(Received 12 Augu?st 1965)

Transcription

1 450 J. Phy8iol. (1966), 183, pp With 11 text-ftgure8 Printed in Great Britain ELECTRICAL RESPONSES OF SMOOTH MUSCLE TO EXTERNAL STIMULATION IN HYPERTONIC SOLUTION BY T. TOMITA From the Department of Pharmacology, University of Oxford (Received 12 Augu?st 1965) SUMMARY 1. The electrical responses of single smooth muscle cells of the guineapig taenia coli to external stimulation were studied in two times hypertonic solution and compared with the responses to intracellular stimulation. 2. Exposure to Krebs solution made two times hypertonic by adding sucrose abolished the mechanical movement and stopped the spontaneous electrical activity. The electrical response to stimulation was essentially similar to that in physiological solution. 3. When the tissue was placed between stimulating electrodes, the cells near the cathode were depolarized and produced spikes, while the cells near the anode were hyperpolarized and produced small spikes only with weak stimuli. The cells near the centre were not polarized but produced spikes with a frequency pattern similar to that near the cathode. 4. When both stimulating electrodes were put close together at one end of the tissue, the intracellularly recorded extrapolar polarization changed its polarity at 1-2 mm distance from the stimulating electrode. When an insulating partition was placed between the stimulating and recording site, the reversed polarity was no longer observed and the electrotonic potential spread decayed roughly exponentially with distance from the stimulating electrode. The time course of the electrotonic potential was similar to that predicted from the cable equation applied to nerve. The space constant was mm (S.E. of mean) and the time constant was msec. The cable properties may be explained by assuming that many fibres, connected in series and in parallel, are aggregated as functional units. 5. The strength-duration curve was a simple hyperbola and the chronaxie was about 20 msec. The relation between extracellularly applied current and intracellularly recorded potential showed that membrane resistance decreased with depolarization and slightly increased with

2 EXTERNAL STIMULATION OF SMOOTH MUSCLE 451 hyperpolarization. The spike was propagated in both directions at the same speed as in physiological solution ( cm/sec). 6. Long anodal current often produced electrical activity of low amplitude which seemed to be due to the spike activity near the cathode, because the same frequency modulation was seen in both activities, and external hyperpolarization reduced the size of the propagated spike. Cessation of a strong and long anodal current was followed by slow depolarization, about 1 sec in duration and up to 10 mv in amplitude, which sometimes triggered a spike. 7. The difference between responses to intracellular and to external stimulation may be explained by assuming that different parts of the cell membrane have different electrical properties. They may be: A, areas of close apposition between cells; B, areas capable of generating the slow component; C, an area capable of producing the spike, but less excitable. INTRODUCTION It is known that, in most visceral smooth muscles, excitation is conducted from cell to cell (see Prosser, 1962). If the propagation of the spike is due to electrical transmission, one would expect a low resistance connexion between cells. There are, however, no reports of direct syncytial connexions between cells, although sites of close apposition, the so-called nexuses, have been described (Dewey & Barr, 1962, 1964). In the circular intestinal muscle of the cat, Nagai & Prosser (1963b) applied current intracellularly and recorded an electrotonic potential in another cell nearby. They concluded that there were relatively low-resistance interfibre junctions. However, Sperelakis & Tarr (1965) using the same preparation reported that current flow through one cell did not have any substantial effect on the transmembrane potentials of adjacent cells. In similar experiments performed in the taenia coli of the guinea-pig, we also failed to confirm Nagai & Prosser's result (Kuriyama & Tomita, unpublished). In the taenia coli of the guinea-pig intracellular stimulation evokes a spike only in very few cells and causes no frequency modulation of spontaneous discharge (Kuriyama & Tomita, 1964a, b). External stimulation, however, easily triggers a spike and changes the spontaneous spike frequency (Biilbring, 1955, 1957; Biilbring, Burnstock & Holman, 1958). In the present experiments the electrical responses to external stimulation were studied and compared with those to intracellular stimulation. By using hypertonic solution the spontaneous electrical activity and the movement of the tissue were eliminated so that the electrical responses could be obtained without any disturbance, as in the frog skeletal muscle (Hodgkin & Horowicz, 1957; Howarth, 1958). The results suggest that the 29-2

