
solution
was decreased
inhibitors.
was used.
Stronger
abolished
treatment
was also decreased.
than that in normal
that by cyclic AMP with theophylline
application
and was similar activities
This inhibition
solution
of phasic contraction.
continued
completely
action
of spike
Cyclic AMP (10 g/ml) during the treat on slow wave and spike activities. Most
activity
activity
inhibition
with
of spike
with caffeine was observed.
more than
During
Spike and mechanical
The inhibitory
weak inhibition
and the tone level was also reduced
the spike
tit ont ly ss ith :u~ additional
in Fig. 3F, spikes sere to be sporadic.
and mechanical
was more considerable
(10-5 g/ml) caused the inhibition
However,
were suppressed
as phosphodiesterase
g/ml) was added to the organ bath after 15 min during the treatment
phosphodiesterase
generation,
papaverine
in
(Fig. 3, A-C).
The magnitude after treatment
of with
to that seen when
in the papaverinc
of cyclic A M P (10 5g/ml).
and small phasic contractions
treatment As shown
wcrc observed
of cyclic AMP in those phosphodiesterase
inhibitors
204
H.
O HKA WA
Fu.. 3. Effect of cyclic AMP combined with theophylline and papaverine on the electrical (upper) and mechanical (lower) activities. A: Control to B and C. B: Theophylline 10_1 g/ml. C: Cyclic AMP 10-6 g~ml after theophylline. D: Control to E and F. E: Papaverine 10 g;ml and F: Cyclic AMP l0-' g'ml after pa paverine. Calibration: I mV, 1 g and 30 sec. was most effective in papaverine. The effect of adrenaline inhibitors. inhibitory action
After treatment action
(10-s-10
activity
after treatment
with theophylline
than that seen in normal
of adrenaline
chanical
was examined
(10-4 g/ml), adrenaline solution.
(10-7 g/ml) on both activities
was completely
with various
blocked.
As shown
exhibited
a stronger
in Fig. 4C, the inhibitory
was considerable,
However,
phosphodiesterase
in particular
the inhibitory
the me
effect of adrenaline
g//1111)in caffeine (10-4 g/ml) was much the same as that seen in normal
solution
(Fig. 4, D-E). The activatory (3). The action amined.
action
on the phosphodiesterase
of cyclic AMP and adrenaline
When imidazole
Fu,. 4.
of imidazole
(10
Eficet of adrenaline
after treatment
activity
with imidazole
is well known was also ex
g/ml) was added to the organ bath, electrical and mechanical
combined
witli theophylline
and caffeine
on the electrical
(upper) and mechanical (lower) activities. A: Theophylline 10 '' glml. B: Adrenaline 10-', g%ml after theophylline. C: Adrenaline 10 g9nil after theophylline D: Caffeine 10-.ti g;'ml. E: Adrenaline 10 1 glml after caffeine and F: Adrenaline 10
g,/nil after caffeine.
Calibration;
I mV, I g and 30 sec.
CYCLIC
AMP
AND
INTESTINAL
SMOOTH
MUSCLE
205
FIG. 5. Effect of cyclic AMP combined with imidazole on the electrical (upper) and mechanical (lower) activities. A: Control to B and C. B: Imidazole 10-5 g/ml. C: Cyclic AMP 10-5 g/ml after imidazole. D: Control to E and F. E: Imidazole 10-5 g/ml and F: Adrenaline 10-7 g/ml after imidazole. Calibration; I mV, I g and 30 sec. activities
were not
altered
as shown
in Fig. 5B and
cyclic AMP (10-s g/ml) or adrenaline g/tnl) was added. activities
E.
After
15 min of imidazole
treatment,
(10-7
Spike and mechanical
were
not
inhibited
by cyclic
AMP (Fig. 5, C) and inhibitory adrenaline
on
mechanical
activity
spike
action
generation was
not
of and
observed
(Fig. 5F). Effects
of catecholainines
under treatment
with adenyl cyclase inhibitor In the dynamic activation brane
theory,
by beta-adrenoceptive
was emphasized been
receptor
reported
hibited various
the
of adenyl cyclase in cell mem
(1, 2, 3). that
periment,
in the
present
A on the electrical
activities
preparations centrations
activity
In the
was
also A in
the effect of externally
concanavalin chanical
(8).
It has
concanavalin
the adenyl cyclase tissues
stimulants
ex
applied and me
examined.
The
were exposed to various con of concanavalin
A (10-7-10-4
g/ml) for 15 min or more. The spike and mechanical activities were, however, not changed.
Neither
of the activities
was
Fiu. 6. Effect of noradrenaline combined with concanavalin A on the electrical (upper) and mechanical (lower) activities. A: Control. 13: Concanavalin A 2.5 10-1 glml. C: Noradrenaline I0 7 g/ml after concanavalin A and D: Noradrenaline 10 5 g/ml after concanavalin A. Cali bration: I mV, I g and 30 sec.
206
H. OHKA WA
altered
by the externally
applied
insulin
(4>,'l0---,-4',,' 10-3 U/nil).
(10-7-10_6 g/ml) was added to the organ bath after treatment g/ml),
the action
generation
of noradrenaline
of the spikes
disappeared
on both activities
was still observed
at the same concentration
When noradrenaline
with concanavalin
was decreased
with noradrenaline
of noradrenaline
A (2.5
(Fig. 6).
10 '6 -/ml
in normal
10-s
That
is, the
while the spikes
solution.
Effects of cyclic GMP awl clibut_r'rvl c.r•clic G.-1IP Cyclic GM P (7:
10.6-7.
