450 J. Phy8iol. (1966), 183, pp. 450-468With 11 text-ftgure8Printed in Great Britain

ELECTRICAL RESPONSES OF SMOOTH MUSCLE TOEXTERNAL STIMULATION IN HYPERTONIC

SOLUTION

BY T. TOMITAFrom the Department of Pharmacology,

University of Oxford

(Received 12 Augu?st 1965)

SUMMARY

1. The electrical responses of single smooth muscle cells of the guinea-pig taenia coli to external stimulation were studied in two times hypertonicsolution and compared with the responses to intracellular stimulation.

2. Exposure to Krebs solution made two times hypertonic by addingsucrose abolished the mechanical movement and stopped the spontaneouselectrical activity. The electrical response to stimulation was essentiallysimilar to that in physiological solution.

3. When the tissue was placed between stimulating electrodes, the cellsnear the cathode were depolarized and produced spikes, while the cells nearthe anode were hyperpolarized and produced small spikes only with weakstimuli. The cells near the centre were not polarized but produced spikeswith a frequency pattern similar to that near the cathode.

4. When both stimulating electrodes were put close together at one endof the tissue, the intracellularly recorded extrapolar polarization changedits polarity at 1-2 mm distance from the stimulating electrode. When aninsulating partition was placed between the stimulating and recording site,the reversed polarity was no longer observed and the electrotonic potentialspread decayed roughly exponentially with distance from the stimulatingelectrode. The time course of the electrotonic potential was similar tothat predicted from the cable equation applied to nerve. The spaceconstant was 1-68 + 0-08 mm (S.E. of mean) and the time constant was60-100 msec. The cable properties may be explained by assuming thatmany fibres, connected in series and in parallel, are aggregated as func-tional units.

5. The strength-duration curve was a simple hyperbola and thechronaxie was about 20 msec. The relation between extracellularlyapplied current and intracellularly recorded potential showed that mem-brane resistance decreased with depolarization and slightly increased with

EXTERNAL STIMULATION OF SMOOTH MUSCLE 451

hyperpolarization. The spike was propagated in both directions at thesame speed as in physiological solution (7.3 + 0 7 cm/sec).

6. Long anodal current often produced electrical activity of lowamplitude which seemed to be due to the spike activity near the cathode,because the same frequency modulation was seen in both activities, andexternal hyperpolarization reduced the size of the propagated spike.Cessation of a strong and long anodal current was followed by slowdepolarization, 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 externalstimulation may be explained by assuming that different parts of the cellmembrane have different electrical properties. They may be: A, areas ofclose apposition between cells; B, areas capable of generating the slowcomponent; C, an area capable of producing the spike, but less excitable.

INTRODUCTION

It is known that, in most visceral smooth muscles, excitation is conductedfrom cell to cell (see Prosser, 1962). If the propagation of the spike is dueto electrical transmission, one would expect a low resistance connexionbetween cells. There are, however, no reports of direct syncytial connex-ions 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 inanother cell nearby. They concluded that there were relatively low-resis-tance interfibre junctions. However, Sperelakis & Tarr (1965) using thesame preparation reported that current flow through one cell did not haveany substantial effect on the transmembrane potentials of adjacent cells.In similar experiments performed in the taenia coli of the guinea-pig, wealso 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 spon-taneous discharge (Kuriyama & Tomita, 1964a, b). External stimulation,however, easily triggers a spike and changes the spontaneous spikefrequency (Biilbring, 1955, 1957; Biilbring, Burnstock & Holman, 1958).

In the present experiments the electrical responses to external stimula-tion were studied and compared with those to intracellular stimulation.By using hypertonic solution the spontaneous electrical activity and themovement of the tissue were eliminated so that the electrical responsescould be obtained without any disturbance, as in the frog skeletal muscle(Hodgkin & Horowicz, 1957; Howarth, 1958). The results suggest that the

29-2

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 membranefrom that which generates the slow component (cf. Kuriyama & Tomita,1965).