3 452 T. TOMITA cells are electrically interconnected at some areas of the cell membrane and, moreover, that the spike is produced in a different part of the membrane from that which generates the slow component (cf. Kuriyama & Tomita, 1965). METHODS The preparation and the Krebs solution were the same as previously described (Biilbring 1954; Kuriyama, 1963). Hypertonic solution was made by adding 10 g sucrose to 100 ml. Krebs solution, making it two times hypertonic. Since the muscle movements were abolished in hypertonic solution, the Perspex ring for fixing the preparation was usually not used. This did not affect the result. Three different arrangements for electrical stimulation were used. (1) About 10 mm taenia was placed longitudinally between two Ag-AgCl plates (4 x 8 mm) at both ends. The distance between the electrodes and the tissue was 1-2 mm at both ends. (2) Two rings of platinum wire, 3-5 mm apart, were placed at one end of the tissue. The potential shift due to the stimulating current was reduced to a minimum by appropriate spacing of the electrodes before inserting the micro-electrode to record the electrotonic potential. If the artifact was still recorded extracellularly its amplitude was subtracted from the intracellularly recorded potential. (3) Two rings of platinum wire were placed at one end of the tissue. This was then put through a small hole of a celluloid film which served as insulating partition between the stimulating site and the remaining length of the tissue from which records were taken. The partition abolished the artifact caused by the external electrical field. Since the stimulating electrodes were immersed in the bathing solution absolute values for stimulating current intensity cannot be given. Relative values of stimulating current intensity were obtained by recording the potential gradient in the solution with two silver electrodes, 2 mm apart, placed in the stimulating bath. The maximum voltage applied to the stimulating electrodes was 30 V. The stimulus duration was specified in the description of experimental results. RESULTS Just after the insertion of the micro-electrode a low resting potential and small spike amplitude were sometimes observed in normal solution (see also Gillespie, 1962). Then, the resting potential and the spike amplitude increased progressively over a period of sec. If during that time external current was applied, the electrotonic potential also increased in a similar manner to the spike. Therefore, if the resting potential or the spike potential represent phenomena related to the properties of the cell membrane, then the electrotonic potential must also be produced at the membrane. The effect of hypertonic solution on electrical activity. Exposure to hypertonic solution hyperpolarized the membrane by mv (mean 12 mv) (Table 1) and reduced the spike frequency. Spontaneous electrical and mechanical activity stopped within 5-10 min. The tissue remained quiescent for several hours. The effect was completely reversible. The response to intracellular stimulation using the Wheatstone bridge method was the same as that in normal Krebs solution, i.e. the membrane time constant was msec (S.E. of mean) (n = 22) and a spike

4 EXTERNAL STIMULATION OF SMOOTH MUSCLE 453 response was as rare as in physiological solution (see Kuriyama & Tomita, 1965). However, a spike was easily triggered by external stimulation, and the fast spike component could be distinguished from the slow component as during spontaneous discharge in normal Krebs solution. The spike parameters in normal and hypertonic Krebs solution are given in Table 1. The amplitude and the maximum rates of rise and fall of the spike were generally greater than those in Krebs solution. However, in preparations in which a very short spike had been observed in Krebs solution hypertonicity increased its duration mainly by decreasing the rate of fall. TABLE 1. Membrane potential, spike parameters and spike frequency, in normal solution and after 15 min exposure to hypertonic solution Solution Membrane Spike Maximum Maximum Spike potential amplitude rate of rise rate of fall frequency (± S.E.) (± S.E.) (± S.E.) ( ± S.E.) ( ± S.E.) (mv) (mv) (V/sec) (V/sec) (spike/sec) Normal Krebs (n = 32) Hypertonic Krebs (n = 27) When the external K-concentration in hypertonic solution was raised to 18 or 24 mm the membrane was depolarized, and then the spontaneous electrical activity reappeared although the mechanical response did not recover. Electrotonic potential. Figure 1 shows the responses to stimuli of 2 sec duration at three different intensities recorded at three different sites when the tissue was placed between two stimulating electrodes at both ends. The cells near the cathode were depolarized, those near the anode were hyperpolarized, while those in the middle part of the tissue showed no polarization. With a weak stimulating current spikes were recorded over the whole length of the tissue. As the stimulus intensity was increased the spike frequency increased near the cathode and in the middle part of the tissue, but spikes were abolished near the anode. Here small potential changes could still be discerned at a frequency which was the same as that of the spikes in the cells near the cathode. The electrotonic potential was also studied when both stimulating electrodes were placed close together (3-5 mm apart) at one end of the tissue. In Fig. 2a the extrapolar polarization was plotted against the distance between the recording site and the nearest stimulating electrode. It was found to change its polarity at about 1-2 mm from the nearest electrode. When the recording electrode was inserted far from the stimulating electrode, for example at a distance of 3 mm or more, a weak current only produced a spike when the nearest electrode was the cathode (cathodal stimulation). When the current intensity was increased, however,

