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The Mechanism of Action of Prostaglandin F2α on the Smooth Muscle of Guinea-Pig Taenia Coli
THE
MECHANISM
THE
OF
SMOOTH
ACTION
MUSCLE
OF Akira
OF
PROSTAGLANDIN
GUINEA-PIG
TAENIA
F2α ON COLI
OUJI
Brain Research Institute, School of Medicine, Chiba Unirersity, Chiba, Japan Accepted March 4, 1974
Abstract-The effect of prostaglandin F2α(PGF2α on isolated smooth muscle of guinea-pig taenia coil as related to cholinergic and adrenergic receptors of the muscle was investigated using the double sucrose-gap method. PGF2α in the concentration of 1.0×10-6 g/ml caused and augmented the contraction of the smooth muscle by depo larizing the membrane, decreasing the membrane resistance, and increasing the fre quency of spike generation. These actions were not abolished by atropine sulfate (10-7 g/ml), phentolamine (10-6 g/ml), propranolol (10-6 g/ml), respectively. PGF2α had the same action as that of acetylcholine on the contraction of the smooth mus cle, but the onset was slower and the duration longer. The inhibitory effects of ad renaline (10-7 g/ml), noradrenaline (10-7 g/ml), and isoproterenol (10-7 g/ml) were not suppressed by treatment with PGF2α. These results indicate that the effect of PG was not mediated either by the cholinergic nor the adrenergic receptors (α and receptors), and it is suggested that PGFα2 may act on sites of the cell membrane β other than adrenergic and cholinergic receptors to increase the membrane conduct ance as well as in the intracellular mechanism for the induction and augmentation of contraction of the tacnia coli smooth muscle. The interrelationship of the PG and Ca ion is also discussed.
It is well known that prostaglandins (PGs) are widely distributed in animal tissues (1-7) and have many biological activities (8).
The presence of PGs in the central and
peripheral nervous systems of the human, ox and other mammalians has been detected (3-5), but exact physiological roles are still unknown. The local presence and release of PGs in organs of smooth muscle by the stimulation of autonomic nerves have also been documented (9-11), but the mechanism of action of PGs on the contraction of smo oth muscle is still obscure.
Regarding the distribution of PGs in nervous system and
release of same by autonomic nervous excitation with the mode of action of autonomic nerves controlling the intestinal smooth muscle mobility, it is reasonable to infer a inter relationship among chemical transmitters in the nerves, PGs and the muscular contrac tion.
Bennet et al. (12) stated that PGE1 and E2 seemed to stimulate both the intrinsic
cholinergic
nerves and
intestinal smooth
muscle cells.
Clegg et al. (13) elucidated
the mechanism of action of Ketogenic PGs (Ei and E2) on the myometriurn, by means of Kymographic, electrical field stimulation and single sucrose-gap methods, and con cluded that PGE1 and E2 have two different kinds of effects; the direct and enhancement. The former is postulated to result from depolarization
at the exposed cell membrane
whereas the latter may result from facilitation of excitation contraction coupling.
The
present experiment was carried out by the application of a double sucrose-gap method to
investigate the mechanism of action of PGF2(, on the smooth muscle of taenia colt, par ticulary as related to cell membrane and cholinergic and adrenergic receptors. MATERIALSAND METHODS Guinea-pigs (non-pregnant) weighing 250 to 350 g were used. Under ethyl ether vapor anesthesia a strip of the guinea-pig taenia colt about 30-40 mm length was dissected from the caecurn. The strip was suspended and bubbled with 100',o 02 in Tyrode solu tion (32'C) for about 40 min to an hour after which it was mounted in the double sucrose gap chamber. The double sucrose-gap method used was an essentially modified version of that de scribed by Stampfli (14), Julian et al (15) and Berger (16). The sucrose-gap chamber can be considered as three separated pools, with two end pools separated from the middle one by high resistance sucrose-gaps, and there is a rubber membrane between each neighbour pool and gap pools (Fig. 1). From left to right (Fig. 1), the pools are designated: cur rent or I-pool; left sucrose-gap; center or C-pool; right sucrose gap; and voltage or V pool, respectively. The muscle strip was mounted in the chamber through the channel (2.0 mm diameter) which runs across the central part of each pool, and exposed to Ty rode solution, isotonic sucrose, test solution, isotonic sucrose and to isotonic K2SO4so lution (see Fig. 1), respectively. One end of the muscle strip was tied and fixed to the sup porting board, and the other end was tied to a fixed point of strain gauge in order to ad just the passive tension and to record the change of tension during the contraction of the muscle. The strip was stretched to a passive tension of 0.5 to 1.0 g.
FIG. 1. Schematic
representation
of the sucrose-gap
chamber.
Dotted
areas
repre
sent sucrose-gap pools separated by rubber membranes. Rub. memb ; rubber membrane. (I) : current pool. (C) : center pool. (V) : voltage pool. I : current. V : voltage. Lower part of the figure is electrical equivalent circuit of the chamber.
