The Effect of Intracellular Current Pulses
in Smooth Muscle Cells of the Guinea Pig
Vas Deferens at Rest and during Transmission
M. R. B E N N E T T
From the Department of Zoology, University of Melbourne, Parkville, N. 2, Victoria,
Australia
ABSTRACT The effect of intracellular current pulses on the membrane of
smooth muscle cells of the guinea pig vas deferens at rest and during transmission was studied. Two main response types were identified: active response cells,
in which a spike was initiated in response to depolarizing currents of sufficient
strength and duration; passive response cells, in which depolarizing currents
gave only electrotonic potential changes. These cells were three times more
numerous than the active response cells. During the crest of the active response
the input resistance fell by about 25 % of the resting value. Comparison of the
active response with the action potential due to stimulating the hypogastric
nerve showed that the former was smaller in amplitude and had a slower rate
of rise and higher threshold. Electrical coupling occurred between the smooth
muscle cells during the propagation of the action potential. Depolarizing current pulses had no effect on the amplitude of the excitatory junction potential
(E.J.P.) in passive response cells, but in general did decrease its amplitude in
active response cells. These results are discussed with respect to the mechanism
of autonomic neuroeffector transmission.
INTRODUCTION
I t has recently been shown t h a t two different responses to intracellular curr e n t pulses c a n be o b t a i n e d f r o m different smooth muscle cells of the taenia
coli a n d the vas deferens. O n e response is a passive electrotonic depolarization to o u t w a r d l y directed currents, which is the response in most cells in
the taenia coli (17). T h e other is a n active response, consisting of a spike
which resembles the action potential (13, 17). T h e active response in single
cells of the vas deferens does n o t cause a contraction of the whole muscle
(6). I t is therefore unlikely t h a t this response is propagated. I n the present
work a q u a n t i t a t i v e study has been m a d e of the different electrical characteristics of these two types of response a n d of their distribution.
D u r i n g chemical transmission, polarization of the cell m e m b r a n e changes
2459
The Journal of General Physiology
0460
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
. ~967
the amplitude of the postsynaptic response (20, 11), whereas during electrical
transmission polarization has no effect on the amplitude of the postsynaptic
response (18, 12). Bennett and Merrillees (6) have reported that depolarizing
current pulses in the smooth muscle cells of the guinea pig vas deferens during
stimulation of the hypogastric nerve, decrease the amplitude of the excitatory
junction potential (E.J.P.) in some cells while not affecting the amplitude in
others. It therefore seemed possible that in some cells of the vas deferens the
E.J.P. is at least partly due to electrical coupling with other cells. Part of the
present study is concerned with determining whether the smooth muscle cells
in this tissue are connected together in an electrical syncytium (8, 23).
METHODS
The isolated guinea pig vas deferens, supplied by the hypogastric nerve, was used in
these experiments. About 2 cm length of outer longitudinal muscle coat was pinned
to a Perspex block on the bottom of a 10 ml bath. A modified Krebs solution (14)
flowed continuously through the bath at a temperature between 34 ° and 37°C. The
composition of the low sodium solution is given by Bennett (6). The hypogastric nerve
was stimulated with pulses of 200 #sec duration, and the resulting potential changes
in the smooth muscle cells were recorded with intracellular microelectrodes (4).
Single microelectrodes were used for both passing current across the muscle cell
membrane and recording the membrane potential. The artefact due to the injected
current passing across the electrode resistance during intracellular recording was
balanced out using the Wheatstone bridge method of Araki and Otani (1). Records
were accepted for analysis if the current-passing bridge was balanced both before
and after penetration of a cell. This criterion seemed satisfactory, because the threshold depolarization for initiation of an active response (see below) was consistent
(31 mv SE 4- 0.9, n = 6) if the bridge remained balanced both before and after ar~
impalement.
RESULTS
Types of Response to Intracellular Current Pulses There were two distinct
types of electrical response to intracellular current flow in the smooth muscle
cells of the vas deferens. Of 200 cells examined, a b o u t 7 5 % gave passive
electrotonic depolarizations to outwardly directed current pulses. A b o u t
2 0 % of the cells gave an active spike-like response to depolarizing pulses.
