ductance in the taenia coli. 0. In view of the fact that muscle from the esophageal. % body was able to respond normally to an exogenous .s 40- excitatory ...
Temperature dependence of responses of esophageal smooth muscle to electrical field stimulation DAVID J. DE CARLE, ALEXANDER C. SZABO, AND JAMES CHRISTENSEN Gastroenterology Research Laboratories, Department of Internal Medicine, University Iowa City, Iowa 52242
DE CARLE, CHRISTENSEN.
DAVID
J., ALEXANDER
C. SZABO,
AND
JAMES
Temperature dependence of responses of esoph-
ageal smooth muscle to electrical field stimulation. Am. J. Physiol. 232(4): E432-E436, 1977 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. l(4): E432-E436, 1977. Strips of smooth muscle, cut transversely from the smoothmuscle segment of opossum esophagus, were superfused with oxygenated Krebs-Ringer solution at 37°C in a system that allowed electrical field stimulation of the intrinsic nerves. Three-tefive-second trains of rectangular pulses (0.5 ms long at 10 Hz) were delivered at 30-s intervals at supramaximal maximal current strength. In strips from the esophageal body, each train resulted in a twitch which followed after the end of the train with a particular latency, the off-response. Strips from the esophagogastric sphincter relaxed during the train. Temperature was varied above and below 37°C to observe the temperature dependence of the responses. Latency of the offresponse varied exponentially with temperature. Amplitude of the off-response showed a linear decline with changes in temperature, both above and below 35”C, the zero-intercepts being 19.6 and 42.3”C, respectively. Amplitude of relaxation of strips from the junction varied little between 20 and 37*C but declined sharply beyond those limits, the zero-intercepts being 14.2 and 42”C, respectively. esophagus;
opossum;
off-response;
lower
esophageal
sphincter
STRIPS of smooth muscle cut from the body of the opossum esophagus respond to electrical field stimulation with a single transient contraction that does not begin until well after the end of the stimulus train. This twitch has been called the off-response. It can be produced by rectangular pulses less than 0.5 ms long, has a frequency optimum of 10 Hz or less, and is sensitive to tetrodotoxin at low concentrations (l-3). Thus, it appears to be nerve mediated. In tubular preparations of the smooth-muscle part of the body of the opossum esophagus in vitro, electromyography reveals bursts of rapid electrical transients occurring only after the end of a stimulus train delivered to the esophageal wall or after deflation of a distending balloon. These brief contractions are also tetrodotoxin sensitive (4). In the intact esophagus of the opossum, examined in vivo under light anesthesia, vagal stimulation induces contraction of the circular muscle layer of the smoothmuscle part of the esophageal body, and this contraction does not occur until after the end of the stimulus train (5). Thus, in all three kinds of preparations, an off-
TRANSVERSE
E432
of Iowa,
response is the principal response of the circular layer of smooth muscle of the esophageal body produced by nerve stimulation, whether intramural nerves or vagal nerves are stimulated. In muscle strips cut from the region of the lower esophageal sphincter in various species, electrical field stimulation induces relaxation during the stimulus train which may or may not be followed by an off-response (3). In studies of strips of smooth muscle cut from the smooth-muscle segment of the opossum esophagus, it was found that the period between the end of the stimulus train (delivered as an electrical field) and the onset of the off-response is not constant along the esophagus (6); this de1 ay or latency of the off-response increases from the most proximal level of smooth muscle to the most distal level. It has been suggested that this gradient in latency of the off-response accounts, at least in part, for the peristaltic nature of the esophageal contraction produced by swallowing. A model of damped oscillations in cellular transmembrane electrical potential has been proposed to account for this latency (71, but the fundamental reason for the latency gradient remains unknown. Nevertheless, it seems reasonable, based on the work reviewed briefly above, to propose that the off-response seen in isolated strips of esophageal smooth muscle may be the in vitro representation of swallowing-induced peristalsis in the smooth-muscle esophagus in vivo. Cooling the esophagus causes slowing of swallowinginduced esophageal peristalsis in normal volunteers. In a common clinical disorder generally called diffuse esophageal spasm, the progressive nature of the swallowing-induced contraction in the smooth muscle segment is lost; instead, swallowing produces forceful, sometimes repetitive contractions of the smooth muscle segment that occur with no fixed order in space. It is a matter of common clinical experience that the disorder, or at least the dysphagia that it produces, is precipitated or made worse when chilled beverages are ingested. This suggests that a reduced temperature in the esophagus has some effect on the mechanisms responsible for swallowing-induced peristalsis. Because the off-response may be related to the peristalsis induced by swallowing, we chose to investigate the effect of temperature alterations on the off-response of strips of esophageal smooth muscle in vitro. The amplitude and latency of the off-response in muscle
ESOPHAGEAL
SMOOTH
MUSCLE
strips from the esophageal body were studied, as was the relaxation of strips cut from the lower esophageal sphincter. The ability of the muscle strips to respond to direct stimulation was assessed by measuring the excitatory response to exogenous acetylcholine and the inhibitory response to exogenous isopropylnorepinephrine. METHODS
