93
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 93- 100
ReGu le 25 mars 1991.
In vitro electro-mechanical activity of the human colon. Simultaneous recording of the electrical patterns of the t w o muscle layers BY
G. RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT
(I)
and I. GIORGIO
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[Istituto scientific0 gastroenterologico Castellana Grotte (Bari) - Italy; ( I ) Laboratoire de Physiologie - Facult6 de Medicine de Tours, France]
(8 figures)
Electrical and mechanical activity on longitudinal and circular layers of the human sigmoid colon were simultaneously studied. Recordings were obtained from two electrode sites spaced 3 cm apart in a piece of colon which had been resected surgically and perfused in an organ bath. Spontaneous electrical activity of the colon showed slow waves and spikes. Slow waves were present for only 24.5% and 12% of the recording time on the longitudinal and circular layers, respectively, and they appeared as localized activity which was irregular in amplitude and varying in frequency. Electrical coupling between the two muscle layers was rarely seen and slow waves were not associated with pressure changes. Spiking activity were recorded as short and long spike bursts on both muscle layers. Short spike bursts were localized activity superimposed on slow waves. The associated mechanical activity, which consisted of single weak pressure changes or prolonged contractions with summation, was determined by slow wave frequency. Long spike bursts were seen at irregular intervals and were either propagated or not propagated activity associated with electrical oscillations ranging from 24 to 46 cpm. Mechanical activity consisted of sustained tonic contractions propagated or not propagated in the same way as the electrical pattern. Coordinated electrical activity of the two muscle layers seldom occurred when spontaneous activity was being recorded. Electrical activity on both muscle layers was very sensitive to stretching and could be initiated or modulated by pharmacological agents. In particular, our findings showed that stimulation induced coordinated spiking activity on the two muscle layers and caused mechanical activity, propagated orally or aborally, which consisted of long lasting, high amplitude contractions. These findings confirm that stretching and pharmacological agents are the most important triggers and regulators of the electrical and mechanical activity of the colon, whose complex physiology has not yet been well understood.
Introduction The highly complex electro-mechanical activity of the colon has still not been thoroughly investigated. Published works may be divided into in vivo and in vitro studies and it is difficult to conciliate the two sets of data. In man, in vivo colonic electrical activity exhibits slow wave activity which is highly irregular in frequency and amplitude, and spiking activity which consists of short spike bursts (< 10 s) and long spike bursts (> 10 s) (SARNAet al., 1982). Short spike bursts (SSB) are related to slow waves and are localized activity. Long spike bursts (LSB), which may be present with or without a definite relationship to slow waves, are seen to propagate over long segments of the colon and are
et al., associated with transit of content (FREXINOS 1985; SCHANG et al., 1986). In vivo studies are usually performed by means of mucosal electrodes. They may be limited by the capacity of the different types of electrode (TAYLOR et al., 1975; COUTURIER et al., 1969; SARNA et al., 1982) to pick up colonic activity, and by the inconsistent relation among slow waves, spike activity and mechanical activity. Zn vitro studies performed on strips of human colonic smooth muscle have contributed to the understanding of the complex electrophysiology of the colon (HUEINGA et al., 1985). Two important in vitro data may provide a partial explanation for the complexity of in vivo recorded activity : a) the longitudinal and circular layers show different patterns of electrical activity
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94
G.
RIEZZO,
(VANMERWYK & DUTHIE,1980; KUBOTA et al., 1983; et al., 1985) and therefore the activity recordHUIZINGA ed in vivo may be a mixture of electrical signals emanating from both the longitudinal and the circular layers; b) colonic electrical activity is highly sensitive to neural and hormonal influence (WIEBECK& CHRISTENSEN, 1971; SNAPEet al., 1977) which may vary during recording sessions leading to changes in the pattern of electrical activity which are apparently spontaneous. To date, the few in vitro studies carried out on the electrical activity of the human colon have not considered the correlated electrical activity of the two muscle layers, its propagation to adjacent segments of tissue, and the relationship between the electrical and mechanical activity of each muscle layer. There is also a lack of comparative studies aimed at characterizing the electrical and motor activity of both muscle layers of the taeniated colon. Finally, in vitro studies are contradictory and variable with regard to the presence of slow waves, their frequency, and the correlation between these and contractions (DUTHIE& KIRK, 1978; VAN MERWYK & DUTHIE,1980; CHAMBERS et al., 1981; KUBOTAet al., 1983). In the present study, experiments were performed on the human sigmoid colon using a technique which makes the simultaneous recording of the electrical activity of the two muscle layers possible. These were performed in order to investigate the in vitro electrical activity of both muscle layers (taenia and intertaenia), their relation to motor activity, and how all this activity is modulated by stretching and pharmacological agents. Materials and Methods
Tissue preparation Thirty-one specimens of human sigmoid colon were obtained surgically from patients with non obstructing rectal carcinoma. Immediately before the resection of the diseased portion, a 15-18 cm segment of normal sigmoid colon was removed, the blood supply to the segment being maintained until the moment of excision. The distal end of this colonic segment was at least 5 cm from the site of the tumour, and the tissue was histologically free of tumour and inflammation. Immediately after resection, two catheters (Vasocan Braunule 18G - 1.2 mm in diameter) were inserted into the isolated segment, one in a sigmoid artery and the other in a vein. The vascular bed was washed by infusing 20 ml of saline solution containing 2500 UI of heparin through the arterial catheter. Thereafter, the sigmoid segment was placed in oxygenated Krebs solution and immediately transferred to the laboratory. Here, the lumen of the sigmoid segment was washed with oxygenated Krebs buffer solution and the artery was immediately connected to the perfusion system. The sigmoid colon was then transferred to an organ bath consisting of a closed chamber half full of warm water. The segment of sigmoid colon was placed on a plexiglass grill which was suspended above the water level and the saturated water vapour atmosphere was maintained at 37°C.
M. A.
MASELLI,
F.
PEZZOLLA,
J. THOUVENOT AND I. GIORGIO
Recording system Electrical activity was recorded by means of 4 couples of bipolar copper electrodes (0.25 mm in diameter) implanted subserously : two couples (3 cm apart) in the longitudinal muscle layer and two couples (3 cm apart) in the circular muscle layer, placed perpendicularly to the direction of the fibre axis. The distance between the electrodes of each couple was 5 mm. The couples of electrodes were implanted at the same level on the longitudinal and circular muscle layers (Fig. 1). electrodes input
I
I
pressure input longitudlnal layer clrcular layer
1 II
warm water
U
7
1 ]I U
FIG. 1. Apparatus f o r luminal perfusion and f o r the electrical and mechanical recording of the isolated sigmoid colon. The specimen was enclosed in a plexiglass chamber. The figure also shows the position of the electrode sites and of the two latex-baloons (dashed line).
Mechanical activity was recorded by means of two thin-walled latex balloons (3 cm long) introduced into the colon at the same level as the recording electrodes, and connected to pressure transducers via polyethylene tubes (made according to our specifications). A contraction of the colon against the surface of the balloon was recorded as a rise in pressure or a fall in the volume of the balloon. Simultaneous recordings of electrical and mechanical activity were made on a 8-channel ReegaMinihuit TR h v m recorder. The paper speed was 12 cm/min. The electrical signals were recorded with AC (time constant set at 0.1 and 6 s) amplification.
Recording session Following the placement of the sigmoid colon in the perfusion chamber and the setting-up of the recording apparatus, the piece was allowed to equilibrate for 30 min, before a 3 h recording was made. At the start of the recording session, the balloons in the lumen of the sigmoid colon were inflated to a pressure of 5 mmHg, which was found to be the pressure which determined the distension at which spontaneous electrical and mechanical activity was greatest. Electromechanical activity measured under these circumstances was called spontaneous activity but this should not be regarded as unstimulated activity, as stretching and/or the activity of enteric neurons may have contributed to the et al., 1985). To measure activity recorded (HUIZINGA the influence of stretching on electrical and mechanical
HUMAN COLON ACTIVITY
in vitro
95
activity, the volume of the balloons was changed during the experiments. In order to verify the viability of the sample in every case, and consequently the validity of the data obtained, at the end of each recording session methylene blue was administered to verify whether perfusion had been optimal. The colonic segments which failed to respond to methylene blue were rejected because this indicated that they had not been well perfused.