5 454 T. TOMITA cathodal stimulation produced hyperpolarization which blocked the spike propagation. On the other hand, with strong currents, only anodal stimulation, which depolarized the cells far away, produced a spike. This phenomenon of reversed polarity seemed to be the same as that observed by Bulbring (1955) when one electrode was touching one end of the tissue and the other electrode was immersed in the bathing solution. a b c Bf lfl *lejem0v a b c 5 sec Fig. 1. Electrical responses recorded intracellularly (upper trace) from three different cells to stimulating currents with three different intensities (lower trace) applied externally. (Arrangement of the stimulating electrodes shown in the inset.) The records of the upper row A were taken when the left stimulating electrode was the cathode and the right electrode the anode; the records of the lower row B were taken at reversed polarity. In each row, the three records on the left a were taken from the same cell near the left stimulating electrode, the three middle records b from a cell near the centre of the tissue, and the three right records c from a cell near the electrode on the right. Zero level indicated on the left of each group of records. The relative current intensities are shown in the lower trace. When a partition was placed between the stimulating and the recording site the reversed polarity was no longer observed and the electrotonic potential decreased roughly exponentially with the distance of the recording electrode from the partition, as shown in the lower diagram of Fig. 2 b. When this arrangement was used, the magnitude and the sign of the electrotonic potential depended on the distance between the nearest stimulating electrode and the partition. As the nearest stimulating electrode was moved further away from the partition (C -* C' -> C"), the amplitude of the electrotonic potential decreased and finally reversed its polarity. This could have been predicted from the relation shown in the lower diagram

6 EXTERNAL STIMULATION OF SMOOTH MUSCLE 455 of Fig. 2a. All the following experiments were done with the stimulating electrode close to the partition. a v DEP. HYP. Anode Cathode b AC" CX' V I ~~~~~~~~~~~~~~x Fig. 2. Diagram ofelectrode arrangement (top) and membrane polarization (bottom). a without, b with, insulating partition. V = voltage, x = distance between nearest stimulating electrode and recording electrode in mm. C = cathode, A = anode. Depolarization upward, DEP.; hyperpolarization downward, HYP. (a) When the stimulating electrode nearest to the recording site was the cathode (-), the membrane polarization labelled 'Cathode' was obtained; when the polarity was reversed, the curve labelled 'Anode' was obtained. (b) Stimulating arrangement with cathode nearest partition. The curve showing membrane polarization changed with the distance of the cathode from the partition (C, C', C"). Figure 3 shows examples of the electrotonic potential produced by anodal stimuli of 250 msec at different intensities and recorded intracellularly at different distances from the partition. The electrotonic potential not only decreased in size roughly exponentially with distance, but also in its rates of rise and fall. It was found to have a time course similar to that predicted from the cable theory applied to a nerve fibre by Hodgkin & Rushton (1946). The space constant A was measured from the