METHODS

The preparation and the Krebs solution were the same as previously described (Biilbring1954; 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 abolishedin 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 mmtaenia 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 ringsof platinum wire, 3-5 mm apart, were placed at one end of the tissue. The potential shiftdue to the stimulating current was reduced to a minimum by appropriate spacing of theelectrodes before inserting the micro-electrode to record the electrotonic potential. If theartifact was still recorded extracellularly its amplitude was subtracted from the intra-cellularly recorded potential. (3) Two rings of platinum wire were placed at one end of thetissue. This was then put through a small hole of a celluloid film which served as insulatingpartition between the stimulating site and the remaining length of the tissue from whichrecords were taken. The partition abolished the artifact caused by the external electricalfield.

Since the stimulating electrodes were immersed in the bathing solution absolute valuesfor stimulating current intensity cannot be given. Relative values of stimulating currentintensity were obtained by recording the potential gradient in the solution with two silverelectrodes, 2 mm apart, placed in the stimulating bath. The maximum voltage applied tothe stimulating electrodes was 30 V. The stimulus duration was specified in the descriptionof experimental results.

RESULTS

Just after the insertion of the micro-electrode a low resting potentialand small spike amplitude were sometimes observed in normal solution(see also Gillespie, 1962). Then, the resting potential and the spike ampli-tude increased progressively over a period of 10-30 sec. If during that timeexternal current was applied, the electrotonic potential also increased in asimilar manner to the spike. Therefore, if the resting potential or the spikepotential represent phenomena related to the properties of the cellmembrane, then the electrotonic potential must also be produced at themembrane.The effect of hypertonic solution on electrical activity. Exposure to hyper-

tonic solution hyperpolarized the membrane by 10-15 mV (mean 12 mV)(Table 1) and reduced the spike frequency. Spontaneous electrical andmechanical activity stopped within 5-10 min. The tissue remained quies-cent 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 membranetime constant was 2-8 + 0-17 msec (S.E. of mean) (n = 22) and a spike

EXTERNAL STIMULATION OF SMOOTH MUSCLE 453response was as rare as in physiological solution (see Kuriyama & Tomita,1965). However, a spike was easily triggered by external stimulation, andthe fast spike component could be distinguished from the slow componentas during spontaneous discharge in normal Krebs solution. The spike para-meters in normal and hypertonic Krebs solution are given in Table 1. Theamplitude and the maximum rates of rise and fall of the spike weregenerally greater than those in Krebs solution. However, in preparations inwhich a very short spike had been observed in Krebs solution hypertonicityincreased its duration mainly by decreasing the rate of fall.

TABLE 1. Membrane potential, spike parameters and spike frequency, in normal solutionand after 15 min exposure to hypertonic solution

Membrane Spike Maximum Maximum Spikepotential amplitude rate of rise rate of fall frequency

Solution (± S.E.) (± S.E.) (± S.E.) ( ± S.E.) ( ± S.E.)(mV) (mV) (V/sec) (V/sec) (spike/sec)

Normal Krebs 51+0 6 55+0 7 7 0+ 0 4 7-2+0-5 12+0-8(n = 32)

Hypertonic Krebs 63+1-1 72+1-6 10-5+0-4 8-8+0-7 0(n = 27)

When the external K-concentration in hypertonic solution was raised to18 or 24 mm the membrane was depolarized, and then the spontaneouselectrical activity reappeared although the mechanical response did notrecover.

Electrotonic potential. Figure 1 shows the responses to stimuli of 2 secduration at three different intensities recorded at three different sites whenthe tissue was placed between two stimulating electrodes at both ends.The cells near the cathode were depolarized, those near the anode werehyperpolarized, while those in the middle part of the tissue showed nopolarization. With a weak stimulating current spikes were recorded overthe whole length of the tissue. As the stimulus intensity was increased thespike frequency increased near the cathode and in the middle part of thetissue, but spikes were abolished near the anode. Here small potentialchanges could still be discerned at a frequency which was the same as thatof 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 thetissue. In Fig. 2a the extrapolar polarization was plotted against thedistance between the recording site and the nearest stimulating electrode.It was found to change its polarity at about 1-2 mm from the nearestelectrode. When the recording electrode was inserted far from the stimu-lating electrode, for example at a distance of 3 mm or more, a weak currentonly produced a spike when the nearest electrode was the cathode (cath-odal stimulation). When the current intensity was increased, however,

cathodal stimulation produced hyperpolarization which blocked the spikepropagation. On the other hand, with strong currents, only anodal stimu-lation, which depolarized the cells far away, produced a spike. Thisphenomenon of reversed polarity seemed to be the same as that observedby Bulbring (1955) when one electrode was touching one end of the tissueand the other electrode was immersed in the bathing solution.