Data presentation Analysis of the slow waves, action potentials and mechanogram was performed by visual inspection of the records for each channel. Slow wave frequency (expressed in cycles per min = cpm) was calculated by counting the number of slow waves present each min; slow wave incidence was calculated as the percentage of the total recording time during which slow waves appeared. The different kinds of spike burst (SSB and LSB) were identified and the recording time occupied by each was measured. The amplitude of slow wave and spike activity was also calculated and expressed in pV. As far as pressure waves were concerned, the amplitude (mmHg) and duration (s) of each pressure wave were measured on the tracing. Of the 31 colonic segments studied, 10 were utilized to study spontaneous activity, 17 to quantify electrical and mechanical activity after excitatory stimulation (carbachol or stretching) and the remaining 4 were rejected because they had not been well perfused. Both the range in which values were observed and the mean values were documented but standard deviation was not as it cannot be stated with certainty that all the specimens came from a homogeneous population.
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Solutions and drugs The Krebs solution (NaCl 117.2 mM; KC15.9 mM; CaC1, 2.5 mM; MgCI, 1.2 mM; NaHC03 15.4 mM; NaH,P04 1.2 mM; glucose 11.5 mM; dextran 2%) was continuously gassed with 95% 0, and 5% CO,. This solution, maintained at 37"C, was delivered to the vascular bed of the sigmoid colon by means of an occlusive roller pump (GILSON MiniPuls 2) at a constant flow rate of 15 ml/min. The pressure of the perfusion was about 60 mmHg. The vascular flow rate was monitored throughout the experiments. The following drugs were used : carbachol (carbamylcholine chloride) and atropine sulfate from Sigma Chemical Co., St Louis, Mo. TABLEI. Spontaneous electrical and mechanical activity.
SLOW WAVES Frequency mean (cpm) range Amplitude mean @V) range Incidence %
SPIKE Amplitude mean @V) range Duration mean ( s ) range Associated oscillations mean (cpm) range Propagation orad mean (cm/s) range aborad mean (cm/s) range
MECHANICAL ACTIVITY Amplitude mean (mmHg) range Duration * mean (s) range
LONG. LA YER
CIRC. LA YER
10.6 (2-16)
(1-14)
110 (50-250)
71.5 (25-180)
24.5
11.9
11.7
SSB
LSB
SSB
LSB
85 (50-200)
131.3 (50-200)
71.7 (25-120)
63 (25-100)
76.2 (45-127)
87 (22.5-172.5)
49.5 (37.5-67.5)
76.8 (22.5-120)
12.4 (1-16)
27.7 (24-46)
12.4 (2-16)
27.7 (24-46)
1 .o (0.9-1.8) 0.4 (0.1-0.6)
0.9 (0.6-1.6) 0.4 (0.2-0.7)
3 (2-6)
3.7 (2.5-6.5)
86.3 (37.5-135)
66 (22.5-105) I
* The duration of mechanical activity is the result of electrical activity recorded both on longitudinal and circular layers. SSB burst; LSB = long spike burst.
=
short spike
96
G. RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT AND I. GIORGIO
In order to study the effect of drugs on colon segments a comparison was made between a 10 min period of activity before the administration of the drug and a 10 min period after. The mean values for the 10 min periods were compared. Results Spontaneous activity
-
Ec 2
.
_ 1 _
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Two patterns of electrical activity were seen : slow waves and action potentials (spikes). SLOW WAVES - Slow waves were found on recordings obtained with a time constant of 6 s. They were found to be absent or lasting no longer than 24.5% of the recording time on the longitudinal layer and 12% on the circular layer. Only in one case were slow waves continually present for the entire recording time. Slow waves were very irregular in amplitude (50-250 pV and 25-180 pV on the longitudinal and circular layers, respectively) and their frequency varied from 2 to 16 cpm (mean 10.6 cpm) on the longitudinal layer and from 1 to 14 cpm (mean 11.7 cpm) on the circular layer (Table I). Two groups of slow wave frequencies with a normal distribution were recorded : a slower rhythm at 2.5 f 0.6 cpm and a faster rhythm at 10.7 & 0.5 cpm on the longitudinal layer; a slower rhythm at 2 -t 0.8 cpm and a faster at rhythm 8.5 0.5 cpm on the circular muscle layer. Electrical coupling between the two muscle layers was rarely seen. Recordings made from adjacent electrode sites 3 cm apart in the same muscle layer, showed that slow waves were usually phase-unlocked. Slow waves were not associated with pressure changes (Fig. 2).
*
FIG. 3. The figure shows short spike bursts simultaneously in Etl and Et2. In P1 and P2 there are weak pressure changes at the same frequency as the electrical activity in Et 1 and Et2. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
Etl Ec 1
1
7
I
100 pv
El2
Ec 2
~~
P 1 I5mmHg
P 2
--
205586
Etl
1 mln
FIG.4. Thefigure shows short spike bursts in Et2 and Ec2 relared to pressure in P2 with summation and “rippling”. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
Ec2 P 1 (6mrnHg
p 2 1 rnin
FIG.2. Thefigure shows aperiod of slow waves in Ecl. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
ACTION POTENTIALS - Short and long spike bursts were recorded. Action potentials consisting of short spike bursts (duration c 10 s) occurred intermittently for a duration of 45 to 127 s (mean 76.2 s) and of 37.5 to 67.5 s (mean 49.5 s) on the longitudinal and circular layers, respectively. The amplitude was 50-200 pV (mean 85 pV) on the longitudinal layer and 25-120 p V (mean 71.7 pV) on the circular layer (Table I).
The short spike bursts were always superimposed on slow waves. When short duration spike activity was present on the longitudinal muscle, the circular muscle showed either electrical quiescence or the same kind of spike potential as the longitudinal layer. The short spike bursts were seen either on only one electrode site at a time, or simultaneously on two adjacent electrode sites without a time lag. These short spike bursts were related to mechanical activity as indicated by the presence of low rhythmic pressure changes (Fig. 3). Contraction frequency was related to spike burst frequency only when slow wave frequency was c 12 cpm. Contractions associated with spike bursts superimposed on slow waves at a frequency higher than 12 cpm showed summation, and a “rippling” at the frequency of the electrical activity was usually present (Fig. 4). Action potentials consisting of long spike bursts (duration > 10 s) were associated with electrical oscillations ranging in frequency from 24 to 46 cpm. The
HUMAN COLON ACTIVITY
in
97
VitrO
cmrbmchol 1Oy 7
Ell
’
- - - -E -C l- -
(100 pv
Et 2
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Ec2
FIG.5. Thefigure shows longspike bursts in EtI and electrical oscillation in Ecl related to electricaloscillations and a sustained tonic contracfion in PI. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P 1 = proximal pressure site; P2 = distal pressure site.
1 man
FIG.6 . Thefigure shows the effect of carbachol I0 pg (see ihe arrow). Long spike bursts without electrical oscillations can be seen. They are propagated in the aboral direction and associated with propagated tonic contractions of long duration (see the arrows). Etl = proximal electrodes on the taenia; Ecl = proximal electrodeson the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P 1 = proximal pressure site; P 2 = distal pressure site.
duration of LSB ranged from 22.5 to 172.5 s (mean 87 s) and from 22.5 to 120 s (mean 76.8 s) on the longitudinal and circular layers, respectively. Amplitude was 50-200 pV (mean 131.3 pV) on the longitudinal layer and 25-100 pV (mean 63 pV) on the circular layer (Fig. 5). LSB appeared in two different patterns : localized either on one electrode or on two electrodes simultaneously, suggesting a local, non propagating activity; or with a time lag, indicating regular
oral or aboral propagating activity (Table I). When LSB were present on the longitudinal muscle, the circular muscle layer showed 3 different patterns : a period of electrical quiescence, short spike bursts, or long spike bursts with electrical oscillations at a frequency of 24-46
TABLE11. Effect of carbachol on electrical activity of the sigmoid colon in vitro. ~
CARBACHOL (I pg)
SLOW WAVE Frequency mean (cpm) range Amplitude mean (JIV) range Incidence
CARBACHOL (10 pgj
CARBACHOL (I00 pg)
LONG. LA YER
LONG. LA YER
CIRC. LA YER
CIRC. LA YER
10.6
(10-11)
8 (5-14)
7.1 (1.8-9)
7 (5-12)
133.3 (100-180)
146.0 (60-200)
132 (80-250)
142 (25-200)
4.5
2.9
3.0
2.8
QJO
SPIKE Amplitude mean (pV) range
116.7 141.7 100-150) (80-200)
135 (70-200)
Duration mean (s) range
82.5 (75-90)
Associated oscillations mean (cpm) range
10.5 (10-12)
SSB
Propagation orad mean (cm/s) range aborad mean (cm/s) range SSB = short spike burst; LSB
LSB
SSB
LSB
SSB
LSB
LSB
SSB
SSB
LSB
130 150 100-165: (50-160)
-
177.5 (100-200)
156.3 166.7 120-200) (100-200)
96.0 75-127.5)
61.5 102.5 270 211 108.8 142.5 (30-90) (75-127.5: 90-127.5 (90-322.5) 127-157: (187-322)
-
347 (270-450)
372.5 156.2 130-200) (300-450)
26 (24-28)
10.8 (6.5-12)
-
26.2 (24-30)
0.7 :0.3-1.6) 0.4 :0.4-0.6) =
long spike burst.