Solution from
the reservoir
closely that
for the three bottles
to the chamber. the
perfused
solution
pools
and
the sucrose
into
polyethylene
The
warming
in each
pool
tubes
bath
was
would
solution passed
for gap through
circulated he maintained
with
pools
flowed
by gravity
a warming
bath
41'C
in such
water
at 32 _'_ l'C
mounted a way
by a thermostat.
Fic,. 2. Experimental apparatus setup sucrose-gap chamber. T : Tyrode irotonic
equipped
for the solution.
double sucrose-gap S : isotonic sucrose
study. SGC solution. K :
K2SO4 solution.
circulator
(Fig. 2).
The rate of flow of the solution
was 2.0 nil/min
in the C
pool and 1.0 ml/min in the I and V-pools. The drugs and the PG were administered via a small vinyl tube with one end inserted in the C-pool, and the other end connected to a micro-infusion
pump (Atom
infusion
pump
of the drugs in a desired concentration 2).
The concentration
PG in contact
AIP-2H),
which controlled
and in a constant
speed into the C-pool
given in the results is at of the final concentration
with the tissue.
Three
Ag-AgCI
pools (I, C, V pools)
by 3 M KCL agar bridges.
located
in order
downstream
to prevent
electrodes
The tip of the electrode
the diffusion
membrane
cle strip to isotonic the electrometer pool
and
applied
potential
K2SO4.
measured
the outside
was established
the potential
at the C-pool.
to the tissue in the I-pool
frequency)
when necessary.
A modified K', 2.7; Mg",
Tyrode
by exposing
Details
solution
between Stimulation
with alternating
to these three
in each pool was
of KCL from contacting
Since the C-pool was maintained
(see Fig.
of the drugs or
were connected
particularly that in the center pool. The membrane potential input impedance preamplifier connected between the V-pool The resting
the administration
the tissue,
was measured with a high and ground (center pool). the V-pool at virtual
end of the mus ground
potential,
the inside of the muscle strip in the V current polarities
pulses (3 sec duration) at constant
interval
were (10 sec
are given in Figs. 1 and 2.
of the following
composition
0.1 ; Ca' ', 1.8; C1-, 141.8; glucose, 5.5.
was used (mM):
The pH of the solution
Na+, 137; was kept
at 7.2-7.3 crystal The
with tris-bulfer.
sucrose
resulting
Isotonic
Isotonic
sucrose
(highly
purified,
Takeda,
sucrose
solution
had
K2SO4 was in concentration
Drugs
used in the present
noradrenaline-HC1
(Sankyo
sulfate
(Tanabe,
Japan),
Tokyo,
Japan).
As n' and
(Sumitomo required
pool of the chamber
by dissolving
of the
order
of
92.5 g
distilled
water.
10-5 ohm-'
cm-1.
of K2SO4 21.1 g per 1 (121.1 mM/I) of distilled water.
Co. Japan),
were adrenaline-HC1
acetylcholine-Cl
prostaglandin
fi blocking
agents,
were used.
in Tyrode
was prepared
in 1000 nil of deionized,
a conductivity
experiments
and crystal
Chem. Co., Japan)
concentration
solution
Japan)
solution
(Sankyo
(Daiichi
Fen (Ono phentolamine
Co.
Japan),
Pharmaceut. (Ciba),
with syringes
dl
atropine
Manuf.
Co.,
and 1-propranolol
The drugs were dissolved and applied
Co. Japan),
and diluted
to the
A, B and C to C
(Fig. 2).
RESULTS Responses
of the tissue to PGF•2,
When the tissue was mounted the three
pools,
spontaneous
in the chamber
spikes
(spike
and Tyrode
potential)
were
solution generally
was perfused recorded.
FIG. 3. Typical responses of the smooth muscle of guinea-pig taenia coli to PGF2 (10-6 g/ml). A line in record : membrane potential changes. B line : membrane potential and electrotonic potential changes with application of stimulation current. Bottom line : current monitor. Bars indicate the application of PGF, (10`6 g/ml) .
Fm. 4. Responses of the smooth muscle of guinea-pig taenia coli to PGF2« (10-` g/ml). Experimental conditions were the same as those cited in the Fig. 3, Here the depolarization effect of PG was not so distinct as that in Fig. 3.
in The
resting
potential
influenced sponse
of the membrane
by depolarization.
~Nas 55__5 mV.
In this
condition,
of the muscle while increasing
tion (Fig. 3A).
The membrane
since the size of electrotonic coil revealed increase
little change
the amplitude
potential
in spike frequency
PGF2n
was depolarized
spikes
of spike
of membrane
Other
genera
of taenia
with the PG, but the
resistance
remained
unaltered
(Fig. 4). The taenia coil did not respond to the PG so fast as that to acetylcholine and there was a latent period of 10-20 sec. Actions
of acetvlcholine
A typical
and cholinergic
ACh (10-6 g/ml) induced
Though
slower,
Fu;.
longer
than
immediately
atropine
effect was completely
effect, the onset
of
the
response
was
that of ACh.
abolished,
than
in the duration.
that of the PG but shorter
Adrenaline
(l0-'
noradrenaline
and adrenergic
g%ml) inhibited
(Fig. 6A), but the inhibitory
tolamine
(10-6 g/ml) and propranolol
renaline,
but did not inhibit
Both noradrenaline
oth muscle,
and these inhibitory
isoproterenol off.
the spontaneous action
activity
action
blockades
effect
was antagonized,
and other
and isoproterenol actions
could
the activating
of ACh
action
of
was more distinct
on the effect of PGF2,
was of short
the mem
duration.