T h e few remaining cells gave responses which were intermediate between
the two main types of response. T h e characteristics of these different cell
types will now be described.
Passive Response Cells In these cells an outward current pulse gave an
electrotonic depolarization of the m e m b r a n e which rose and fell exponentially
with a time constant of about 1.8 msec (SE 4- 0.17, n = 9) as is shown in Fig.
1 a. There was a linear relationship between current and voltage in these cells
for depolarizations up to 80 m y (Fig. 2), and the input resistance of the cells
246,
M. R. BENNETT Current Pulses in Smooth Muscle
was a b o u t 15 m~2 (SE 4- 0.9, n = 9). W h e n the hypogastric nerve to these cells
was stimulated s u p r a m a x i m a l l y at 60 cycles/see, the resulting action potential
h a d a n o r m a l overshoot (Fig. 1 b).
It was often possible to obtain a p p a r e n t electrotonie responses by pressing
the microelectrode against the cells w i t h o u t penetrating them. Such responses
were probably due to current passing between the tip of the electrode a n d the
m e m b r a n e . It was therefore always necessary w h e n identifying a passive
a
b
I
I
FIGURE 1. The effect of intracellular current pulses on passive response cells, a, responses to current pulses of different intensity. The amplitude of the current pulse is given
by the horizontal bars. b, action potential fired in the same cell as a during supramaxima] stimulation of the hypogastric nerve. Zero membrane potential is given by the
horizontal line at the top. Hypogastric nerve stimulated at 60 c/sec. Horizontal calibration: 20 msec; vertical calibration: 50 mv, 5 namp.
response to m a k e sure t h a t the electrode was inside the cell by stimulating the
hypogastric nerve a n d firing an action potential.
Active Response Cells In these cells the electrical response to intracellular
c u r r e n t pulses of sufficient strength a n d duration, consisted of a fast depolarizing response which lasted for a b o u t 5 msec (Fig. 3) a n d resembled the action
potential. T h e r e was a threshold for firing of the response which occurred after
a depolarization of a b o u t 31 m v (SE 4- 0.9, n = 6). Near the threshold level
there was a characteristic creep in the depolarization d u r i n g a steady current,
before the cell 'developed the full active response (Fig. 4).
~There was a linear relationship between current a n d voltage in these cells,
until the threshold depolarization was a p p r o a c h e d (Figs. 2 a n d 3). T h e input
resistance in this range of depolarizations was a b o u t 22 Mf~ (SE 4- 2, n ---- 6),
2462
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
•
1967
mv
(D
C
123
..Q
60-
E
0)
/
E
40
/
/
FIGURE 2. Current-voltage relationship for a passive response
cell and an active response cell
Open circles give the values for
a passive response cell, filled
circles for an active response
cell. At about 30 mv depolarization an active response was
fired whose amplitude at threshold was about 55 mv as given
by the closed circle at 1.7 namp.
/
O
./
o
N
~_ 20
/
~D
-6
0)
0
namp
0
i
Current intensity
t h a t is s o m e 300-/0 g r e a t e r t h a n
to see w h e t h e r t h e r e w a s a n y
r e s p o n s e . I t is p r o b a b l e t h a t
r e s p o n s e is c o n s t a n t b e c a u s e a n
for t h e passive r e s p o n s e cells. A test w a s m a d e
c h a n g e in i n p u t resistance d u r i n g the active
t h e r e s i s t a n c e d u r i n g t h e crest of t h e a c t i v e
increase in the current strength above thresh-
|
i
.[
FIGURE 3. The effect ofintracellular current pulses on active response cells, a to d shows
the effect of increasing the current intensity of two closely spaced pulses. At c the response develops a hump which on increasing the current intensity in d develops into the
full active response. The horizontal bars give tbe amplitude of the current pulse in
each case. Horizontal calibration: 20 msec; vertical calibration: 50 mv, 5 namp.
M. R. BENNETT Current Pulses in Smooth Muscle
2463
old displaced the response in the depolarizing direction without much alteration of its form (Fig. 5). The graph of Fig. 6 shows the relation between current
and voltage displacement before threshold was reached, during the crest of the
!