121.7 150 (80-200) [100-200
29.2 (24-34) 0.7 (0.4-1.2) 0.4 (0.3-0.6)
7.5 (7-8)
158.3 (50-250)
32.3 (28-48) 0.7 (0.4-1.7) 0.35 (0.2-0.6)
SSB
7.5 (7-8)
33 (30-36) 0.7 (0.3-1 .O) 0.4 (0.2-0.6)
-
0.7 (0.5-1.8) 0.4 (0.2-0.6)
7 (6-12)
LSB
26.5 (24-30) 0.75 (0.5-1.0) 0.6 (0.31.0)
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MECHANICAL ACTIVITY Amplitude mean (mmHg) range Duration * mean (s) range
CARBACHOL (I pg)
CARBACHOL (10 pg)
SSB
LSB
SSB
LSB
SSB
LSB
4.2 (3-6)
5 (4.5-5.5)
7 (4-9)
7.5 (4.5-9.5)
7.8 (5.5-9.5)
(5.5-9.5)
112.5 (80-130)
116.3 (97.5-135)
123.8 (82.5-165)
131.3 (75- 187.5)
131.8 (85- 156)
330 (247-465)
cpm. The mechanical activity which corresponded to the LSB consisted of a prolonged tonic contraction propagated orally, aborally, or not propagated at all, like its corresponding electrical activity (Table I & Fig. 5 ) . No associated mechanical activity was found when a period of electrical oscillations at a frequency of about 28 cpm without superimposed spikes was recorded on the taenia .
CARBACHOL (100 pg)
8.3
atropine 1Oy
v
Cholinergic-mediated activity
In both muscle layers, cholinergic stimulation caused an increase in spike activity associated with an increase in the strength of contractions. Electrical and mechanical activity was greatly affected by the degree of concentration of carbachol (Fig. 6). Carbachol (1 - 100 pg) caused an increase in slow wave amplitude and a decrease in slow wave incidence and frequency both on the longitudinal and circular layers. After the administration of carbachol(1 and 10pg), there was an increase in SSB amplitude and duration on both muscle layers but after carbachol 100 pg SSB were only present on the circular layer. SSB were sometimes seen on only one electrode site and sometimes on both sites simultaneously. Furthermore, the slow waves associated to SSB showed a decrease in frequency. Similarly, the administration of carbachol (1-100 pg) caused an increase in the amplitude and duration of the mechanical activity associated with this kind of spike activity. After carbachol(1-100 pg), an increase in LSB amplitude and duration and an associated increase in pressure amplitude and duration was seen (Tables I1 and 111). Long spike bursts were present on the two muscle layers and were always propagated orally or aborally as was true of the related mechanical activity. Atropine (10 pg) affected the spiking activity of the longitudinal and circular layers as well as slow wave activity in 8 of the 17 specimens studied. In 5 specimens spiking activity and slow wave frequencies disappeared but reappeared upon the administration of carbachol or the application of stretching (Fig. 7). In the remaining three specimens the amplitude and duration of spiking activity were reduced after the administration of atropine. On the longitudinal layer, the duration of LSB changed from 95 s (range 22-65 s) to 57 s (37-59 s) and their amplitude from 150 pV (range 142-175 pV)
1 rnin
FIG.7. The figure shows the effect of atropine (see the arrow) on spiking activity andpressure waves. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; PI = proximal pressure site; P 2 = distal pressure site.
to 130 pV (range 125-145 pV). The frequency of electrical oscillations reduced from 26 cpm (range 22-30 cpm) to 17 cpm (range 15-25 cpm). Similarly, on the circular layer the duration of LSB changed from 90 s (range 60-102 s) to 60 s (range 40-72 s) and their amplitude from 55 pV (range 28-65 pV) to 32 pV (range 18-38 pV). Electrical oscillations decreased in frequency from 28 cpm (range 22-35 cpm) to 22 cpm (range 18-28 cpm). The amplitude of mechanical activity changed from 3.5 mmHg (range 2.5-5 mmHg) to 2 mmHg (1.6-2.3 mmHg) and the change in duration corresponded to that of the electrical pattern. Stretch-mediated activity
In both muscle layers, electrical as well as mechanical activity were highly influenced by the distension induced by the balloons placed in the lumen of the colon. In the longitudinal muscle layer, slow waves and spikes disappeared in 7 segments and were greatly reduced in amplitude in the remaining 10 segments when the volume in the balloons was reduced to zero. Similarly, in the circular muscle layer, slow waves and spikes either disappeared ( n = 12) or their amplitude was greatly diminished (n = 5). When the balloons were blown up again (n = 8), electrical and
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HUMAN COLON ACTIVITY
in vitro
FIG. 8 . Thefigure shows the effect of stretch (see the arrow). Long spike bursts without electrical oscillations can be seen. They are propagated in the aboral direction and associated with propagated tonic contractions of long duration. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
mechanical activity reappeared on both muscle layers. Moreover, when further distension was applied to the segments (n = 5 ) both the electrical and mechanical patterns changed. Before stretching, the taenia showed LSB lasting 82 s (range 34-145 s) and the circular layer showed slow waves or electrical quiescence. After stretching, bursts of long spike activity lasting 102 s (range 60-180 s) without electrical oscillations, were simultaneously present on the longitudinal and circular layers. The related mechanical activity consisted of long lasting tonic contractions (duration 100 s; range 60-180 s) with an amplitude of 6.3 mmHg (range 4-9 mmHg) (Fig. 8). Discussion
Spontaneous activity Our findings showed that slow waves were present for only about 25% of the recording time on the longitudinal muscle layer and about 12% on the circular muscle layer, as reported by other authors (DUTHIE&KIRK, 1978; VANMERWYK & DUTHIE,1980; BUENOet al., 1980; KUBOTAet al., 1983; HUIZINGA & DANIEL,1986). On each muscle layer, we found multiple frequency components of slow waves present in two ranges, as reported by other in vivo and in vitro studies (SNAPEet al., 1977; BUENOet al., 1980; SARNAet al., 1980, 1982; FRIERIet al., 1983; ALTAPARMAKOV & WIENBECK,1984) and in vitro (VAN MERWYK& DUTHIE,1980; C H A ~ ~ EetRa/. S, 1981 ;HUIZINGA et al., 1985). The simultaneous recording of electrical activity on the longitudinal and circular muscle layers in adjacent sites, demonstrated that the frequency and amplitude of the slow waves of the two muscular layers differed for the greater part of the recording time and that electrical coupling did not often occur. Moreover, slow waves were never found to be phase-locked at adjacent electrode sites which were 3 cm apart in the same muscle layer. This supports the theory that in the human sigmoid colon, coupling between adjacent oscillators is generally poor (CHAMBERS et al., 1981; SARNAet al., 1982). Neither in the longitudinal nor
99
circular muscle layers of the in vitro human colon did we see mechanical activity when slow waves without superimposed spiking activity were present, as did DUTHIEand KIRK (1978). Although in vivo (SNAPEet al., 1977) and in vitro (HUIZINGA et al., 1985) experiments have suggested that some mechanical contractions are correlated to slow waves without spikes, we agree with other authors that the apparent finding of contractions without spikes in fact merely reflects the inability of recording apparatus to detect the presence of spikes (BUENOet al., 1980). Spike burst activity is the most common and mechanically effective activity of the colon (BUENOet al., 1980; DANIEL& HUIZINGA, 1984; HUIZINGA et al., 1985). Regardless of the name given to these spike bursts, there are always long-duration and shortet al., 1980; SAFWAet al., 1981, duration bursts (BUENO 1982; SCHANG& DEVROEDE, 1983; DAPOIGNY et al., 1988). Short spike bursts are localized activity (BUENO et al., 1980; SARNAet al., 1981), in fact, even when they are simultaneously recorded at the same frequency in adjacent electrode sites, there is no time-lag. When spike bursts are superimposed on slow waves at a frequency lower than 12 cpm, the associated mechanical activity consists of single weak pressure changes. When frequency exceeds 12 cpm, there is a prolonged contraction showing summation and “rippling”. Long spike bursts were the dominant type of electrical activity recorded in our samples. As others have mainet al., 1981,1982; FREXINOS et al., 1985; tained (SARNA SCHANG et al., 1986; SZURSZEWSKI et al., 1987), we found that long spike bursts had no clear relationship to slow wave activity. They occurred at random and were either isolated in one site or were found to propagate orally and aborally. The rate of propagation was similar to that reported in vivo by SARNA(1982) but lower than the rate observed by SCHANG and DEVROEDE (1983); SCHANG et al. (1986) and BUENOet al. (1980). We found that mechanical activity consisted of sustained tonic contractions which were sometimes propagated or sometimes not in the same way as its corresponding electrical pattern. In our samples, long spike bursts were almost always continually superimposed on electrical oscillations ranging from 24 to 46 cpm. It has been reported that the development of segmental and propulsive contractions can occur in association with an increase in the amplitude of these electrical oscillations without et al., 1986). On the contrary, we did spikes (HUIZINGA not observe pressure changes when electrical oscillations were present without superimposed spikes. It is possible, however, that contractions arose from a spiked zone which was not recorded electrically.
