AMERICAN JOURNAL OF PHYSIOLOGY Vol. 229, No. 6, December 1975. Printed
Stimulation atropine,
in U.S.A.
of intestinal procaine,
ALEX Department
smooth muscle by
and tetrodotoxin
BORTOFF AND RICHARD of Physiology, State University
MULLER of New
York,
BORTOFF, ALEX, AND RICHARD MULLER. Stimulation of intestinal smooth muscle by atropine, procaine, and tetrodotoxin. Am. J. Physiol. 229(6): 1609-1613. 1975.-In order to determine whether or not atropine, procaine, and tetrodotoxin (TTX) can stimulate intestinal smooth muscle directly, we examined the effects of these drugs on the mechanical and electrical activities of several types of cat intestinal smooth muscle preparations. The preparations consisted of isolated rings of I) intact intestinal wall, 2) intact longitudinal and circular muscle, 3) ganglion-free circular muscle, and 4) ganglion-free circular muscle devoid of its dense layer and plexus muscularis profundus. Atropine and procaine ( > 10m4 M) stimulated all- four types of preparation. On the other hand, TTX (up to 5 x lo- 6 M) stimulated only preparations I and 2. It is concluded that whereas atropine and procaine can directly stimulate intestinal smooth muscle, the excitatory effect of TTX is neurally mediated. intestinal
motility;
electrophysiology
IT IS WELL ESTAB LISHED that intestinal smooth muscle can be stimulated by certain drugs which are know n primarily for their inhibitory effects on neural processes. Among these are high concentrations of muscarinic blocking agents such as atropine (3, 4, 15-17), local anesthetics such as procaine and lidocaine (1, 15- 17), and tetrodotoxin (I, 9, 12, 15, 17). In several recent studies the excitatory effects of such drugs have been used as evidence to support the concept that, at least in the case of the small intestine, the intrinsic excitability of visceral smooth muscle is held in abeyance by spontaneously active inhibitory neurons of the intramural plexus. Thus, Wood (15) has interpreted the results of his experiments demonstrating excitation of isolated segments of cat jejunum by procaine, lidocaine, and tetrodotoxin in terms of “blockade of a spontaneously active inhibitory nervous system,” which permits the intestinal muscle to exhibit its intrinsic activity. In an attempt to explain the excitatory effect of atropine, Wood has suggested that at least two neurons are involved, a driver neuron which is spontaneously active and a follower inhibitory neuron which is activated via cholinergic synapses; atropine, according to this scheme, releases the muscle from inhibition by synaptic blockade. A similar explanation for the excitatory effects of tetrodotoxin, lidocaine, and procaine has more recently been proposed by Biber and Fara (l), who studied the effects of these drugs on the small intestines of anesthetized cats. In contrast, other investigators have proposed a direct
Upstate
A4edical
Center,
Syracuse,
New
York
13210
excitatory effect of atropine and local anesthetics on smooth muscle. A direct effect of atropine was suggested by Christensen and Lund (4), since synaptic blockade by nicotine or hexamethonium did not affect the excitatory response of opossum esophageal smooth muscle to high concentrations of atropine. Atropine has also been shown to stimulate the smooth muscle of chick amnion (5), which is devoid of nerves. The excitatory effects of local anesthetics, including procaine and lidocaine, are unaffected by antagonists to excitatory neurotransmitters on smooth muscle from either in these rat vas deferens (14) or rabbit uterus (6), although studies the possibility of blocking neural inhibition was not considered. Thus, the question of whether atropine, local anesthetics, and tetrodotoxin exert their excitatory effect on intestinal smooth muscle directly, or indirectly by release of neural inhibition, is still unresolved. The following experiments were undertaken in an attempt to resolve this issue. METHODS
AND
MATERIALS
All experiments were performed on segments of jejunum obtained from young adult cats under pentobarbital anesthesia. Four types of preparation were used. The first consisted of intact rings of jejunum approximately 5 mm wide. The second consisted of similar rings of intestine from which the mucosa and submucosa had been removed. These are referred to below as “longitudinal-circular IIIUScle” and were prepared in the following manner: segments of jejunum approximately 2 cm long were gently everted and slipped onto a glass rod. Under a dissecting microscope, a longitudinal incision was made through the mucosa and submucosa without penetrating the circular muscle layer. Opposing edges of the cut mucosa-submucosa were then grasped by blunt forceps and gently pulled apart. When the edges were completely separated so that circular muscle was visible through the incision along the entire length of was completely rethe segment, the mucosa-submucosa moved by gently pulling it away from the rest of the segment, force being applied in the circular direction. For use as longitudinal-circular muscle preparations, the segments were removed from the glass rod, inverted, and cut into rings approximately 5 mm wide. Two types of isolated circular muscle preparations were also prepared from these segments. The first was prepared by removing the longitudinal muscle layer from segments which had been slipped back onto the glass rod with the longitudinal muscle layer in its normal orientation. Under
1609
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1610 the dissecting microscope, a longitudinal incision was made with a scalpel along the length of the segment, deep enough to just penetrate the circular muscle layer. The longitudinal muscle layer, myenteric plexus, and the outer portion of circular muscle were then removed by peeling back the tissues as a single sheet, beginning at one edge of the longitudinal incision and continuing around the circumference to the other edge. Strands of connective tissue, blood vessels, and nerves which pass through the segment were cut with iris scissors during the peeling process. The resulting preparations consisted of ganglion-free circular muscle as originally described by Evans and Schild (8). Two rings, 5-10 mm wide, were usually cut from each preparation for use in the following experiments ; the ends of the segments were cut off and discarded. Microscopic examination of complete serial sections of three such circular muscle preparations stained with Mallory’s triple revealed no ganglion cells. A second type of circular muscle preparation was also In these preparations the innerused in some experiments. most portion of the circular muscle layer was removed in order to eliminate the dense circular muscle layer together muscularis profundus,” with the nerve plexus, “plexus which lies between the dense layer and the main circular muscle layer. Both the dense layer and the nerve plexus have been most recently described by Duchon et al. (7). These layers were removed from the intestinal segments immediately after removal of the mucosa-submucosa. After making a superficial longitudinal incision in the circular muscle layer, one edge of the cut was gently teased away from the other edge until enough circular muscle was freed to enable grasping it along the entire length of the intestinal segment with a pair of forceps. This procedure was facilitated by the use of a pair of long, gently tapering forceps held almost parallel to the longitudinal axis of the segment. In successful trials the innermost portion of circular muscle could be removed as a single sheet by gently peeling it back. It is estimated that 15-25 % of the circular muscle layer was removed by this method. The longitudinal with