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RELAXANT EFFECT OF ASPIRIN-LIKE DRUGS ON ISOLATED GUINEA PIG TRACHEAL CHAIN
STUDIES FROM
ON
THE
NERVE
ACTION
ELEMENTS Soichi YOSHIDA
POTENTIAL
ORIGINATED
IN
PIG
GUINEA
URETER
and Tetsuro KUGA
Department of Experimental Pharmacology, Research Institute for Chemobiodynamics, Chiba University, Chiba 280, Japan Accepted July 4, 1977
In 1955, Prosser et al. (1) recorded the action potential of rat ureter which they termed `prespike' . Recently, light and electron microscopical studies on the presence of nervous elements and plexuses in mammalian ureter have provided evidence of essentially similar patterns of ureteric innervation in various species (2, 3, 4, 5). The present study was an attempt to clarify the physiological and pharmacological properties of the action potential of the nervous elements in the ureter. Male guinea pigs weighing about 400 g were used. The ureter was isolated according to the procedure of Kobayashi (6) and the preparation consisted of renal pelvis and upper ureter (20 mm in length). removed.
To exclude spontaneous activity of the ureter, the calyx was
The apparatus used herein was that described by Kosterlitz and Lydon (7) with
slight modification.
The experiments were carried out in a chamber in which two pairs of
electrodes and a single one were used for stimulation, recording and grounding, respectively. The stimulating electrodes were placed on the pelvis of the preparation and recording elec trodes on the opposite side.
In the chamber, liquid paraffin was superimposed on the
Tyrode solution, equilibrated with 100% 02.
When action potential was recorded, the
preparation was placed in liquid paraffin. The stimulation, applied every 5 sec in a single stimulus and at I min intervals in a train of stimuli, was carried out with rectangular pulses from a stimulator with an isolator unit.
The action potentials were displayed on an oscil
loscope through an AC-preamplifier, photographed by a tracing camera, and simultaneously registrated on a pen recorder. When the stimulus consisted of a single pulse (2.5 volts, 0.5 msec) was applied, only one biphasic action potential shown in Fig. 1-A was obtained.
By the train stimuli (2.5 volts,
0.5 cosec, 50 Hz, 20 pulses or more) a series of the biphasic action potentials which cor responded to the number of pulses were evoked (Fig. 2-A) and after a prolonged latency of over 2.5 sec, a multiphasic high amplitude action potential was recorded (Fig. 2-B).
This
prolonged latency was shortened by increasing the number of pulses (Fig. 2-C). This multiphasic action potential was abolished by 2 x 10-3 g/ml MnC12, which suppressed generation of Ca-dependent muscle action potentials. The former biphasic action potential was abolished by 5 x 10-7 g/ml tetrodotoxin (Fig. 1-B) and dibucaine which blocked the nerve excitation, but was not abolished by 2 x 10-3 g/ml MnC12. This biphasic action
Fig. 1. Effect of tetrodotoxin on the evoked nerve action potentials. A, control; B, in the presence of tetrodotoxin. C, strength-duration relationship of the stimulus pulse for evocation of the action potential in the nerve elements. Abcissa, pulse duration (0.05-3000 msec); ordinate, current required to evoke a threshold response (mA).
Fig. 2. Nerve action potentials and smooth muscle action potentials evoked by a train stimuli (intensity 2.5 volts, duration 0.5 cosec, frequency 50 Hz). A, a series of nerve action potentials; B, smooth muscle action potential evoked by 20 pulses;
potential
C, the same potential
was not abolished
phenoxybenzamine,
by 30 pulses.
by autonomic
propranolol
and
nervous
blocking
hexamethonium.
Therefore,
by a single pulse and train pulses appear to have originated in the muscle tissue, and the high amplitude have originated
from the smooth
The physiological were 0.40-0.45
properties
msec (Fig. 1-C).
response
substances
such as atropine,
the responses
evoked
from nervous elements distributed
after a prolonged
latency appears
to
muscle of the ureter. on the nervous The absolute
elements
refractory
were as follows; period was 2.0-2.5
the chronaxie msec and the
relative
refractory
period
about
6 msec.
The conduction
velocity
120 cm/sec.
values were similar to those in C-fiber which is classified as a mammalian It is not clear why there is such a prolonged and the smooth
muscle response.
a slow conduction showed
city of excitation
muscle
the electrical
stimulation
however, it is possible to consider
potential
in the pelvis.