Stimulated activity Our results show that cholinergic stimulation of the longitudinal and the circular muscle layers of the human sigmoid colon not only enhances spiking activity (SSB and LSB) and mechanical activity but also causes a change in slow wave activity. In fact, the administration of carbachol(1-100 pg) reduced the incidence and frequency of slow waves and increased their amplitude. After the drug, slow waves without spikes were still
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100
G . RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT AND I. GIORGIO
found without electrical coupling between the two muscle layers but slow waves and electrical oscillations associated to spiking activity had the same frequency on the two muscle layers, suggesting that there was coordinated activity. In our study cholinergic stimulation did not induce different patterns of spiking activity in the two muscle layers. These results are not in acet al. 's cordance with the data reported by HUIZINGA in vitro study in man (1986) but this could partly be due to the fact that our experimental conditions were very different from those of the above authors who used isolated longitudinal and circular tissue strip preparations. Ou samples consisted of segments of sigmoid colon and hence there was no clear borderline between the two muscle layers and communications were possible via bundles of longitudinal layers penetrating into the circular muscle layers and becoming histologically indistinguishable from them (HUIZINGA et a]., 1985). The human colon is not only sensitive to neural ac& KIRK, 1978; VAN tivity but also to stretching (DUTHIE MERWYK & DUTHIE, 1980; HUIZINGA et al., 1985). The circular layer in particular is sensitive to adequate et al., 1985). When distension stimulation (HUIZINGA was applied to our preparations, the electrical activity of the circular layers changed from electrical quiescence or slow waves to LSB with electrical oscillations. On the other hand, when distension was reduced to zero, slow wave and spiking activity disappeared on both muscle layers. These findings confirm that the electrical activity of the human colon depends on the extent of stretching and explain the fact that it is not possible to record consistent slow wave and spike activity from et al., 1975). the colon in vivo (TAYLOR The results of the present study help to explain the multiple patterns of electrical activity recorded in vivo from the human colon. Isoelectric periods observed in vivo are physiologic phenomena because they can be recorded in vitro on the two muscle layers. Slow waves present a wide range of amplitudes and frequencies and do not show electrical coupling between longitudinal and circular layers. Spiking activity consists of short and long spike bursts, and cholinergic stimulation as well as stretching can modulate both slow wave and spike potential activity. Long spike bursts are controlled by periodic, high frequency, slow wave activity called electrical oscillations. Finally, coordinated and propagated electrical activity of the two muscle layers only occurs when specific stimulation, such as a cholinergic agent or stretch, is applied to the sigmoid colon. References ALTAPARMAKOV, I. & WIENBECK, M. (1984) Local inhibition of myoelectrical activity of human colon by loperamide. Dig. Dis. Sci. 29, 232-238.
BUENO,L., FIORAMONTI, J., RUCKEBUSCH, Y., FREXINOS,J. & COULOM, P. (1980) Evaluation of colonic myoelectrical activity in health and functional disorders. Gut 21, 480-485. CHAMBERS, M. M., BOWES,K. L., KINGMA,J. L., BANNISTER, C. & COTE,K. R. (1981) In vitro electrical activity in human colon. Gastroenterology 81, 502-508. COUTURIER, C., ROZE,C., COUTURIER-TURPIN, M. H. & DEBRAY, C. (1969) Electromyography of the colon in situ. An experimental study in man and in rabbit. Gastroenterology 56, 317-322. DANIEL,E. E. & HUIZINGA,J. D. (1984) Physiology of human colonic motility. Ed. Poitras P. Montreal, 69-84. DAPOIGNY, M., TROLESE,J. F., BOMMELAER, G. & TOURNUT, R. (1988) Myoelectric spiking activity of right colon, left colon and rectosigmoid of healthy humans. Dig. Dis. Sci. 33, 1007-1012. DUTHIE,H. L. & KIRK,D. (1978) Electrical activity of human colonic smooth muscle in vitro. J. Physiol. (Lond.) 283,319-330. FREXINOS, J., BUENO,L. & FIORAMONTI, J. (1985) Diurnal changes in myoelectric spiking activity of the human colon. Gastroenterology 88, 1104-1110. FRIERI,G., PARISI,F., CORAZZIARI, E. & CAPRILLI,R. (1983) Colonic electromyography in chronic costipation. Gastroenterology 84, 737-740. HUIZINGA, J. D., STERN,H. S., CHOW,E., D W T, N. E. &ELSHARKAWY, T. Y. (1985) Electrophysiologic control of motility in the human colon. Gastroenterology 88, 500-511. HUIZINGA, J. D. & DANIEL,E. E. (1986) Control of human colonic motor function. Dig. Dis. Sci. 31, 865-877. HUIZINGA, J. D., STERN,H. S., CHOW,E., DIAMANT, N. E. & ELSHARKAWY, T. Y. (1986) Electrical basis of excitation and inhibition of human colonic smooth muscle. Gastroenterology90, 1197-1204. KUBOTA, M., ITO,Y. & IKEDA,K. (1983) Membrane properties and innervation of smooth muscle cells in Hirschsprung's disease. Am. J. Physiol. 244, G406-G415. SARNA,S. K . , BARDAKJIAN, B. L., WATERFALL, W. E. & LIND,J. F. (1980) Human colonic electrical control activity (ECA). Gastroenterology 18, 1526-1536. SARNA,S. K., WATERFALL, W. E., BARDAKJIAN, B. L. & LIND,J. F. (1981) Types of human colonic electrical activities recorded postoperatively. Gastroenterology 81, 61-70. SARNA,S. K., LATIMER, P., CAWBELL,D. & WATERFALL, W. E. (1982) Electrical and contractile activities of the human rectosigmoid. Gut 23, 698-705. SCHANG,J. C. & DEVROEDE, G. (1983) Fasting and postprandial myoelectric spiking activity in the human sigmoid colon. Gastroenterology 85, 1048-1053. SCHANG,J. C., HEMOND,M., HEBERT,M. & PLOTE, M. (1986) Myoelectrical activity and intraluminal flow in human sigmoid colon. Dig. Dis. Sci. 31, 1331-1337. SNAPE,W. J., CARLSON, G. M. & COHEN,S. (1977) Human colonic myoelectric activity in response to prostigmin and the gastrointestinal hormones. Dig. Dis. 22, 881-887. SZURSZEWSKI, J. H. (1987) Electrophysiological basis of gastrointestinal motility. In : JOHNSON, L. R. ed. Physiology of the gastrointestinaltract. Volume 1.2nd ed. Raven Press New York, 383-422. TAYLOR,I., DUTHIE,H. L., SMALLWOOD, R. & LINKENS, D. (1975) Large bowel myoelectrical activity in man. Gut 16, 808-814. VAN MERWYK,A. J. & DUTHIE,H. L. (1980) Characteristics of human colonic smooth muscle in vitro. In : CHRISTENSEN, J. ed. GastrointestinalMotility. Raven Press New York, 473-478. WIENBECK, M. & CHRISTENSEN, J. (1971) Effects of some drugs on electrical activity of the isolated colon of the cat. Gastroenterology 61, 470-478. Dott. G. RIEZZO lstituto Scientific0 Gastroenterologico via F. Valente, 4 70013 Castellana Grotte (BA) - Italy.
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 93- 100
ReGu le 25 mars 1991.