myenteric plexus and some muscle layer, together circular muscle, was then removed as described above. The remaining circular muscle was quite thin and usually separated into natural rings or tight spirals. These were separated on the glass rod by gently moving them apart with a pair of forceps and cutting any connecting strands with iris scissors. The resulting preparations consisted of rings of circular muscle approximately 5-6 mm wide. TO summarize, the four preparations were rings of I) intact intestinal wall, 2) longitudinal-circular muscle (containing myen teric plexus), 3) circular muscle, apparently devoid of ganglion cells but containing the dense layer and plexus muscularis profundus (referred to below as “intact circular muscle”), and 4) circular muscle devoid of both the dense layer and the plexus layer (referred to below as “stripped circular muscle”). Recordings of electrical and mechanical activity were taken from intestinal rings immersed in a loo-ml bath of oxygenated Tyrode solution maintained at 37°C. The composition of the Tyrode solution, in millimoles per liter, was: NaCl, 120.8; KCl, 5.9; NaHC03, 15.5; NaHzPO4, 1.2 ; CaCl2, 2.5 ; MgC12, 1.2 ; and glucose, 11.5. The buffer system was completed by aeration with a mixture of 95 % 02-5 % CO%
A. BORTOFF
AND
R. MULLER
The recording systems were of two kinds. In one, only mechanical activity was recorded, in which case up to four muscle rings were placed on a small glass rod which was immersed and anchored in the bath. A silk thread was attached to each muscle ring, and the other end was tied to a force-displacement transducer (Grass Instrument Co., model Ft .03). Records of mechanical activity were displayed on a four-channel Grass polygraph. In the second type of recording system, electrical activity was recorded, usually together with mechanical activity, using pressure electrodes attached to force-displacement transducers (2). Pressure electrode recordings are similar to those obtained with suction electrodes, i.e., they are monophasic and qualitatively resemble transmembrane recordings. By attaching the pressure electrode to the force-displacement transducer, a record of contractile activity in the region of the electrode tip is also obtained. Drugs were added to the bath with a hypodermic syringe. The drugs used in these experiments included atropine sulfate, procaine hydrochloride, tetrodotoxin (TTX), BaCl2, and acetylcholine chloride. RESULTS
Before studying the effects of atropine, procaine, and tetrodotoxin, stimulation of mechanical activity by acetylcholine and Ba++ was recorded, since these drugs are known to have a direct excitatory effect on intestinal smooth muscle. As indicated by the tracings of mechanical activity in Figs. 1 and 2, both drugs produced essentially the same effect. Spontaneous, rhythmic contractions were usually present in the rings containing both longitudinal and circular muscle, while the rings containing only circular muscle were usually quiescent. Following the addition to the bath of either acetylcholine or Ba ++, the magnitude of contractions in the longitudinal-circular muscle rings increased while rhythmic contractions were elicited in the intact circular muscle preparations. Rarely was a contracture elicited in circular muscle rings unless the concentration of acetylcholine or Ba++ was very high, even though acetylcholine often produced a partial contracture in the longitudinal circular preparations as in Fig. 1. The latency of the response to acetylcholine was shorter than that to Ba++, and the latency for both drugs was usually shorter for the longitudinal circular preparations. The rhythmical contractions elicited in circular muscle occurred either as bursts (Fig. 1, Fig. 2, Cr and C,> or as long trains (Fig. 2, C,). L’CC
# Ach IO-% FIG. 1. Effect tudinal-circular of cat jejunum. 4 X low6 M 10 In this and the because of large 36°C.
t4xlo-6M
‘l
of acetylcholine on mechanical activity of longi(L + C) and intact circular (C) muscle preparations Concentration of acetylcholine in bath was raised to min after initial concentration of 10B6 M was added. following figures, tension records are not calibrated variation in sizes of preparations used. Temperature,
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EXCITATORY
EFFECTS
OF
ATROPINE,
PROCAINE,
AND
1611
TTX
f Atropine
5 x lO*M
‘Imin
3. Typical mechanical response to atropine of longitudinalcircular (L + C) and intact circular (C) muscle preparations of cat jejunum. A low level of spontaneous activity was present only in longitudinal-circular preparation prior to addition of atropine. Temperature, 37°C. FIG.
f BaC12
5x lO’5 g/ml
‘I
FIG. 2. Effect of Ba++ on mechanical activity of longitudinalcircular (L + C) and intact circular (Cl, Cz, and C,) muscle preparations of cat jejunum. All 4 preparations occupied same bath. Prior to addition of BaCl2, longitudinal-circular preparation exhibited spontaneous activity, the 3 circular muscle rings were quiescent. Temperature, 36°C.
The excitatory effect of large concentrations of atropine was similar to that of acetylcholine or Ba++, except that the latency was longer and more variable. Of 30 intact circular muscle preparations used. in one series of experiments, 27 responded to atropine at a threshold concentration between 1 and 4 X 10V4 M, whereas three did not respond up to 2 X UP2 M. A typical m-opine response is shown in Fig. 3. After the addition of atropine to the bath, the contraction amplitudes of the longitudinal circular preparation gradually increased in size ; suddenly after 2 min, 30 s, contractions 15-30 times greater in amplitude appeared. Intact circular muscle preparations responded with similarly large contractions after variable latencies, sometimes as long as 4-5 min, in the case of Fig. 3, after 2 min, 40 s. Once initiated, the contractions continued in both types of preparation as bursts or prolonged trains until after the atropine was washed out of the bath. Contractions usually persisted for a time after the bath was washed, and after subsiding could be reelicited, showing no apparent tachyphylaxis. In response to the concentrations of atropine used in these experiments, the contractions of both types of preparation were rhythmical, showing little if any contracture. The contractions of intact circular muscle were sometimes surprisingly regular, but were usually of a frequency lower than the maximal frequency exhibited by the intact preparation, i.e., the slow-wave frequency. Procaine also stimulated both intact circular and longitudinal circular preparations from 8 of 11 cats (Fig. 4). The latent periods, although variable, tended to be shorter than those for atropine. The mechanical response consisted of rhythmic contractions which occasionally fused into partial contractures (Fig. 9). Excitatory responses to atropine and to procaine were not affected by prior addition to the bath of up to 5 X lo+ g/ml TTX. Stripped circular muscle reacted to atropine and procaine in much the same way as intact circular muscle. A somewhat surprising finding, however, was that it appeared to be even more sensitive to atropine, procaine, and Ba++ than the intact circular preparation (Figs. 5, 6, and 7). Relative sensitivity of the two preparations to these drugs was not studied by means of dose-response curves, since the main purpose of this study was simply to deter-
+ Procai ne
2 x loo4 M
‘I
4. Effect of procaine on mechanical activity of 1 longitudinalcircular (L + C) and 3 intact circular (Cl, Cp, C,) muscle preparations of cat jejunum. All 4 preparations were present in bath at same time. Records to left of break were taken ca. 5 min prior to addition of procaine. Temperature, 36°C. FIG.