Kobayashi
of the cat that the conduction
velo
muscle is 2 -4 mmn, sec (6, 8). In the present experiments,
was the pelvis and recordings
these two pairs of electrodes
velocity of the action potential is 2 mm'see,
action
using a pelvis ureter specimen
of the smooth
area stimulated between
By way of explanation
velocity of smooth
in experiments
latency between
These
nerve fiber.
were from the upper
v,-is 5 mm.
Consequently,
ureter.
the
The distance
even if the conduction
in the pelvis of guinea pigs used in the present experiments
it will take only about
electrodes
to recording
electrodes,
observed.
Therefore,
the prolonged
2.5 sec for the excitation
to reach from stimulating
but in a few preparations,
4 sec or more latency
latency can hardly be explained
was
only by the conduction
velocity in the pelvis. In intestinal
smooth
with a prolonged
muscle or in the nerve suppling
latency have been reported. by field stimulation,
small intestine
triggered
depolarization,
i.e. an early and a delayed
depolarization
might be related to a peripheral
Kuriyama
system in the intestinal
discharge
potentials
onset of stimulation that
nervous
circuits
neurons
was evoked
sidered.
experiments,
On the other
cells in the pacemaker
hand,
the possibility
a pacemaker
time in some part of a bundle,
course
of onset
of the
for synchronization
that there
potential.
Many propagate
pulses
were required
excitation
(11) concluded
excitatory
to the ureter.
response
From
may
of action potentials.
a pacemaker
potential.
When
cells tends to synchronize,
it is considered
be determined
muscle
physiological were about
by
action
the
studies,
potential Burnstock
100 ,,, in diameter;
a short time that
the time
time required
muscle "as
which could and
Prosser
when the diameter
reduced to below 100 , , there
These facts suggest that excitation
muscle of the pelvis does not reach the efTector muscle bundle to the nerve fibers.
by which the
muscle cells within a bundle.
smooth
of strips of guinea pig taenia coil or cat intestinal was no propagation
latency recorded
exceeds a critical value at a particular
Consequently,
to produce
nervous
above may be con
This change is then followed
of all smooth
that effector muscle bundles
They considered
hypothetical
is some mechanism
change in individual
potential.
of the activity
a
cannot be ruled out. It is generally considered
the potential
of action
several
to those as described
thus affecting cells in other parts of the bundle. later by the generation
after
muscle has the ability to produce
of cells producing
plexus or
Hirst et al. (10) recorded
With regard to the prolonged similar
region are synchronized
that each cell in smooth the number
possibilities
that the delayed
only after a delay of 2-11 sec from the
pathways
had been made to fire repetitively.
in the present
nerve network.
of the
two types of slow
They explained
of guinea pig small intestine.
in some
activity
reflex at the level of the myenteric
from some neurons
in myenteric
activity
smooth muscle, responses
et al. (9) observed
depolarization.
the positive feedback of synaptic
intestinal
In studies on the membrane
of the smooth
until many pulses are applied
Another possible explanation for the beginning of the smooth muscle action potential in the pelvis is that the smooth muscle cells required many pulses for excitation as can be seen in vas deferens (12). When membrane potential changes of single smooth muscle cells of the guinea pig vas deferens are recorded intracellularly during repetitive stimulation of the hypogastric nerve, the response of each stimulus to the nerve is a depolarization termed a `junction potential'.
Individual junction potentials sum with each other until
at a critical over-all depolarization, a spike is initiated. Although the action potential of the nervous elements was also recorded from other areas of the upper ureter, the smooth muscle action potential was recorded after prolonged latency only when the pelvis was stimulated by a low voltage stimulation.
These obser
vations suggested that there is a relationship between the nerve action potential and smooth muscle action potential in the pelvis.
It is tempting to speculate that there is a transmission
from autonomic nerve to smooth muscle.
In fact, substances such as tetrodotoxin and
dibucaine which block nerve excitation also abolished the smooth muscle action potential. In addition, Kuriyama (13) showed in the hypogastric nerve-vas deferens preparation
of
the guinea pig that calcium increases the transmitter output during nerve stimulation and magnesium interferes with the release of the transmitter.