7 456 P. POMIPA spatial decay of the electrotonic potential along the tissue. It ranged from 1-4 to 1-9 mm (mean 1P (s.e.) mm, n = 6). The electrotonic potentials obtained near the insulating partition had the time constant of (s.e.) msec (n = 8, range: msec). However, this value could not be taken as the true value of the time constant because current flow near the partition might be complicated due to imperfect insulation around Distance Intensity -* _j ~~~~~~~~~~b-50 min 0 c ~~~~~~~~~~~~~~~~~~~~~~~ 300 msec I;;-50 mv Fig. 3. Intracellular records of electronic potentials (lower trace) produced by externally applied anodal current with three different intensities (upper trace). (a) at 0-7 mm, (b) at 2-2 mm, (c) at 3-3 mm from the partition. the tissue and the distance between the stimulating electrode and the partition. The time constant (Tm) was calculated from the following cable equation (Hodgkin & Rushton, 1946) inserting an appropriate value for T to get the theoretical curve which fitted the actual records of electrotonic potentials at three different distances from the partition: V ) (ex [ + erf (4T+ VT) - e-x[l +erf( X -VT)]} where Vm = electrotonic potential obtained at a given distance from the stimulating electrode; (V)t= = the steady level of the electrotonic potential x=o at zero distance from the stimulating electrode; T = titm; X = x/a. (V)t= was obtained by extrapolating the relationship between the electrotonic

8 EXTERNAL STIMULATION OF SMOOTH MUSCLE 457 potential and the distance from the partition to zero distance. The values from two preparations were calculated in this way. They were A = 16 mm, Tm = 60 msec and A = 1-4 mm, TMn = 80 msec. When the time to reach half-maximum of the electrotonic potential was plotted against distance, the relation was a straight line, as would be expected from the cable property. From this, somewhat longer time constants were obtained, i.e. 70 and 100 msec. These values are consistent with Nagai & Prosser's (1963 b) observations in the circular intestinal muscle of the cat in which they used pressure electrodes; they found the space constant to be 1*03 mm and the time constant 133 msec. Some slight deviations from the time course of the electrotonic potential which was predicted by the cable theory were observed. For example, with strong anodal stimulation, electrical activity often appeared as a result of the conducted activity produced near the cathode, as will be described later. Furthermore, if the current pulse was shorter than msec the rate of fall of the electrotonic potential was usually slower than the rate of rise. An increase of the membrane resistance due to hyperpolarization may be one of the reasons. Another complication was the offresponse which was produced on cessation of a long and strong anodal current which will also be described later. Cathodal stimulation. If the stimulus was sufficiently long (more than 1 sec) repetitive spikes usually appeared. Figure 4 shows the responses to cathodal stimulation of 2 sec duration and different intensities recorded at different distances from the partition. Up to a certain limit, the number of spikes produced by a stimulus increased with its intensity, but, beyond this, increasing the intensity reduced the number of spikes to one (also cf. Fig. 9). The observed maximum frequency was usually about 1/sec. Far away from the partition the electrotonic potential was small, but the frequency pattern of the spikes was similar to that in the cell near the partition. After cessation of a long cathodal stimulus, there was a transient hyperpolarization, the time course of which was very similar to the positive afterpotential following a spike. The relation between extracellularly applied current and intracellularly recorded electrotonic potential as well as action potential is shown in Fig. 5. Since the amplitude of the spike remained almost constant, it may be assumed that a true electrotonic potential across the membrane was actually recorded. The observed rectification shows that membrane resistance decreased with strong depolarization. In some preparations the resistance increased slightly with strong hyperpolarization. Since the strength-duration curve was a simple hyperbola, and the chronaxie was msec in four experiments, a nervous contribution to excitation was unlikely, although there were some qualitative differences

