J. Physiol. (1977), 266, pp. 751-764 With 7 text-ftgure8 Printed in Great Britain
751
SOME PROPERTIES OF THE SMOOTH MUSCLE OF MOUSE VAS DEFERENS
BY MOLLIE E. HOLMAN, G. S. TAYLOR AND T. TOMITA* From the Department of Physiology, Monash University,
Clayton, Victoria, 3168, Australia (Received 13 September 1976) SUMMARY
1. Contractions of the mouse vas deferens in response to electrical stimulation differ from those recorded from the guinea-pig vas deferens in that they are abolished by tetrodotoxin. 2. Changes in membrane potential were recorded from the smooth muscle of both preparations in response to stimulation with current pulses applied by an intracellular electrode and by large extracellular plate electrodes. 3. Both preparations behaved similarly in response to intracellular stimulation. Electrotonic potentials in response to extracellular current pulses spread in a longitudinal direction in the guinea-pig vas deferens in accordance with the cable-like properties of this preparation. In contrast, no longitudinal spread of electrotonus was observed in the mouse vas deferens. 4. Responses to nerve stimulation differed in the two preparations. In the guinea-pig, single stimuli caused excitatory junction potentials (e.j.p.s) which gave rise to action potentials. Some cells from the mouse vas deferens showed similar e.j.p.s and action potentials, although the threshold for the initiation of action potentials was lower and more variable. 5. The majority of cells in the mouse vas deferens failed to show action potentials in response to a single stimuli even though the amplitude of e.j.p.s was from 35 to 40 mV. This was probably due to the large resting membrane potential of these cells, as all-or-nothing action potentials could be evoked if successive e.j.p.s were allowed to sum with each other or if a depolarizing current pulse was applied at the peak of an e.j.p. 6. The nature of the response to nerve stimulationrecorded from different * Present address: Japan.
School
of Medicine, Fukuoka University, Fukuoka 814,
752 M. E. HOLMAN, C. S. TA YLOR AND T. TOMITA cells in the mouse vas deferens could be correlated with the amplitude and time course of the response of the same cell to intracellular stimulation. 7. It is concluded that individual smooth muscle cells in both preparations are probably coupled electrically but that there are few, if any, low resistance pathways in the longitudinal direction in the mouse vas deferens. INTRODUCTION
It has been shown that many smooth muscles have cable-like properties when current is applied with large extracellular electrodes and membrane potentials are measured with intracellular electrodes (Tomita, 1970; Bennett, 1972; Tomita, 1975). The conduction of action potentials in a variety of smooth muscles, including the ureter, taenia coli and vas deferens of the guinea-pig can be explained by these cable properties which appear to be independent of the pattern or density of innervation. Similar cable properties have also been observed in smooth muscles which do not normally appear to generate action potentials (Mekata, 1971). Conducted action potentials may be readily evoked with large extracellular electrodes in the guinea-pig vas deferens but it is difficult to initiate an action potential by intracellular injection of depolarizing current (Hashimoto, Holman & Tille, 1966; Bennett, 1967; Tomita, 1967). This observation may be explained by the theory that current injected by an intracellular electrode spreads in three-dimensions through the interconnexions between cells which give smooth muscle its cable properties (Noble, 1966; Tomita, 1970; Bennett, 1972). The electrical behaviour of the mouse vas deferens in response to intracellular stimulation appears to be qualitatively similar to that of the guinea-pig vas deferens (see Hashimoto & Holman, 1967). However, there is some doubt as to whether or not the mouse vas deferens also has cable-like properties when current is applied with large external electrodes, since contractions appear to be limited to the region of the stimulating electrodes (Furness & Burnstock, 1969). In the present experiments, responses of the mouse vas deferens to the application of current pulses by intracellular and external electrodes were compared with those of the guinea-pig in an attempt to clarify the possible differences of the electrical properties of these two smooth muscles. An attempt has also been made to account for the differences in the responses of these two preparations to nerve stimulation. Some of the results have been communicated to the Australian Physiological and Pharmacological Society (Tomita, Taylor & Holman, 1974).
