Clayton, Victoria, 3168, Australia

Transcription

1 J. Physiol. (1977), 266, pp With 7 text-ftgure8 Printed in Great Britain 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: School of Medicine, Fukuoka University, Fukuoka 814, Japan.

2 752 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 ofthe 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).

3 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 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.

4 754 M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA 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 - _ - 3 -L t g jj0-5 g 5jl~ g (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.

5 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 50 msec ~ ~ JS0mV 200 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,

6 756 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 cellresponded 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

7 MOUSE VAS DEFERENS 757 (a) (b) (c) A n C - _ B~~~~~~~~~~~~~~ D J_ 10-9 A 100 msec - e =< - -- ]so mv 50 msec " 200 msec Fig. 4. Records from two different cells (A, B from one cell and C, D from another cell) from mouse vas deferens. Responses to intracellular polarization (a), to polarization with external large electrodes (b) and to intramural nerve stimulation (c). Depolarizing currents are applied in A(b) and C (b) and hyperpolarizing current in B(b) and D(b) but no polarization was observed even though current intensity was the same as in A(c) and B(c) in Fig. 2. For nerve stimulation, 1 msec pulses of different intensities were used. Although polarity of nerve stimulation was different in this experiment for A(c) and C(c), the pattern of the response was independent of polarity and was correlated with the way in which the cell responded to intracellular stimulation.

8 758 M. E. HOLMAN, a. S. TA YLOR AND T. TOMITA stimulation and often responded repetitively during prolonged depolarization. Action potentials elicited by intracellular stimulation were mv in amplitude and 3-5 msec in duration at half-maximum amplitude (see Figs. 3B and C, 4C). The proportion of penetrations during which action potentials could be recorded was higher (about 30%) in the mouse than in the guinea-pig vas deferens. Cells with a lower input resistance and faster electrotonic potentials generally failed to give action potentials in response to intracellular stimulation (see Figs. 3A and 4A). As in the guinea-pig vas deferens intermediate types of response were also observed. In the mouse vas deferens, no electrotonic potentials could be observed when polarizing current pulses were applied externally using the same large plate electrodes as those used for the guinea-pig vas deferens. Fig. 4(b) shows the failure to detect electrotonic potentials in two different cells (at a distance of 0 3 mm from the polarizing plate). Even a potential field about three times that which gave a polarization of 30 mv in the guinea-pig vas deferens failed to produce a clear polarization in the mouse vas deferens. Excitatory junction potentials Furness & Burnstock (1969) have drawn attention to differences in the response to nerve stimulation of guinea-pig and mouse vas deferens. Their results were confirmed and extended in our experiments. In the guinea-pig vas deferens, there was no difference in the excitatory junctional potentials (e.j.p.s) and the action potentials evoked by the e.j.p. in different cells. In every instance an increase in stimulus intensity caused a progressive increase in the amplitude of the e.j.p. E.j.p.s which exceeded about 25 mv in amplitude caused 'all-or-nothing' action potentials. Fig. 5 shows e.j.p.s and action potentials recorded from an inactive cell (A) and from an active cell (B) in response to nerve stimulation. The difference in the time course of electrotonic potentials produced by intracellular polarization was clearly evident (A(a) and B(a)). Although the configuration of e.j.p.s in cell (A) and cell (B) was slightly different in this experiment, such differences were within the range of variations in each type of cell. These responses to intramural nerve stimulation confirmed previous reports (Burnstock & Holman, 1961; Kuriyama, 1963). In the mouse vas deferens, the configuration of e.j.p.s and active responses differed in different types of cells according to their response to intracellular stimulation. In an inactive cell, large slow e.j.p.s were generally initiated (Figs. 3(c), 4(c)). As stimulus intensity was increased the amplitude of e.j.p.s increased, reaching an amplitude of mv in a graded manner. The rate of rise of the e.j.p. also increased as the stimulus was

