mcmaster university, hamilton, ontario, canada 4. spiking activity

14
J. Physiol. (1987),392, pp. 21-34 21 With 8 text-figures Printed in Great Britain MYOGENIC ELECTRICAL CONTROL ACTIVITY IN LONGITUDINAL MUSCLE OF HUMAN AND DOG COLON BY EDWIN CHOW AND JAN D. HUIZINGA From the Intestinal Disease Research Unit and the Department of Neurosciences, McMaster University, Hamilton, Ontario, Canada (Received 13 May 1986) SUMMARY 1. The myogenic electrical activities of longitudinal muscle cells of the dog and human colon were investigated using intracellular microelectrodes. 2. The resting membrane potentials of dog and human longitudinal muscle cells at the serosal side of the muscle layer were -49-4 + 0-9 and - 44-8 + 1.3 mV respectively. 3. Spontaneous electrical activity consisted of electrical oscillations of 13-7 + 1 1 mV and 8-6 + 2-1 mV amplitude, and 19-8 + 1I0 cycles/min and 2641 + 1-6 cycles/min frequency for dog and human cells respectively. 4. Spiking activity only occurred superimposed on the electrical oscillations; the mean rate of rise of spikes was - 150 mV/s in the dog and - 260 mV/s in human cells and that of the oscillations was - 18 mV/s in the dog and - 16 mV in human cells. 5. Spiking activity was abolished by calcium influx blockers and 0;01 mM-calcium Krebs solution. The amplitude of the electrical oscillations was reduced to 0-2-1-0 mV 30 min after calcium influx blockade or 30 min in 0-01 mM-calcium Krebs sol- ution. 6. Because of the high frequency of the oscillation-spike complexes, there was summation of associated contractile events in such a way that contraction frequency corresponded to frequency of bursts of oscillations and not to the frequency of the individual oscillations. 7. The resting membrane potential of the longitudinal muscle cells at the my- enteric plexus side of the layer was -44-9 + 1I0 mV, significantly lower than at the serosal side. 8. A gradient in membrane potential and slow-wave amplitude exists in circular muscle of dog colon, with the highest value at the mucosal side (-68-4 and 28-1 mV respectively) and the lowest at the myenteric side (- 62-5 and 8-6 mV) of the muscle layer. 9. Differences between resting membrane potential and electrical activity of longitudinal and circular muscle cells of the dog colon measured at the myenteric side of both muscle layers suggests absence of electrotonic coupling between the two types of cells. 10. Similarity of resting membrane potentials of longitudinal and circular muscle cells of the human colon suggests possible electronic coupling. 11. Since the electrical oscillations in longitudinal muscle control occurrence of

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J. Physiol. (1987),392, pp. 21-34 21With 8 text-figuresPrinted in Great Britain

MYOGENIC ELECTRICAL CONTROL ACTIVITY IN LONGITUDINALMUSCLE OF HUMAN AND DOG COLON

BY EDWIN CHOW AND JAN D. HUIZINGAFrom the Intestinal Disease Research Unit and the Department of Neurosciences,

McMaster University, Hamilton, Ontario, Canada

(Received 13 May 1986)

SUMMARY

1. The myogenic electrical activities of longitudinal muscle cells of the dog andhuman colon were investigated using intracellular microelectrodes.

2. The resting membrane potentials of dog and human longitudinal muscle cells atthe serosal side of the muscle layer were -49-4 + 0-9 and - 44-8 + 1.3 mV respectively.

3. Spontaneous electrical activity consisted of electrical oscillations of 13-7 + 1 1mV and 8-6 + 2-1 mV amplitude, and 19-8 + 1I0 cycles/min and 2641 + 1-6 cycles/minfrequency for dog and human cells respectively.

4. Spiking activity only occurred superimposed on the electrical oscillations; themean rate of rise of spikes was - 150 mV/s in the dog and - 260 mV/s in humancells and that of the oscillations was - 18 mV/s in the dog and - 16 mV in humancells.

5. Spiking activity was abolished by calcium influx blockers and 0;01 mM-calciumKrebs solution. The amplitude of the electrical oscillations was reduced to 0-2-1-0mV 30 min after calcium influx blockade or 30 min in 0-01 mM-calcium Krebs sol-ution.

6. Because of the high frequency of the oscillation-spike complexes, there wassummation of associated contractile events in such a way that contraction frequencycorresponded to frequency of bursts of oscillations and not to the frequency of theindividual oscillations.

7. The resting membrane potential of the longitudinal muscle cells at the my-enteric plexus side of the layer was -44-9 + 1I0 mV, significantly lower than at theserosal side.