In vitro electro-mechanical activity of the human colon. Simultaneous recording of the electrical patterns of the t w o muscle layers BY
G. RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT
(I)
and I. GIORGIO
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[Istituto scientific0 gastroenterologico Castellana Grotte (Bari) - Italy; ( I ) Laboratoire de Physiologie - Facult6 de Medicine de Tours, France]
(8 figures)
Electrical and mechanical activity on longitudinal and circular layers of the human sigmoid colon were simultaneously studied. Recordings were obtained from two electrode sites spaced 3 cm apart in a piece of colon which had been resected surgically and perfused in an organ bath. Spontaneous electrical activity of the colon showed slow waves and spikes. Slow waves were present for only 24.5% and 12% of the recording time on the longitudinal and circular layers, respectively, and they appeared as localized activity which was irregular in amplitude and varying in frequency. Electrical coupling between the two muscle layers was rarely seen and slow waves were not associated with pressure changes. Spiking activity were recorded as short and long spike bursts on both muscle layers. Short spike bursts were localized activity superimposed on slow waves. The associated mechanical activity, which consisted of single weak pressure changes or prolonged contractions with summation, was determined by slow wave frequency. Long spike bursts were seen at irregular intervals and were either propagated or not propagated activity associated with electrical oscillations ranging from 24 to 46 cpm. Mechanical activity consisted of sustained tonic contractions propagated or not propagated in the same way as the electrical pattern. Coordinated electrical activity of the two muscle layers seldom occurred when spontaneous activity was being recorded. Electrical activity on both muscle layers was very sensitive to stretching and could be initiated or modulated by pharmacological agents. In particular, our findings showed that stimulation induced coordinated spiking activity on the two muscle layers and caused mechanical activity, propagated orally or aborally, which consisted of long lasting, high amplitude contractions. These findings confirm that stretching and pharmacological agents are the most important triggers and regulators of the electrical and mechanical activity of the colon, whose complex physiology has not yet been well understood.
Introduction The highly complex electro-mechanical activity of the colon has still not been thoroughly investigated. Published works may be divided into in vivo and in vitro studies and it is difficult to conciliate the two sets of data. In man, in vivo colonic electrical activity exhibits slow wave activity which is highly irregular in frequency and amplitude, and spiking activity which consists of short spike bursts (< 10 s) and long spike bursts (> 10 s) (SARNAet al., 1982). Short spike bursts (SSB) are related to slow waves and are localized activity. Long spike bursts (LSB), which may be present with or without a definite relationship to slow waves, are seen to propagate over long segments of the colon and are
et al., associated with transit of content (FREXINOS 1985; SCHANG et al., 1986). In vivo studies are usually performed by means of mucosal electrodes. They may be limited by the capacity of the different types of electrode (TAYLOR et al., 1975; COUTURIER et al., 1969; SARNA et al., 1982) to pick up colonic activity, and by the inconsistent relation among slow waves, spike activity and mechanical activity. Zn vitro studies performed on strips of human colonic smooth muscle have contributed to the understanding of the complex electrophysiology of the colon (HUEINGA et al., 1985). Two important in vitro data may provide a partial explanation for the complexity of in vivo recorded activity : a) the longitudinal and circular layers show different patterns of electrical activity
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94
G.
RIEZZO,
(VANMERWYK & DUTHIE,1980; KUBOTA et al., 1983; et al., 1985) and therefore the activity recordHUIZINGA ed in vivo may be a mixture of electrical signals emanating from both the longitudinal and the circular layers; b) colonic electrical activity is highly sensitive to neural and hormonal influence (WIEBECK& CHRISTENSEN, 1971; SNAPEet al., 1977) which may vary during recording sessions leading to changes in the pattern of electrical activity which are apparently spontaneous. To date, the few in vitro studies carried out on the electrical activity of the human colon have not considered the correlated electrical activity of the two muscle layers, its propagation to adjacent segments of tissue, and the relationship between the electrical and mechanical activity of each muscle layer. There is also a lack of comparative studies aimed at characterizing the electrical and motor activity of both muscle layers of the taeniated colon. Finally, in vitro studies are contradictory and variable with regard to the presence of slow waves, their frequency, and the correlation between these and contractions (DUTHIE& KIRK, 1978; VAN MERWYK & DUTHIE,1980; CHAMBERS et al., 1981; KUBOTAet al., 1983). In the present study, experiments were performed on the human sigmoid colon using a technique which makes the simultaneous recording of the electrical activity of the two muscle layers possible. These were performed in order to investigate the in vitro electrical activity of both muscle layers (taenia and intertaenia), their relation to motor activity, and how all this activity is modulated by stretching and pharmacological agents. Materials and Methods
Tissue preparation Thirty-one specimens of human sigmoid colon were obtained surgically from patients with non obstructing rectal carcinoma. Immediately before the resection of the diseased portion, a 15-18 cm segment of normal sigmoid colon was removed, the blood supply to the segment being maintained until the moment of excision. The distal end of this colonic segment was at least 5 cm from the site of the tumour, and the tissue was histologically free of tumour and inflammation. Immediately after resection, two catheters (Vasocan Braunule 18G - 1.2 mm in diameter) were inserted into the isolated segment, one in a sigmoid artery and the other in a vein. The vascular bed was washed by infusing 20 ml of saline solution containing 2500 UI of heparin through the arterial catheter. Thereafter, the sigmoid segment was placed in oxygenated Krebs solution and immediately transferred to the laboratory. Here, the lumen of the sigmoid segment was washed with oxygenated Krebs buffer solution and the artery was immediately connected to the perfusion system. The sigmoid colon was then transferred to an organ bath consisting of a closed chamber half full of warm water. The segment of sigmoid colon was placed on a plexiglass grill which was suspended above the water level and the saturated water vapour atmosphere was maintained at 37°C.
M. A.
MASELLI,
F.
PEZZOLLA,
J. THOUVENOT AND I. GIORGIO
Recording system Electrical activity was recorded by means of 4 couples of bipolar copper electrodes (0.25 mm in diameter) implanted subserously : two couples (3 cm apart) in the longitudinal muscle layer and two couples (3 cm apart) in the circular muscle layer, placed perpendicularly to the direction of the fibre axis. The distance between the electrodes of each couple was 5 mm. The couples of electrodes were implanted at the same level on the longitudinal and circular muscle layers (Fig. 1). electrodes input
I
I
pressure input longitudlnal layer clrcular layer
1 II
warm water
U
7
1 ]I U
FIG. 1. Apparatus f o r luminal perfusion and f o r the electrical and mechanical recording of the isolated sigmoid colon. The specimen was enclosed in a plexiglass chamber. The figure also shows the position of the electrode sites and of the two latex-baloons (dashed line).
Mechanical activity was recorded by means of two thin-walled latex balloons (3 cm long) introduced into the colon at the same level as the recording electrodes, and connected to pressure transducers via polyethylene tubes (made according to our specifications). A contraction of the colon against the surface of the balloon was recorded as a rise in pressure or a fall in the volume of the balloon. Simultaneous recordings of electrical and mechanical activity were made on a 8-channel ReegaMinihuit TR h v m recorder. The paper speed was 12 cm/min. The electrical signals were recorded with AC (time constant set at 0.1 and 6 s) amplification.
Recording session Following the placement of the sigmoid colon in the perfusion chamber and the setting-up of the recording apparatus, the piece was allowed to equilibrate for 30 min, before a 3 h recording was made. At the start of the recording session, the balloons in the lumen of the sigmoid colon were inflated to a pressure of 5 mmHg, which was found to be the pressure which determined the distension at which spontaneous electrical and mechanical activity was greatest. Electromechanical activity measured under these circumstances was called spontaneous activity but this should not be regarded as unstimulated activity, as stretching and/or the activity of enteric neurons may have contributed to the et al., 1985). To measure activity recorded (HUIZINGA the influence of stretching on electrical and mechanical
HUMAN COLON ACTIVITY
in vitro
95
activity, the volume of the balloons was changed during the experiments. In order to verify the viability of the sample in every case, and consequently the validity of the data obtained, at the end of each recording session methylene blue was administered to verify whether perfusion had been optimal. The colonic segments which failed to respond to methylene blue were rejected because this indicated that they had not been well perfused.