MOUSE VAS DEFERENS
753
METHODS
Male guinea-pigs and mice were stunned, bled and the vasa deferentia were dissected out. Contractile activity was recorded via a strain gauge from whole vasa deferentia (about 1*5 cm long) suspended vertically in an organ bath of 4 ml. capacity and perfused continuously with physiological saline at 350 C. For electrical stimulation of the preparation, Ag-AgCl ring electrodes (diameter 3 mm) were placed at each end of the tissue. For external current application, a method similar to that described by Abe & Tomita (1968) was used. The bath (35 mm long, 10 mm wide and 6 mm deep) was divided into two sections, one for recording and the other for polarizing the preparation, by a thin silver plate (200 /sm thick) which had two holes having different diameters. This plate was used to polarize the smooth muscle fibres with long current pulses of weak intensity and also to stimulate intramural nerve fibres with short current pulses of strong intensity (transmural stimulation). A segment from the central region of a guinea-pig vas deferens or mouse vas deferens was put through the hole fitting their diameter. The side of the plate facing the recording chamber was insulated. In some experiments, guinea-pig and mouse vasa deferentia were set up simultaneously using both holes. For intracellular stimulation, a single micro-electrode was used to pass current and to record the potential produced. The voltage drop across the micro-electrode was compensated by means of a circuit incorporated in the WPI 4A Electrometer. Micro-electrodes were filled with 3m-KCI and had resistances between 40 and 60 Mn. The input resistance for intracellular current injection was determined from the ratio of the steady-state change in membrane potential and the amplitude of the current pulse. When the time course of the change in membrane potential in response to an intracellular current pulse was plotted logarithmically as a function of time, it was apparent that the time course of the onset and decay of the change in membrane potential was approximated by an exponential function; the time constant of this function will be referred to as r. The time constant of the smooth muscle membrane, determined from the application of current by large external electrodes (Abe & Tomita, 1968) will be referred to as r,. An analysis of the changes in membrane potential caused by the injection of current at a point in a syncytial structure has been described by Jack, Noble & Tsien (1975). They derived equations for a model (two infinite parallel sheets of membrane) in which the membrane area increased as a function of r2 where r was the distance from the point of current injection. Fig. 5-3a of Jack et al. (1975) shows their computed data for the time course of changes in membrane potential (V). In this figure, V is plotted as a function of T = t/Tm, where Tm is the membrane time constant given by the product of membrane resistance (Rm; Q cm2) and membrane capacitance (Cm; uzF/cm2). Values of V are plotted for various values of R = n/A2. In this model, A2 = Rm.b/2R1 where R. is the specific resistivity of the material between the sheets of membrane and b is the distance between the membranes. Wben these data were plotted logarithmically it was apparent that when R = 0-1 the relation between V and T could be approximated by a single exponential function. According to Jack et al. (1975) the time constant of this function T. should be one or two orders of magnitude shorter than the membrane time constant Tm. Physiological saline contained (mm): NaCl 120, KCl 5.9, CaCl2 2-5, MgCl2 1-2, NaHCO3 25-0, and glucose 11-5. The solution was in a reservoir and aerated with gas mixture of 5 % CO2 and 95% 02, and flowed through the chamber at 0-5 ml./min. The temperature of solution in the chamber was kept constant at 35 ± 0.50 C.
M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA
754
RESULTS
Mechanical responses in the guinea-pig and mouse vasa deferentia Fig. 1 shows effects of tetrodotoxin (4 x 10-7 g/ml.) on contractions of the guinea-pig and mouse vasa deferentia. Contractions produced by repetitive current pulses (20 Hz) of less than 1 msec in duration were completely abolished by tetrodotoxin, which is known to block the
(a)
A -
B
_
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0-5 g jj0-5 g 5jl~
1
_
__
-101 01g
(b) Tetrodotoxin
_Jk
.