9 458 T. TOMITA between the responses to long and short stimuli. When the stimulating current was short (less than 10 msec), the spike was often produced in a graded manner with short latency, as shown in Fig. 6. When the stimulating current was long (more than 30 msec) the spike appeared in an all-or-none E u.~~~~~~~~~ mmii Eu,]M 50m ]50 mv Fig. 4. Intracellular records (lower trace) of responses to external cathodal stimulation with different intensities (upper trace). Top row: at 0-2 mm; second row: at 0-7 mm; third row: at 1-5 mm; fourth row: at 2-2 mm; bottom row: at 3*3 mm from the partition. Note that the size of the electrotonic potential diminished but the spike frequency pattern remained the same at different recording distances. manner and its latency was sometimes very long (up to 1 sec) at the threshold intensity. The threshold potential at which the spike arose was higher for the spike with short latency than for that with long latency (Fig. 6). The spike was propagated over the whole tissue in both directions at the same speed. The conduction velocity was measured with two micro-

10 EXTERNAL STIMULATION OF SMOOTH MUSCLE 459 electrodes which were inserted 3-5 mm apart. The conduction velocity was 7X (S.E. of mean) cm/sec (n = 57), the same as that obtained in Krebs solution by Bulbring et al. (1958). The refractory period of the conducted spike was much longer than that of the spike which was generated near the recording electrode. The absolute refractory period of the spike which was directly produced by the stimulating current was nearly as short as the duration of the spike (22-36 msec, mean 29 msec in five experiments), while that of the conducted spike was msec (mean 620 msec) in five experiments. mv 40 (80) 'a. 00 / $ 30 (60), Fig. 5. Relation between externally applied current 1 and intracellularly recorded potential mv shown by closed circles. Depolarization upward, hyperpolarization downward. Open circles = peak of the spike potential, voltage scale in brackets.

11 460 T. TOMITA Reducing the width of the tissue by cutting the muscle longitudinally, parallel to the fibre axis, did not affect spike generation, provided the strips were wider than 150,t. However, in a strip of less than 100 It width, the spike was produced in a graded manner (Fig. 7). This result might be I I 50 my'v 100 tmisec I 0 I m 250 rmlsec - 50 m9 L.J 500 mi'scc Fig. 6. Threshold responses recorded intracellularly from the same cell (lower trace) to external current pulses with different duration and amplitude (upper trace). From left to right: Top row 2, 3, 3, 3 msec; middle row 3, 5, 30, 30 msec; bottom row 30, 400, 400, 400 msec. Note different time bases. A,J I -50 l,v' J I -50 mv 200 msec Fig. 7. Intracellular records. Effect on spike generation of reducing the width of the tissue by cutting longitudinally. a and b, superimposed responses to increasing stimulus intensity in a strip of 220,u width; c and d, 130,u. The four records in e were obtained from a strip of 70,u width with increasing stimulus duration, and in f with increasing intensity. The stimulus intensity was adjusted to near threshold.

12 EXTERNAL STIMULATION OF SMOOTH MUSCLE 461 due either to damage or to the small size of the tissue being a limiting factor. The importance of size was suggested by the fact that a graded spike was also produced when a monopolar stimulating electrode of 50 It diameter was used, while an electrode of 100 It in diameter produced a full spike in an all-or-none manner, confirming observations by Nagai & Prosser (1963a). a b c d e 1F g h 1111ll IPIIIiml] somv L 3 sec Fig. 8. Effect of cutting the tissue transversely. The electrotonic potential was evoked by stimulating electrodes A and followed by a conducted spike evoked by electrodes B fr#n the opposite end of the tissue. A small part of the tissue was cut transversely at 0 3 mm, and the micro-electrode was inserted at 1-5 mm from the partition. Records a-d were taken from an intact bundle and, by moving it less than 50,u across, e-i from the cut bundle. Upper trace = stimulus, lower trace = intracellular record. When part of the tissue was cut transversely, at right angles to the axis, the electrotonic potential seen when recordings were taken from an intact bundle (Fig. 8, a-d), could not be recorded when the electrode was inserted into the bundle which was cut (Fig. 8, e-i). As far as passive electrical connexion is concerned, therefore, each bundle appeared to be independent suggesting a very poor electrotonic spread across side connexions. In contrast, the spike evoked by the electrotonic potential (Fig. 8, c, d) was recorded even in the transversely cut bundle (Fig. 8, i) suggesting that spike conduction occurs across side connexions. Anodal stimulation. During a strong and long anodal current pulse small repetitive potential changes were often seen, especially near the stimulating electrode, as shown in Figs. 9 and 11. In the upper row of Fig. 9 are