Data presentation Analysis of the slow waves, action potentials and mechanogram was performed by visual inspection of the records for each channel. Slow wave frequency (expressed in cycles per min = cpm) was calculated by counting the number of slow waves present each min; slow wave incidence was calculated as the percentage of the total recording time during which slow waves appeared. The different kinds of spike burst (SSB and LSB) were identified and the recording time occupied by each was measured. The amplitude of slow wave and spike activity was also calculated and expressed in pV. As far as pressure waves were concerned, the amplitude (mmHg) and duration (s) of each pressure wave were measured on the tracing. Of the 31 colonic segments studied, 10 were utilized to study spontaneous activity, 17 to quantify electrical and mechanical activity after excitatory stimulation (carbachol or stretching) and the remaining 4 were rejected because they had not been well perfused. Both the range in which values were observed and the mean values were documented but standard deviation was not as it cannot be stated with certainty that all the specimens came from a homogeneous population.
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Solutions and drugs The Krebs solution (NaCl 117.2 mM; KC15.9 mM; CaC1, 2.5 mM; MgCI, 1.2 mM; NaHC03 15.4 mM; NaH,P04 1.2 mM; glucose 11.5 mM; dextran 2%) was continuously gassed with 95% 0, and 5% CO,. This solution, maintained at 37"C, was delivered to the vascular bed of the sigmoid colon by means of an occlusive roller pump (GILSON MiniPuls 2) at a constant flow rate of 15 ml/min. The pressure of the perfusion was about 60 mmHg. The vascular flow rate was monitored throughout the experiments. The following drugs were used : carbachol (carbamylcholine chloride) and atropine sulfate from Sigma Chemical Co., St Louis, Mo. TABLEI. Spontaneous electrical and mechanical activity.
SLOW WAVES Frequency mean (cpm) range Amplitude mean @V) range Incidence %
SPIKE Amplitude mean @V) range Duration mean ( s ) range Associated oscillations mean (cpm) range Propagation orad mean (cm/s) range aborad mean (cm/s) range
MECHANICAL ACTIVITY Amplitude mean (mmHg) range Duration * mean (s) range
LONG. LA YER
CIRC. LA YER
10.6 (2-16)
(1-14)
110 (50-250)
71.5 (25-180)
24.5
11.9
11.7
SSB
LSB
SSB
LSB
85 (50-200)
131.3 (50-200)
71.7 (25-120)
63 (25-100)
76.2 (45-127)
87 (22.5-172.5)
49.5 (37.5-67.5)
76.8 (22.5-120)
12.4 (1-16)
27.7 (24-46)
12.4 (2-16)
27.7 (24-46)
1 .o (0.9-1.8) 0.4 (0.1-0.6)
0.9 (0.6-1.6) 0.4 (0.2-0.7)
3 (2-6)
3.7 (2.5-6.5)
86.3 (37.5-135)
66 (22.5-105) I
* The duration of mechanical activity is the result of electrical activity recorded both on longitudinal and circular layers. SSB burst; LSB = long spike burst.
=
short spike
96
G. RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT AND I. GIORGIO
In order to study the effect of drugs on colon segments a comparison was made between a 10 min period of activity before the administration of the drug and a 10 min period after. The mean values for the 10 min periods were compared. Results Spontaneous activity
-
Ec 2
.
_ 1 _
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Two patterns of electrical activity were seen : slow waves and action potentials (spikes). SLOW WAVES - Slow waves were found on recordings obtained with a time constant of 6 s. They were found to be absent or lasting no longer than 24.5% of the recording time on the longitudinal layer and 12% on the circular layer. Only in one case were slow waves continually present for the entire recording time. Slow waves were very irregular in amplitude (50-250 pV and 25-180 pV on the longitudinal and circular layers, respectively) and their frequency varied from 2 to 16 cpm (mean 10.6 cpm) on the longitudinal layer and from 1 to 14 cpm (mean 11.7 cpm) on the circular layer (Table I). Two groups of slow wave frequencies with a normal distribution were recorded : a slower rhythm at 2.5 f 0.6 cpm and a faster rhythm at 10.7 & 0.5 cpm on the longitudinal layer; a slower rhythm at 2 -t 0.8 cpm and a faster at rhythm 8.5 0.5 cpm on the circular muscle layer. Electrical coupling between the two muscle layers was rarely seen. Recordings made from adjacent electrode sites 3 cm apart in the same muscle layer, showed that slow waves were usually phase-unlocked. Slow waves were not associated with pressure changes (Fig. 2).
*
FIG. 3. The figure shows short spike bursts simultaneously in Etl and Et2. In P1 and P2 there are weak pressure changes at the same frequency as the electrical activity in Et 1 and Et2. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
Etl Ec 1
1
7
I
100 pv
El2
Ec 2
~~
P 1 I5mmHg
P 2
--
205586
Etl
1 mln
FIG.4. Thefigure shows short spike bursts in Et2 and Ec2 relared to pressure in P2 with summation and “rippling”. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
Ec2 P 1 (6mrnHg
p 2 1 rnin
FIG.2. Thefigure shows aperiod of slow waves in Ecl. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
ACTION POTENTIALS - Short and long spike bursts were recorded. Action potentials consisting of short spike bursts (duration c 10 s) occurred intermittently for a duration of 45 to 127 s (mean 76.2 s) and of 37.5 to 67.5 s (mean 49.5 s) on the longitudinal and circular layers, respectively. The amplitude was 50-200 pV (mean 85 pV) on the longitudinal layer and 25-120 p V (mean 71.7 pV) on the circular layer (Table I).
The short spike bursts were always superimposed on slow waves. When short duration spike activity was present on the longitudinal muscle, the circular muscle showed either electrical quiescence or the same kind of spike potential as the longitudinal layer. The short spike bursts were seen either on only one electrode site at a time, or simultaneously on two adjacent electrode sites without a time lag. These short spike bursts were related to mechanical activity as indicated by the presence of low rhythmic pressure changes (Fig. 3). Contraction frequency was related to spike burst frequency only when slow wave frequency was c 12 cpm. Contractions associated with spike bursts superimposed on slow waves at a frequency higher than 12 cpm showed summation, and a “rippling” at the frequency of the electrical activity was usually present (Fig. 4). Action potentials consisting of long spike bursts (duration > 10 s) were associated with electrical oscillations ranging in frequency from 24 to 46 cpm. The
HUMAN COLON ACTIVITY
in
97
VitrO
cmrbmchol 1Oy 7
Ell
’
- - - -E -C l- -
(100 pv
Et 2
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Ec2
FIG.5. Thefigure shows longspike bursts in EtI and electrical oscillation in Ecl related to electricaloscillations and a sustained tonic contracfion in PI. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P 1 = proximal pressure site; P2 = distal pressure site.
1 man
FIG.6 . Thefigure shows the effect of carbachol I0 pg (see ihe arrow). Long spike bursts without electrical oscillations can be seen. They are propagated in the aboral direction and associated with propagated tonic contractions of long duration (see the arrows). Etl = proximal electrodes on the taenia; Ecl = proximal electrodeson the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P 1 = proximal pressure site; P 2 = distal pressure site.
duration of LSB ranged from 22.5 to 172.5 s (mean 87 s) and from 22.5 to 120 s (mean 76.8 s) on the longitudinal and circular layers, respectively. Amplitude was 50-200 pV (mean 131.3 pV) on the longitudinal layer and 25-100 pV (mean 63 pV) on the circular layer (Fig. 5). LSB appeared in two different patterns : localized either on one electrode or on two electrodes simultaneously, suggesting a local, non propagating activity; or with a time lag, indicating regular
oral or aboral propagating activity (Table I). When LSB were present on the longitudinal muscle, the circular muscle layer showed 3 different patterns : a period of electrical quiescence, short spike bursts, or long spike bursts with electrical oscillations at a frequency of 24-46
TABLE11. Effect of carbachol on electrical activity of the sigmoid colon in vitro. ~
CARBACHOL (I pg)
SLOW WAVE Frequency mean (cpm) range Amplitude mean (JIV) range Incidence
CARBACHOL (10 pgj
CARBACHOL (I00 pg)
LONG. LA YER
LONG. LA YER
CIRC. LA YER
CIRC. LA YER
10.6
(10-11)
8 (5-14)
7.1 (1.8-9)
7 (5-12)
133.3 (100-180)
146.0 (60-200)
132 (80-250)
142 (25-200)
4.5
2.9
3.0
2.8
QJO
SPIKE Amplitude mean (pV) range
116.7 141.7 100-150) (80-200)
135 (70-200)
Duration mean (s) range
82.5 (75-90)
Associated oscillations mean (cpm) range
10.5 (10-12)
SSB
Propagation orad mean (cm/s) range aborad mean (cm/s) range SSB = short spike burst; LSB
LSB
SSB
LSB
SSB
LSB
LSB
SSB
SSB
LSB
130 150 100-165: (50-160)
-
177.5 (100-200)
156.3 166.7 120-200) (100-200)
96.0 75-127.5)
61.5 102.5 270 211 108.8 142.5 (30-90) (75-127.5: 90-127.5 (90-322.5) 127-157: (187-322)
-
347 (270-450)
372.5 156.2 130-200) (300-450)
26 (24-28)
10.8 (6.5-12)
-
26.2 (24-30)
0.7 :0.3-1.6) 0.4 :0.4-0.6) =
long spike burst.