Phen
the ce and fl-effects of ad
activating
effects of the PG (Fig.
had an inhibitory
not be suppressed
of the PG was blocked
and the effect of PG was restored
on
spike and hyperpolarized
of adrenaline
(10-6 g/ml) antagonized
the depolarizing
6B and C).
Fig. 7, the depolarizing
ACh
but there was no influence The depolarization
of' adrenaline,
the
sulfate (10-' g/ml) to C-pool for 10 min, the ACh (10-6 g/ml)
the PG on the muscle (Fig. 5C).
worn
and burst
5. Comparison of effects of acetylcholine (ACh) and PGF2«. A line in record changes of membrane potential under treatment with ACh (l0' g/ml). B line under treatment with PGF2 (10' g/ml) --control. C line : under pretreatment
After perfusing
brane
of membrane
when the ACh in the C-pool was washed
with atropine sulfate (10' g;'ml) for 10 mm, but the PG effect was not abolished.
Actions
of taenia coil can be seen in Fig. 5.
by depolarization
the PG had the same contraction
but lasted
(ACh),
on the affect of PGF2
muscle
the muscle to contract
of spikes, and the effect disappeared out.
blockade
effect of ACh on the smooth
re
resistance,
preparations
when in contact
in membrane
were not
the contraction
and the frequency
(Fig. 3B).
potential
and the decrease
enhanced
with decrease
decreased
in membrane
The spontaneous
by PG.
transiently
after the action
action
on the smo As shown
by noradrenaline
in and
of these two agents had
Fic,. 6. Comparison of effects of adrenaline (Ad) and PGF2a. A line in record : changes of membrane potentials under treatment with Ad (10-' g, ml) and PGF2a (10-6 g'ml). B line : under pretreatment with phentolamine (10-° g ml) for 10 min. C line : under pretreatment with both phentolamine (10-6 g,`ml) and propranolol (10-6 g, ml) for 10 min, the effects of Ad were abolished, but the effect of PGF2a remained.
Fi<,. 7. Effects of PGF-, isoproterenol and noradrenaline on the electrical activities of taenia coli smooth muscle. PG : PGF2,1 (10`' 9/ml). I : isoproterenol (10-' g. ml). NA : noradrenaline (10' g'ml). A line in record : PG control. B line : the PG effect was suppressed by I and recovered after the I effect wore off. C line : the PG effect was suppressed by NA and recovered after the NA effect had worn off.
DISCUSSION The results of the experiment effect of prominent of smooth brane
inducing
contraction
revealed of the
that
smooth
PGF2,,
(10-6 -/nil)
muscle
of taenia
had the coll.
remarkable
The elfect
was
in two respects. The first was manifested by the induction of the contraction muscle with depolarizing action on the membrane and an increase of the mem
conductance.
contraction
The second
while increasing
of PG may not be required slightly depolarized of membrane be determined ACh equilibrium
the frequency
potential
might
of contraction
of other preparations
and spike
by the equilibrium
by the induction
of spike generation.
for the induction
the membrane
conductance
was manifested
frequency
potential
tions of taenia coli used in the experiment
of
had a resting
The depolarizing of the
muscle,
of taenia coli without
(Fig. 4).
of the PG.
be in the range
and augmentation
Bennett
membrane
PG
any change effect may
(17) estimated mV.
action
as the
The depolarizing
20 to -26
of
that
the
All the prepara
potential
higher than
the equilibrium fect.
potential
of ACh, so that ACh always showed
In the same way, when the resting membrane
the equlibrium could
potential
of PGF2a (
not be observed
seen in Fig. 4.
lasted
and even reversal
potential
of spike
longer, the actions of PG and ACh appeared
this point of view, it can be concluded nergic
receptors
nerves
and that
of taenia
coil
muscle as described mechanical bring
and Tomita
and blocked
(17).
(20, 21).
discharge
is the result
of the membrane
by phentolamine
ade),
experiment
respectively.
suppress
The present
the inhibitory
isoproterenol
(l0-'
PG between
adrenergic
as
adrenaline
of the suppression
or inhibited action
the
receptors
intrinsic
that
on
cholinergic
intestinal
smooth
on the electrical extensively
hyperpolarized
and
by Bul
the membrane
that the hyperpolarization
(a-effect)
and the blockade
of the pace
maker
(c-blockade)
and propranolol
potential
of
(a-effect) , (s-block
gave the same results (Fig. 6), but the effect
by the adrenergic
of adrenaline
g/ml) (Fig. 7).