13 462 T. TOMITA responses to cathodal stimulation. With increasing stimulus intensity repetitive discharge of increasing frequency was observed, though with too strong intensity this was absent. The lower row shows responses to anodal stimulation. When the anodal stimulus intensity was weak, a large spike was observed though its amplitude was smaller than the spike produced by the cathodal stimulus. As the intensity was stepped up, small repetitive responses appeared. These responses seemed to be due to the spike activity produced near the cathode because the frequency patterns were similar, as may be seen by comparing the top and bottom records shown in Fig. 9. r-w EU 'nil I~~~~~~~~~~~~~~~~~-5 * 50 3 sec Fig. 9. Upper trace, stimulus; lower trace, intracellular records. The responses to cathodal (top row) and anodal stimulations (bottom row) from the same cell close to the partition (about 200, distance). For description see text. In order to study these small responses the following experiment was done. Two sets of the stimulating electrodes were placed at both ends of the tissue and an insulating partition separated one end with one pair of electrodes from the remaining part of the tissue from which records were taken, as shown in the inset of Fig. 10. The electrodes A in the recording part (left) were used for triggering the spike and those B in the stimulating part (right) were used for application of the electrotonic potential. As shown in Fig. 10, the amplitude of the conducted spike was decreased when the membrane was hyperpolarized. This reduction of the spike size may be due to a process similar to that operating during long hyperpolarization as shown in Fig. 9. The middle row of Fig. 10 shows a similar response to that in the top row taken at fast sweep to show that the time course was almost the same though the spike amplitude was reduced. If the spike was

14 EXTERNAL STIMULATION OF SMOOTH MUSCLE 463 mv ] 0-5C 10 3 sec -50 mv APAM B 300 msec ]J-50 mv Fig. 10. Effects of external polarization on the spike. (Upper trace = stimulus, lower trace = intracellular record.) The recording micro-electrode (ME) was inserted at about 500 /, from the partition. During hyperpolarization applied with electrodes B, the spike was triggered by electrodes A (top and middle rows), or by electrodes B (bottom row). In the top and middle rows, the polarizing current intensity was successively increased, while current intensity for triggering the spike was kept constant. In the bottom row, the stimulating current intensity was adjusted to threshold intensity whenever the polarizing current was increased. I 0-50 mv a-d -50 mv [ e, f -SO mv g, h _ 2 sec Fig. 11. Intracellular records of responses (lower trace) to long external anodal current pulses of increasing intensity (upper trace). a-d at 0-2 mm; e-h at 0 7 mm from the partition. Note the small potential changes during hyperpolarization and, after cessation of current pulse, the slow depolarization which, in the last record (h), triggered a spike. s

15 464 T. TOMITA generated by the same electrode as that used for the membrane polarization, the spike amplitude remained constant during anodal polarization as shown in the bottom row of Fig. 10. On the cessation of a strong and long anodal current pulse, a slow depolarization, about 1 sec in duration and up to 10 mv in size, was produced. The size of the slow depolarization increased in a graded manner with the strength of the applied current. Sometimes, on cessation of a strong current pulse, it triggered a spike (Fig. 11). DISCUSSION It has been reported that hypertonic solution decreased the nexal area and electrotonic coupling between the cells of the taenia coli and abolished the propagation of the action potential (Barr, Dewey & Evans, 1965). In the present experiments, however, in two times hypertonic solution, although the spontaneous electrical activity stopped, the spike was still propagated at almost the same speed as in physiological solution. In electron micrographs (K. Shoenberg, unpublished observations) the number of nexuses was not noticeably reduced though the intervening extracellular spaces seemed to be enlarged and the cells shrunk. The cessation of the spontaneous activity may be due to hyperpolarization produced by a loss of cell water and an increase of the internal K-concentration (R. Casteels & J. Setekleiv, personal communication). It may also be due to structural changes of the membrane near the nexus where the slow component (pace-maker activity) is probably produced. The fact that increasing the external K-concentration restores the spontaneous electrical discharge in hypertonic solution suggests that the main factor is hyperpolarization. When current is applied externally, an electronic potential can be easily observed from every single cell within 2-3 mm distance from the stimulating electrode. Using external recording, similar observations have been made in the smooth muscle of frog stomach (Shuba, 1961). The behaviour of the electrotonic potential agrees roughly with that which would be expected from a tissue with cable properties. This result is very surprising because the structure of the tissue is very different from that of a simple core conductor, e.g. a nerve fibre, even if low-resistance connexions between cells are taken into account. According to the cable theory, the space constant, A, is proportional to the square root of the radius of the fibre if the specific resistance of the membrane and the cytoplasm are assumed to be constant (Hodgkin & Rushton, 1946). The space constant of the smooth muscle fibre having a diameter of 5,u should be i of that of the skeletal muscle fibre of 80,u