a b c

Bf|lfl *leJEm0Va b c

5 secFig. 1. Electrical responses recorded intracellularly (upper trace) from three differ-ent cells to stimulating currents with three different intensities (lower trace)applied externally. (Arrangement of the stimulating electrodes shown in theinset.) The records of the upper row A were taken when the left stimulatingelectrode was the cathode and the right electrode the anode; the records of thelower row B were taken at reversed polarity. In each row, the three records on theleft a were taken from the same cell near the left stimulating electrode, the threemiddle records b from a cell near the centre of the tissue, and the three rightrecords c from a cell near the electrode on the right. Zero level indicated on theleft of each group of records. The relative current intensities are shown in thelower trace.

When a partition was placed between the stimulating and the recordingsite the reversed polarity was no longer observed and the electrotonicpotential decreased roughly exponentially with the distance of the record-ing 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 elec-trotonic potential depended on the distance between the nearest stimulat-ing electrode and the partition. As the nearest stimulating electrode wasmoved further away from the partition (C -* C' -> C"), the amplitude ofthe electrotonic potential decreased and finally reversed its polarity. Thiscould have been predicted from the relation shown in the lower diagram

454 T. TOMITA

EXTERNAL STIMULATION OF SMOOTH MUSCLE 455

of Fig. 2a. All the following experiments were done with the stimulatingelectrode close to the partition.

a

b

v

DEP. Anode

HYP. Cathode

AC" CX'

V

I ~~~~~~~~~~~~~~x

Fig. 2. Diagramofelectrode arrangement (top) and membrane polarization (bottom).a without, b with, insulating partition. V = voltage, x = distance between neareststimulating 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 thecathode (-), the membrane polarization labelled 'Cathode' was obtained; whenthe polarity was reversed, the curve labelled 'Anode' was obtained.

(b) Stimulating arrangement with cathode nearest partition. The curve showingmembrane polarization changed with the distance of the cathode from the partition(C, C', C").

Figure 3 shows examples of the electrotonic potential produced byanodal stimuli of 250 msec at different intensities and recorded intra-cellularly at different distances from the partition. The electrotonicpotential 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 coursesimilar to that predicted from the cable theory applied to a nerve fibre byHodgkin & Rushton (1946). The space constant A was measured from the

spatial decay of the electrotonic potential along the tissue. It ranged from1-4 to 1-9 mm (mean 1P62 + 0-08 (s.E.) mm, n = 6). The electrotonicpotentials obtained near the insulating partition had the time constant of55 + 3 (s.E.) msec (n = 8, range: 38-85 msec). However, this value couldnot be taken as the true value of the time constant because current flownear the partition might be complicated due to imperfect insulation around

0

Intensity -*

Distance

-50 min_j ~~~~~~~~~~bI;;-50 mV

c ~~~~~~~~~~~~~~~~~~~~~~~300 msec

Fig. 3. Intracellular records of electronic potentials (lower trace) produced byexternally 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 thepartition. The time constant (Tm) was calculated from the following cableequation (Hodgkin & Rushton, 1946) inserting an appropriate value for Tto get the theoretical curve which fitted the actual records of electrotonicpotentials 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 thestimulating electrode; (V)t= = the steady level ofthe electrotonic potential

x=Oat zero distance from the stimulating electrode; T = tITm; X = x/A. (V)t=

was obtained by extrapolating the relationship between the electrotonic

456 P. POMIPA

EXTERNAL STIMULATION OF SMOOTH MUSCLE 457

potential and the distance from the partition to zero distance. The valuesfrom two preparations were calculated in this way. They were A = 16mm, Tm = 60 msec and A = 1-4 mm, TMn = 80 msec. When the time toreach half-maximum of the electrotonic potential was plotted againstdistance, the relation was a straight line, as would be expected from thecable 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 whichthey used pressure electrodes; they found the space constant to be 1*03 mmand 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 aresult of the conducted activity produced near the cathode, as will bedescribed later. Furthermore, if the current pulse was shorter than 200-300 msec the rate of fall of the electrotonic potential was usually slowerthan the rate of rise. An increase of the membrane resistance due to hyper-polarization may be one of the reasons. Another complication was the off-response which was produced on cessation of a long and strong anodalcurrent which will also be described later.