In the present experiments, the
smooth muscle action potential was abolished by raising the concentration or lowering that of calcium.
of magnesium
Direct stimulation to the smooth muscle cells of the pelvis
does, however, initiate a propagating
smooth
muscle action
potential
(unpublished
observation). Recent histochemical studies (2, 3, 14, 15) have demonstrated that the intrinsic in nervation of the ureter consists of a fine network of nerves in the adventitial, muscular, and submucosal layers.
It is considered that the action potential evoked by stimulation of the
pelvic region is due to the excitation of this nerve network in the adventitial and muscular layers. REFERENCES 1) PROSSER, C.L.. SMITH,C.E. ANDMELTON, C.E.: Am.J. Phvsiol. 181, 651 (1955): 2) ELBADAwI, A. ANDSCHENK,E.A.: Am. J. Anat. 126, 103 (1969); 3) AUNG-KHiN,M.: Invest. Urol. 10, 307 (1972); 4) HoYES,A.D., BOURNL, R. ANDMARTIN,B.G.H.: J. Lond. 119, 123 (1975): 5) HOPES,AD., BARBER, P. ANDMARTIN, B.G.H.: Cell Tiss. Res. 160, 151(1975): 6) KOBAYASHI, M.: Ann J. Phvsiol. 208, 715 (1965), 7) KosTERLITZ, H.W. ANDLYDON,R.J.: Brit. J. Pharmacol. 43, 74 (1971); 8) KOBAYASHI, M.: Japan. J. Smooth Muscle Res. 11, 165 (1975) (in Japanese); 9) KURIYAMA, H., OSA,T. ANDTOIDA,N.: J. Physiol. 119, 275 (1967); 10) HIRST,G.D.S., HOLMAN,M.E. AND McKIRDY,H.C.: J. Physiol. 244, 113 (1975): 11) BURNSTOCK, G. AND PROSSER, C.L.: Am. J. Physiol. 199, 553 (1960): 12) BURNSTOCK, G. ANDHOLMAN, M.E.: J. Physiol. 155, 115 ("1961); 13) KuRIYAvIA, H.: J. Physiol. 175. 211 (1964); 14) NOTLEY,R.G.: Brit. J. Urol. 40, 37 (1968); 15) DUARTE-ESCALANTE, 0., LABAY, P. ANDBOYARSKY, S.: J. OWL, Baltimore 101, 803 (1969)
ON
THE
NERVE
ACTION
ELEMENTS Soichi YOSHIDA
POTENTIAL
ORIGINATED
IN
PIG
GUINEA
URETER
and Tetsuro KUGA
Department of Experimental Pharmacology, Research Institute for Chemobiodynamics, Chiba University, Chiba 280, Japan Accepted July 4, 1977
In 1955, Prosser et al. (1) recorded the action potential of rat ureter which they termed `prespike' . Recently, light and electron microscopical studies on the presence of nervous elements and plexuses in mammalian ureter have provided evidence of essentially similar patterns of ureteric innervation in various species (2, 3, 4, 5). The present study was an attempt to clarify the physiological and pharmacological properties of the action potential of the nervous elements in the ureter. Male guinea pigs weighing about 400 g were used. The ureter was isolated according to the procedure of Kobayashi (6) and the preparation consisted of renal pelvis and upper ureter (20 mm in length). removed.
To exclude spontaneous activity of the ureter, the calyx was
The apparatus used herein was that described by Kosterlitz and Lydon (7) with
slight modification.
The experiments were carried out in a chamber in which two pairs of
electrodes and a single one were used for stimulation, recording and grounding, respectively. The stimulating electrodes were placed on the pelvis of the preparation and recording elec trodes on the opposite side.
In the chamber, liquid paraffin was superimposed on the
Tyrode solution, equilibrated with 100% 02.
When action potential was recorded, the
preparation was placed in liquid paraffin. The stimulation, applied every 5 sec in a single stimulus and at I min intervals in a train of stimuli, was carried out with rectangular pulses from a stimulator with an isolator unit.
The action potentials were displayed on an oscil
loscope through an AC-preamplifier, photographed by a tracing camera, and simultaneously registrated on a pen recorder. When the stimulus consisted of a single pulse (2.5 volts, 0.5 msec) was applied, only one biphasic action potential shown in Fig. 1-A was obtained.