I
FIGURE 4. All-or-none character of the active response, a, two closely spaced current
pulses of threshold intensity were delivered to the same cell. T h e first pulse did not cause
a n active response, b u t a h u m p developed on the passive response. T h e second pulse
gave the full active response, b, very slight increase in the c u r r e n t intensity over t h a t
which gave a passive response with a h u m p gives the full active response. Horizontal
lines give the current intensity. Horizontal calibration: 20 msec; vertical calibration:
50 mv, 5 n a m p .
active response, and during the afterpotential. In all three cases the currentvoltage relation was linear, indicating that the input resistance at any particular phase, though not equal to that at other phases, is probably independent
of the membrane voltage or applied current. The input resistance during the
crest of the response was about 18 Mr1 (SE -4- 3.4, n = 6), that is less than 7 0 %
2464
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
" VOLUME
5°
•
1967
of the input resistance before the threshold depolarization was reached. T h e
input resistance during the afterpotential was 19 MQ (SE 4- 4, n = 5) which
is also less than the subthreshold input resistance.
Other Responses to Intracellular Current Pulses In less than 5~0 of the cells
examined, an intracellular current pulse gave a passive response until depolarizations of about 40-50 mv were reached. At this level of depolarization
[
I
FIGURE 5. Effect of increasing the current intensity during the active response. Increasing the depolarizing current during the active response displaced it in the depolarizing direction and caused it to occur earlier during the response, without much alteration
of its form. Horizontal calibration: 20 msee; vertical calibration: 50 mv, 5 namp.
the response became nonlinear (Fig. 7), and a h u m p developed. This h u m p in
the response increased in amplitude with further depolarization but did not
show the all-or-none characteristics of the active response.
Effect of Changing the Duration of the Current Pulse on Active Response Cells
When the effect of different current strengths on the active response was
examined, the duration of the pulse was maintained between 20-40 msec, so
that the full active response was displayed. If the current pulse was removed
at any time during the active response, the m e m b r a n e immediately repolarized to its resting value. This effect is illustrated in Fig. 8, which shows that
as the duration of the current pulse decreases so does that of the active response, no matter at what time the pulse is terminated during the response.
M. R. BENNETT CurrentPulses in SmoothMuscle
2465
W h e n the d u r a t i o n b e c o m e s v e r y small, it is difficult to distinguish b e t w e e n
the passive electrotonic p o t e n t i a l c h a n g e s a n d the active response (Fig. 8 d).
S u p r a t h r e s h o l d c u r r e n t pulses of long d u r a t i o n (40-100 msec) g e n e r a l l y
fired o n l y a single active response, as is s h o w n in Fig. 9 a, a l t h o u g h it is
possible t h a t longer d u r a t i o n c u r r e n t pulses m a y h a v e a l l o w e d a second active
response to occur. O n a few occasions the first spike-like response to the c u r r e n t
pulse was followed b y a series of d a m p e d oscillations (Fig. 9 b).
Comparison of the Characteristics of the Active Response with Those of the Action
Potential A c o m p a r i s o n was m a d e b e t w e e n the characteristics of the
mv
8O
¢-
.£ 60
FIGURE 6. Current-voltage relations during an active response. Filled circles give the
current-voltage relation up to
the threshold depolarization.
Open circles give the displacement of the crest of the local
response by currents of different intensity. Filled squares
give the displacement of the
afterP0tential by currents of
different intensity. Note that
the current-voltage relation is
linear in each case.
0
a
%
O.