PG were
in nature . From of PG between choli
coli were investigated
and both effects were abolished
of PG was not abolished
on
same
of the muscle, and concluded
K-conductance
the
to be quite different
the
that
conduc
sulfate and the effects
The effect of catecholamines
They stated
the spike discharge
of ACh and
by atropine
effect of PGF2a
taenia
of this is
of membrane
there is no interaction
be considered
of the quinea-pig
is due to the increased spikes
the stimulating
cannot
by Bennet
activities
that
effect of the PG
One evidence
increase
(18, 19), the actions
But as the effect of PG was not influenced
ef
was near or even lower than
has to be considered.
generation
depolarizing
50 mV), the depolarizing
In respect of the effects of depolarization,
tance, and increase identical.
45 to
a marked
blockades,
(10-' g/ml),
These results
indicate
nor did PG (10-6 g/ml)
noradrenaline that
and the sites of adrenergic
(l0-'
g/ml)
and
there is no interaction action.
The results
of
of ex
perinment as shown in Fig. 7 may be explained by the fact that PGF2„ and catecholamines acted on different sites of the cell membrane and the result was the summation of actions of these two substances. centration overcome hibited,
(10-' g/ml)
When
noradrenaline
the inhibitory
the contractor
action
and was restored
action
and isoproterenol of these two amines
of PG, so that the contraction
after
were moderate
the noradrenaline
caused
potential
as the cell was exposed
to the PG.
in the size of electrotonic
potential
in the membrane
resistance.
by the PG was in
independent
of the cell of taenia coil smooth Kuriyama
of smooth
By means
to
isoproterenol
(23) concluded that PGE1 lowers blood pressure by an action lomine. Our present results confirm this conclusion. The size of electrotonic
enough
effects diminished , Carlson and Oro (22), and Strong and Bohr
since the effect of PG was more long lasting.
and
was strong
in con
and Tomita
of catecho
muscle was reduced
(24) reported
that
a change
muscle cells of taenia coil indicates a change
of this analysis,
the
reduction
of electrotonic
potential of the smooth muscle cells caused by the PG expressed the decrease of mem brane resistance of the cell caused by the PG, i.e. the increase of permeability of the mem brane
was induced
by the action
one of the most significant
of PG.
actions
al (13) may not be due to the result
of PG.
Changing Thus,
the membrane
permeability
the direct effect described
of depolarization
but rather
may be
by Clegg et
to the increase
of con
ductance brane
of the cell membrane.
conductance,
The fact that Ca ions play a role in controlling
and the spike in taenia cob is due to Ca ion entry
tion of PG F2 and Ca ion may be reduced. present
experiments
which would
Though
there was no other
support
this deduction,
the effect of PGF2„ on the cell membrane
is to decrease
zation
fix Ca ions.
of Ca ions at sites which normally
and Ca may be a most interesting of the action of prostaglandins sec) in the onset of action that of ACh.
gic receptor
on the smooth
mechanism
of action
nor the suppression
of alpha
and
sites of the cell membrane
at specific receptors
and on the intracellular
membrane
of the taenia coil smooth
and
in the intracellular
Acl;nor,/cdgenrents: Chiba
University,
Medicine,
muscle.
There
Gratitude
for guidance
Kitazato
are also due to Ono
regarding was a latent
of the PG
period (10-20
University,
mechanism
muscle.
sacroplasma
The mobilization
is due to Prof.
and encouragement for instruction
Pharmaceutical
Company,
for induction
by PG should
of choliner
receptors.
the membrane
also
PGF2n
of Ca ions on the he considered.
and to Prof.
M. Kano,
Tokyo,
Japan,
or
and augmentation
School
and
may
conductance
Y. Hagihara,
in physiology
with
be disregarded.
by the stimulation
beta adrenergic to increase
that
by mobili
the mechanism
of the PG cannot
that the effect of PG is not mediated
act on the special
of contraction
to assume
resistance
This interrelationship
investigation
in the
of the PG and the PG action lasted longer in comparison
The intracellular
It is concluded
one for further
evidence
it is reasonable membrane
mem
(24), the interac
of Medicine,
technology.
for providing
School
of
Thanks PGF2a.