9 Atropine
5x 10s4 M
w-
I min
FIG. 5. Effect of atropine on mechanical activity simultaneously recorded from 2 stripped - - circular muscle rings (C 1 and C,) and 2 intact circular muscle rings (C, and C,). Contractile responses occurred in intact rings after washout (w) of atropine. Temperature, 37°C.
mine whether or not atropine, procaine, and tetrodotoxin could directly stimulate circular smooth muscle at the same concentrations used by previous investigators to stimulate intact intestine. However, at the same dose of atropine (5 X 1OW4M) and of procaine (5 X low4 M), the response of stripped circular muscle was greater than that of intact circular muscle in three out of four and four out of four preparations, respectively. An interesting observation for
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1612
A.
4 Procaine 6. Effect of C,) and stripped 37°C.
FIG.
and ture,
W
5 x low4 M procaine (C,
on and
Cd)
mechanical circular
activity muscle
of rings.
b
4
I min
intact (Cl Tempera-
BORTOFF
AND
R.
MULLER
circular muscle, evidence of pacemaker activity should be apparent in circular muscle during exposure to these drugs. This was indeed found to be the case when the tissue was examined for electrical activity with pressure electrodes. Pacemaker activity consisted, for the most part, of “diastolic depolarization” which gave rise to single-spike potentials at regular intervals. Examples of diastolic depolarizations are shown in the top tracing of Fig. 8 during exposure to atropine and in the top tracing of Fig. 9 in response to procaine. The top tracing of Fig. 8 was taken from what was presumably the pacemaker area of a ring of circular muscle, whereas the bottom tracing was taken from another region of the same tissue approximately 10 min later. The configuration of the latter is more characteristic of follower cells, since the depolarization between spikes is minimal or absent altogether. The tracings of Fig. 9 were obtained from intact circular muscle in the presence of 5 X 10e4 M procaine. This was one of a series of spike potential bursts that occurred in this preparation during exposure to this drug. The first spike appeared at the end of a gradual depolarization, and the intervals between subsequent spikes appear to be inversely
c2
t TTX
5~lO-~g/mI
+ BaCI,
3xlO-“g/mI
-
lmin
7. Effect of ‘ITX on intact ring of intestinal wall (INT), longitudinal-circular muscle ring (L + C), intact circular muscle ring (C I), and stripped circular muscle ring (C 2). Elapsed time during break in records: 2 min. Note that ‘ITX augmented contractions only in innervated preparations, although Ba++ stimulated all 4 preparations. Temperature, 37OC. FIG.
which no explanation is immediately obvious is that preparations occasionally exhibited a greater contractile response when a drug was washed out from the bath than when it was administered (Fig. 5). This occurred most frequently in response to atropine, but was also occasionally observed following washout of Ba++ and of procaine. Administration of TTX alone produced somewhat equivocal results. In 8 of 10 preparations consisting of either intact intestinal wall or longitudinal plus circular muscle layers, addition of TTX to the bath at a concentration of 5 X lo-” g/ml resulted in an augmentation of spontaneous contractions (Fig. 7). However, TTX at this concentration produced an apparent stimulation in only three of nine intact circular preparations (in one of these only a single contraction) and failed to stimulate any of four stripped circular muscle preparations, although the latter responded vigorously to subsequent administration of Ba++ (Fig . 7) . It would appear then that TTX, in order to produce its full excitatory effect, requires the presence of the myenteric plexus or longitudinal muscle, or both. If, as indicated by the previous records, atropine and procaine can induce rhythmic contractions in intestinal
Atropine
10°3M
‘5
FIG. 8. Electrical and mechanical activity induced in circular muscle preparations of cat jejunum by lOA M atropine, recorded with a pressure electrode. Atropine was added to bath ca. 30 min prior to time top records (Cl) were obtained. Pacemaker potentials (diastolic depolarizations) are evident in Cl. Each spike is associated with a contraction. Lower tracing (Cz) was obtained from another point on same preparation approximately 10 min later. This tracing is more characteristic of “follower” cells, no pacemaker potential being evident. Mechanical record is not included because contractions recorded by pressure electrode were not displayed. Temperature, 37°C.
Pfocaine
5X10w4 M
miz
FIG. 9. Electrical (top) and mechanical activity recorded with a pressure electrode from intact circular muscle preparation of cat jejunum in presence of 5 X 10e4 M procaine. Prior to addition of procaine to bath, muscle was quiescent. Several bursts of activity appeared followed by a long train of spikes and associated contractions. Note pacemaker potential preceding each spike. Temperature, 37 “C.