8. A gradient in membrane potential and slow-wave amplitude exists in circularmuscle of dog colon, with the highest value at the mucosal side (-68-4 and 28-1 mVrespectively) and the lowest at the myenteric side (- 62-5 and 8-6 mV) of the musclelayer.

9. Differences between resting membrane potential and electrical activity oflongitudinal and circular muscle cells of the dog colon measured at the myenteric sideof both muscle layers suggests absence of electrotonic coupling between the twotypes of cells.

10. Similarity of resting membrane potentials of longitudinal and circular musclecells of the human colon suggests possible electronic coupling.

11. Since the electrical oscillations in longitudinal muscle control occurrence of

E. CHOW AND J. D. HUIZINGA

spiking activity and type of contraction, they may be called 'electrical controlactivity'.

INTRODUCTION

The circular and longitudinal muscle layers of the colon have different myogenicelectrical activities (Huizinga & Daniel, 1986). However, only the circular musclelayer of the colon of the cat (Christensen, Caprilli & Lund, 1969) and the dog (El-Sharkawy, 1983) have been investigated extensively using intracellular electrodetechniques. No studies directly comparing intracellular activities from both musclelayers are available to date. The electrical activity of the circular muscle layer of thedog colon (El-Sharkawy, 1983; Chambers, Kingma & Bowes, 1984; Huizinga, Chang,Diamant & El-Sharkawy, 1984) consists of regular high-amplitude slow waves atfixed frequency. This activity is not sensitive to stretch or inhibitory substances. Itperforms a control function in that it determines the occurrence of spiking activitywhich only appears on the most depolarized phase of the slow wave. Such anomnipresent slow wave is not reported to occur in the longitudinal muscle layer.Intracellular recordings showed electrical quiescence only (Durdle, Kingma, Bowes& Chambers, 1983). With extracellular techniques, electrical oscillatory activity ofirregular amplitude and frequency (El-Sharkawy, 1983; Huizinga & Daniel, 1986)was observed. The present study will give a complete description of intracellularlymeasured longitudinal muscle activity, and will compare this with circular muscleactivity.The circular muscle electrical activities of normal human colon have not been

recorded using intracellular recording techniques. Impalements were either unsuc-cessful (Van Merwyk & Duthie, 1980) or showed sporadic oscillatory activity(Huizinga, Stern, Chow, Diamant & El-Sharkawy, 1985). In tissue from Hirschprung'sdisease patients (Kubota, Itoh & Ikeda, 1983), intracellular electrical activity wasobserved, which was similar to electrical activity observed in normal tissue recordedusing extracellular techniques (Huizinga et al. 1985). Some data on intracellularactivity of longitudinal muscle cells of human colon have been obtained (Duthie &Kirk, 1978; Van Merwyk & Duthie, 1980; Huizinga et al. 1985). The present studywill describe the intracellular electrical activity of longitudinal muscle cells in detailand will compare it with longitudinal muscle activity from the dog colon.An important reason for the undertaking of this study was to answer the question

as to whether or not the colonic longitudinal muscle has myogenic 'electrical controlactivity'.

METHODS

Tissue preparationHuman. Segments of human tissue from the sigmoid and proximal colon were obtained from

individuals undergoing surgery for cancer. Strips of muscle were taken at least 5 cm away from thesite of cancer, and the tissue in between was histologically free of tumour and inflammation. Thesegments were immediately put into oxygenated Krebs solution and transported to the laboratoryfor preparation and immediate experimentation. With the mucosal side up, the tissue was pinnedflat to the Sylgard floor of a dish containing continuously oxygenated Krebs solution. The mucosawas removed by sharp dissection.

Dog. Dogs, either male or female, were anaesthetized using pentobarbitone (35 mg/kg) given i.v.The colon was exposed by a mid-line abdominal incision and a 10-12 cm segment of proximal or

22

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON

distal colon was removed. The segment was placed on cotton gauze moistened with oxygenatedKrebs solution and opened flat. Colonic contents were carefully removed. The segment was thenpinned flat to the Sylgard bottom of a dissecting dish filled with oxygenated Krebs solution andthe mucosa was removed. The Krebs solution was oxygenated continuously and changed every10 min.