121.7 150 (80-200) [100-200
29.2 (24-34) 0.7 (0.4-1.2) 0.4 (0.3-0.6)
7.5 (7-8)
158.3 (50-250)
32.3 (28-48) 0.7 (0.4-1.7) 0.35 (0.2-0.6)
SSB
7.5 (7-8)
33 (30-36) 0.7 (0.3-1 .O) 0.4 (0.2-0.6)
-
0.7 (0.5-1.8) 0.4 (0.2-0.6)
7 (6-12)
LSB
26.5 (24-30) 0.75 (0.5-1.0) 0.6 (0.31.0)
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MECHANICAL ACTIVITY Amplitude mean (mmHg) range Duration * mean (s) range
CARBACHOL (I pg)
CARBACHOL (10 pg)
SSB
LSB
SSB
LSB
SSB
LSB
4.2 (3-6)
5 (4.5-5.5)
7 (4-9)
7.5 (4.5-9.5)
7.8 (5.5-9.5)
(5.5-9.5)
112.5 (80-130)
116.3 (97.5-135)
123.8 (82.5-165)
131.3 (75- 187.5)
131.8 (85- 156)
330 (247-465)
cpm. The mechanical activity which corresponded to the LSB consisted of a prolonged tonic contraction propagated orally, aborally, or not propagated at all, like its corresponding electrical activity (Table I & Fig. 5 ) . No associated mechanical activity was found when a period of electrical oscillations at a frequency of about 28 cpm without superimposed spikes was recorded on the taenia .
CARBACHOL (100 pg)
8.3
atropine 1Oy
v
Cholinergic-mediated activity
In both muscle layers, cholinergic stimulation caused an increase in spike activity associated with an increase in the strength of contractions. Electrical and mechanical activity was greatly affected by the degree of concentration of carbachol (Fig. 6). Carbachol (1 - 100 pg) caused an increase in slow wave amplitude and a decrease in slow wave incidence and frequency both on the longitudinal and circular layers. After the administration of carbachol(1 and 10pg), there was an increase in SSB amplitude and duration on both muscle layers but after carbachol 100 pg SSB were only present on the circular layer. SSB were sometimes seen on only one electrode site and sometimes on both sites simultaneously. Furthermore, the slow waves associated to SSB showed a decrease in frequency. Similarly, the administration of carbachol (1-100 pg) caused an increase in the amplitude and duration of the mechanical activity associated with this kind of spike activity. After carbachol(1-100 pg), an increase in LSB amplitude and duration and an associated increase in pressure amplitude and duration was seen (Tables I1 and 111). Long spike bursts were present on the two muscle layers and were always propagated orally or aborally as was true of the related mechanical activity. Atropine (10 pg) affected the spiking activity of the longitudinal and circular layers as well as slow wave activity in 8 of the 17 specimens studied. In 5 specimens spiking activity and slow wave frequencies disappeared but reappeared upon the administration of carbachol or the application of stretching (Fig. 7). In the remaining three specimens the amplitude and duration of spiking activity were reduced after the administration of atropine. On the longitudinal layer, the duration of LSB changed from 95 s (range 22-65 s) to 57 s (37-59 s) and their amplitude from 150 pV (range 142-175 pV)
1 rnin
FIG.7. The figure shows the effect of atropine (see the arrow) on spiking activity andpressure waves. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; PI = proximal pressure site; P 2 = distal pressure site.
to 130 pV (range 125-145 pV). The frequency of electrical oscillations reduced from 26 cpm (range 22-30 cpm) to 17 cpm (range 15-25 cpm). Similarly, on the circular layer the duration of LSB changed from 90 s (range 60-102 s) to 60 s (range 40-72 s) and their amplitude from 55 pV (range 28-65 pV) to 32 pV (range 18-38 pV). Electrical oscillations decreased in frequency from 28 cpm (range 22-35 cpm) to 22 cpm (range 18-28 cpm). The amplitude of mechanical activity changed from 3.5 mmHg (range 2.5-5 mmHg) to 2 mmHg (1.6-2.3 mmHg) and the change in duration corresponded to that of the electrical pattern. Stretch-mediated activity
In both muscle layers, electrical as well as mechanical activity were highly influenced by the distension induced by the balloons placed in the lumen of the colon. In the longitudinal muscle layer, slow waves and spikes disappeared in 7 segments and were greatly reduced in amplitude in the remaining 10 segments when the volume in the balloons was reduced to zero. Similarly, in the circular muscle layer, slow waves and spikes either disappeared ( n = 12) or their amplitude was greatly diminished (n = 5). When the balloons were blown up again (n = 8), electrical and
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HUMAN COLON ACTIVITY
in vitro
FIG. 8 . Thefigure shows the effect of stretch (see the arrow). Long spike bursts without electrical oscillations can be seen. They are propagated in the aboral direction and associated with propagated tonic contractions of long duration. Etl = proximal electrodes on the taenia; Ecl = proximal electrodes on the circular muscle layer; Et2 = distal electrodes on the taenia; Ec2 = distal electrodes on the circular muscle layer; P1 = proximal pressure site; P2 = distal pressure site.
mechanical activity reappeared on both muscle layers. Moreover, when further distension was applied to the segments (n = 5 ) both the electrical and mechanical patterns changed. Before stretching, the taenia showed LSB lasting 82 s (range 34-145 s) and the circular layer showed slow waves or electrical quiescence. After stretching, bursts of long spike activity lasting 102 s (range 60-180 s) without electrical oscillations, were simultaneously present on the longitudinal and circular layers. The related mechanical activity consisted of long lasting tonic contractions (duration 100 s; range 60-180 s) with an amplitude of 6.3 mmHg (range 4-9 mmHg) (Fig. 8). Discussion
Spontaneous activity Our findings showed that slow waves were present for only about 25% of the recording time on the longitudinal muscle layer and about 12% on the circular muscle layer, as reported by other authors (DUTHIE&KIRK, 1978; VANMERWYK & DUTHIE,1980; BUENOet al., 1980; KUBOTAet al., 1983; HUIZINGA & DANIEL,1986). On each muscle layer, we found multiple frequency components of slow waves present in two ranges, as reported by other in vivo and in vitro studies (SNAPEet al., 1977; BUENOet al., 1980; SARNAet al., 1980, 1982; FRIERIet al., 1983; ALTAPARMAKOV & WIENBECK,1984) and in vitro (VAN MERWYK& DUTHIE,1980; C H A ~ ~ EetRa/. S, 1981 ;HUIZINGA et al., 1985). The simultaneous recording of electrical activity on the longitudinal and circular muscle layers in adjacent sites, demonstrated that the frequency and amplitude of the slow waves of the two muscular layers differed for the greater part of the recording time and that electrical coupling did not often occur. Moreover, slow waves were never found to be phase-locked at adjacent electrode sites which were 3 cm apart in the same muscle layer. This supports the theory that in the human sigmoid colon, coupling between adjacent oscillators is generally poor (CHAMBERS et al., 1981; SARNAet al., 1982). Neither in the longitudinal nor
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circular muscle layers of the in vitro human colon did we see mechanical activity when slow waves without superimposed spiking activity were present, as did DUTHIEand KIRK (1978). Although in vivo (SNAPEet al., 1977) and in vitro (HUIZINGA et al., 1985) experiments have suggested that some mechanical contractions are correlated to slow waves without spikes, we agree with other authors that the apparent finding of contractions without spikes in fact merely reflects the inability of recording apparatus to detect the presence of spikes (BUENOet al., 1980). Spike burst activity is the most common and mechanically effective activity of the colon (BUENOet al., 1980; DANIEL& HUIZINGA, 1984; HUIZINGA et al., 1985). Regardless of the name given to these spike bursts, there are always long-duration and shortet al., 1980; SAFWAet al., 1981, duration bursts (BUENO 1982; SCHANG& DEVROEDE, 1983; DAPOIGNY et al., 1988). Short spike bursts are localized activity (BUENO et al., 1980; SARNAet al., 1981), in fact, even when they are simultaneously recorded at the same frequency in adjacent electrode sites, there is no time-lag. When spike bursts are superimposed on slow waves at a frequency lower than 12 cpm, the associated mechanical activity consists of single weak pressure changes. When frequency exceeds 12 cpm, there is a prolonged contraction showing summation and “rippling”. Long spike bursts were the dominant type of electrical activity recorded in our samples. As others have mainet al., 1981,1982; FREXINOS et al., 1985; tained (SARNA SCHANG et al., 1986; SZURSZEWSKI et al., 1987), we found that long spike bursts had no clear relationship to slow wave activity. They occurred at random and were either isolated in one site or were found to propagate orally and aborally. The rate of propagation was similar to that reported in vivo by SARNA(1982) but lower than the rate observed by SCHANG and DEVROEDE (1983); SCHANG et al. (1986) and BUENOet al. (1980). We found that mechanical activity consisted of sustained tonic contractions which were sometimes propagated or sometimes not in the same way as its corresponding electrical pattern. In our samples, long spike bursts were almost always continually superimposed on electrical oscillations ranging from 24 to 46 cpm. It has been reported that the development of segmental and propulsive contractions can occur in association with an increase in the amplitude of these electrical oscillations without et al., 1986). On the contrary, we did spikes (HUIZINGA not observe pressure changes when electrical oscillations were present without superimposed spikes. It is possible, however, that contractions arose from a spiked zone which was not recorded electrically.