_
.s
_a
_
_
30 sec
Fig. 1. A, mechanical responses of guinea-pig and B, mouse vasa deferentia to electrical stimulation in the absence (a) and in the presence (b) of tetrodotoxin (5 x 10-7 g/ml.). Repetitive stimulation with 0-5 msec pulses (5V for guinea-pig and IOV for mouse) produced a sustained contraction in the guinea-pig but a bimodal response in mouse vas deferens. A single pulse of 1 see duration (3-5 and 7 V) caused contractions in both preparations but contractions in the mouse were small. Tetrodotoxin blocked responses to repetitive stimulation in both preparations and to single pulse stimulation in mouse but did not affect contractions produced by single pulses in
guinea-pig.
effects of nerve stimulation without affecting the action potentials of smooth muscles (Builbring & Tomita, 1967; Hashimoto, Holman & McLean, 1967; Kuriyama, Osa & Toida, 1966). When the duration of the current pulses was increased to more than 10 msec, contractions of the guinea-pig vas deferens could still be evoked in the presence of tetrodotoxin, probably due to direct stimulation of the muscle. On the other hand, in the mouse vas deferens, it was not possible to produce a detectable contraction by external stimulation after application of tetrodotoxin, even if the pulse duration was increased to 100 msec or more.
MOUSE VAS DEFERENS
755
Electrical responses in the guinea-pig vas deferens Responses of the guinea-pig vas deferens to intracellular stimulation were the same as observed previously (Hashimoto et al. 1966; Bennett, 1967; Tomita, 1967). In Fig. 2, the responses to intracellular stimulation (a) and (b), were compared with those to external stimulation, (c) and (d), in two different cells (A and B). In general, responses to depolarizing (a)
(b)
(c)
(d)
B ] 10-9 A ______________
~
~
JS0mV
200 msec
50 msec
Fig. 2. Intracellular records from two different cell types (A and B) in guinea-pig vas deferens. (a) responses to hyperpolarizing current and (b) responses to depolarizing current applied by an intracellular electrode; upper traces show current, lower traces, changes in membrane potential. (c) and (d) show responses to hyperpolarizing and depolarizing current pulses applied with large extracellular plate electrodes. The current calibration (10-9 A) applies only to intracellular polarization.
current applied with an intracellular electrode, could be classified into two types. One type of cell showed a lower input resistance (from 10 to 30 MQ), a faster time course (or ranged from 1 to 5 msec) and no active response, as shown in Fig. 2A. The other type of cell was characterized by a higher input resistance (from 20 to 50 MQ) a slower time course (Tr ranged from 5 to 15 msec) and an active response (Fig. 2B). An active response could be obtained from about 15 % of penetrations and an inactive response from about 80%. For convenience we will refer to cells as either 'active' or 'inactive'. However some intermediate forms of response were also observed. When the preparation was polarized with external large electrodes, as shown in Fig. 2 (c) and (d) the time course of electrotonic potentials recorded at a distance of 0-3 mm from the polarizing plate was ten to thirty times slower than that produced by intracellular polarization,
756 M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA and action potentials were always initiated, even in cells which did not respond actively to intracellular stimulation (Fig. 2A (d)), as reported previously (Tomita, 1967). It was not possible to detect any consistent difference in the electrotonic potentials due to extracellular polarization between cells which responded or failed to respond to intracellular stimulation. (a)
A
(b)
(C)
-"_
B
C
] 10-9A
j50mV 100 msec
Fig. 3. Responses to intracellular polarization, (a) and (b), and to transmural nerve stimulation (c) from three different cells (A, B and C) from mouse vas deferens. The upper traces are records of current, the lower traces, membrane potential. Responses to transmural stimulation, with 0 5 msec pulses of varying intensity, were correlated with the wayinwhich the cell responded to intracellular stimulation.