Microelectrode experimentsFor intracellular recordings of the electrical activity, a small segment was pinned to the floor of

a continuously perfused muscle chamber. Microelectrodes filled with 3 M-KCl were used as describedpreviously (Huizinga et al. 1985). With human tissue, a segment with the mucosal side up from theintertaenial region was used for penetration of circular cells; longitudinal muscle was removed. Asegment from one of the taenia with the serosal side up was used for penetration of longitudinalmuscle cells; the circular muscle layer was removed. With dog tissue, penetrations were carried outon both sides of each muscle layer. The circular muscle layer was penetrated from the 'mucosal'side with mucosa, and most of longitudinal muscle removed, and from the 'myenteric plexus' sidewith mucosa, and longitudinal muscle removed. The longitudinal muscle layer was penetrated fromthe 'serosal' side with the circular muscle removed, and from the 'myenteric plexus' side, withcircular muscle removed. Electrical recordings were made from the superficial cells only. Electrodeswere never driven through a cell to reach a subsequent layer of cells. The size of the segment was15 x 5 mm. Half of the segment was pinned on a Sylgard bottom to prevent excessive movement;the other half of the preparation was attached to a force transducer to record tension.

Suction electrode experimentsDose-response relationships to D600, nitrendipine and extracellular calcium were carried out

with suction electrodes. The procedure has been described previously (Huizinga et al. 1985).

Solution and drugsAll experiments were performed at 37 'C. The composition of the Krebs solution was (mM): NaCl,

120-3; KCl, 5-9; CaCl2, 2-5; MgCl2, 1-2; NaHCO3, 15-4; NaH2PO4, 1-2; glucose, 11 5. The solution wasequilibrated with 95% 02-5% CO2. The pH of the solution was 7 3-7 4. Tetrodotoxin (TTX) wasobtained from Sigma, St Louis, U.S.A.; D600 was obtained from Knoll, F.R.G., and nitrendipinewas a gift from R. A. Janis of the Miles Institute for Preclinical Research.

TerminologyColonic smooth muscle generates electrical oscillatory activities at various frequencies and with

different rates of rise. Using accepted terminology where possible, short-lasting (50-100 ms dur-ation) oscillations are referred to as spikes or spike action potentials. Slower oscillations (1-2 sduration) are referred to as 'electrical oscillations'. Oscillations lasting > 2 s, at - 6 cycles/min,only recorded in circular muscle of dog colon, are referred to as slow waves.

Data presentationData are presented as mean values+ standard error of the means. Mean rate of rise was

measured. With a spike superimposed on a slower electrical oscillation, two separate measurementswere made. Significance of difference was calculated by the t test.

RESULTS

Dog colon longitudinal muscleThe resting membrane potential of the dog colon longitudinal muscle cells,

measured at the serosal side of the muscle layer, was -49 4+09 mV (Table 1 andFig. 1). Spontaneous electrical oscillations (for terminology, see Methods) occurred,with an amplitude which varied between tissues from 9-3 to 24-3 mV and a frequencyfrom 12-0 to 25-5 cycles/min. Mean rate of rise of the oscillations was 18-2 + 2-5 mV/s,superimposed on which spiking activity could occur with a mean rate of rise of1509 + 31-0 mV/s. The threshold for spiking activity was - 35-1 +I1 mV. This

23

E. CHOW AND J. D. HUIZINGA

Circular mucosal side

-68 mV

r Circular myenteric plexus side-62 mV

F Longitudinal myenteric plexus side-42 mV

Longitudinal serosal side

-50 mV

1 minFig. 1. Comparison between intracellularly recorded electrical activity of the circular andlongitudinal muscle of the dog colon, recorded at both sides of the muscle layers. See alsoTable 1.

TABLE 1. Comparison of intracellularly recorded electrical activity of the colon at different sites

Oscillation/slow wave

m.p. Amplitude Frequencyn (mV) (mV) (cycles/min)

Dog colon longitudinal (serosal) 19 (57) -494+09 13-7+1-1 19-8+1-0Dog colon longitudinal (myenteric) 6 (18) -449 + IOa 14-2+ 1*6 16-9+0*9Dog colon circular (myenteric) 6 (28) -62-5+19 8&6+2-1 5-5+0-2Dog colon circular (mucosal) 8 (24) -68-4+1 3b 28-1 + 1-1c 51+0.2Human colon longitudinal (serosal) 9 (25) -44-8+±13 8-5+0-9 26-1 + 1P6Human colon circular (mucosal) 6 (18) -42-3 ±0-8 3.3+1.4 Irregularn = number of subjects, with number ofimpalements in parentheses; m.p. = resting membrane

potential.a Significantly different from dog longitudinal (serosal) value, P < 0 05.b Significantly different from dog circular (mucosal) value, P < 0 05.e Significantly different from dog circular (mucosal) value, P < 0 001.