Cathodal stimulation. If the stimulus was sufficiently long (more than 1sec) repetitive spikes usually appeared. Figure 4 shows the responses tocathodal stimulation of 2 sec duration and different intensities recorded atdifferent distances from the partition. Up to a certain limit, the number ofspikes 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 awayfrom the partition the electrotonic potential was small, but the frequencypattern of the spikes was similar to that in the cell near the partition. Aftercessation of a long cathodal stimulus, there was a transient hyperpolariza-tion, the time course of which was very similar to the positive after-potential following a spike.The relation between extracellularly applied current and intracellularly

recorded electrotonic potential as well as action potential is shown inFig. 5. Since the amplitude of the spike remained almost constant, it maybe assumed that a true electrotonic potential across the membrane wasactually recorded. The observed rectification shows that membraneresistance decreased with strong depolarization. In some preparations theresistance increased slightly with strong hyperpolarization.

Since the strength-duration curve was a simple hyperbola, and thechronaxie was 16-23 msec in four experiments, a nervous contribution toexcitation was unlikely, although there were some qualitative differences

458 T. TOMITA

between the responses to long and short stimuli. When the stimulatingcurrent was short (less than 10 msec), the spike was often produced in agraded manner with short latency, as shown in Fig. 6. When the stimulatingcurrent was long (more than 30 msec) the spike appeared in an all-or-none

E u.~~~~~~~~~

mmii 50m]50 mV

Eu,]M

Fig. 4. Intracellular records (lower trace) of responses to external cathodalstimulation with different intensities (upper trace). Top row: at 0-2 mm; secondrow: at 0-7 mm; third row: at 1-5 mm; fourth row: at 2-2 mm; bottom row: at3*3 mm from the partition. Note that the size of the electrotonic potentialdiminished but the spike frequency pattern remained the same at differentrecording distances.

manner and its latency was sometimes very long (up to 1 sec) at thethreshold intensity. The threshold potential at which the spike arose washigher 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-

EXTERNAL STIMULATION OF SMOOTH MUSCLE 459

electrodes which were inserted 3-5 mm apart. The conduction velocity was7X3 + 0 7 (S.E. of mean) cm/sec (n = 57), the same as that obtained inKrebs 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. Theabsolute refractory period of the spike which was directly produced by thestimulating current was nearly as short as the duration of the spike(22-36 msec, mean 29 msec in five experiments), while that of the con-ducted spike was 180-1300 msec (mean 620 msec) in five experiments.

mV

40 (80) 'a. °° /00$

30 (60) ,

32 11120-

40

Fig. 5. Relation between externally applied current 1 and intracellularly recordedpotential mV shown by closed circles. Depolarization upward, hyperpolarizationdownward. Open circles = peak of the spike potential, voltage scale in brackets.

Reducing the width of the tissue by cutting the muscle longitudinally,parallel to the fibre axis, did not affect spike generation, provided thestrips 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

II

I 0

50 my'v

100 tmisec

I 0

501m

250 rmlsec

- 50 m9

L_ __.J 500 mI'ScCFig. 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; bottomrow 30, 400, 400, 400 msec. Note different time bases.

A

I°

I

-50 l,V'

-50 mV

200 msecFig. 7. Intracellular records. Effect on spike generation of reducing the width ofthe tissue by cutting longitudinally. a and b, superimposed responses to increasingstimulus intensity in a strip of 220 ,u width; c and d, 130 ,u. The four records in ewere obtained from a strip of 70 ,u width with increasing stimulus duration, and in fwith increasing intensity. The stimulus intensity was adjusted to near threshold.