19
-o 40
19
7O
/
E 2O
0
namp 0
i
Current intensity
active response a n d those of the action p o t e n t i a l d u e to s t i m u l a t i o n of the
h y p o g a s t r i e nerve. T h i s c o m p a r i s o n was m a d e to see w h e t h e r there was a n y
difference b e t w e e n a response w h i c h is p r o b a b l y l i m i t e d to a single cell a n d
the action p o t e n t i a l w h i c h is p r o b a b l y initiated in m a n y cells at once. T h e
t h r e s h o l d d e p o l a r i z a t i o n for firing a n active response (31 m v , SE 4- 0.9, n = 5)
was a b o u t 10 m v g r e a t e r t h a n t h a t for initiating a n action p o t e n t i a l (21 m v ,
SE 4- 0.6, n ---- 10). T h e t h r e s h o l d d e p o l a r i z a t i o n for initiation of the action
p o t e n t i a l was d e t e r m i n e d b y s t i m u l a t i n g the h y p o g a s t r i c nerves a t frequencies
g r e a t e r t h a n 50 cycles/see, so as to ensure local initiation of the action p o t e n tial. T h e m a x i m u m r a t e of rise of the active response (10 v/see, SE 4- 0.6,
n = 5) was less t h a n t h a t of the action p o t e n t i a l (15 v/see, SE 4- 1, n = 6), a n d
the a b s o l u t e a m p l i t u d e of the active response ( - 3 m v , SE ± 1.5, n = 5) w i t h
,~466
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
a
• VOLUME
5°
•
1967
b
I
I
FIGURE 7. Effect of intracellular current pulses on nearly active response cells, a, tbe
normal response from a passive response cell to intracellular current flow. b, graded
response to current pulses of increasing amplitude. Horizontal bars give the current
intensity. Horizontal calibration: 20 msec; vertical calibration: 50 mv, 5 namp.
t
I
FIGURE 8. Effect of changing the duration of the current pulse on active response cells.
From a to d the duration of a just suprathreshold current pulse was decreased. Two
closely spaced pulses were given in each ease. The duration and strength of the pulses
are given by the horizontal bars. Note the undershoot of the membrane potential during
the repolarization in a, b, and c indicating an increase in permeability to potassium
during the recovery phase of the response. Horizontal calibration: 20 msec; vertical
calibration: 50 my, 5 namp.
M. R. BENNETT Current Pulses zn Smooth Muscle
2467
just s u p r a t h r e s h o l d c u r r e n t pulses was a b o u t 20 m v less t h a n t h a t of the action
potential (16 mv, SE 4- 1, n = 30). T h e active response is thus slower a n d
smaller t h a n the action potential a n d has a h i g h e r firing threshold.
!
!
FIGURE 9. Suprathreshold current pulses of long duration, a, usual response to a long
duration current pulse. Initial active response and then a slow depolarization, b, response sometimes seen during long duration pulses, active response followed by damped
oscillations. Horizontal bars give the current intensity. Horizontal calibration: 20 msec;
vertical calibration: 50 mv, 5 namp.
Electrical Connections between Smooth Muscle Cells T h e easiest w a y to test
w h e t h e r t h e r e are electrical connections between the s m o o t h muscle cells, is to
observe the c o n d u c t i o n of an action potential t h r o u g h o u t the s m o o t h muscle
tissue. It is not possible to do this b y s u p r a m a x i m a l stimulation of the h y p o gastric nerves, because it is difficult to distinguish b e t w e e n an action potential
w h i c h has been initiated locally, a n d one w h i c h has b e e n c o n d u c t e d over some
distance. I t is not possible to stimulate the muscle cells directly w i t h o u t also
exciting the i n t r a m u r a l extensions of the hypogastric n e r v e (16). T h e s e diffi-
2468
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
"
1967
culties were resolved by b a t h i n g the vas deferens in a solution in w h i c h the
s o d i u m activity was r e d u c e d to 28 mM. R e d u c i n g the s o d i u m activity to this
value does not alter the characteristics of the s m o o t h muscle action potential,
b u t does block transmission f r o m the hypogastric n e r v e (3).
Action potentials could easily be r e c o r d e d at distances u p to 0.5 c m f r o m
the external stimulating electrode, w h e n the muscle was excited with single
pulses of 6 msec duration. Fig. 10 shows two action potentials r e c o r d e d at
a
b
I
I
FIGURE 10. Propagation of action potentials through the smooth nmscle cells in low
sodium solution, a, action potential recorded 2 mm from large external stimulating
electrode, b, 4 mm from the same electrode. Horizontal calibration: 20 msec; vertical
calibration: 50 mv.
distances of 2 a n d 4 m m f r o m the stimulating electrode, in low s o d i u m solutions. A r o u g h m e a s u r e gave the velocity of p r o p a g a t i o n of the action potential
as 20 c m / s e c . T h e action potential could not be r e c o r d e d at distances greater
t h a n 0.5 c m f r o m the stimulating electrode, because the m i c r o e l e c t r o d e was disl o d g e d by the c o n t r a c t i o n of the tissue before the action potential h a d arrived.