activity occurred in basically three different patterns (see Table 2). The mostcommon type (twenty-seven out of fifty-seven impalements) showed continuousoscillatory activity, either with constant amplitude of oscillation (sixteen out oftwenty-seven; Fig. 2B) or with variable amplitude (eleven out of twenty-seven;Fig. 1). Spikes occurred superimposed on the oscillations. Another type showed inter-mittent activity (seventeen out of fifty-seven; Fig. 2A). Bursts of oscillations alter-nated with periods of relative electrical quiescence. The burst duration was274+ 74 s; the burst frequency was 0-7 + 01 bursts/min. Spikes occurred super-imposed on the oscillations. Interaction between intrinsic nerve activity and longi-tudinal muscle activity was reported previously (Huizinga et al. 1985), and weconfirmed in this study that periodic activity is myogenic and not necessarily a

24

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON 25

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Fig. 2. Different patterns of dog colonic longitudinal muscle activity. Top tracings of eachpanel are intracellularly recorded electrical activity; lower tracings are simultaneouslymeasured mechanical activity. A, burst-type activity. Note that contractions fuse, andconsequently contraction frequency equals burst frequency and not the frequency of theelectrical oscillations. B, continuous activity. The associated mechanical activity is asustained tonic contraction. Similar activity with superimposed spikes is seen in Fig. 1 C,'quiescence'. Small-amplitude oscillations (< 1 mV) are often seen but are sometimesreduced to noise level. There is virtually no tone when electrical activity is 'quiescent'.

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5 sFig. 3. Intracellularly recorded activity from human colonic longitudinal muscle. A,recording from sigmoid colon; continuous oscillatory activity with superimposed spikes;the associated contractile activity is a tonic contraction; the trace shows that the con-traction is a summation of contractions initiated by the individual oscillation-spikecomplexes. B, recording from proximal colon; continuous electrical oscillatory activitywithout spiking activity; mechanical activity was not recorded. C, activity from thesigmoid colon; three oscillation-spike complexes are shown at low and high recordingspeed.

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON

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1 minFig. 4. Effect of calcium entry blockade on electrical activity in dog colonic longitudinalmuscle. A, spontaneous, intracellularly recorded, electrical activity. The oscillatory ac-tivity is continuous but oscillation amplitude is variable and the occurrence of spikesirregular. B, 5 min after addition of D600 (5 x 10-7 M), spikes are abolished and oscillationsare reduced in amplitude; after 30 min presence of D600 (10-8 M), the electrical oscillatoryactivity further reduced to noise level.

consequence of periodic neural activity: TTX (5 x 10- M) did not affect burst typeactivity (n = 3). The third type showed quiescence during 5 min or more continuousrecording (thirteen out of fifty-seven; Fig. 2C). Occasionally small oscillations werevisible (Table 2). There was no significant difference between resting membranepotential of quiescent and active muscle cells.

Differences in characteristics of electrical activity between proximal and distalcolon were not observed. Therefore data from both sections are combined in Tables1 and 2.Each oscillation spike complex was associated with contractile activity (Fig. 2).

Also, the oscillation itself contributed to contraction (Fig. 2B). Because of the highoscillation frequency, the contractions did not relax completely before the nextcontraction arrived and the contractions were summed (Fig. 2A). Therefore theoscillation frequency did not correspond to the contraction frequency. When theoscillatory activity was continuous, the contraction was a sustained tonic one. Whenthe electrical activity was periodic, then the contraction frequency corresponded tothe electrical burst frequency.A minimal amount of stretch was necessary to observe spontaneous electrical

activity. There was no consistent relationship between the amount of stretch and thepattern of electrical activity.The resting membrane potential of the muscle cells at the myenteric side of the

muscle layer was -449+1I0 mV which is significantly lower than the resting po-tential at the serosal side. The oscillation amplitude and frequency were not sig-nificantly different (see Table 1). The preparation showed either continuous or bursttype activities, similar to the above described patterns.

Human colon longitudinal muscleThe human colon longitudinal muscle cells measured at the serosal side of the

muscle layer had a resting membrane potential of -44-8 mV (Table 1 and Fig. 3).

27

E. CHOW AND J. D. HUIZINGA

0.5 mV

C

30 sFig. 5. Effect ofnitrendipine on human colon longitudinal muscle activity. A, spontaneouselectrical activity recorded with an extracellular suction electrode; one or more spikes areseen superimposed on the oscillations. B, activity 15 min after addition of nitrendipine(3 x 10-8 M); note reduction of spiking activity, burst duration and oscillation amplitude.C, after 25 min in nitrendipine (10-7 M) spikes are abolished; some low-amplitude oscil-lations remain.