16 EXTERNAL STIMULATION OF SMOOTH MUSCLE 465 diameter. However, the actual value of the space constant of the taenia coli of the guinea-pig is 1-6 mm, which is the same as that of the skeletal muscle fibre (Fatt & Katz, 1951). This could be due to a very high membrane resistance or a very low internal resistance, but this is unlikely because the specific membrane resistance was calculated to be rather lower than that of the skeletal muscle fibre (Kuriyama & Tomita, 1965). It can be assumed that the internal resistance is more or less similar in all excitable tissues. One possible explanation for the long space constant may be that many fibres, connected in series and in parallel, are aggregated in bundles of large diameter (cf. Builbring, 1954). Such bundles might act as functional units. The conception of a functional bundle is also suggested by the observation that the production of the spike becomes graded if longitudinal strips of less than 100 It in width are dissected or if the stimulating electrode is less than 50,u in diameter (cf. Nagai & Prosser, 1963a). If conduction is brought about by a local circuit current in a fibre with cable properties, the conduction velocity (v) is proportional to a factor v = S'IV(2R,) x Vradius/CmVRi (Katz, 1948), where S' is a safety factor, R'm is the active membrane resistance, Cm is the specific membrane capacitance and Ri is the specific internal resistance. The conduction velocity of taenia coli is 7 cm/sec, which is about 2-l of that of skeletal muscle (1.6 m/sec, Katz, 1948). The small fibre diameter could explain the slow conduction velocity, but it was rejected for the above explanation of A in which a large bundle diameter was assumed. However, the slow conduction velocity may also be explained by a large Cm, a low safety factor and high active membrane resistance. Though the Cm of single taenia coli cells was similar to that of skeletal muscle cells (Kuriyama & Tomita, 1965), the Cm of a bundle may be large owing to many aggregated interconnected fibres. The safety factor may be low because of the poorly developed Na-carrier mechanism and the slow rate of rise of the spike (Holman, 1958; Kuriyama & Tomita, 1965). Nevertheless, the above equation might still be insufficient to express all the properties of smooth muscle because the interconnections of the fibres and the non-homogeneity of the cell membrane (to be discussed later) can also modify the conduction velocity. The strength-duration curve can be calculated from the cable theory (Hodgkin & Rushton, 1946) by the following equation, if it is assumed that the critical depolarization (V) is constant: I = V/Re x I/erf /ItIr, where I = strength, t = duration, Re = the effective membrane resistance, and r = the time constant. From this equation the chronaxie is 0-24 x T. From the values of T obtained in the present experiments ( msec), the calculated chronaxie is msec, which agrees well with the 30 Physiol. 183