460 T. TOMITA

,J

J

EXTERNAL STIMULATION OF SMOOTH MUSCLE 461

due either to damage or to the small size of the tissue being a limitingfactor. The importance of size was suggested by the fact that a gradedspike was also produced when a monopolar stimulating electrode of 50 Itdiameter was used, while an electrode of 100 It in diameter produced a fullspike in an all-or-none manner, confirming observations by Nagai &Prosser (1963a).

a b c d e 1F g h

1111ll IPIIIiml]°somVL

3 sec

Fig. 8. Effect of cutting the tissue transversely. The electrotonic potential wasevoked by stimulating electrodes A and followed by a conducted spike evoked byelectrodes B fr#n the opposite end of the tissue. A small part of the tissue was cuttransversely at 0 3 mm, and the micro-electrode was inserted at 1-5 mm from thepartition. Records a-d were taken from an intact bundle and, by moving it less than50 ,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 intactbundle (Fig. 8, a-d), could not be recorded when the electrode was insertedinto the bundle which was cut (Fig. 8, e-i). As far as passive electricalconnexion is concerned, therefore, each bundle appeared to be independentsuggesting a very poor electrotonic spread across side connexions. Incontrast, the spike evoked by the electrotonic potential (Fig. 8, c, d) wasrecorded even in the transversely cut bundle (Fig. 8, i) suggesting thatspike 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 stimulat-ing electrode, as shown in Figs. 9 and 11. In the upper row of Fig. 9 are

responses to cathodal stimulation. With increasing stimulus intensityrepetitive discharge of increasing frequency was observed, though with toostrong intensity this was absent. The lower row shows responses to anodalstimulation. When the anodal stimulus intensity was weak, a large spikewas observed though its amplitude was smaller than the spike producedby the cathodal stimulus. As the intensity was stepped up, small repetitiveresponses appeared. These responses seemed to be due to the spike activityproduced near the cathode because the frequency patterns were similar, asmay be seen by comparing the top and bottom records shown in Fig. 9.

EU 'nil I~~~~~~~~~~~~~~~~~-50r-W* 50

3 sec

Fig. 9. Upper trace, stimulus; lower trace, intracellular records. The responses tocathodal (top row) and anodal stimulations (bottom row) from the same cellclose to the partition (about 200 , distance). For description see text.

In order to study these small responses the following experiment wasdone. Two sets of the stimulating electrodes were placed at both ends of thetissue and an insulating partition separated one end with one pair ofelectrodes from the remaining part of the tissue from which records weretaken, as shown in the inset of Fig. 10. The electrodes A in the recordingpart (left) were used for triggering the spike and those B in the stimulatingpart (right) were used for application of the electrotonic potential. Asshown in Fig. 10, the amplitude of the conducted spike was decreased whenthe membrane was hyperpolarized. This reduction of the spike size maybe due to a process similar to that operating during long hyperpolarizationas shown in Fig. 9. The middle row of Fig. 10 shows a similar response tothat in the top row taken at fast sweep to show that the time course wasalmost the same though the spike amplitude was reduced. If the spike was

462 T. TOMITA

EXTERNAL STIMULATION OF SMOOTH MUSCLE 463

3 sec

10-50 mV

300 msec

mV

] 0

- 5C

APAM£ B

]J-50 mV

Fig. 10. Effects of external polarization on the spike. (Upper trace = stimulus,lower trace = intracellular record.) The recording micro-electrode (ME) wasinserted at about 500 /, from the partition. During hyperpolarization applied withelectrodes B, the spike was triggered by electrodes A (top and middle rows), orby electrodes B (bottom row). In the top and middle rows, the polarizing currentintensity was successively increased, while current intensity for triggering thespike was kept constant. In the bottom row, the stimulating current intensity wasadjusted to threshold intensity whenever the polarizing current was increased.

-50 mV [

e, f

I 0

-50 mV

a-d

-SO mV

g, h

_

2 sec

Fig. 11. Intracellular records of responses (lower trace) to long external anodalcurrent pulses of increasing intensity (upper trace). a-d at 0-2 mm; e-h at 0 7 mmfrom 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

generated by the same electrode as that used for the membrane polariza-tion, the spike amplitude remained constant during anodal polarization asshown 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 pro-duced. The size of the slow depolarization increased in a graded mannerwith the strength of the applied current. Sometimes, on cessation of astrong current pulse, it triggered a spike (Fig. 11).