The electrical oscillations of the longitudinal muscle cells were variable in amplitudeand frequency comparing different tissues (Table 2). Spiking activity only occurredsuperimposed on the oscillations (Fig. 3). The mean rate of rise of the oscillation was15-6+ 4 0 mV/s, and that of the spike was 260+ 77 mV/s. The threshold for spikingactivity was -37-1 + 0 9 mV, very similar to the dog colon. The different patterns ofelectrical activity described for the dog colon were also observed in the human colon:continuous activity (six out of nine preparations), burst type activity (two out ofnine) and electrical quiescence (one out of nine).

Longitudinal muscle strips were obtained from proximal colon (n = 3) and sigmoidcolon (n = 6). No differences were observed and data were combined. The relationshipbetween electrical activity and contractile activity was as observed in the dogcolon.

Involvement of calcium in longitudinal muscle activityCalcium influx blockade by D600 and nitrendipine, and calcium-free Krebs sol-

ution abolished contractile activity in both dog and human longitudinal muscle.In the dog colon, D600 (1 x 10--5 X 10-7 M) abolished spiking activity, decreased

amplitude of oscillations from 0-32 + 0-11 mV to 0 09+ 0-03 mV (n = 1O, P < 0 05,suction electrode experiments) and reduced burst duration from 25+5 s to 12+3 s(n = 4, P < 0-05). Similar results were obtained with nitrendipine (3 x 10-8-1 x 10-7 M).Oscillatory activity was reduced to noise level after 50 min in D600 (10-6 M, n = 10).The sensitivity of the oscillations and spikes to calcium entry blockers was confirmedwith intracellular measurements. However, pharmacological experiments with micro-electrodes in tissue in which spiking activity occurred was difficult since spiking-induced contractions frequently caused disconnection of cell and electrode. Never-theless, Fig. 4 shows abolishment of spiking activity by D600 (5 x 10-7 M) andreduction of the oscillation amplitude from 10 to 5 mV. After 30 min in D600 theoscillation amplitude was reduced further. Some oscillatory activity remained but

28

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON 29

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1 miFig. 6. Effect of D600 on activity of human colonic longitudinal muscle. Upper tracingsof each panel show intracellular electrical activity, lower tracings mechanical activity. A,spontaneous electrical activity; the oscillatory activity is continuous with superimposedspikes. B, 5 min after addition of D600 (5 x 10-1 m) spikes are abolished. C, after 20 minin D600 oscillations are reduced in amplitude. D, after 30 min presence of D600 (10-6 m)the electrical oscillatory activity is further reduced in amplitude; however, oscillations <,1 mV remain. B, C and D show that the oscillatory activity contributes to contractileactivity.

the amplitude approached noise level and accurate measurement of amplitude andfrequency was not possible. Very similar results were obtained with nitrendipine(IO-' m; n = 2). Five minutes after nitrendipine application (IO-' m; n = 2) spikingactivity was abolished and the oscillation amplitude reduced from 15-9 +04 mV to4-5 + 0-5 mV, with concomitant decrease in membrane potential from 49-5+13 mVto 43-5 + 0-5 mV. After 15 min the oscillation amplitude decreased to VS5 + 0-5 mVand the membrane potential to - 42-0+I 0 mV. The oscillation frequency did notchange. After 30 min the oscillation amplitude was reduced to < 0-2 mV.

In the human colon, nitrendipine (3 x 10-'-10-' m; suction electrode experiments)reduced the frequency of spiking activity by 80-100%; the oscillation amplitude was0-6+02 mV before and 0-4+0 mV 10 min after nitrendipine (10-v m; n = 7; Fig. 5).This was not significantly different. In tissues which showed burst-type activity(n = 4), the burst duration reduced from 30-0+3-5 s to 14-0+2-1 s (P < 0-01). After30 min in nitrendipine (10-v m) spiking activity was abolished and oscillation am-

plitude reduced to < 01 mV. Intracellular measurements confirmed these data. After5 min in the presence of nitrendipine (10-1 m; n = 2), the oscillation amplitude hadreduced from 8-7 + 0-7 mV to 2-7 + 2-0 mV, with a decrease in membrane potentialfrom 45-0 ± 341 mV to 39-2±+34 mV. After 30 min the oscillation amplitude had

E. CHOW AND J. D. HUIZINGA

1 mV

ig

C

D

1 minFig. 7. Effect of lowering extracellular calcium on activity of the human colon longitudinalmuscle. A, activity in normal Krebs solution; electrical activity is extracellularly re-corded. B, activity after 5 min in 0-2 mM-Ca2". C, activity after 5 min in 005 mM-Ca2".Low-amplitude oscillations remain. D, recovery in 2-5 mM-Ca2+.

reduced to < 0-2 mV without further decrease in membrane potential. D600(5 x 10-7M) gave similar results (Fig. 6): spikes disappeared within 5 min; after30 min in D600 (10-6 M) continuous oscillatory activity remained at 0-2-1-0 mVamplitude.