By the train stimuli (2.5 volts,
0.5 cosec, 50 Hz, 20 pulses or more) a series of the biphasic action potentials which cor responded to the number of pulses were evoked (Fig. 2-A) and after a prolonged latency of over 2.5 sec, a multiphasic high amplitude action potential was recorded (Fig. 2-B).
This
prolonged latency was shortened by increasing the number of pulses (Fig. 2-C). This multiphasic action potential was abolished by 2 x 10-3 g/ml MnC12, which suppressed generation of Ca-dependent muscle action potentials. The former biphasic action potential was abolished by 5 x 10-7 g/ml tetrodotoxin (Fig. 1-B) and dibucaine which blocked the nerve excitation, but was not abolished by 2 x 10-3 g/ml MnC12. This biphasic action
Fig. 1. Effect of tetrodotoxin on the evoked nerve action potentials. A, control; B, in the presence of tetrodotoxin. C, strength-duration relationship of the stimulus pulse for evocation of the action potential in the nerve elements. Abcissa, pulse duration (0.05-3000 msec); ordinate, current required to evoke a threshold response (mA).
Fig. 2. Nerve action potentials and smooth muscle action potentials evoked by a train stimuli (intensity 2.5 volts, duration 0.5 cosec, frequency 50 Hz). A, a series of nerve action potentials; B, smooth muscle action potential evoked by 20 pulses;
potential
C, the same potential
was not abolished
phenoxybenzamine,
by 30 pulses.
by autonomic
propranolol
and
nervous
blocking
hexamethonium.
Therefore,
by a single pulse and train pulses appear to have originated in the muscle tissue, and the high amplitude have originated
from the smooth
The physiological were 0.40-0.45
properties
msec (Fig. 1-C).
response
substances
such as atropine,
the responses
evoked
from nervous elements distributed
after a prolonged
latency appears
to
muscle of the ureter. on the nervous The absolute
elements
refractory
were as follows; period was 2.0-2.5
the chronaxie msec and the
relative
refractory
period
about
6 msec.
The conduction
velocity
120 cm/sec.
values were similar to those in C-fiber which is classified as a mammalian It is not clear why there is such a prolonged and the smooth
muscle response.
a slow conduction showed
city of excitation
muscle
the electrical
stimulation
however, it is possible to consider
potential
in the pelvis.
Kobayashi
of the cat that the conduction
velo
muscle is 2 -4 mmn, sec (6, 8). In the present experiments,
was the pelvis and recordings
these two pairs of electrodes
velocity of the action potential is 2 mm'see,
action
using a pelvis ureter specimen
of the smooth
area stimulated between
By way of explanation
velocity of smooth
in experiments
latency between
These
nerve fiber.
were from the upper
v,-is 5 mm.
Consequently,
ureter.
the
The distance
even if the conduction
in the pelvis of guinea pigs used in the present experiments
it will take only about
electrodes
to recording
electrodes,
observed.
Therefore,
the prolonged
2.5 sec for the excitation
to reach from stimulating
but in a few preparations,
4 sec or more latency
latency can hardly be explained
was
only by the conduction
velocity in the pelvis. In intestinal
smooth
with a prolonged
muscle or in the nerve suppling
latency have been reported. by field stimulation,
small intestine
triggered
depolarization,
i.e. an early and a delayed
depolarization
might be related to a peripheral
Kuriyama
system in the intestinal
discharge
potentials
onset of stimulation that
nervous
circuits
neurons
was evoked
sidered.
experiments,
On the other
cells in the pacemaker
hand,
the possibility
a pacemaker
time in some part of a bundle,
course
of onset
of the
for synchronization
that there
potential.
Many propagate
pulses
were required
excitation
(11) concluded
excitatory
to the ureter.
response
From
may
of action potentials.
a pacemaker
potential.
When
cells tends to synchronize,
it is considered
be determined
muscle
physiological were about
by
action
the
studies,
potential Burnstock
100 ,,, in diameter;
a short time that
the time
time required
muscle "as
which could and
Prosser
when the diameter
reduced to below 100 , , there
These facts suggest that excitation
muscle of the pelvis does not reach the efTector muscle bundle to the nerve fibers.
by which the
muscle cells within a bundle.
smooth
of strips of guinea pig taenia coil or cat intestinal was no propagation
latency recorded
exceeds a critical value at a particular
Consequently,
to produce
nervous
above may be con
This change is then followed
of all smooth
that effector muscle bundles
They considered
hypothetical
is some mechanism
change in individual
potential.