DISCUSSION

It has been reported that hypertonic solution decreased the nexal areaand electrotonic coupling between the cells of the taenia coli and abolishedthe propagation of the action potential (Barr, Dewey & Evans, 1965). Inthe present experiments, however, in two times hypertonic solution,although the spontaneous electrical activity stopped, the spike was stillpropagated at almost the same speed as in physiological solution. Inelectron micrographs (K. Shoenberg, unpublished observations) thenumber of nexuses was not noticeably reduced though the interveningextracellular spaces seemed to be enlarged and the cells shrunk. Thecessation of the spontaneous activity may be due to hyperpolarizationproduced by a loss of cell water and an increase of the internal K-concen-tration (R. Casteels & J. Setekleiv, personal communication). It mayalso be due to structural changes of the membrane near the nexus wherethe slow component (pace-maker activity) is probably produced. The factthat increasing the external K-concentration restores the spontaneouselectrical discharge in hypertonic solution suggests that the main factoris hyperpolarization.When current is applied externally, an electronic potential can be easily

observed from every single cell within 2-3 mm distance from the stimu-lating electrode. Using external recording, similar observations have beenmade in the smooth muscle of frog stomach (Shuba, 1961). The behaviourof the electrotonic potential agrees roughly with that which would beexpected from a tissue with cable properties. This result is very surprisingbecause the structure of the tissue is very different from that of a simplecore conductor, e.g. a nerve fibre, even if low-resistance connexionsbetween cells are taken into account.

According to the cable theory, the space constant, A, is proportional tothe square root of the radius of the fibre if the specific resistance of themembrane and the cytoplasm are assumed to be constant (Hodgkin &Rushton, 1946). The space constant of the smooth muscle fibre having adiameter of 5 ,u should be i of that of the skeletal muscle fibre of 80 ,u

T. TOMITA464

EXTERNAL STIMULATION OF SMOOTH MUSCLE 465

diameter. However, the actual value of the space constant of the taeniacoli of the guinea-pig is 1-6 mm, which is the same as that of the skeletalmuscle fibre (Fatt & Katz, 1951). This could be due to a very high mem-brane resistance or a very low internal resistance, but this is unlikelybecause the specific membrane resistance was calculated to be rather lowerthan that of the skeletal muscle fibre (Kuriyama & Tomita, 1965). It canbe assumed that the internal resistance is more or less similar in all excit-able tissues. One possible explanation for the long space constant may bethat many fibres, connected in series and in parallel, are aggregated inbundles of large diameter (cf. Builbring, 1954). Such bundles might act asfunctional units.The conception of a functional bundle is also suggested by the observa-

tion that the production of the spike becomes graded if longitudinal stripsof less than 100 It in width are dissected or if the stimulating electrode isless than 50 ,u in diameter (cf. Nagai & Prosser, 1963a).

If conduction is brought about by a local circuit current in a fibre withcable properties, the conduction velocity (v) is proportional to a factorv = 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 membranecapacitance and Ri is the specific internal resistance. The conductionvelocity of taenia coli is 7 cm/sec, which is about 2-l of that of skeletalmuscle (1.6 m/sec, Katz, 1948). The small fibre diameter could explain theslow conduction velocity, but it was rejected for the above explanationof A in which a large bundle diameter was assumed. However, the slowconduction velocity may also be explained by a large Cm, a low safetyfactor and high active membrane resistance. Though the Cm of singletaenia coli cells was similar to that of skeletal muscle cells (Kuriyama &Tomita, 1965), the Cm of a bundle may be large owing to many aggregatedinterconnected fibres. The safety factor may be low because of the poorlydeveloped Na-carrier mechanism and the slow rate of rise of the spike(Holman, 1958; Kuriyama & Tomita, 1965). Nevertheless, the aboveequation might still be insufficient to express all the properties of smoothmuscle because the interconnections of the fibres and the non-homo-geneity of the cell membrane (to be discussed later) can also modify theconduction velocity.The strength-duration curve can be calculated from the cable theory

(Hodgkin & Rushton, 1946) by the following equation, if it is assumedthat the critical depolarization (V) is constant: I = V/Rex 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 (60-100 msec),the calculated chronaxie is 14-24 msec, which agrees well with the