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EXCITATORY
EFFECTS
OF
ATROPINE,
PROCAINE,
AND
related to the rate of slow depolarization associated with them. In this particular preparation, the spike potential bursts eventually gave rise to a prolonged train of single spikes similar to those in Fig. 8. DISCUSSION
The results of these experiments indicate that the excitatory effect of TTX requires the presence of myenteric plexus and/or longitudinal muscle; it is virtually impossible to mechanically separate the longitudinal muscle layer from the myenteric plexus. However, TTX has no excitatory (or inhibitory) effect on intestinal spike potentials, which apparently utilize Ca++ as the charge carrier, or on intestinal slow waves (10). Indeed, the only well-documented effect of TTX is to block fast sodium channels (11). It seems reasonable to conclude, then, that the excitatory effect -of TTX on intestinal smooth muscle is mediated via the myenteric plexus, probably by eliminating spontaneous neural inhibition as suggested by Wood (15) and by Biber and Fara (1). In contrast to TTX, atropine and procaine consistently stimulated both types of circular muscle preparations used The intact circular muscle rings, in these experiments. prepared essentially according to the method of Evans and Schild (8), contained no ganglion cells which could be detected by our own histologic examination, a finding in agreement with observations made by others on similar preparations of circular muscle (8, 13). In addition, we used circular muscle rings from which the innermost layers, containing both the dense layer and the plexus muscularis profundus, had been removed. In most cases this stripped circular muscle preparation was even more sensitive to the stimulating effects of atropine and procaine than were the intact circular muscle preparations. On the basis of these results, it is concluded that atropine and procaine, at the concentrations used in these experiments, have a direct excitatory effect on intestinal circular muscle. There are, of course, severed nerve terminals in both types of circular muscle preparations, but it is unlikely that they could exert the same tonic inhibitory influence on
1613
3-2-X
intestinal muscle that has been attributed to neural elements of the myenteric plexus (15, 17). The fact that the presence of TTX in concentrations up to 5 X 1O-6 g/ml did not alter the response to either atropine or procaine also indicates that neural elements are not involved in this response. It could be argued that, even though these results provide evidence for a direct excitatory effect of atropine and procaine on intestinal smooth muscle, they do not exclude the possibility that in the intact preparation the effect of these drugs may be partly due to a release of neural inhibition. Although this may in fact occur, the threshold concentrations of atropine and procaine required for stimulation of the circular muscle preparations in our experiments were essentially the same as those necessary to stimulate the longitudinal plus circular preparations and also those indicated by Wood (15) for his intact in vitro preparations. Since it is unlikely that the threshold concentrations of procaine and atropine which directly stimulate circular muscle would be the same as those required to block inhibitory neural activity, a direct excitatory effect is indicated as the primary mode of action of these drugs. The rhythmic contractions elicited in circular muscle by these drugs were related to the appearance of pacemaker potentials, appearing in pressure electrode records as diastolic depolarizations between rather prolonged single spikes. This is contrasted to bursts of spikes which have been recorded at the peaks of similar depolarizations in response to Ba+f and also to hyoscine (unpublished observations). It appears that such agents can induce membrane potential oscillations in normally quiescent isolated circular muscle and that the oscillations generate spike potentials which propagate through the tissue triggering coordinated contractile responses. In conclusion, whereas excitation of intestinal muscle by TTX appears to be neuronally mediated, excitation by atropine and procaine can occur directly. This work search Grant Metabolism, Received
was supported AM 06958 and Digestive
in part from the Diseases.
for publication
9 September
1
by Public National
Health Institute
Service Reof Arthritis,
1974.
I
REFERENCES motility increased by tetro1. BIBER, B., AND J. FARA. Intestinal dotoxin, lidocaine, and procaine. Experientia 29 : 551-552, 1973. 2. BORTOFF, A. Electrical activity of intestine recorded with pressure electrode. Am. J. Physiol. 201 : 209-Z 12, 196 1. 3. BORTOFF, A., AND J. SACCO. Myogenic control of intestinal peristalsis. In : Proceedings of the Fourth International Symposium on Gastrointestinal Motility, edited by E. E. Daniel. Vancouver: Mitchell, 1974, p. 53-60. 4. CHRISTENSEN, J., AND G. F. LUND. Atropine excitation of esophageal smooth muscle. J. Pharmacol. Exptl. Therap. 163 : 287-289, 1968. 5. CUTHBERT, A. W. Some effects of atropine on smooth muscle. Brit. J. Pharmacol. 2 1 : 285-294, 1963. 6. DANIEL, E. E., AND M. WOLOWYK. The contractile response of the uterus to cocaine. Can. J. Physiol. Pharmacol. 44: 72 l-730, 1966. 7. DUCHON, G., R. HENDERSON, AND E. E. DANIEL. Circular muscle In : Proceedings of the Fourth Internalayers in the small intestine. tional Symposium on Gastrointestinal Motility, edited by E. E. Daniel. Vancouver: Mitchell, 1974, p. 635-646. 8. EVANS, D. H. L., AND H. 0. SCHILD. The reactions of plexusfree circular muscle of cat jejunum to drugs. J. Physiol., London 119: 376-399, 1953. 9. KURIYAMA, H., T. OSA, AND N. TOIDA. Effect of tetrodotoxin on
smooth
muscle
Chemotherap. 10.
11. 12. 13.
14.
of the
guinea-pig
taenia
Brit. J. Pharmacol.
coli.
! 966.
Lru, J., C. C. PROSSER, AND D. JOB. Ionic dependence of slow waves and spikes in intestinal muscle. Am. J. Physiol. 2 17: 15421547, 1969. NARAHASHI, T. Mechanism of action of tetrodotoxin and saxitoxin on excitable membranes. Federation Proc. 3 1 : 1 124-l 132, 1972. PERSSON, C. G. A. Excitatory effect of tetrodotoxin on an isolated smooth muscle organ. J. Pharm. Pharmacol. 23 : 986-987, 1971. PROSSER, C. L., AND N. SPERELAKIS. Transmission in ganglionfree circular muscle from the cat intestine. Am. J. Physiol. 187 : 536-545, 1956. VOHRA, M. M. An analysis of the contractile responses of the rat vas deferens to Xylocaine (lidocaine) and procaine. European J. Pharmacol. 9 : 14-20, 1970.
1-5. WOOD, dotoxin, 16. WOOD,
17.
cells
27 : 366-376,
J. D. Excitation and Xylocaine. J. F., AND B.
testinal
muscularis:
Physiol.
32 : 734-737,
of intestinal
muscle
by
atropine, tetro1972. relationships of the in-
Am. J. PhysioZ. 222 : 118-125, R.
effects
HARRIS. of
atropine
Phase
and
Xylocaine.
J. APPZ.
1972.