REFERENCES 1) BERGSTROM,S., AND SAMUELSSON, B.:J. biol. Chem., 237, 3005 (1962) 2) BERGSTROM,S., AND SAMUELSSON, B.: Acta chem. scand., 17, 282 (1963) 3) SAMUELSSON, B.: Biochim, biophys. Acta, 84, 707 (1964) 4) SAMUELSSON, B.: Biochim. biophys. Acta, 84, 218 (1964) 5) DANIELS,E.G., HINMAN,J.W., LFACH, B.E., AND MUIRHEAD,E.E.: Nature, 215, 1298 (1967) 6) LEE, J.B., COVINO,G., TAKMAN,B. H. AND SMITH, E.R.: Circulation Res., 17, 57 (1965) 7) LEE, J.B., CROWSHAW,K., TAKMAN,B.H. ANDATTERP, K.A.: Biochem. J. 105, 1251 (1967) 8) HORTON, E.W.: Physiol. Rev., 49, 122 (1969) 9) DAVIES,B.N., HORTON,E.W. AND WITHRINGTON,P.G.: J. Physiol., 188, 38 (1966) 10) COCEANI,F., PACE-.ASCIAK, C., VOLTA,F. ANDWOLFEL.S.: Am, J. Physiol., 213, 1056 (1967)11 ) RAMWELL,P.W. AND SHAW, H.E.: Am. J. Physiol., 211, 125 (1966) 12) BENNET,A., ELEG. K,G. AND SCHOLES,G.B.: Br. J. Pharmacol. Chemother. 34, 630 (1968) 13) CLEGG, P.C., HALL, W.J., AND PICKLES,V.R.: J. Physiol. 183, 123 (1966) 14) STÄMPFLI, R.: Experientia, 10, 508 (1954) 15) JULIAN, F.J., MOORE,J.W. AND GOLDMAN,D.E.: J. gen. Physiol., 45, 1955 (1962) 16) BERGER, W.: Arch. ges. Physiol., 277, 570 (1963) 17) BENNET, M.R.: Nature, Lond., 211, 1149 (1966) 18) BÜLBRING, E.: J. Physiol., 128, 200 (1955) 19) BÜLBRING, E. AND BURNSTOCK,G.: Br. J. Pharmacol. Chemother.. 15, 611 (1960) 20) BÜLBRING, E. AND TOMITA, T.: Proc. R. Soc. B., 172, 89 (1969) 21) BÜLBRING, E. AND TOMITA,T.: Proc. R. Soc. B., 172, 103 (1969) 22) CARLSON,L., AND ORO, L.: Acta physiol. scand, 67, 89 (1966) 23) STRONG,G. AND BOHR, D.F.: Am. J. Physiol., 213, 725 (1967) 24) KURIYAMA, H. AND TOMITA,T.: J. gen. Physiol., 55, 147 (1970)
MECHANISM
THE
OF
SMOOTH
ACTION
MUSCLE
OF Akira
OF
PROSTAGLANDIN
GUINEA-PIG
TAENIA
F2α ON COLI
OUJI
Brain Research Institute, School of Medicine, Chiba Unirersity, Chiba, Japan Accepted March 4, 1974
Abstract-The effect of prostaglandin F2α(PGF2α on isolated smooth muscle of guinea-pig taenia coil as related to cholinergic and adrenergic receptors of the muscle was investigated using the double sucrose-gap method. PGF2α in the concentration of 1.0×10-6 g/ml caused and augmented the contraction of the smooth muscle by depo larizing the membrane, decreasing the membrane resistance, and increasing the fre quency of spike generation. These actions were not abolished by atropine sulfate (10-7 g/ml), phentolamine (10-6 g/ml), propranolol (10-6 g/ml), respectively. PGF2α had the same action as that of acetylcholine on the contraction of the smooth mus cle, but the onset was slower and the duration longer. The inhibitory effects of ad renaline (10-7 g/ml), noradrenaline (10-7 g/ml), and isoproterenol (10-7 g/ml) were not suppressed by treatment with PGF2α. These results indicate that the effect of PG was not mediated either by the cholinergic nor the adrenergic receptors (α and receptors), and it is suggested that PGFα2 may act on sites of the cell membrane β other than adrenergic and cholinergic receptors to increase the membrane conduct ance as well as in the intracellular mechanism for the induction and augmentation of contraction of the tacnia coli smooth muscle. The interrelationship of the PG and Ca ion is also discussed.
It is well known that prostaglandins (PGs) are widely distributed in animal tissues (1-7) and have many biological activities (8).
The presence of PGs in the central and
peripheral nervous systems of the human, ox and other mammalians has been detected (3-5), but exact physiological roles are still unknown. The local presence and release of PGs in organs of smooth muscle by the stimulation of autonomic nerves have also been documented (9-11), but the mechanism of action of PGs on the contraction of smo oth muscle is still obscure.
Regarding the distribution of PGs in nervous system and
release of same by autonomic nervous excitation with the mode of action of autonomic nerves controlling the intestinal smooth muscle mobility, it is reasonable to infer a inter relationship among chemical transmitters in the nerves, PGs and the muscular contrac tion.
Bennet et al. (12) stated that PGE1 and E2 seemed to stimulate both the intrinsic
cholinergic
nerves and
intestinal smooth
muscle cells.
Clegg et al. (13) elucidated
the mechanism of action of Ketogenic PGs (Ei and E2) on the myometriurn, by means of Kymographic, electrical field stimulation and single sucrose-gap methods, and con cluded that PGE1 and E2 have two different kinds of effects; the direct and enhancement. The former is postulated to result from depolarization
at the exposed cell membrane
whereas the latter may result from facilitation of excitation contraction coupling.