10-5 g;'ml) and dibutyryl
no striking action on the electrical activity caused
by cyclic GMP at a concentration
was no considerable (10-8-10
and mechanical
change in the mechanical
g//1111)in dibutyryl
activities.
A slight inhibition
of the spike
of 7
g ml vvas recorded
'pile
activity.
cyclic GMP
cyclic G M P (10 E; 10-1 g 1111)had
10
The inhibitory
(10-5 g,'ml) vas similar
there
action of noradrenaline to that seen in normal
solution. DISCUSSION Cyclic AMP the intestinal increases
and beta-stimulants
smooth
muscle.
inhibited
According
the adenyl cyclase
activity
the electrical
to the dynamic
and mechanical
receptor
in cell membrane
hypothesis,
and consequently
level of cyclic AMP increases.
This increase of cyclic AMP content
relaxation
muscle.
of intestinal
smooth
by cyclic AMP which indicates smooth
muscle in a manner
AMP may be partially
As shown in results,
due to such inhibition
spike activity
activity.
to the
was suppressed
activity
Relaxation
of the membrane
of
the intracellular
relates positively
that cyclic AMP affects the membrane
similar to that of beta-stimulants.
activities beta-stimulant
of intestinal
caused
by cyclic
In previous
papers
(4, 5), the effect of dibutyryl cyclic AMP on the membrane activity was reported. The effects of cyclic AMP and its dibutyryl derivative on the membrane activity of intestinal smooth muscle were found
to be similar.
The phosphodiesterase theophylline
involved
on the phosphodiesterase data
suggest
treatment
that
activity
the actions
action
is considered
Under
treatment
herein.
Results
However,
In fact, results
to have an activatory
as mentioned
the interaction
AMP or beta-stimulants hibitors and activators,
stimulants inhibited
after treatment
(I, 2, 3).
above appear
between
with caffeine,
to support
in themselves,
modified
of intestinal
activity
receptor
inhibitors,
activators
he excluded
smooth
(3).
was decreased
the dynamic
cannot
under showed
and papaverine.
and adrenaline
hypothesis. and cyclic
because
these in
caused
by beta
the electrical activity.
role in the relaxation
It had been reported A, however,
activity
activity
theophylline
by
(6). These
are potentiated
on the phosphodiesterase
these phosphodiesterase
on the membrane
inhibited
of papaverine
in the present experiment
the effect of cyclic AMP
plays an important
by concanavalin
and cyclic AMP obtained
action
action
than that of theophylline
of cyclic AMP on the mechanical
with Imidazole,
Adenyl cyclasc
greater
of beta-stimulants
muscle was indeed potentiated
is reportedly
(6, 7) and the inhibitory
is apparently
with those inhibitors.
that the inhibitory
Imidazole
in cyclic AMP breakdown
(3), caffeine (3) and papaverine
that in various
the inhibitory
mechanism
tissues this enzyme activity
action
on adenyl cyclase activity
vas by
CYCLIC A:AIP AND INTESTINAL SMOOTH
MUSCLE
207
this agent was limited to within a certain range (8). In the present work, concentrations of 10-7-10-1 g/ml were used. Electrical and mechanical activities were not affected at various concentrations of concanavalin A. of noradrenaline
However, after concanavalin A, the inhibitory action
was decreased suggesting that the inhibition of adenyl cyclase activity
may be caused by externally applied concanavalin A. It had been reported that the intracellular level of cyclic AMP is reciprocally related to the intracellular content of cyclic GMP (10). Though the excitatory action of dibutyryl cyclic GMP on the smooth muscle strip of guinea-pig ileum and rat stomach has been reported (Il,
12), cyclic GMP and dibutyryl cyclic GMP showed no obvious effects on
either activity in the present experiment.
The relationship of cyclic GMP to cholinergic
transmission (12) and Ca ` supply to the contractile element during dibutyryl cyclic GMP treatment (11) was suggested for the mechanism of action of cyclic GMP. The discrepancy between the obtained results and the former data on the effect of cyclic GMP was not eluci dated and further study concerning the action of cyclic GMP on the activity of smooth muscle is now underway in our laboratory. REFERENCES 1) BELLEAU, B.: Pharmacol. Rcr., 21, 131 (1966) 2) ROBINSON, G.A., BUTCHER, R.W. ANDSUTHERLAND, E.W.: Ann. N. Y. Acad. Sci., 139, 703 (1967) 3) BUTCHER, R.W. ANDSUTHERLAND, E.W.: J. biol. Chem., 237, 1244 (1962) 4) TAKAGI, K. et al.: Japan. J. Pharinacol., 21, 477 (1971) 5) OHKAWA, H.: Bull. YamaguchiAled. Sch., 22, 185 (1975) 6) TRINER,L., VULLIEMOZ, Y., SCH\VATL, 1.ANDNAHAS,G.G.: Iioche,n. biophvs.Res. Common., 40, 64 (1970) 7) PocH, G. ANDKUKOVETZ, W.R.: Life Sci., 10, 133 (1971) 8) CUATRECASAS, P. ANDTELL,G.P.E.: Proc. natn. Acad. Sci U.S.A., 70, 485 (1973) 9) OHKAWA, H. ANDWATANABE, M.: TohokuJ. exp. Med., 117, 69 (1975) 10) ILLIANO, G., TELL,G.P.E., SIEGEL,M.I. ANDCUATRECASAS, P.: Proc. natn. Acad. Sci. U.S.A., 70, 2443 (1973) 11) TAKAYANAGI, 1. ANDTAKAGI,K.: Japan. J. Pharmacol., 23, 573 (1973) 12) PUGLISI, L., BERTI,F. ANDPAOLETTI, R.: Experientia,27, 1187 (1971)