of the activity
a
cannot be ruled out. It is generally considered
the potential
of action
several
to those as described
thus affecting cells in other parts of the bundle. later by the generation
after
muscle has the ability to produce
of cells producing
plexus or
Hirst et al. (10) recorded
With regard to the prolonged similar
region are synchronized
that each cell in smooth the number
possibilities
that the delayed
only after a delay of 2-11 sec from the
pathways
had been made to fire repetitively.
in the present
nerve network.
of the
two types of slow
They explained
of guinea pig small intestine.
in some
activity
reflex at the level of the myenteric
from some neurons
in myenteric
activity
smooth muscle, responses
et al. (9) observed
depolarization.
the positive feedback of synaptic
intestinal
In studies on the membrane
of the smooth
until many pulses are applied
Another possible explanation for the beginning of the smooth muscle action potential in the pelvis is that the smooth muscle cells required many pulses for excitation as can be seen in vas deferens (12). When membrane potential changes of single smooth muscle cells of the guinea pig vas deferens are recorded intracellularly during repetitive stimulation of the hypogastric nerve, the response of each stimulus to the nerve is a depolarization termed a `junction potential'.
Individual junction potentials sum with each other until
at a critical over-all depolarization, a spike is initiated. Although the action potential of the nervous elements was also recorded from other areas of the upper ureter, the smooth muscle action potential was recorded after prolonged latency only when the pelvis was stimulated by a low voltage stimulation.
These obser
vations suggested that there is a relationship between the nerve action potential and smooth muscle action potential in the pelvis.
It is tempting to speculate that there is a transmission
from autonomic nerve to smooth muscle.
In fact, substances such as tetrodotoxin and
dibucaine which block nerve excitation also abolished the smooth muscle action potential. In addition, Kuriyama (13) showed in the hypogastric nerve-vas deferens preparation
of
the guinea pig that calcium increases the transmitter output during nerve stimulation and magnesium interferes with the release of the transmitter.
In the present experiments, the
smooth muscle action potential was abolished by raising the concentration or lowering that of calcium.
of magnesium
Direct stimulation to the smooth muscle cells of the pelvis
does, however, initiate a propagating
smooth
muscle action
potential
(unpublished
observation). Recent histochemical studies (2, 3, 14, 15) have demonstrated that the intrinsic in nervation of the ureter consists of a fine network of nerves in the adventitial, muscular, and submucosal layers.
It is considered that the action potential evoked by stimulation of the
pelvic region is due to the excitation of this nerve network in the adventitial and muscular layers. REFERENCES 1) PROSSER, C.L.. SMITH,C.E. ANDMELTON, C.E.: Am.J. Phvsiol. 181, 651 (1955): 2) ELBADAwI, A. ANDSCHENK,E.A.: Am. J. Anat. 126, 103 (1969); 3) AUNG-KHiN,M.: Invest. Urol. 10, 307 (1972); 4) HoYES,A.D., BOURNL, R. ANDMARTIN,B.G.H.: J. Lond. 119, 123 (1975): 5) HOPES,AD., BARBER, P. ANDMARTIN, B.G.H.: Cell Tiss. Res. 160, 151(1975): 6) KOBAYASHI, M.: Ann J. Phvsiol. 208, 715 (1965), 7) KosTERLITZ, H.W. ANDLYDON,R.J.: Brit. J. Pharmacol. 43, 74 (1971); 8) KOBAYASHI, M.: Japan. J. Smooth Muscle Res. 11, 165 (1975) (in Japanese); 9) KURIYAMA, H., OSA,T. ANDTOIDA,N.: J. Physiol. 119, 275 (1967); 10) HIRST,G.D.S., HOLMAN,M.E. AND McKIRDY,H.C.: J. Physiol. 244, 113 (1975): 11) BURNSTOCK, G. AND PROSSER, C.L.: Am. J. Physiol. 199, 553 (1960): 12) BURNSTOCK, G. ANDHOLMAN, M.E.: J. Physiol. 155, 115 ("1961); 13) KuRIYAvIA, H.: J. Physiol. 175. 211 (1964); 14) NOTLEY,R.G.: Brit. J. Urol. 40, 37 (1968); 15) DUARTE-ESCALANTE, 0., LABAY, P. ANDBOYARSKY, S.: J. OWL, Baltimore 101, 803 (1969)