17 466 T. TOMITA experimental results (about 20 msec). This theoretical description can only be regarded as a rough approximation because only one time factor is considered for the development of the electrotonic potential as well as for an active process, i.e. the local potential. However, the order of magnitude of the calculated and experimentally found values seems to be substantially correct. There are many differences between the responses to external and intracellular stimulation. Intracellular stimulation causes no frequency modulation of the spontaneous discharge, it evokes a spike only in a few cells; and the spike thus triggered has no slow component. An electrotonic spread cannot be detected if current is applied to a nearby cell through an intracellular micro-electrode. The time constant measured with intracellular current application using the Wheatstone bridge method is 2-8 msec. On the other hand, external stimulation produces frequency modulation of the spontaneous activity. A spike with a slow component is produced in all cells, and electrotonic spread is easily observed. The space constant is 1*6 mm and the time constant is msec. Some of these differences may be due to differences in current distribution. Intracellularly applied current might decrease very sharply with distance from the stimulating micro-electrode because of the short space constant, 0*1-0-2 mm, calculated from the electrical properties of a single muscle fibre (Barr, 1961; Sperelakis & Tarr, 1965), or because of the threedimensional current spread through side connexions of the fibres, as in heart muscle (Woodbury & Crill, 1961; Noble, 1962). Externally applied current decays more slowly because of the long space constant (1-6 mm) and the one-dimensional current flow. For these reasons, intracellular stimulation may activate only a very small area, while externally applied current may stimulate a much larger area and many cells. Since intracellular polarization affects only the spike and not the slow component of the spontaneous discharge, it has been suggested that they are produced in different parts of the cell membrane (Kuriyama & Tomita, 1965). The results of the present experiments may also be explained by assuming that a cell membrane is not homogeneous but is composed of areas with different properties. Areas A may be patches of membrane very close to neighbouring cells, perhaps the nexuses. When a potential field is applied longitudinally to the tissue, most of the current flows through these areas from cell to cell. Areas B may surround A. They would produce the slow component of the electrical activity and have the longer time constant. Current passing through A might affect B. Area C may be the remaining part of the membrane. It would produce the spike when the slow component has reached threshold intensity. It

18 EXTERNAL STIMULATION OF SMOOTH MUSCLE 467 would have a shorter time constant and might be electrically less excitable. During intracellular stimulation, most of the current would flow through this membrane. These areas may not be strictly differentiated from each other but may merge gradually. It is also possible that their sizes may change, when the extracellular space, especially the distance between the adjoining cell membranes, is changed by deformation of the cell, for example, due to applied tension or contraction in physiological conditions. It may be assumed that the slow component triggers the spike in physiological conditions. Hence, on the basis of the present hypothesis, when a long current pulse is applied through the external electrode, area B which has a long time constant is excited and a slow component is produced which generates an all-or-none spike. However, when a short current pulse is applied through an external electrode, this stimulates area C which has a short time constant and may produce a spike in a graded manner. Areas B and C may correspond to the different membranes of two different crustacean muscle fibres studied by Dorai Raj (1964). One type of fibre has a long time constant (usually at least 100 msec) and produces no spike. The other type has a short time constant (about 10 msec) and responds to depolarization sometimes in a graded manner and sometimes with an all-or-none spike. The fibres which respond to direct stimulation with only a graded spike produce a full-size spike in an all-ornone manner in response to indirect (nerve) stimulation. Another instance of non-uniformity of a muscle membrane has been reported in Romalea microptera by Werman, McCann & Grundfest (1961). The spike generation in taenia coli seems, sometimes, to be all or none, sometimes more or less graded. The question arises whether the spike size depends on the number of active cells participating, or on the excitability of the single cell. From the effect of external hyperpolarization on the amplitude of the conducted spike (Fig. 10) the second explanation is more likely because the spike amplitude is reduced without change in time course. This observation makes it unlikely that the size of the action potential is determined by electrotonic spread from distant active cells, the conduction of which is blocked during the hyperpolarization. Moreover, intracellular stimulation produces a spike almost always in a graded manner and its amplitude is also increased by conditioning hyperpolarization. Graded excitability of a single cell would also explain the graded responses produced by short current pulses and the fluctuations in spike size during spontaneous activity in normal solution. I wish to thank Dr E. Biilbring for much help and advice, and the U.S. Public Health Service for financial support (Grant No. GM 10404). 30-2

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