9 MOUSE VAS DEFERENS 759 increased. After the e.j.p. exceeded about 30 mv faster secondary depolarizations often appeared on the rising phase or during the peak of the e.j.p. (Fig. 4A(c)). These may have been aborted action potentials. In active cells from the mouse vas deferens it was more difficult to demonstrate gradation of the amplitude of a single e.j.p. with stimulus A (a) (b) (C) 8 ~~~~~~~~~~A ] A _< t ~~~~~~~~~~~~~~~50 MV 50 msec 200 msec Fig. 5. Records from an inactive cell (A) and an active cell (B) from guinea. pig vas deferens. (a) and (b) show responses to current pulses applied with an intracellular electrode and (c) responses to transmuial nerve stimulation at two different intensities with 05 msec pulses. strength. The amplitude of e.j.p.s in response to a threshold stimulus showed marked fluctuations and in some cells these e.j.p.s ranged from 10 to 25 mv in amplitude. Action potentials were initiated when the e.j.p. amplitude exceeded mv. A further increase in stimulus strength caused a decrease in the delay before the action potential was initiated and an increase in the amplitude of the falling phase of the e.j.p. which followed the action potential. The time course of the falling phase of e.j.p.s in active cells was relatively fast and in some cells, the time course of the e.j.p. was similar to that of spontaneous e.j.p.s. Some cells showed responses which were intermediate between those described for typical active and inactive cells. Since it was impossible to elicit an action potential in inactive cells by extracellular or intracellular stimulation or by a single e.j.p. it seemed possible that the membrane of these cells might be electrically inexcitable. The results shown in Fig. 6 appear to rule out this possibility. If a large

10 760 M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA depolarizing current pulse was applied to an inactive cell towards the end of the peak of the e.j.p., or during its falling phase, an action potential could always be initiated. Furthermore if one or more e.j.p.s were elicited by repetitive stimulation at frequencies which allowed successive e.j.p.s to sum with each other an action potential was always elicited (see Fig. 7) when the membrane was depolarized to about -25 mv. ] 10-9A 5OmV 200 msec Fig. 6. Large slow excitatory junction potentials (e.j.p.) recorded from an inactive cell from the mouse vas deferens. The records on the right show the response to a pulse of depolaiizing current (monitored by the upper trace) which was passed through the recording electrode during the e.j.p. DISCUSSION One explanation for the behaviour of both guinea-pig and mouse vas deferens in response to intracellular stimulation might be the existence of intracellular connexions between neighbouring cells (Bennett, 1967; Tomita, 1967). Further evidence for such connexions in the guinea-pig vas deferens follows from the cable-like properties of this preparation. In the mouse, however, there was no evidence for the spread of electrotonic potentials or action potentials in a longitudinal direction.

11 MOUSE VAS DEFERENS 761 If it is assumed that the cells of the mouse vas deferens are completely uncoupled electrically and that their dimensions are similar to the cells of the guinea-pig vas deferens, it is possible to estimate the values of Rm and Cm (see Hashimoto et at. 1966). If it is further assumed that the current from an intracellular electrode is uniformly distributed over the cell membrane, Rm is equal to the product of the input resistance and the surface area of the cell and rx is equal to the product of Rm and Cm. Merrillees (1968) found that the surface area of cells of the guinea-pig vas deferens was about 5 x 1O-5 cm2. The value of Rm calculated in this 50 mv 50mV L 200 msec Fig. 7. The effect of three transmural stimuli on two inactive cells from mouse vas deferens. Summation of the depolarization caused by successive e.j.p.s lead to the initiation of active responses. I