In calcium-free medium with 1 mM-ETGA present, spikes disappeared and oscil-lations reduced to noise level in 7-2 + 1-0 min in the dog colon (n = 5) and 8-7 + 3-0 minin the human colon (n = 3) longitudinal muscle. The effect of gradual reduction ofextracellular calcium was studied in dog (n = 6) and human (n = 5) tissue. Reductionof calcium to 0-5 mm did not affect activities (Figs 7 and 8). With the extracellularcalcium concentration < 041 mm, spiking activity was inhibited. Oscillation ampli-tude was reduced to < 041 mV, often not distinguishable from noise. Figure 7 showscontinuous presence ofthe oscillatory activity at - 0-4 mV amplitude. The oscillationfrequency was reduced to 22 cycles/min from 27 cycles/min in normal Krebssolution.

Dog colon circular muscleCircular muscle activity is traditionally measured from cells situated at the mu-

cosal side of the circular muscle layer. The activities and membrane potentials weobserved from these cells were similar to those reported previously (El-Sharkawy,1983; Chambers et al. 1984). The resting membrane potential was - 68-4+ 1-3 mV.Electrical slow-wave activity was recorded at constant frequency (5-1 cycles/min)and amplitude (28-1 mV) (Fig. 1 and Table 1).We also recorded from cells at the myenteric plexus side of the circular muscle

30

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON

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Fig. 8. Effect on lowering extracellular calcium on some parameters of extracellularelectrical activity of the human (n = 5) and dog (n = 6) colon longitudinal muscle.

layer, to compare characteristics of these cells with those of the myenteric side of thelongitudinal muscle layer. The resting potential was - 625 + 1P9 mV, the slow-waveamplitude 8-6 + 21 mV and its frequency 5-5 cycles/min.Comparison of this activity with that of the longitudinal muscle cells at the

myenteric plexus side showed that there was significant difference in resting mem-brane potential and frequency of electrical oscillations.

Human colon circular muscleThe circular muscle cells of the human colon had a resting membrane potential of

-42-3+0O8 mV. Measured intracellularly the membrane was either electricallyquiescent or showed small oscillatory activity with occasional spiking activity butvery irregular (Table 1). Electrical oscillatory and spiking activity have been des-cribed in this tissue using other techniques (Huizinga et al. 1985; Huizinga, 1986). Aregular slow-wave activity as described for the dog colon circular muscle was notobserved.

DISCUSSION

The present study shows that colonic longitudinal muscle activity is similar in thedog and human with respect to the characteristics of the oscillatory activity, spikingactivity and threshold for spiking activity. Thus, for longitudinal muscle activity,

31

E. CHOW AND J. D. HUIZINGA

the dog colon may be used as a model for the human colon. Activity consists of aslow oscillation (mean rate of rise - 17 mV/s) occurring within the frequency rangeof 12-0-25-5 cycles/min (dog) and 20O0-31-8 cycles/min (human). This oscillationdetermines the type -of contraction. With a continuous oscillation, the musclepresents a tonic contraction. With a burst-type oscillation pattern, the musclepresents a phasic contractile activity at the electrical burst frequency. The oscillationis myogenic in nature, since it is present with nerve conduction blockage. Thecontractile force is enhanced by spiking activity superimposed on the sloweroscillations. Since the spiking activity is restricted to the depolarized phase of theoscillations, the oscillatory activity can be called 'control activity'. We reportedpreviously (Huizinga et al. 1985) that intrinsic nervous activity can change burst-type activity into continuous activity or vice versa. However, we also showed, andconfirmed in this study using intracellular microelectrodes, that periodic activityoccurs in the presence of nerve conduction blockade. In these circumstances stretchis probably the major stimulant, suggesting that a continuous stimulation of the,myogenic oscillator can cause periodic activity. This is in complete agreement withresults obtained with a recently developed computer model. A multiport-synthesizedrelaxation oscillator (Bardakjian, El-Sharkawy & Diamant, 1982) gives periodic ac-tivity upon continuous (d.c.) stimulation of one of its input portals (B. J. Barjakjian,personal communication).The present study describes for the first time the differences between longitudinal