H o w e v e r , the muscle was seen to c o n t r a c t at distances of 1.5 c m f r o m the
stimulating electrode. T h e s e results s u p p o r t the conclusion of Burnstock,
H o l m a n , a n d K u r i y a m a (10) a n d of T o m i t a (22), t h a t there are electrical
connections b e t w e e n the smooth muscle cells of the vas deferens, w h i c h allow
the action potential to be p r o p a g a t e d t h r o u g h o u t the tissue.
Since the velocity of p r o p a g a t i o n a n d the d u r a t i o n at half the height of the
action potential are a b o u t 20 c m / s e c and 6 msec respectively, a b o u t 1200 # of
M. R. BENNETT
24-69
CurrentPulses in Smooth Muscle
!
!
FmURE 11. Lack of effect of depolarizing or hyperpolarizing pulses on the amplitude
of the E.J.P. in a passive response cell. a, response of the membrane to intracellular current pulses of different intensity and of about 25 msec duration. The horizontal bars give
the amplitude of the current pulse, b, depolarization of the membrane by about 60 rnv
during the E.J.P. The lower E.J.P. was recorded immediately before the current pulse,
whose amplitude is given by the horizontal lines, c, hyperpolarization of the membrane
by about 30 mv during the E.J.P. The upper E.J.P. was recorded immediately before
the current pulse. Records retouched. Horizontal calibration: a, 20 msec, b and c, 100
msec; vertical calibration: 50 mv, 5 namp.
tissue a r e d e p o l a r i z e d b y o v e r 30 m y a t t h e s a m e t i m e . M e r r i l l e e s 1 h a s estim a t e d t h e a v e r a g e l e n g t h of these s m o o t h m u s c l e cells a t 4 5 0 / z . T h u s a l e n g t h
of tissue a b o u t five s m o o t h m u s c l e cells l o n g will b e s i m u l t a n e o u s l y d e p o l a r i z e d
o v e r 30 m v d u r i n g t h e a c t i o n p o t e n t i a l .
1 Merrillees, N. C. R. The nervous environment of individual smooth muscle cells reconstructed
from serial sampling with the electron microscope. Paper to be published.
2470
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
•
VOLUME
5 °
'
i967
Effect of Polarization of the Cell Membrane of Passive Response Cells during the
E.J.P. Cells were impaled in which the responses to current pulses of
short duration (30 msec) were only electrotonic, for depolarizations of up to
60 mv. To make sure that the microelectrode had penetrated these cells an
action potential was initiated by stimulating the hypogastric nerve. If the
action potential did not have a normal overshoot, the observations were rejected. Depolarizations and hyperpolarizations of up to 50 mv had no consistent effect on the amplitude of the E.J.P. in these cells, as is shown in Fig.
11. The effects of polarization on the amplitude of the E.J.P. in nine passive
response cells are shown in Fig. 12. The largest change in the amplitude of the
E.J.P. during the current pulse was =t=20% of the size of the control E.J.P.
140-
g04
c -'3
~w
b0C
60
mE
T
mv
,
-40
-20
0
+20
+40
Amplitude of polarization during the E.JP
,
*60
FIGURE 12. Effect of polarization of the cell membrane on the amplitude of the E.J.P.
in passive response cells. The effect of current pulses on the amplitude of the E.J.P. in
nine different cells is shown, each symbol giving the responses for one cell. Note that the
amplitude of the E.J.P. never varied by more than 20%.
Effect of Polarization of lhe Cell Membrane of Active Response Cells during the
E.J.P. Cells were identified as capable of giving active responses, and
then polarizing currents were passed during the fully facilitated E.J.P. This
was followed by further current pulses of short duration to check that the cell
was still capable of giving an active response. Finally an action potential was
initiated by stimulating the hypogastric nerve with suprathreshold pulses and
the characteristics of this action potential were checked.
The amplitude of the E.J.P. generally decreased during the depolarization
of active response cells. The depolarization of about 60 mv shown in Fig. 13
almost reduced the E.J.P to zero in this active response cell. Depolarizations
greater than this caused fluctuations in the potential recording which are
attributed to a change in electrode resistance. It was therefore not possible to
depolarize the m e m b r a n e by amounts which m a y have reversed the E.J.P.