The
present experiment was carried out by the application of a double sucrose-gap method to
investigate the mechanism of action of PGF2(, on the smooth muscle of taenia colt, par ticulary as related to cell membrane and cholinergic and adrenergic receptors. MATERIALSAND METHODS Guinea-pigs (non-pregnant) weighing 250 to 350 g were used. Under ethyl ether vapor anesthesia a strip of the guinea-pig taenia colt about 30-40 mm length was dissected from the caecurn. The strip was suspended and bubbled with 100',o 02 in Tyrode solu tion (32'C) for about 40 min to an hour after which it was mounted in the double sucrose gap chamber. The double sucrose-gap method used was an essentially modified version of that de scribed by Stampfli (14), Julian et al (15) and Berger (16). The sucrose-gap chamber can be considered as three separated pools, with two end pools separated from the middle one by high resistance sucrose-gaps, and there is a rubber membrane between each neighbour pool and gap pools (Fig. 1). From left to right (Fig. 1), the pools are designated: cur rent or I-pool; left sucrose-gap; center or C-pool; right sucrose gap; and voltage or V pool, respectively. The muscle strip was mounted in the chamber through the channel (2.0 mm diameter) which runs across the central part of each pool, and exposed to Ty rode solution, isotonic sucrose, test solution, isotonic sucrose and to isotonic K2SO4so lution (see Fig. 1), respectively. One end of the muscle strip was tied and fixed to the sup porting board, and the other end was tied to a fixed point of strain gauge in order to ad just the passive tension and to record the change of tension during the contraction of the muscle. The strip was stretched to a passive tension of 0.5 to 1.0 g.
FIG. 1. Schematic
representation
of the sucrose-gap
chamber.
Dotted
areas
repre
sent sucrose-gap pools separated by rubber membranes. Rub. memb ; rubber membrane. (I) : current pool. (C) : center pool. (V) : voltage pool. I : current. V : voltage. Lower part of the figure is electrical equivalent circuit of the chamber.
Solution from
the reservoir
closely that
for the three bottles
to the chamber. the
perfused
solution
pools
and
the sucrose
into
polyethylene
The
warming
in each
pool
tubes
bath
was
would
solution passed
for gap through
circulated he maintained
with
pools
flowed
by gravity
a warming
bath
41'C
in such
water
at 32 _'_ l'C
mounted a way
by a thermostat.
Fic,. 2. Experimental apparatus setup sucrose-gap chamber. T : Tyrode irotonic
equipped
for the solution.
double sucrose-gap S : isotonic sucrose
study. SGC solution. K :
K2SO4 solution.
circulator
(Fig. 2).
The rate of flow of the solution
was 2.0 nil/min
in the C
pool and 1.0 ml/min in the I and V-pools. The drugs and the PG were administered via a small vinyl tube with one end inserted in the C-pool, and the other end connected to a micro-infusion
pump (Atom
infusion
pump
of the drugs in a desired concentration 2).
The concentration
PG in contact
AIP-2H),
which controlled
and in a constant
speed into the C-pool
given in the results is at of the final concentration
with the tissue.
Three
Ag-AgCI
pools (I, C, V pools)
by 3 M KCL agar bridges.
located
in order
downstream
to prevent
electrodes
The tip of the electrode
the diffusion
membrane
cle strip to isotonic the electrometer pool
and
applied
potential
K2SO4.
measured
the outside
was established
the potential
at the C-pool.
to the tissue in the I-pool
frequency)
when necessary.
A modified K', 2.7; Mg",
Tyrode
by exposing
Details
solution
between Stimulation
with alternating
to these three
in each pool was
of KCL from contacting
Since the C-pool was maintained
(see Fig.
of the drugs or
were connected
particularly that in the center pool. The membrane potential input impedance preamplifier connected between the V-pool The resting
the administration
the tissue,
was measured with a high and ground (center pool). the V-pool at virtual
end of the mus ground
potential,
the inside of the muscle strip in the V current polarities
pulses (3 sec duration) at constant
interval
were (10 sec
are given in Figs. 1 and 2.
of the following
composition
0.1 ; Ca' ', 1.8; C1-, 141.8; glucose, 5.5.
was used (mM):
The pH of the solution
Na+, 137; was kept
at 7.2-7.3 crystal The
with tris-bulfer.
sucrose
resulting
Isotonic
Isotonic
sucrose
(highly
purified,
Takeda,
sucrose
solution
had
K2SO4 was in concentration
Drugs
used in the present
noradrenaline-HC1
(Sankyo
sulfate
(Tanabe,
Japan),
Tokyo,
Japan).
As n' and
(Sumitomo required
pool of the chamber
by dissolving
of the
order
of
92.5 g
distilled
water.
10-5 ohm-'
cm-1.
of K2SO4 21.1 g per 1 (121.1 mM/I) of distilled water.
Co. Japan),
were adrenaline-HC1
acetylcholine-Cl
prostaglandin
fi blocking
agents,
were used.
in Tyrode
was prepared
in 1000 nil of deionized,
a conductivity
experiments
and crystal
Chem. Co., Japan)
concentration
solution
Japan)
solution
(Sankyo
(Daiichi
Fen (Ono phentolamine
Co.
Japan),
Pharmaceut. (Ciba),
with syringes
dl
atropine
Manuf.
Co.,
and 1-propranolol
The drugs were dissolved and applied
Co. Japan),
and diluted
to the
A, B and C to C
(Fig. 2).
RESULTS Responses
of the tissue to PGF•2,
When the tissue was mounted the three
pools,
spontaneous
in the chamber
spikes
(spike
and Tyrode
potential)
were
solution generally
was perfused recorded.
FIG. 3. Typical responses of the smooth muscle of guinea-pig taenia coli to PGF2 (10-6 g/ml). A line in record : membrane potential changes. B line : membrane potential and electrotonic potential changes with application of stimulation current. Bottom line : current monitor. Bars indicate the application of PGF, (10`6 g/ml) .