WOOD, J. D., AND D. R. MARSH. Effects of atropine, tetrodotoxin and lidocaine on rebound excitation of guinea-pig small testine. J. Pharmacol. ExptZ. Therap. 184 : 590-598, 1973.
in-
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Stimulation atropine,
in U.S.A.
of intestinal procaine,
ALEX Department
smooth muscle by
and tetrodotoxin
BORTOFF AND RICHARD of Physiology, State University
MULLER of New
York,
BORTOFF, ALEX, AND RICHARD MULLER. Stimulation of intestinal smooth muscle by atropine, procaine, and tetrodotoxin. Am. J. Physiol. 229(6): 1609-1613. 1975.-In order to determine whether or not atropine, procaine, and tetrodotoxin (TTX) can stimulate intestinal smooth muscle directly, we examined the effects of these drugs on the mechanical and electrical activities of several types of cat intestinal smooth muscle preparations. The preparations consisted of isolated rings of I) intact intestinal wall, 2) intact longitudinal and circular muscle, 3) ganglion-free circular muscle, and 4) ganglion-free circular muscle devoid of its dense layer and plexus muscularis profundus. Atropine and procaine ( > 10m4 M) stimulated all- four types of preparation. On the other hand, TTX (up to 5 x lo- 6 M) stimulated only preparations I and 2. It is concluded that whereas atropine and procaine can directly stimulate intestinal smooth muscle, the excitatory effect of TTX is neurally mediated. intestinal
motility;
electrophysiology
IT IS WELL ESTAB LISHED that intestinal smooth muscle can be stimulated by certain drugs which are know n primarily for their inhibitory effects on neural processes. Among these are high concentrations of muscarinic blocking agents such as atropine (3, 4, 15-17), local anesthetics such as procaine and lidocaine (1, 15- 17), and tetrodotoxin (I, 9, 12, 15, 17). In several recent studies the excitatory effects of such drugs have been used as evidence to support the concept that, at least in the case of the small intestine, the intrinsic excitability of visceral smooth muscle is held in abeyance by spontaneously active inhibitory neurons of the intramural plexus. Thus, Wood (15) has interpreted the results of his experiments demonstrating excitation of isolated segments of cat jejunum by procaine, lidocaine, and tetrodotoxin in terms of “blockade of a spontaneously active inhibitory nervous system,” which permits the intestinal muscle to exhibit its intrinsic activity. In an attempt to explain the excitatory effect of atropine, Wood has suggested that at least two neurons are involved, a driver neuron which is spontaneously active and a follower inhibitory neuron which is activated via cholinergic synapses; atropine, according to this scheme, releases the muscle from inhibition by synaptic blockade. A similar explanation for the excitatory effects of tetrodotoxin, lidocaine, and procaine has more recently been proposed by Biber and Fara (l), who studied the effects of these drugs on the small intestines of anesthetized cats. In contrast, other investigators have proposed a direct
Upstate
A4edical
Center,
Syracuse,
New
York
13210
excitatory effect of atropine and local anesthetics on smooth muscle. A direct effect of atropine was suggested by Christensen and Lund (4), since synaptic blockade by nicotine or hexamethonium did not affect the excitatory response of opossum esophageal smooth muscle to high concentrations of atropine. Atropine has also been shown to stimulate the smooth muscle of chick amnion (5), which is devoid of nerves. The excitatory effects of local anesthetics, including procaine and lidocaine, are unaffected by antagonists to excitatory neurotransmitters on smooth muscle from either in these rat vas deferens (14) or rabbit uterus (6), although studies the possibility of blocking neural inhibition was not considered. Thus, the question of whether atropine, local anesthetics, and tetrodotoxin exert their excitatory effect on intestinal smooth muscle directly, or indirectly by release of neural inhibition, is still unresolved. The following experiments were undertaken in an attempt to resolve this issue. METHODS
AND
MATERIALS
All experiments were performed on segments of jejunum obtained from young adult cats under pentobarbital anesthesia. Four types of preparation were used. The first consisted of intact rings of jejunum approximately 5 mm wide. The second consisted of similar rings of intestine from which the mucosa and submucosa had been removed. These are referred to below as “longitudinal-circular IIIUScle” and were prepared in the following manner: segments of jejunum approximately 2 cm long were gently everted and slipped onto a glass rod. Under a dissecting microscope, a longitudinal incision was made through the mucosa and submucosa without penetrating the circular muscle layer. Opposing edges of the cut mucosa-submucosa were then grasped by blunt forceps and gently pulled apart. When the edges were completely separated so that circular muscle was visible through the incision along the entire length of was completely rethe segment, the mucosa-submucosa moved by gently pulling it away from the rest of the segment, force being applied in the circular direction. For use as longitudinal-circular muscle preparations, the segments were removed from the glass rod, inverted, and cut into rings approximately 5 mm wide. Two types of isolated circular muscle preparations were also prepared from these segments. The first was prepared by removing the longitudinal muscle layer from segments which had been slipped back onto the glass rod with the longitudinal muscle layer in its normal orientation. Under
1609
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1610 the dissecting microscope, a longitudinal incision was made with a scalpel along the length of the segment, deep enough to just penetrate the circular muscle layer. The longitudinal muscle layer, myenteric plexus, and the outer portion of circular muscle were then removed by peeling back the tissues as a single sheet, beginning at one edge of the longitudinal incision and continuing around the circumference to the other edge. Strands of connective tissue, blood vessels, and nerves which pass through the segment were cut with iris scissors during the peeling process. The resulting preparations consisted of ganglion-free circular muscle as originally described by Evans and Schild (8). Two rings, 5-10 mm wide, were usually cut from each preparation for use in the following experiments ; the ends of the segments were cut off and discarded. Microscopic examination of complete serial sections of three such circular muscle preparations stained with Mallory’s triple revealed no ganglion cells. A second type of circular muscle preparation was also In these preparations the innerused in some experiments. most portion of the circular muscle layer was removed in order to eliminate the dense circular muscle layer together muscularis profundus,” with the nerve plexus, “plexus which lies between the dense layer and the main circular muscle layer. Both the dense layer and the nerve plexus have been most recently described by Duchon et al. (7). These layers were removed from the intestinal segments immediately after removal of the mucosa-submucosa. After making a superficial longitudinal incision in the circular muscle layer, one edge of the cut was gently teased away from the other edge until enough circular muscle was freed to enable grasping it along the entire length of the intestinal segment with a pair of forceps. This procedure was facilitated by the use of a pair of long, gently tapering forceps held almost parallel to the longitudinal axis of the segment. In successful trials the innermost portion of circular muscle could be removed as a single sheet by gently peeling it back. It is estimated that 15-25 % of the circular muscle layer was removed by this method. The longitudinal with myenteric plexus and some muscle layer, together circular muscle, was then removed as described above. The remaining circular muscle was quite thin and usually separated into natural rings or tight spirals. These were separated on the glass rod by gently moving them apart with a pair of forceps and cutting any connecting strands with iris scissors. The resulting preparations consisted of rings of circular muscle approximately 5-6 mm wide. TO summarize, the four preparations were rings of I) intact intestinal wall, 2) longitudinal-circular muscle (containing myen teric plexus), 3) circular muscle, apparently devoid of ganglion cells but containing the dense layer and plexus muscularis profundus (referred to below as “intact circular muscle”), and 4) circular muscle devoid of both the dense layer and the plexus layer (referred to below as “stripped circular muscle”). Recordings of electrical and mechanical activity were taken from intestinal rings immersed in a loo-ml bath of oxygenated Tyrode solution maintained at 37°C. The composition of the Tyrode solution, in millimoles per liter, was: NaCl, 120.8; KCl, 5.9; NaHC03, 15.5; NaHzPO4, 1.2 ; CaCl2, 2.5 ; MgC12, 1.2 ; and glucose, 11.5. The buffer system was completed by aeration with a mixture of 95 % 02-5 % CO%
A. BORTOFF
AND
R. MULLER
The recording systems were of two kinds. In one, only mechanical activity was recorded, in which case up to four muscle rings were placed on a small glass rod which was immersed and anchored in the bath. A silk thread was attached to each muscle ring, and the other end was tied to a force-displacement transducer (Grass Instrument Co., model Ft .03). Records of mechanical activity were displayed on a four-channel Grass polygraph. In the second type of recording system, electrical activity was recorded, usually together with mechanical activity, using pressure electrodes attached to force-displacement transducers (2). Pressure electrode recordings are similar to those obtained with suction electrodes, i.e., they are monophasic and qualitatively resemble transmembrane recordings. By attaching the pressure electrode to the force-displacement transducer, a record of contractile activity in the region of the electrode tip is also obtained. Drugs were added to the bath with a hypodermic syringe. The drugs used in these experiments included atropine sulfate, procaine hydrochloride, tetrodotoxin (TTX), BaCl2, and acetylcholine chloride. RESULTS
Before studying the effects of atropine, procaine, and tetrodotoxin, stimulation of mechanical activity by acetylcholine and Ba++ was recorded, since these drugs are known to have a direct excitatory effect on intestinal smooth muscle. As indicated by the tracings of mechanical activity in Figs. 1 and 2, both drugs produced essentially the same effect. Spontaneous, rhythmic contractions were usually present in the rings containing both longitudinal and circular muscle, while the rings containing only circular muscle were usually quiescent. Following the addition to the bath of either acetylcholine or Ba ++, the magnitude of contractions in the longitudinal-circular muscle rings increased while rhythmic contractions were elicited in the intact circular muscle preparations. Rarely was a contracture elicited in circular muscle rings unless the concentration of acetylcholine or Ba++ was very high, even though acetylcholine often produced a partial contracture in the longitudinal circular preparations as in Fig. 1. The latency of the response to acetylcholine was shorter than that to Ba++, and the latency for both drugs was usually shorter for the longitudinal circular preparations. The rhythmical contractions elicited in circular muscle occurred either as bursts (Fig. 1, Fig. 2, Cr and C,> or as long trains (Fig. 2, C,). L’CC
# Ach IO-% FIG. 1. Effect tudinal-circular of cat jejunum. 4 X low6 M 10 In this and the because of large 36°C.
t4xlo-6M
‘l
of acetylcholine on mechanical activity of longi(L + C) and intact circular (C) muscle preparations Concentration of acetylcholine in bath was raised to min after initial concentration of 10B6 M was added. following figures, tension records are not calibrated variation in sizes of preparations used. Temperature,
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EXCITATORY
EFFECTS
OF
ATROPINE,
PROCAINE,
AND
1611
TTX
f Atropine
5 x lO*M
‘Imin
3. Typical mechanical response to atropine of longitudinalcircular (L + C) and intact circular (C) muscle preparations of cat jejunum. A low level of spontaneous activity was present only in longitudinal-circular preparation prior to addition of atropine. Temperature, 37°C. FIG.
f BaC12
5x lO’5 g/ml
‘I
FIG. 2. Effect of Ba++ on mechanical activity of longitudinalcircular (L + C) and intact circular (Cl, Cz, and C,) muscle preparations of cat jejunum. All 4 preparations occupied same bath. Prior to addition of BaCl2, longitudinal-circular preparation exhibited spontaneous activity, the 3 circular muscle rings were quiescent. Temperature, 36°C.
The excitatory effect of large concentrations of atropine was similar to that of acetylcholine or Ba++, except that the latency was longer and more variable. Of 30 intact circular muscle preparations used. in one series of experiments, 27 responded to atropine at a threshold concentration between 1 and 4 X 10V4 M, whereas three did not respond up to 2 X UP2 M. A typical m-opine response is shown in Fig. 3. After the addition of atropine to the bath, the contraction amplitudes of the longitudinal circular preparation gradually increased in size ; suddenly after 2 min, 30 s, contractions 15-30 times greater in amplitude appeared. Intact circular muscle preparations responded with similarly large contractions after variable latencies, sometimes as long as 4-5 min, in the case of Fig. 3, after 2 min, 40 s. Once initiated, the contractions continued in both types of preparation as bursts or prolonged trains until after the atropine was washed out of the bath. Contractions usually persisted for a time after the bath was washed, and after subsiding could be reelicited, showing no apparent tachyphylaxis. In response to the concentrations of atropine used in these experiments, the contractions of both types of preparation were rhythmical, showing little if any contracture. The contractions of intact circular muscle were sometimes surprisingly regular, but were usually of a frequency lower than the maximal frequency exhibited by the intact preparation, i.e., the slow-wave frequency. Procaine also stimulated both intact circular and longitudinal circular preparations from 8 of 11 cats (Fig. 4). The latent periods, although variable, tended to be shorter than those for atropine. The mechanical response consisted of rhythmic contractions which occasionally fused into partial contractures (Fig. 9). Excitatory responses to atropine and to procaine were not affected by prior addition to the bath of up to 5 X lo+ g/ml TTX. Stripped circular muscle reacted to atropine and procaine in much the same way as intact circular muscle. A somewhat surprising finding, however, was that it appeared to be even more sensitive to atropine, procaine, and Ba++ than the intact circular preparation (Figs. 5, 6, and 7). Relative sensitivity of the two preparations to these drugs was not studied by means of dose-response curves, since the main purpose of this study was simply to deter-
+ Procai ne
2 x loo4 M
‘I
4. Effect of procaine on mechanical activity of 1 longitudinalcircular (L + C) and 3 intact circular (Cl, Cp, C,) muscle preparations of cat jejunum. All 4 preparations were present in bath at same time. Records to left of break were taken ca. 5 min prior to addition of procaine. Temperature, 36°C. FIG.