The effect of depolarization during the E.J.P of four active response cells is
shown in the graph of Fig. 14. In three cases the E.J.P decreased in amplitude
during depolarization, but in one case the E.J.P was unchanged by depolariza-
M. R. BENNETT Current Pulses in Smooth Muscle
247~
t i o n s of u p to 60 m v . I t t h e r e f o r e s e e m s l i k e l y t h a t , if t h e a m p l i t u d e o f t h e
E . J . P is c h a n g e d a t a l l b y p o l a r i z a t i o n o f t h e cell m e m b r a n e , t h e n t h e E . J . P
h a s o c c u r r e d in a n a c t i v e r e s p o n s e t y p e o f cell. I n a f e w c a s e s t h e e f f e c t o f
I
I
FIGURE 13. Effect of depolarization on the amplitude of the E.J.P. in an active response
cell. a, response of the membrane to intracellular current pulses of different intensity and
of about 30 msec duration. The horizontal bars give the amplitude of the current pulse.
The membrane was depolarized during the E.J.P. by about 30 mv in b and 60 my in c.
The lower E.J.P. in both b and c was recorded immediately before the current pulse.
Horizontal lines in b and c give the intensity of the current pulse. Records retouched.
Horizontal calibration: a, 20 msec, b and c, 100 msec; vertical calibration: 50 my, 5
namp.
h y p e r p o l a r i z a t i o n o n t h e E . J . P in a c t i v e r e s p o n s e cells w a s t e s t e d , a n d s h o w n
not to alter the amplitude of the E.J.P.
I t s e e m s u n l i k e l y t h a t t h e d i f f e r e n t effects o f p o l a r i z a t i o n o n t h e E . J . P .
c o u l d b e d u e to b r i d g e u n b a l a n c e . A s m e n t i o n e d p r e v i o u s l y , t h e t h r e s h o l d f o r
2472
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
-
VOLUME
5°
"
i967
initiation of a n active response was v e r y consistent b e t w e e n cells if the c u r r e n t
b r i d g e r e m a i n e d b a l a n c e d b o t h before a n d after a n i m p a l e m e n t . F u r t h e r m o r e ,
p o l a r i z a t i o n s of u p to 60 m v did not h a v e a n y effect on the E . J . P . in a n y passive response cells. I t is i m p r o b a b l e t h a t the bridge was u n b a l a n c e d d u r i n g the
i m p a l e m e n t of one t y p e of cell b u t n o t of the other.
18 cells w e r e i m p a l e d in w h i c h no a t t e m p t was m a d e to identify the cells as
either active or passive response type. T h e a m p l i t u d e of the E.J.P. in nine of
-~
120]
c° ~
80~~"
gcL
g-~
_c uJ
c~
0
\
40
~
"-
_
t-
(1)
EL_
m v0 0
'
2'0
'
4'0
60
"'
Amplitude of depolarization during theEJ.P.
FIGURE 14. Effect of depolarization on the amplitude of the E.J.P. in active response
cells. The effect of current pulses on the amplitude of the E.J.P. in four different cells
is shown, each symbol giving the response for one cell. Note that the amplitude of the
EJ.P. was reduced by over 50% during depolarization in three cells, but was not affected in one cell (open triangles).
these cells v a r i e d b y only 4 - 2 0 % for d e p o l a r i z a t i o n s of u p to 60 mv. T h e E.J.P.
in six of the cells was r e d u c e d to n e a r l y zero b y d e p o l a r i z a t i o n s of 60 mv. T h e
r e m a i n i n g t h r e e cells g a v e inconsistent results.
DISCUSSION
~Ihe o b s e r v a t i o n (17) t h a t s o m e s m o o t h muscle cells are not c a p a b l e of giving
a n active response to i n t r a c e l l u l a r c u r r e n t pulses has b e e n c o n f i r m e d . T h e r e
are a n u m b e r of striated muscle fibers w h i c h give only passive electrotonic
p o t e n t i a l c h a n g e s to intracellular c u r r e n t pulses (9, 2) a n d these cells are
c o n t r a c t e d directly b y the p o s t s y n a p t i c p o t e n t i a l (15). H o w e v e r , the passive
response cells in the vas deferens are c a p a b l e of s u p p o r t i n g a n action p o t e n t i a l
d u e to s t i m u l a t i o n of the h y p o g a s t r i c n e r v e as well as a p o s t s y n a p t i c potential.