Stimulated activity Our results show that cholinergic stimulation of the longitudinal and the circular muscle layers of the human sigmoid colon not only enhances spiking activity (SSB and LSB) and mechanical activity but also causes a change in slow wave activity. In fact, the administration of carbachol(1-100 pg) reduced the incidence and frequency of slow waves and increased their amplitude. After the drug, slow waves without spikes were still
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G . RIEZZO, M. A. MASELLI, F. PEZZOLLA, J. THOUVENOT AND I. GIORGIO
found without electrical coupling between the two muscle layers but slow waves and electrical oscillations associated to spiking activity had the same frequency on the two muscle layers, suggesting that there was coordinated activity. In our study cholinergic stimulation did not induce different patterns of spiking activity in the two muscle layers. These results are not in acet al. 's cordance with the data reported by HUIZINGA in vitro study in man (1986) but this could partly be due to the fact that our experimental conditions were very different from those of the above authors who used isolated longitudinal and circular tissue strip preparations. Ou samples consisted of segments of sigmoid colon and hence there was no clear borderline between the two muscle layers and communications were possible via bundles of longitudinal layers penetrating into the circular muscle layers and becoming histologically indistinguishable from them (HUIZINGA et a]., 1985). The human colon is not only sensitive to neural ac& KIRK, 1978; VAN tivity but also to stretching (DUTHIE MERWYK & DUTHIE, 1980; HUIZINGA et al., 1985). The circular layer in particular is sensitive to adequate et al., 1985). When distension stimulation (HUIZINGA was applied to our preparations, the electrical activity of the circular layers changed from electrical quiescence or slow waves to LSB with electrical oscillations. On the other hand, when distension was reduced to zero, slow wave and spiking activity disappeared on both muscle layers. These findings confirm that the electrical activity of the human colon depends on the extent of stretching and explain the fact that it is not possible to record consistent slow wave and spike activity from et al., 1975). the colon in vivo (TAYLOR The results of the present study help to explain the multiple patterns of electrical activity recorded in vivo from the human colon. Isoelectric periods observed in vivo are physiologic phenomena because they can be recorded in vitro on the two muscle layers. Slow waves present a wide range of amplitudes and frequencies and do not show electrical coupling between longitudinal and circular layers. Spiking activity consists of short and long spike bursts, and cholinergic stimulation as well as stretching can modulate both slow wave and spike potential activity. Long spike bursts are controlled by periodic, high frequency, slow wave activity called electrical oscillations. Finally, coordinated and propagated electrical activity of the two muscle layers only occurs when specific stimulation, such as a cholinergic agent or stretch, is applied to the sigmoid colon. References ALTAPARMAKOV, I. & WIENBECK, M. (1984) Local inhibition of myoelectrical activity of human colon by loperamide. Dig. Dis. Sci. 29, 232-238.
BUENO,L., FIORAMONTI, J., RUCKEBUSCH, Y., FREXINOS,J. & COULOM, P. (1980) Evaluation of colonic myoelectrical activity in health and functional disorders. Gut 21, 480-485. CHAMBERS, M. M., BOWES,K. L., KINGMA,J. L., BANNISTER, C. & COTE,K. R. (1981) In vitro electrical activity in human colon. Gastroenterology 81, 502-508. COUTURIER, C., ROZE,C., COUTURIER-TURPIN, M. H. & DEBRAY, C. (1969) Electromyography of the colon in situ. An experimental study in man and in rabbit. Gastroenterology 56, 317-322. DANIEL,E. E. & HUIZINGA,J. D. (1984) Physiology of human colonic motility. Ed. Poitras P. Montreal, 69-84. DAPOIGNY, M., TROLESE,J. F., BOMMELAER, G. & TOURNUT, R. (1988) Myoelectric spiking activity of right colon, left colon and rectosigmoid of healthy humans. Dig. Dis. Sci. 33, 1007-1012. DUTHIE,H. L. & KIRK,D. (1978) Electrical activity of human colonic smooth muscle in vitro. J. Physiol. (Lond.) 283,319-330. FREXINOS, J., BUENO,L. & FIORAMONTI, J. (1985) Diurnal changes in myoelectric spiking activity of the human colon. Gastroenterology 88, 1104-1110. FRIERI,G., PARISI,F., CORAZZIARI, E. & CAPRILLI,R. (1983) Colonic electromyography in chronic costipation. Gastroenterology 84, 737-740. HUIZINGA, J. D., STERN,H. S., CHOW,E., D W T, N. E. &ELSHARKAWY, T. Y. (1985) Electrophysiologic control of motility in the human colon. Gastroenterology 88, 500-511. HUIZINGA, J. D. & DANIEL,E. E. (1986) Control of human colonic motor function. Dig. Dis. Sci. 31, 865-877. HUIZINGA, J. D., STERN,H. S., CHOW,E., DIAMANT, N. E. & ELSHARKAWY, T. Y. (1986) Electrical basis of excitation and inhibition of human colonic smooth muscle. Gastroenterology90, 1197-1204. KUBOTA, M., ITO,Y. & IKEDA,K. (1983) Membrane properties and innervation of smooth muscle cells in Hirschsprung's disease. Am. J. Physiol. 244, G406-G415. SARNA,S. K . , BARDAKJIAN, B. L., WATERFALL, W. E. & LIND,J. F. (1980) Human colonic electrical control activity (ECA). Gastroenterology 18, 1526-1536. SARNA,S. K., WATERFALL, W. E., BARDAKJIAN, B. L. & LIND,J. F. (1981) Types of human colonic electrical activities recorded postoperatively. Gastroenterology 81, 61-70. SARNA,S. K., LATIMER, P., CAWBELL,D. & WATERFALL, W. E. (1982) Electrical and contractile activities of the human rectosigmoid. Gut 23, 698-705. SCHANG,J. C. & DEVROEDE, G. (1983) Fasting and postprandial myoelectric spiking activity in the human sigmoid colon. Gastroenterology 85, 1048-1053. SCHANG,J. C., HEMOND,M., HEBERT,M. & PLOTE, M. (1986) Myoelectrical activity and intraluminal flow in human sigmoid colon. Dig. Dis. Sci. 31, 1331-1337. SNAPE,W. J., CARLSON, G. M. & COHEN,S. (1977) Human colonic myoelectric activity in response to prostigmin and the gastrointestinal hormones. Dig. Dis. 22, 881-887. SZURSZEWSKI, J. H. (1987) Electrophysiological basis of gastrointestinal motility. In : JOHNSON, L. R. ed. Physiology of the gastrointestinaltract. Volume 1.2nd ed. Raven Press New York, 383-422. TAYLOR,I., DUTHIE,H. L., SMALLWOOD, R. & LINKENS, D. (1975) Large bowel myoelectrical activity in man. Gut 16, 808-814. VAN MERWYK,A. J. & DUTHIE,H. L. (1980) Characteristics of human colonic smooth muscle in vitro. In : CHRISTENSEN, J. ed. GastrointestinalMotility. Raven Press New York, 473-478. WIENBECK, M. & CHRISTENSEN, J. (1971) Effects of some drugs on electrical activity of the isolated colon of the cat. Gastroenterology 61, 470-478. Dott. G. RIEZZO lstituto Scientific0 Gastroenterologico via F. Valente, 4 70013 Castellana Grotte (BA) - Italy.