30 Physiol. 183

experimental results (about 20 msec). This theoretical description canonly be regarded as a rough approximation because only one timefactor is considered for the development of the electrotonic potentialas well as for an active process, i.e. the local potential. However, theorder of magnitude of the calculated and experimentally found valuesseems to be substantially correct.There are many differences between the responses to external and intra-

cellular stimulation. Intracellular stimulation causes no frequency modu-lation of the spontaneous discharge, it evokes a spike only in a few cells;and the spike thus triggered has no slow component. An electrotonicspread cannot be detected if current is applied to a nearby cell through anintracellular micro-electrode. The time constant measured with intra-cellular current application using the Wheatstone bridge method is 2-8msec. On the other hand, external stimulation produces frequency modu-lation of the spontaneous activity. A spike with a slow component isproduced in all cells, and electrotonic spread is easily observed. The spaceconstant is 1*6 mm and the time constant is 60-100 msec.Some of these differences may be due to differences in current distri-

bution. Intracellularly applied current might decrease very sharply withdistance from the stimulating micro-electrode because of the short spaceconstant, 0*1-0-2 mm, calculated from the electrical properties of a singlemuscle fibre (Barr, 1961; Sperelakis & Tarr, 1965), or because of the three-dimensional current spread through side connexions of the fibres, as inheart muscle (Woodbury & Crill, 1961; Noble, 1962). Externally appliedcurrent decays more slowly because of the long space constant (1-6 mm)and the one-dimensional current flow. For these reasons, intracellularstimulation may activate only a very small area, while externally appliedcurrent may stimulate a much larger area and many cells.

Since intracellular polarization affects only the spike and not the slowcomponent of the spontaneous discharge, it has been suggested that theyare produced in different parts of the cell membrane (Kuriyama & Tomita,1965). The results of the present experiments may also be explained byassuming that a cell membrane is not homogeneous but is composed ofareas 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 thetissue, most of the current flows through these areas from cell to cell.

Areas B may surround A. They would produce the slow component ofthe electrical activity and have the longer time constant. Current passingthrough 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

T. TOMITA466

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 throughthis membrane.

These areas may not be strictly differentiated from each other but maymerge gradually. It is also possible that their sizes may change, when theextracellular space, especially the distance between the adjoining cellmembranes, is changed by deformation of the cell, for example, due toapplied tension or contraction in physiological conditions.

It may be assumed that the slow component triggers the spike inphysiological conditions. Hence, on the basis of the present hypothesis,when a long current pulse is applied through the external electrode, area Bwhich has a long time constant is excited and a slow component isproduced which generates an all-or-none spike. However, when a shortcurrent pulse is applied through an external electrode, this stimulatesarea C which has a short time constant and may produce a spike in agraded manner. Areas B and C may correspond to the different membranesof two different crustacean muscle fibres studied by Dorai Raj (1964). Onetype of fibre has a long time constant (usually at least 100 msec) andproduces no spike. The other type has a short time constant (about 10msec) and responds to depolarization sometimes in a graded manner andsometimes with an all-or-none spike. The fibres which respond to directstimulation with only a graded spike produce a full-size spike in an all-or-none manner in response to indirect (nerve) stimulation. Another instanceof non-uniformity of a muscle membrane has been reported in Romaleamicroptera 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 sizedepends on the number of active cells participating, or on the excitabilityof the single cell. From the effect of external hyperpolarization on theamplitude of the conducted spike (Fig. 10) the second explanation is morelikely because the spike amplitude is reduced without change in timecourse. This observation makes it unlikely that the size of the actionpotential is determined by electrotonic spread from distant active cells,the conduction of which is blocked during the hyperpolarization. More-over, intracellular stimulation produces a spike almost always in a gradedmanner and its amplitude is also increased by conditioning hyperpolariza-tion. Graded excitability of a single cell would also explain the gradedresponses produced by short current pulses and the fluctuations in spikesize during spontaneous activity in normal solution.

I wish to thank Dr E. Biilbring for much help and advice, and the U.S. Public HealthService for financial support (Grant No. GM 10404).

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468 T. TOMITA

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