Electrical responses in the mouse vas deferens The responses of the mouse vas deferens to intracellular current injection were similar to those of the guinea-pig vas deferens. However, the input resistance measured in this way was greater than that of the guineapig vas deferens, ranging from 20 to 200 M. Electrotonic potentials were slower and their time course was more variable. In the mouse, rx ranged from 3 to 25 msec. In cells with a high input resistance T. was longer than that of cells with a low input resistance (see Figs. 3 and 4). Cells with a high input resistance were readily excited by intracellular
757
MOUSE VAS DEFERENS (c)
(b)
(a)
A
C
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751
SOME PROPERTIES OF THE SMOOTH MUSCLE OF MOUSE VAS DEFERENS
BY MOLLIE E. HOLMAN, G. S. TAYLOR AND T. TOMITA* From the Department of Physiology, Monash University,
Clayton, Victoria, 3168, Australia (Received 13 September 1976) SUMMARY
1. Contractions of the mouse vas deferens in response to electrical stimulation differ from those recorded from the guinea-pig vas deferens in that they are abolished by tetrodotoxin. 2. Changes in membrane potential were recorded from the smooth muscle of both preparations in response to stimulation with current pulses applied by an intracellular electrode and by large extracellular plate electrodes. 3. Both preparations behaved similarly in response to intracellular stimulation. Electrotonic potentials in response to extracellular current pulses spread in a longitudinal direction in the guinea-pig vas deferens in accordance with the cable-like properties of this preparation. In contrast, no longitudinal spread of electrotonus was observed in the mouse vas deferens. 4. Responses to nerve stimulation differed in the two preparations. In the guinea-pig, single stimuli caused excitatory junction potentials (e.j.p.s) which gave rise to action potentials. Some cells from the mouse vas deferens showed similar e.j.p.s and action potentials, although the threshold for the initiation of action potentials was lower and more variable. 5. The majority of cells in the mouse vas deferens failed to show action potentials in response to a single stimuli even though the amplitude of e.j.p.s was from 35 to 40 mV. This was probably due to the large resting membrane potential of these cells, as all-or-nothing action potentials could be evoked if successive e.j.p.s were allowed to sum with each other or if a depolarizing current pulse was applied at the peak of an e.j.p. 6. The nature of the response to nerve stimulationrecorded from different * Present address: Japan.
School
of Medicine, Fukuoka University, Fukuoka 814,
752 M. E. HOLMAN, C. S. TA YLOR AND T. TOMITA cells in the mouse vas deferens could be correlated with the amplitude and time course of the response of the same cell to intracellular stimulation. 7. It is concluded that individual smooth muscle cells in both preparations are probably coupled electrically but that there are few, if any, low resistance pathways in the longitudinal direction in the mouse vas deferens. INTRODUCTION
It has been shown that many smooth muscles have cable-like properties when current is applied with large extracellular electrodes and membrane potentials are measured with intracellular electrodes (Tomita, 1970; Bennett, 1972; Tomita, 1975). The conduction of action potentials in a variety of smooth muscles, including the ureter, taenia coli and vas deferens of the guinea-pig can be explained by these cable properties which appear to be independent of the pattern or density of innervation. Similar cable properties have also been observed in smooth muscles which do not normally appear to generate action potentials (Mekata, 1971). Conducted action potentials may be readily evoked with large extracellular electrodes in the guinea-pig vas deferens but it is difficult to initiate an action potential by intracellular injection of depolarizing current (Hashimoto, Holman & Tille, 1966; Bennett, 1967; Tomita, 1967). This observation may be explained by the theory that current injected by an intracellular electrode spreads in three-dimensions through the interconnexions between cells which give smooth muscle its cable properties (Noble, 1966; Tomita, 1970; Bennett, 1972). The electrical behaviour of the mouse vas deferens in response to intracellular stimulation appears to be qualitatively similar to that of the guinea-pig vas deferens (see Hashimoto & Holman, 1967). However, there is some doubt as to whether or not the mouse vas deferens also has cable-like properties when current is applied with large external electrodes, since contractions appear to be limited to the region of the stimulating electrodes (Furness & Burnstock, 1969). In the present experiments, responses of the mouse vas deferens to the application of current pulses by intracellular and external electrodes were compared with those of the guinea-pig in an attempt to clarify the possible differences of the electrical properties of these two smooth muscles. An attempt has also been made to account for the differences in the responses of these two preparations to nerve stimulation. Some of the results have been communicated to the Australian Physiological and Pharmacological Society (Tomita, Taylor & Holman, 1974).