9 Atropine
5x 10s4 M
w-
I min
FIG. 5. Effect of atropine on mechanical activity simultaneously recorded from 2 stripped - - circular muscle rings (C 1 and C,) and 2 intact circular muscle rings (C, and C,). Contractile responses occurred in intact rings after washout (w) of atropine. Temperature, 37°C.
mine whether or not atropine, procaine, and tetrodotoxin could directly stimulate circular smooth muscle at the same concentrations used by previous investigators to stimulate intact intestine. However, at the same dose of atropine (5 X 1OW4M) and of procaine (5 X low4 M), the response of stripped circular muscle was greater than that of intact circular muscle in three out of four and four out of four preparations, respectively. An interesting observation for
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1612
A.
4 Procaine 6. Effect of C,) and stripped 37°C.
FIG.
and ture,
W
5 x low4 M procaine (C,
on and
Cd)
mechanical circular
activity muscle
of rings.
b
4
I min
intact (Cl Tempera-
BORTOFF
AND
R.
MULLER
circular muscle, evidence of pacemaker activity should be apparent in circular muscle during exposure to these drugs. This was indeed found to be the case when the tissue was examined for electrical activity with pressure electrodes. Pacemaker activity consisted, for the most part, of “diastolic depolarization” which gave rise to single-spike potentials at regular intervals. Examples of diastolic depolarizations are shown in the top tracing of Fig. 8 during exposure to atropine and in the top tracing of Fig. 9 in response to procaine. The top tracing of Fig. 8 was taken from what was presumably the pacemaker area of a ring of circular muscle, whereas the bottom tracing was taken from another region of the same tissue approximately 10 min later. The configuration of the latter is more characteristic of follower cells, since the depolarization between spikes is minimal or absent altogether. The tracings of Fig. 9 were obtained from intact circular muscle in the presence of 5 X 10e4 M procaine. This was one of a series of spike potential bursts that occurred in this preparation during exposure to this drug. The first spike appeared at the end of a gradual depolarization, and the intervals between subsequent spikes appear to be inversely
c2
t TTX
5~lO-~g/mI
+ BaCI,
3xlO-“g/mI
-
lmin
7. Effect of ‘ITX on intact ring of intestinal wall (INT), longitudinal-circular muscle ring (L + C), intact circular muscle ring (C I), and stripped circular muscle ring (C 2). Elapsed time during break in records: 2 min. Note that ‘ITX augmented contractions only in innervated preparations, although Ba++ stimulated all 4 preparations. Temperature, 37OC. FIG.
which no explanation is immediately obvious is that preparations occasionally exhibited a greater contractile response when a drug was washed out from the bath than when it was administered (Fig. 5). This occurred most frequently in response to atropine, but was also occasionally observed following washout of Ba++ and of procaine. Administration of TTX alone produced somewhat equivocal results. In 8 of 10 preparations consisting of either intact intestinal wall or longitudinal plus circular muscle layers, addition of TTX to the bath at a concentration of 5 X lo-” g/ml resulted in an augmentation of spontaneous contractions (Fig. 7). However, TTX at this concentration produced an apparent stimulation in only three of nine intact circular preparations (in one of these only a single contraction) and failed to stimulate any of four stripped circular muscle preparations, although the latter responded vigorously to subsequent administration of Ba++ (Fig . 7) . It would appear then that TTX, in order to produce its full excitatory effect, requires the presence of the myenteric plexus or longitudinal muscle, or both. If, as indicated by the previous records, atropine and procaine can induce rhythmic contractions in intestinal
Atropine
10°3M
‘5
FIG. 8. Electrical and mechanical activity induced in circular muscle preparations of cat jejunum by lOA M atropine, recorded with a pressure electrode. Atropine was added to bath ca. 30 min prior to time top records (Cl) were obtained. Pacemaker potentials (diastolic depolarizations) are evident in Cl. Each spike is associated with a contraction. Lower tracing (Cz) was obtained from another point on same preparation approximately 10 min later. This tracing is more characteristic of “follower” cells, no pacemaker potential being evident. Mechanical record is not included because contractions recorded by pressure electrode were not displayed. Temperature, 37°C.
Pfocaine
5X10w4 M
miz
FIG. 9. Electrical (top) and mechanical activity recorded with a pressure electrode from intact circular muscle preparation of cat jejunum in presence of 5 X 10e4 M procaine. Prior to addition of procaine to bath, muscle was quiescent. Several bursts of activity appeared followed by a long train of spikes and associated contractions. Note pacemaker potential preceding each spike. Temperature, 37 “C.