T h e existence of two different types of response to intracellular c u r r e n t
M. R . B~.N~a"r
CurrentPulses in Smooth Muscle
2473
pulses does not necessarily m e a n that there are two different types of cell. T h e
different responses m a y reflect differences in the degree of electrical coupling
between the cells. T h e smooth muscle cells of the vas deferens are arranged in
an approximately hexagonal close-packed array, with various degrees of
overlap between the cells.1 Thus every smooth muscle cell has about 12 other
cells in close contact with it. In some cases the contact between the cells is so
close as to resemble a tight junction (19). If there is extensive coupling between
passive response cells and adjacent cells, the electrical loading m a y be so great
that it is not possible to initiate an active response in these cells with an intracellular electrode. According to this interpretation the active response cells
would not be as extensively coupled to adjacent cells, although some electrical
loading of these cells is indicated by the fact that the active response is slower
and smaller than the action potential and is not self-maintaining.
T h e r e was no significant change in the amplitude of the E.J.P. in passive
response cells during intracellular current pulses, although the E.J.P was in
general decreased in active response cells. T h e r e are a n u m b e r of possible
explanations for these effects. First, if passive response cells are extensively
coupled to adjacent cells, the E.J.P. will be little affected in these cells by
current injection, because there is a rapid decrement of the voltage in an
electrical syncytium (22, 24). Hence the E.J.P. in the passive cells would not
be influenced as m u c h as the E.J.P. in active cells by injected current because
a larger spread of E.J.P. current from local, relatively uninfluenced, cells
should occur in the more heavily loaded passive cells. Second, there is a great
variation in the density of nerves within a few thousand angstroms of muscle
cells in the vas deferens, some cells having apparently no innervation at all.l
It is therefore possible that some cells are very little affected by chemical
transmitter, the E.J.P. in these cells being predominantly due to electrical
coupling between the cells during transmission. K u r i y a m a and T o m i t a (17)
have reported that m a n y of the cells in the taenia coli do not give an active
response to intracellular current pulses. T o m i t a (21) has shown that electrical
connections must exist between the smooth muscle cells of the taenia coli. If
the cells of the taenia coli are polarized during transmission from either the
perivascular (4) or intramural (5) inhibitory nerves, there is usually no change
in the amplitude of the inhibitory junction potentials (7). It therefore seems
possible that the junction potentials in some cells of the taenia coli are in part
due to electrical coupling between the cells.
It has been suggested that transmitter must be released from the axon
varicosities as well as the axon termination, as every smooth muscle cell in the
vas deferens is affected by at least six axons, but no smooth muscle cell has
more than two axons terminating on it, and most have none (4). If there is
electrical coupling between the cells this argument is no longer correct, as some
axons m a y effect their action via electrical coupling between the muscle cells.
~474
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
5°
•
1967
H o w e v e r , the v e r y few a x o n t e r m i n a t i o n s o b s e r v e d b y M e r r i l l e e s ' in the vas
deferens m a k e it unlikely t h a t t r a n s m i t t e r is o n l y released f r o m t h e t e r m i n a l
varicosity.
I should like to thank Professor G. Burnstoek and Dr. G. Campbell for their criticisms of the manuscript and D. Yorke for technical assistance.
During this work the author held the Gilmour Research Scholarship.
The work was supported by Public Health Research Grant (NB 2902) from the National Institute
of Neurological Diseases and Blindness, United States Public Health Service, and the National
Health and Medical Research Council of Australia.
Receivedfor publication 13 Febnggy 1967.
REFERENCES
I. ARAKI, r., and T. OTANL 1955. Responses of single motoneurones to direct
stimulation in toad's spinal cord. J. Neurophysiol.18:472.
2. ATWOOD, H. L., G. HOYLE, and J. SMYTH. 1965. Mechanical and electrical
responses of single innervated crab-muscle fibres, or. P1~siol.,180:449.