MOUSE VAS DEFERENS
753
METHODS
Male guinea-pigs and mice were stunned, bled and the vasa deferentia were dissected out. Contractile activity was recorded via a strain gauge from whole vasa deferentia (about 1*5 cm long) suspended vertically in an organ bath of 4 ml. capacity and perfused continuously with physiological saline at 350 C. For electrical stimulation of the preparation, Ag-AgCl ring electrodes (diameter 3 mm) were placed at each end of the tissue. For external current application, a method similar to that described by Abe & Tomita (1968) was used. The bath (35 mm long, 10 mm wide and 6 mm deep) was divided into two sections, one for recording and the other for polarizing the preparation, by a thin silver plate (200 /sm thick) which had two holes having different diameters. This plate was used to polarize the smooth muscle fibres with long current pulses of weak intensity and also to stimulate intramural nerve fibres with short current pulses of strong intensity (transmural stimulation). A segment from the central region of a guinea-pig vas deferens or mouse vas deferens was put through the hole fitting their diameter. The side of the plate facing the recording chamber was insulated. In some experiments, guinea-pig and mouse vasa deferentia were set up simultaneously using both holes. For intracellular stimulation, a single micro-electrode was used to pass current and to record the potential produced. The voltage drop across the micro-electrode was compensated by means of a circuit incorporated in the WPI 4A Electrometer. Micro-electrodes were filled with 3m-KCI and had resistances between 40 and 60 Mn. The input resistance for intracellular current injection was determined from the ratio of the steady-state change in membrane potential and the amplitude of the current pulse. When the time course of the change in membrane potential in response to an intracellular current pulse was plotted logarithmically as a function of time, it was apparent that the time course of the onset and decay of the change in membrane potential was approximated by an exponential function; the time constant of this function will be referred to as r. The time constant of the smooth muscle membrane, determined from the application of current by large external electrodes (Abe & Tomita, 1968) will be referred to as r,. An analysis of the changes in membrane potential caused by the injection of current at a point in a syncytial structure has been described by Jack, Noble & Tsien (1975). They derived equations for a model (two infinite parallel sheets of membrane) in which the membrane area increased as a function of r2 where r was the distance from the point of current injection. Fig. 5-3a of Jack et al. (1975) shows their computed data for the time course of changes in membrane potential (V). In this figure, V is plotted as a function of T = t/Tm, where Tm is the membrane time constant given by the product of membrane resistance (Rm; Q cm2) and membrane capacitance (Cm; uzF/cm2). Values of V are plotted for various values of R = n/A2. In this model, A2 = Rm.b/2R1 where R. is the specific resistivity of the material between the sheets of membrane and b is the distance between the membranes. Wben these data were plotted logarithmically it was apparent that when R = 0-1 the relation between V and T could be approximated by a single exponential function. According to Jack et al. (1975) the time constant of this function T. should be one or two orders of magnitude shorter than the membrane time constant Tm. Physiological saline contained (mm): NaCl 120, KCl 5.9, CaCl2 2-5, MgCl2 1-2, NaHCO3 25-0, and glucose 11-5. The solution was in a reservoir and aerated with gas mixture of 5 % CO2 and 95% 02, and flowed through the chamber at 0-5 ml./min. The temperature of solution in the chamber was kept constant at 35 ± 0.50 C.
M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA
754
RESULTS
Mechanical responses in the guinea-pig and mouse vasa deferentia Fig. 1 shows effects of tetrodotoxin (4 x 10-7 g/ml.) on contractions of the guinea-pig and mouse vasa deferentia. Contractions produced by repetitive current pulses (20 Hz) of less than 1 msec in duration were completely abolished by tetrodotoxin, which is known to block the
(a)
A -
B
_
° 0 -
-
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-L
t
0-5 g jj0-5 g 5jl~
1
_
__
-101 01g
(b) Tetrodotoxin
_Jk
.