Fm. 4. Responses of the smooth muscle of guinea-pig taenia coli to PGF2« (10-` g/ml). Experimental conditions were the same as those cited in the Fig. 3, Here the depolarization effect of PG was not so distinct as that in Fig. 3.
in The
resting
potential
influenced sponse
of the membrane
by depolarization.
~Nas 55__5 mV.
In this
condition,
of the muscle while increasing
tion (Fig. 3A).
The membrane
since the size of electrotonic coil revealed increase
little change
the amplitude
potential
in spike frequency
PGF2n
was depolarized
spikes
of spike
of membrane
Other
genera
of taenia
with the PG, but the
resistance
remained
unaltered
(Fig. 4). The taenia coil did not respond to the PG so fast as that to acetylcholine and there was a latent period of 10-20 sec. Actions
of acetvlcholine
A typical
and cholinergic
ACh (10-6 g/ml) induced
Though
slower,
Fu;.
longer
than
immediately
atropine
effect was completely
effect, the onset
of
the
response
was
that of ACh.
abolished,
than
in the duration.
that of the PG but shorter
Adrenaline
(l0-'
noradrenaline
and adrenergic
g%ml) inhibited
(Fig. 6A), but the inhibitory
tolamine
(10-6 g/ml) and propranolol
renaline,
but did not inhibit
Both noradrenaline
oth muscle,
and these inhibitory
isoproterenol off.
the spontaneous action
activity
action
blockades
effect
was antagonized,
and other
and isoproterenol actions
could
the activating
of ACh
action
of
was more distinct
on the effect of PGF2,
was of short
the mem
duration.
Phen
the ce and fl-effects of ad
activating
effects of the PG (Fig.
had an inhibitory
not be suppressed
of the PG was blocked
and the effect of PG was restored
on
spike and hyperpolarized
of adrenaline
(10-6 g/ml) antagonized
the depolarizing
6B and C).
Fig. 7, the depolarizing
ACh
but there was no influence The depolarization
of' adrenaline,
the
sulfate (10-' g/ml) to C-pool for 10 min, the ACh (10-6 g/ml)
the PG on the muscle (Fig. 5C).
worn
and burst
5. Comparison of effects of acetylcholine (ACh) and PGF2«. A line in record changes of membrane potential under treatment with ACh (l0' g/ml). B line under treatment with PGF2 (10' g/ml) --control. C line : under pretreatment
After perfusing
brane
of membrane
when the ACh in the C-pool was washed
with atropine sulfate (10' g;'ml) for 10 mm, but the PG effect was not abolished.
Actions
of taenia coil can be seen in Fig. 5.
by depolarization
the PG had the same contraction
but lasted
(ACh),
on the affect of PGF2
muscle
the muscle to contract
of spikes, and the effect disappeared out.
blockade
effect of ACh on the smooth
re
resistance,
preparations
when in contact
in membrane
were not
the contraction
and the frequency
(Fig. 3B).
potential
and the decrease
enhanced
with decrease
decreased
in membrane
The spontaneous
by PG.
transiently
after the action
action
on the smo As shown
by noradrenaline
in and
of these two agents had
Fic,. 6. Comparison of effects of adrenaline (Ad) and PGF2a. A line in record : changes of membrane potentials under treatment with Ad (10-' g, ml) and PGF2a (10-6 g'ml). B line : under pretreatment with phentolamine (10-° g ml) for 10 min. C line : under pretreatment with both phentolamine (10-6 g,`ml) and propranolol (10-6 g, ml) for 10 min, the effects of Ad were abolished, but the effect of PGF2a remained.
Fi<,. 7. Effects of PGF-, isoproterenol and noradrenaline on the electrical activities of taenia coli smooth muscle. PG : PGF2,1 (10`' 9/ml). I : isoproterenol (10-' g. ml). NA : noradrenaline (10' g'ml). A line in record : PG control. B line : the PG effect was suppressed by I and recovered after the I effect wore off. C line : the PG effect was suppressed by NA and recovered after the NA effect had worn off.
DISCUSSION The results of the experiment effect of prominent of smooth brane
inducing
contraction
revealed of the
that
smooth
PGF2,,
(10-6 -/nil)
muscle
of taenia
had the coll.
remarkable
The elfect
was
in two respects. The first was manifested by the induction of the contraction muscle with depolarizing action on the membrane and an increase of the mem
conductance.
contraction
The second
while increasing
of PG may not be required slightly depolarized of membrane be determined ACh equilibrium
the frequency
potential
might
of contraction
of other preparations
and spike
by the equilibrium
by the induction
of spike generation.
for the induction
the membrane
conductance
was manifested
frequency
potential
tions of taenia coli used in the experiment
of
had a resting
The depolarizing of the
muscle,
of taenia coli without
(Fig. 4).
of the PG.
be in the range
and augmentation
Bennett
membrane
PG
any change effect may
(17) estimated mV.
action
as the
The depolarizing
20 to -26
of
that
the
All the prepara
potential
higher than
the equilibrium fect.
potential
of ACh, so that ACh always showed
In the same way, when the resting membrane
the equlibrium could
potential
of PGF2a (
not be observed
seen in Fig. 4.
lasted
and even reversal
potential
of spike
longer, the actions of PG and ACh appeared
this point of view, it can be concluded nergic
receptors
nerves
and that
of taenia
coil
muscle as described mechanical bring
and Tomita
and blocked
(17).