and circular muscle cells of the dog colon using intracellularly recorded signals fromthe myenteric plexus side of the layers. Comparison shows that their electricalactivities are quite different, in particular the oscillation (slow wave) frequency. Theresting membrane potential of the longitudinal muscle cells is 18 mV depolarizedcompared to the circular muscle cells. The difference in resting membrane potentialand electrical activity between the muscle layers make it unlikely that there iselectrotonic coupling between the layers. It is possible that the intrinsic nervoussystem (El-Sharkawy, 1983) or interstitial cells of Cajal (Thuneberg, 1982) play a rolein co-ordination of activities of both muscle layers.The present study shows that the circular muscle layer does not exhibit uniform

electrical activity. The cells at the mucosal side have the highest resting potentialand the highest amplitude of slow waves. Therefore, it is likely that the circularpacemaker cells are located at the mucosal side of the muscle layer, which wassuggested previously by Durdle et al. (1983). In the dog stomach there is also agradient in slow-wave amplitude and resting membrane potential, with the myentericplexus side having the highest resting membrane potential and the highest slow-wave amplitude (Bauer, Reed & Sanders, 1985). The pacemaker cells may be circularmuscle cells or special cells such as the interstitial cells of Cajal (Thuneberg, 1982).It is also possible that the differences in electrical activity of the cells are primarilycaused by dissimilarity in mechanisms for excitation. Permeability characteristics ofchannels or density of channels may be different.Data from circular muscle of the human colon obtained using the sucrose gap

technique (Huizinga et al. 1985; Huizinga, 1986) and longitudinal muscle obtained inthis study, show that differences exist between circular and longitudinal muscle ofthe human colon but there are more similarities than between their dog colonic

32

LONGITUDINAL MUSCLE ACTIVITY OF THE COLON

counterparts. The main difference is that the oscillation frequency in the longitudinalcells is between 20-0 and 31-8 cycles/min, while the circular muscle has a much widerfrequency range. The resting membrane potential is similar, and therefore electro-tonic coupling between the muscle layers seems possible, especially since fibres fromone layer run into the other layer (Huizinga et al. 1985).

In the oscillation-spike complex, the mean rate of rise of the spike is - 150 mV/s inthe dog and - 260 mV/s in the human; that of the electrical oscillations - 18 mV/sand - 16 mV/s in the dog and human colonic longitudinal muscle cells respectively.A sharp boundary between oscillation and spike was usually not observed, mostprobably because both oscillation amplitude and spike are dependent on extracellularcalcium. The spike is abolished within minutes by calcium-free Krebs and calciuminflux blockers, and is probably due to inward calcium current as in other smoothmuscle (Tomita, 1970). The electrical oscillation amplitude is greatly reduced (> 90 %)by calcium influx blockers and calcium-free solutions. Some oscillatory activityremains in many preparations after 30 min in D600 (10-6 M) or nitrendipine (10-v M).In others, oscillations are not distinguishable from 'noise'. It is likely that part of theoscillation is due to inward calcium current. The fact that the oscillation is associatedwith contractile activity supports this hypothesis. It is also likely that part of theoscillation is due to another process which may be in part dependent on extracellularor intracellular calcium. Sodium inward current may be involved since preliminarydata indicate that the oscillation amplitude is sensitive to reduction of sodium in thesuperfusion fluid (J. D. Huizinga, unpublished). An oscillatory metabolic activitymay also be involved in the origin of the electrical oscillations (Prosser & Mangel,1982).An interesting characteristic of colonic muscle is that no slow-wave frequency

gradient along the long axis of the colon is observed in dog colon longitudinal (thisstudy) and circular (El-Sharkawy, MacDonald & Diamant, 1980; Chambers et al.1984) muscle, or in human colon longitudinal (this study) and circular (Waterfall,Huizinga & Shannon, 1986) muscle. In the small intestine, the slow-wave gradient isthought to be the driving force behind propagating electrical activity which isassociated with propulsive contractile activity (Daniel & Sarna, 1978). Absence of afrequency gradient in the colon is consistent with the idea that propulsion is not theonly function of the colon. It suggests that propulsion may be associated with othertypes of electrical activity. The burst type activity as seen in Figs 2 and 5 may fulfilthis function. Such activity has been associated with propulsive activity in vivo in thedog (Kocylowski, Bowes & Kingma, 1979) and human colon (Schang & Hemond,1985).The authors acknowledge financial support from the Medical Research Council of Canada and the

Canadian Foundation for Ileitis and Colitis. We appreciated the assistance of Ms Anne Shin. Wethank Dr E. E. Daniel for discussion, Mr Colin Ikeson for technical assistance and Mrs ElizabethGuy for secretarial assistance.