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EXCITATORY
EFFECTS
OF
ATROPINE,
PROCAINE,
AND
related to the rate of slow depolarization associated with them. In this particular preparation, the spike potential bursts eventually gave rise to a prolonged train of single spikes similar to those in Fig. 8. DISCUSSION
The results of these experiments indicate that the excitatory effect of TTX requires the presence of myenteric plexus and/or longitudinal muscle; it is virtually impossible to mechanically separate the longitudinal muscle layer from the myenteric plexus. However, TTX has no excitatory (or inhibitory) effect on intestinal spike potentials, which apparently utilize Ca++ as the charge carrier, or on intestinal slow waves (10). Indeed, the only well-documented effect of TTX is to block fast sodium channels (11). It seems reasonable to conclude, then, that the excitatory effect -of TTX on intestinal smooth muscle is mediated via the myenteric plexus, probably by eliminating spontaneous neural inhibition as suggested by Wood (15) and by Biber and Fara (1). In contrast to TTX, atropine and procaine consistently stimulated both types of circular muscle preparations used The intact circular muscle rings, in these experiments. prepared essentially according to the method of Evans and Schild (8), contained no ganglion cells which could be detected by our own histologic examination, a finding in agreement with observations made by others on similar preparations of circular muscle (8, 13). In addition, we used circular muscle rings from which the innermost layers, containing both the dense layer and the plexus muscularis profundus, had been removed. In most cases this stripped circular muscle preparation was even more sensitive to the stimulating effects of atropine and procaine than were the intact circular muscle preparations. On the basis of these results, it is concluded that atropine and procaine, at the concentrations used in these experiments, have a direct excitatory effect on intestinal circular muscle. There are, of course, severed nerve terminals in both types of circular muscle preparations, but it is unlikely that they could exert the same tonic inhibitory influence on
1613
3-2-X
intestinal muscle that has been attributed to neural elements of the myenteric plexus (15, 17). The fact that the presence of TTX in concentrations up to 5 X 1O-6 g/ml did not alter the response to either atropine or procaine also indicates that neural elements are not involved in this response. It could be argued that, even though these results provide evidence for a direct excitatory effect of atropine and procaine on intestinal smooth muscle, they do not exclude the possibility that in the intact preparation the effect of these drugs may be partly due to a release of neural inhibition. Although this may in fact occur, the threshold concentrations of atropine and procaine required for stimulation of the circular muscle preparations in our experiments were essentially the same as those necessary to stimulate the longitudinal plus circular preparations and also those indicated by Wood (15) for his intact in vitro preparations. Since it is unlikely that the threshold concentrations of procaine and atropine which directly stimulate circular muscle would be the same as those required to block inhibitory neural activity, a direct excitatory effect is indicated as the primary mode of action of these drugs. The rhythmic contractions elicited in circular muscle by these drugs were related to the appearance of pacemaker potentials, appearing in pressure electrode records as diastolic depolarizations between rather prolonged single spikes. This is contrasted to bursts of spikes which have been recorded at the peaks of similar depolarizations in response to Ba+f and also to hyoscine (unpublished observations). It appears that such agents can induce membrane potential oscillations in normally quiescent isolated circular muscle and that the oscillations generate spike potentials which propagate through the tissue triggering coordinated contractile responses. In conclusion, whereas excitation of intestinal muscle by TTX appears to be neuronally mediated, excitation by atropine and procaine can occur directly. This work search Grant Metabolism, Received
was supported AM 06958 and Digestive
in part from the Diseases.
for publication
9 September
1
by Public National
Health Institute
Service Reof Arthritis,
1974.
I
REFERENCES motility increased by tetro1. BIBER, B., AND J. FARA. Intestinal dotoxin, lidocaine, and procaine. Experientia 29 : 551-552, 1973. 2. BORTOFF, A. Electrical activity of intestine recorded with pressure electrode. Am. J. Physiol. 201 : 209-Z 12, 196 1. 3. BORTOFF, A., AND J. SACCO. Myogenic control of intestinal peristalsis. In : Proceedings of the Fourth International Symposium on Gastrointestinal Motility, edited by E. E. Daniel. Vancouver: Mitchell, 1974, p. 53-60. 4. CHRISTENSEN, J., AND G. F. LUND. Atropine excitation of esophageal smooth muscle. J. Pharmacol. Exptl. Therap. 163 : 287-289, 1968. 5. CUTHBERT, A. W. Some effects of atropine on smooth muscle. Brit. J. Pharmacol. 2 1 : 285-294, 1963. 6. DANIEL, E. E., AND M. WOLOWYK. The contractile response of the uterus to cocaine. Can. J. Physiol. Pharmacol. 44: 72 l-730, 1966. 7. DUCHON, G., R. HENDERSON, AND E. E. DANIEL. Circular muscle In : Proceedings of the Fourth Internalayers in the small intestine. tional Symposium on Gastrointestinal Motility, edited by E. E. Daniel. Vancouver: Mitchell, 1974, p. 635-646. 8. EVANS, D. H. L., AND H. 0. SCHILD. The reactions of plexusfree circular muscle of cat jejunum to drugs. J. Physiol., London 119: 376-399, 1953. 9. KURIYAMA, H., T. OSA, AND N. TOIDA. Effect of tetrodotoxin on
smooth
muscle
Chemotherap. 10.
11. 12. 13.
14.
of the
guinea-pig
taenia
Brit. J. Pharmacol.
coli.
! 966.
Lru, J., C. C. PROSSER, AND D. JOB. Ionic dependence of slow waves and spikes in intestinal muscle. Am. J. Physiol. 2 17: 15421547, 1969. NARAHASHI, T. Mechanism of action of tetrodotoxin and saxitoxin on excitable membranes. Federation Proc. 3 1 : 1 124-l 132, 1972. PERSSON, C. G. A. Excitatory effect of tetrodotoxin on an isolated smooth muscle organ. J. Pharm. Pharmacol. 23 : 986-987, 1971. PROSSER, C. L., AND N. SPERELAKIS. Transmission in ganglionfree circular muscle from the cat intestine. Am. J. Physiol. 187 : 536-545, 1956. VOHRA, M. M. An analysis of the contractile responses of the rat vas deferens to Xylocaine (lidocaine) and procaine. European J. Pharmacol. 9 : 14-20, 1970.
1-5. WOOD, dotoxin, 16. WOOD,
17.
cells
27 : 366-376,
J. D. Excitation and Xylocaine. J. F., AND B.
testinal
muscularis:
Physiol.
32 : 734-737,
of intestinal
muscle
by
atropine, tetro1972. relationships of the in-
Am. J. PhysioZ. 222 : 118-125, R.
effects
HARRIS. of
atropine
Phase
and
Xylocaine.
J. APPZ.
1972.
WOOD, J. D., AND D. R. MARSH. Effects of atropine, tetrodotoxin and lidocaine on rebound excitation of guinea-pig small testine. J. Pharmacol. ExptZ. Therap. 184 : 590-598, 1973.
in-
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