12 762 M. E. HOLMAN, G. S. TAYLOR AND T. TOMITA way for cells of the mouse vas deferens having the lowest input resistance and shortest time constant (inactive cells), was about 1000 Q cm2 and the value of Cm for these cells was about 3 gf/cm2. The corresponding values of Rm and Cm for cells having the highest input impedance and longest time constant (active cells) were 10,000 Q cm2 and 2 5 #uf/cm2 respectively. The values of Rm and Cm calculated for other smooth muscles from their cable properties are about 100,000 Q cm2 and 1,tF/cm2 respectively and STm is about 100 msec (Bennett, 1972; Tomita, 1975). Hence it must be concluded that if the inactive cells of the mouse vas deferens are uncoupled from their neighbours, their membrane resistance is substantially lower than that of other smooth muscles. It seems more likely that the low value of rx for these cells is due to the spread of current into neighbouring cells (see Methods). For active cells, the estimated value of Rm was higher and rx was longer (up to 25 msec). Perhaps this may indicate that these cells are less extensively coupled to their neighbours than are inactive cells. When intracellular recordings were made from the guinea-pig vas deferens it was most probable that the cells whose activity was sampled were part of the outer longitudinal layer where there is evidence that cells are arranged in well-organized bundles which branch and anastomose with each other (Merrillee, 1968). Throughout our experiments on the mouse vas deferens we gained the impression that most active cells were impaled soon after the electrode touched the surface of the preparation, whereas inactive cells were found most commonly during deeper penetrations. We have considered the possibility that active cells may be part of the longitudinal smooth muscle and inactive cells may occur in the circular coat. Preliminary histological studies have indicated that the arrangement of the longitudinal muscle of wall of the mouse vas deferens differs from that of the guinea-pig. In transverse sections of mouse vas deferens the longitudinal muscle was usually confined to two crescentshaped bands, one on either side of the vas deferens. In most sections these bands failed to cover the whole of the circular layer. The thickness of these bands was variable but in some transverse sections the maximum width of the band was comparable with the width of the circular layer. The smooth muscle cells within the longitudinal bonds appeared to be organized into bundles which were smaller than those of the longitudinal layer of the guinea-pig vas deferens. In the present experiments no systematic attempt was made to verify the site from which recordings were made histologically so that no conclusion can be drawn at present as to whether active and inactive cells are arranged into the two different layers. Furness & Burnstock (1969) have drawn attention to differences in the behaviour of the guinea-pig and mouse vas deferens in response to nerve

13 MOUSE VAS DEFERENS 763 stimulation. Bennett (1972) has provided a model which accounts for the time course of the e.j.p. in the guinea-pig vas deferens which lasts for about 1 sec. In his model the time course of the e.j.p. is dictated mainly by the diffusion of noradrenaline from its release sites within a bundle of smooth muscle cells into the extracellular space surrounding the bundle. When such e.j.p.s reach mv an all-or-nothing action potential is initiated. During transmural stimulation of the guinea-pig vas deferens this probably occurs, more or less simultaneously, over a sufficiently large distance to ensure that a conducted action potential is initiated. Active cells in the mouse vas deferens responded to nerve stimulation in a somewhat similar way though the time course of the e.j.p. was approximately a tenth that recorded in the guinea-pig. This may be due to a more rapid decay in the concentration of transmitter perhaps because the bundles of smooth muscle cells are smaller than in the guinea-pig vas deferens. If active cells are sufficiently uncoupled from their neighbours, release of noradrenaline from relatively few varicosities might cause sufficient depolarization to evoke an action potential. This view is supported by the findings that action potentials could be induced by relatively small depolarizing currents applied by an intracellular electrode and that just threshold e.j.p.s in these cells fluctuated very markedly. It is puzzling that under the conditions of these experiments a single e.j.p. recorded from an inactive cell in the mouse vas deferens failed to evoke an action potential. It is difficult to be sure of the value of the resting membrane potential when one is recording below the surface of the preparation; however we have consistently recorded values of -70 mv from deeper cells. Hence we suggest that the depolarization associated with a single e.j.p. is subthreshold for the initiation of an action potential in inactive cells. It is also possible that the initiation of an action potential by a second e.j.p. or a depolarizing current pulse may have been facilitated by the preceding depolarization (Anderson & Ramon 1976). It will be interesting to discover whether or not there are other mammalian smooth muscles whose properties resemble those of the inactive cells of the mouse vas deferens and if these properties can be correlated with the pattern and density of innervation. We wish to thank Mrs V. Bernath for her skilful assistance with some of these experiments. REFERENCES ABE, Y. & TOMITA, T. (1968). Cable properties of smooth muscle. J. Physiol. 196, ANDERSON, N. C. & RAMON, F. (1976). Interaction between pacemaker electrical behaviour and action potential mechanism in uterine smooth muscle. In Physiology

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