_
.s
_a
_
_
30 sec
Fig. 1. A, mechanical responses of guinea-pig and B, mouse vasa deferentia to electrical stimulation in the absence (a) and in the presence (b) of tetrodotoxin (5 x 10-7 g/ml.). Repetitive stimulation with 0-5 msec pulses (5V for guinea-pig and IOV for mouse) produced a sustained contraction in the guinea-pig but a bimodal response in mouse vas deferens. A single pulse of 1 see duration (3-5 and 7 V) caused contractions in both preparations but contractions in the mouse were small. Tetrodotoxin blocked responses to repetitive stimulation in both preparations and to single pulse stimulation in mouse but did not affect contractions produced by single pulses in
guinea-pig.
effects of nerve stimulation without affecting the action potentials of smooth muscles (Builbring & Tomita, 1967; Hashimoto, Holman & McLean, 1967; Kuriyama, Osa & Toida, 1966). When the duration of the current pulses was increased to more than 10 msec, contractions of the guinea-pig vas deferens could still be evoked in the presence of tetrodotoxin, probably due to direct stimulation of the muscle. On the other hand, in the mouse vas deferens, it was not possible to produce a detectable contraction by external stimulation after application of tetrodotoxin, even if the pulse duration was increased to 100 msec or more.
MOUSE VAS DEFERENS
755
Electrical responses in the guinea-pig vas deferens Responses of the guinea-pig vas deferens to intracellular stimulation were the same as observed previously (Hashimoto et al. 1966; Bennett, 1967; Tomita, 1967). In Fig. 2, the responses to intracellular stimulation (a) and (b), were compared with those to external stimulation, (c) and (d), in two different cells (A and B). In general, responses to depolarizing (a)
(b)
(c)
(d)
B ] 10-9 A ______________
~
~
JS0mV
200 msec
50 msec
Fig. 2. Intracellular records from two different cell types (A and B) in guinea-pig vas deferens. (a) responses to hyperpolarizing current and (b) responses to depolarizing current applied by an intracellular electrode; upper traces show current, lower traces, changes in membrane potential. (c) and (d) show responses to hyperpolarizing and depolarizing current pulses applied with large extracellular plate electrodes. The current calibration (10-9 A) applies only to intracellular polarization.
current applied with an intracellular electrode, could be classified into two types. One type of cell showed a lower input resistance (from 10 to 30 MQ), a faster time course (or ranged from 1 to 5 msec) and no active response, as shown in Fig. 2A. The other type of cell was characterized by a higher input resistance (from 20 to 50 MQ) a slower time course (Tr ranged from 5 to 15 msec) and an active response (Fig. 2B). An active response could be obtained from about 15 % of penetrations and an inactive response from about 80%. For convenience we will refer to cells as either 'active' or 'inactive'. However some intermediate forms of response were also observed. When the preparation was polarized with external large electrodes, as shown in Fig. 2 (c) and (d) the time course of electrotonic potentials recorded at a distance of 0-3 mm from the polarizing plate was ten to thirty times slower than that produced by intracellular polarization,
756 M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA and action potentials were always initiated, even in cells which did not respond actively to intracellular stimulation (Fig. 2A (d)), as reported previously (Tomita, 1967). It was not possible to detect any consistent difference in the electrotonic potentials due to extracellular polarization between cells which responded or failed to respond to intracellular stimulation. (a)
A
(b)
(C)
-"_
B
C
] 10-9A
j50mV 100 msec
Fig. 3. Responses to intracellular polarization, (a) and (b), and to transmural nerve stimulation (c) from three different cells (A, B and C) from mouse vas deferens. The upper traces are records of current, the lower traces, membrane potential. Responses to transmural stimulation, with 0 5 msec pulses of varying intensity, were correlated with the wayinwhich the cell responded to intracellular stimulation.
Electrical responses in the mouse vas deferens The responses of the mouse vas deferens to intracellular current injection were similar to those of the guinea-pig vas deferens. However, the input resistance measured in this way was greater than that of the guineapig vas deferens, ranging from 20 to 200 M. Electrotonic potentials were slower and their time course was more variable. In the mouse, rx ranged from 3 to 25 msec. In cells with a high input resistance T. was longer than that of cells with a low input resistance (see Figs. 3 and 4). Cells with a high input resistance were readily excited by intracellular
757
MOUSE VAS DEFERENS (c)
(b)
(a)
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