REFERENCES

BARDAKJIAN, B. J., EL-SHARKAWY, T. Y. & DIAMANT, N. E. (1982). On a multiport synthesizedrelaxation oscillator representing a bioelectrical rhythm. Proceedings of the Institute of Electricaland Electronics Engineering, volume ofBiomedical Engineering, 4th annual conference, pp. 433-436,Philadelphia, PA: IEEE.

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BAUER, A. J., REED, J. B. & SANDERS, K. M. (1985). Slow wave heterogeneity within the circularmuscle of the canine gastric antrum. Journal of Physiology 366, 221-232.

CHAMBERS, M. M., KINGMA, Y. J. & BOWES, K. L. (1984). Intracellular electrical activity in circularmuscle of canine colon. Gut 25, 1268-1270.

CHRISTENSEN, J., CAPRILLI, R. & LUND, G. F. (1969). Electrical slow waves in circular muscle ofcat colon. American Journal of Physiology 217, 971-976.

DANIEL, E. E. & SARNA, S. K. (1978). The generation and conduction of activity in smooth muscle.Annual Review of Pharmacology and Toxicology 18, 145-166.

DURDLE, N. G., KINGMA, Y. J., BowEs, K. K. & CHAMBERS, M. M. (1983). Origin of slow waves inthe canine colon. Gastroenterology 84, 375-382.

DUTHIE, H. L. & KIRK, D. (1978). Electrical activity of human colonic smooth muscle in vitro.Journal of Physiology 283, 319-330.

EL-SHARKAWY, T. Y. (1983). Electrical activities of the muscle layers of the canine colon. Journalof Physiology 342, 67-83.

EL-SHARKAWY, T. Y., MACDONALD, W. M. & DIAMANT, N. E. (1980). Characteristics of the slowwave activity of the canine colon. In Gastrointestinal Motility, ed. CHRISTENSEN, J., pp. 415-423.New York: Raven Press.

HUIZINGA, J. D. (1986). Electrophysiology of human colon motility in health and disease. Clinicsin Gastroenterology 15, 879-890.

HUIZINGA, J. D. & DANIEL, E. E, (1986). Control ofhuman colon motor function: a review. DigestiveDisease Sciences 31, 865-877.

HUIZINGA, J. D., CHANG, G., DIAMANT, N. E. & EL-SHARKAWY, T. Y. (1984). Electrophysiologicalbasis of excitation of canine colonic circular muscle by cholinergic agents and Substance P.Journal of Pharmacology and Experimental Therapeutics 231, 692-699.

HUIZINGA, J. D., STERN, H. S., CHOW, E., DIAMANT, N. E. & EL-SHARKAWY, T. Y. (1985).Electrophysiologic control of motility in the human colon. Gastroenterology 88, 500-511.

KOCYLOWSKI, M., BoWES, K. L. & KINGMA, Y. J. (1979). Electrical and mechanical activity in theex vivo perfused total canine colon. Gastroenterology 77, 1021-1026.

KUBOTA, M., ITO, Y. & IKEDA, K. (1983). Membrane properties and innervation of smooth musclecells in Hirschsprung's disease. American Journal of Physiology 244, G406-415.

PROSSER, C. L. & MANGEL, A. W. (1982). Mechanisms of spike and slow wave pacemaker activityin smooth muscle cells. In Cellular Pacemakers vol. 1, Mechanisms of Pacemaker Generation, ed.CARPENTER, D. O., pp. 273-301. New York: Wiley & Sons.

SCHANG, J. C. & HEMOND, M. (1985). Myoelectric spike bursts associated with colonic propulsion.Digestive Disease and Sciences 30, 791.

TOMITA, T. (1970). Electrical properties of mammalian smooth muscle. In Smooth muscle, ed.BULBRING, E., BRADING, A. F., JONES, A. W. & TOMITA, T., pp. 197-243. Baltimore: Williams& Wilkins.

THUNEBERG, L. (1982). Interstitial cells of Cajal: intestinal pacemaker cells. In Advances inAnatomy, Embryology and Cell Biology, pp. 1-130. Berlin: Spinger Verlag.

VAN MERWYK, A. J. & DUTHIE, H. L. (1980). Characteristics of human colonic smooth muscle invitro. In Gastrointestinal Motility, ed. CHRISTENSEN, J., pp. 473-478. New York: Raven Press.

WATERFALL, W. E., HUIZINGA, J. D. & SHANNON, S. (1986). The absence of a frequency gradientin electrical slow waves in the human colon. Gastroenterology 90, 1685 (abstract).

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