(20, 21).
discharge
is the result
of the membrane
by phentolamine
ade),
experiment
respectively.
suppress
The present
the inhibitory
isoproterenol
(l0-'
PG between
adrenergic
as
adrenaline
of the suppression
or inhibited action
the
receptors
intrinsic
that
on
cholinergic
intestinal
smooth
on the electrical extensively
hyperpolarized
and
by Bul
the membrane
that the hyperpolarization
(a-effect)
and the blockade
of the pace
maker
(c-blockade)
and propranolol
potential
of
(a-effect) , (s-block
gave the same results (Fig. 6), but the effect
by the adrenergic
of adrenaline
g/ml) (Fig. 7).
PG were
in nature . From of PG between choli
coli were investigated
and both effects were abolished
of PG was not abolished
on
same
of the muscle, and concluded
K-conductance
the
to be quite different
the
that
conduc
sulfate and the effects
The effect of catecholamines
They stated
the spike discharge
of ACh and
by atropine
effect of PGF2a
taenia
of this is
of membrane
there is no interaction
be considered
of the quinea-pig
is due to the increased spikes
the stimulating
cannot
by Bennet
activities
that
effect of the PG
One evidence
increase
(18, 19), the actions
But as the effect of PG was not influenced
ef
was near or even lower than
has to be considered.
generation
depolarizing
50 mV), the depolarizing
In respect of the effects of depolarization,
tance, and increase identical.
45 to
a marked
blockades,
(10-' g/ml),
These results
indicate
nor did PG (10-6 g/ml)
noradrenaline that
and the sites of adrenergic
(l0-'
g/ml)
and
there is no interaction action.
The results
of
of ex
perinment as shown in Fig. 7 may be explained by the fact that PGF2„ and catecholamines acted on different sites of the cell membrane and the result was the summation of actions of these two substances. centration overcome hibited,
(10-' g/ml)
When
noradrenaline
the inhibitory
the contractor
action
and was restored
action
and isoproterenol of these two amines
of PG, so that the contraction
after
were moderate
the noradrenaline
caused
potential
as the cell was exposed
to the PG.
in the size of electrotonic
potential
in the membrane
resistance.
by the PG was in
independent
of the cell of taenia coil smooth Kuriyama
of smooth
By means
to
isoproterenol
(23) concluded that PGE1 lowers blood pressure by an action lomine. Our present results confirm this conclusion. The size of electrotonic
enough
effects diminished , Carlson and Oro (22), and Strong and Bohr
since the effect of PG was more long lasting.
and
was strong
in con
and Tomita
of catecho
muscle was reduced
(24) reported
that
a change
muscle cells of taenia coil indicates a change
of this analysis,
the
reduction
of electrotonic
potential of the smooth muscle cells caused by the PG expressed the decrease of mem brane resistance of the cell caused by the PG, i.e. the increase of permeability of the mem brane
was induced
by the action
one of the most significant
of PG.
actions
al (13) may not be due to the result
of PG.
Changing Thus,
the membrane
permeability
the direct effect described
of depolarization
but rather
may be
by Clegg et
to the increase
of con
ductance brane
of the cell membrane.
conductance,
The fact that Ca ions play a role in controlling
and the spike in taenia cob is due to Ca ion entry
tion of PG F2 and Ca ion may be reduced. present
experiments
which would
Though
there was no other
support
this deduction,
the effect of PGF2„ on the cell membrane
is to decrease
zation
fix Ca ions.
of Ca ions at sites which normally
and Ca may be a most interesting of the action of prostaglandins sec) in the onset of action that of ACh.
gic receptor
on the smooth
mechanism
of action
nor the suppression
of alpha
and
sites of the cell membrane
at specific receptors
and on the intracellular
membrane
of the taenia coil smooth
and
in the intracellular
Acl;nor,/cdgenrents: Chiba
University,
Medicine,
muscle.
There
Gratitude
for guidance
Kitazato
are also due to Ono
regarding was a latent
of the PG
period (10-20
University,
mechanism
muscle.
sacroplasma
The mobilization
is due to Prof.
and encouragement for instruction
Pharmaceutical
Company,
for induction
by PG should
of choliner
receptors.
the membrane
also
PGF2n
of Ca ions on the he considered.
and to Prof.
M. Kano,
Tokyo,
Japan,
or
and augmentation
School
and
may
conductance
Y. Hagihara,
in physiology
with
be disregarded.
by the stimulation
beta adrenergic to increase
that
by mobili
the mechanism
of the PG cannot
that the effect of PG is not mediated
act on the special
of contraction
to assume
resistance
This interrelationship
investigation
in the
of the PG and the PG action lasted longer in comparison
The intracellular
It is concluded
one for further
evidence
it is reasonable membrane
mem
(24), the interac
of Medicine,
technology.
for providing
School
of
Thanks PGF2a.
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