3. BENNETT, M. R. 1967. The effect of cations on the electrical properties of the
smooth muscle cells of the guinea-pig vas deferens. J. Physiol. (London).
190:465.
4. BENNETT, M. R., G. BURNSTOCK, and M. E. HOLMAN. 1966 a. Transmission from
perivascular inhibitory nerves to the smooth muscle cells of the guinea-pig
taenia coli. J. Physiol., (London). 182:527.
5. BENNETT, M. R., G. BImNSTOCK, and M. E. HOLMS. 1966 b. Transmission from
intramural inhibitory nerves to the smooth muscle cells of the guinea-pig
taenia coil J. Physiol., (London). 182:541.
6. BENNETT, M. R., and N. C. R. M.ERRILLEES.1966. An analysis of the transmission
of excitation from autonomic nerves to smooth muscle. J. Physiol., (London).
185:520.
7. BENNETT, M. R., and D. C. ROGERS. 1967. A study of the innervation of the taenia coli. ,7. Cell Biol. 33:573.
8. BOZLER, E. 1942. The action potentials accompanying conducted responses in
visceral smooth muscles. Am. J. Physiol. 136:553.
9. BURKE, W., and B. L. GINSBORO. 1956. Electrical properties of slow fibres. J.
Physiol., (London). 132:586.
10. BimNSTOCK, G., M. E. HOLMAN, and H. KURIYAMA. 1964. Facilitation of transmission from autonomic nerve to smooth muscle of guinea-pig vas deferens.
J. Physiol., (London). 172:31.
11. ECCLES, J. C. 1964. T h e Physiology of Synapses. Springer-Verlag, Berlin.
12. FURSHPAN, E. J. 1964. Electrical transmission at an excitatory synapse in a vertebrate brain. Science. 144:878.
13. HASmMOTO, Y., M. E. HOLMAN, a n d J . TILLE. 1966. Electrical properties of the
smooth muscle membrane of the guinea-pig vas deferens. J. Physiol., (London).
186:27.
14. HuKovIc, S. 1961. Responses of the isolated sympathetic nerve-ductus deferens
preparation of the guinea-pig. Brit. J. Pharmacol. 16:188.
M. R. B~s~ma'T CurrentPulses in Smooth Muscle
2475
15. KUFFLER,S. W., and E. M. VAUGHANWILLIAMS. 1953. Small-nerve junctional
potentials. The distributions of small motor nerves to frog skeletal muscle,
and the membrane characteristics of the fibres they innervate. J. Physiol.,
(London). 121:289.
16. KUR1YAMA,H. 1963. Electrophysiological observations on the motor innervation
of the smooth muscle cells in the guinea-pig vas deferens, or. Physiol., (London). 169:213.
17. KURIYAMA,H., and T. TOM.ITA. 1965. The responses of single smooth muscle
cells of guinea-pig taenia coli to intra-cellularly applied currents, and their
effect on the spontaneous electrical activity. J. Physiol., (London). 178:270.
18. MARTIN, A. R., and G. PmAR. 1963. Transmission through the ciliary ganglion
of the chick. J. Physiol., (London). 168:464.
19. MeRRmta~S, N. C. R., G. Bm~STOCK, and M. E. HOLM~a~. 1963. Correlation
of fine structure and physiology of the innervation of smooth muscle in the
guinea-pig vas deferem. J. Cell Biol. 19:529.
20. TAm~UCHI,A., and N. T^m~ucm. 1960. Further analysis of relationsMp between
end-plate potential and end-plate current. J. Neurophysiol. 23:397.
21. TO~ITA, T. 1966. Electrical responses of smooth muscle to external stimulation
in hypertonic solutions. J. Physiol., (London). 183:450.
22. ToMrr^, T. 1966. Membrane capacity and resistance of mammalian smooth
muscle. J. Theoret. Biol. 12:216.
23. VAYO, H. W. 1965. Determination of the electrical parameters of vertebrate
visceral smooth muscle, or. Theoret. Biol. 9:263.
24. WooD,traY, J. W., and W. E. GRILL. 1961. On the problem of impulse conduction in the atrium. In Nervous Inhibition. E. Florey, editor. Pergamon Press,
New York. 244.