E433 37°C. In other experiments, the temperature was raised in approximately 1°C steps to 4O*C and then reduced to 37*C. At each temperature level, the off-response latency was measured repeatedly; it usually became stable within 5 min of the time when the temperature became stable. The stable values achieved at each stage were then tabulated. Only the values obtained during the initial cooling or heating treatment were used for calculation of results, although very little hysteresis was noted when the results of a full cycle of temperature changes were examined. Ten strips were used in all, and mean latency in seconds at each temperature was plotted against temperature. Effect of temperature on off-response amplitude and LES relaxation. In a separate set of experiments, smooth muscle strips were prepared and studied in a similar manner except that a pair of strips was cut from each esophagus, one strip coming from the esophagogastric junction and one strip coming from the midesophageal body. The strips from the esophagogastric junction invariably maintained tension of from 3 to 6 g when stretched to 150% of resting length and relaxed during electrical field stimulation. Temperature was varied as described above, and the amplitude of relaxation of the junction strip and off-response of the body strip were measured after stabilization at each temperature. The value at each temperature for each strip was expressed as a percentage of the maximum value obtained for that strip. These values were then used to calculate a mean amplitude at each temperature, and a plot of temperature against mean amplitude was obtained. The effect of 10 ~1 of acetylcholine 10m4M injected into the superfusion stream was tested at 37*C and at the extremes of temperature variation. This volume and concentration result in a final concentration in the bath of approximately 6 x lo- 7 M. Complete dose-response curves for acetylcholine were obtained in four body strips at 37, 32.6, 28.2, and 23.5OC. Dose-response curves for isopropylnorepinephrine were obtained in four junctional strips at 37, 32.6, 27.2, and 23.l”C.
Opossums of either sex weighing between 1.9 and 4.2 kg were anesthetized with intraperitoneal sodium pentobarbital. The stomach and esophagus were removed en bloc and opened along the greater curvature of the stomach. The mucosa was removed by sharp dissection through the submucosa, and transverse strips of smooth muscle approximately 2 mm x 1.5 cm were cut from the esophageal body. In experiments designed to examine strips from the esophageal body, no strips were cut less than 1 cm from the gross esophagogastric junction or closer than 1 cm to the grossly evident junction between striated and smooth muscle. One to four strips were cut from each animal. They were suspended in a tissue bath with one end fixed and the other attached to an isometric force/displacement transducer (Grass model FT. 03) connected to a strip-chart recorder (Beckman type RM Dynog-raph). They were then stretched to about 150% of initial length. The bath, which has been described in detail elsewhere (8), was a small block of Plexiglas with a central channel. Two vertical channels 3 mm in diameter allowed thermistor probes to be passed into the central channel adjacent to the two suspended muscle strips. At intervals along the central channel, there were platinum rings connected to the exterior to allow electrical field stimulation of the contained muscle strips. Krebs solution, containing sodium 138.5 mM, potassium 4.6 mM, calcium 2.5 mM, chloride 125.0 mM, phosphate 1.2 mM, and bicarbonate 21.9 mM, and aerated with 95% O,-5% CO*, was pumped through the bath from a jacketed reservoir at 6.0 ml/min. A jacketed heating coil immediately proximal to the bath, and connected in RESULTS Effects of temperature variation on latency of offparallel with the reservoir to a circulating water bath, allowed temperature within the tissue bath to be accu- response. In all strips, lowering the temperature below rately regulated. Temperature adjacent to the tissues 37*C increased the latency, whereas raising the temperature above 37*C reduced it. A representative example within the tissue bath was monitored with two thermistor probes (Yellow Springs Instruments model 402) con- of the off-response at 37 and 26*C is shown in Fig. 1. The plot of mean latency in all eight strips against temperanected to a telethermometer (Yellow Springs Instruments model 46 TUC). ture was a single exponential curve (Fig. 2). At 37*C the The strips were stimulated with rectangular pulses mean latency was 0.44 t 0.05 s (SE), whereas at 24°C 0.5 ms long, at 10 Hz. Pulse trains lasted 2 s and were the mean latency was 2.66 -+ 0 .29 s (SE). The points fit repeated continuously every 30 s. The time from the end the function y = aebx where y is latency in seconds, x is of the stimulus train to the onset of the off-response was the temperature in degrees centigrade, and a, e, and b measured at 5-min intervals using a chart paper speed are the constants 57.57, 2.72, and -0.13, respectively. The r value is 0.99. The standard error of the mean for of 25 mm/s. The onset of the off-response was considered to occur at the point where the base line had risen by 1 each temperature, shown in Fig. 2, ranges from 0.01 at mm, using a gain such as to give a pen deflection of at 37°C to 0.29 at 24°C. In these experiments, all strips came from the proximal end of the esophagus, and this least 1 g/cm. Effect of temperature variation on latency of off-reaccounts for the short latency at 37*C. When strips from sponse. Measurements were first made during 1 h at the distal esophagus, with a much longer latency at 37°C. Then, the temperature in the bath was lowered to 37”C, were tested, a similar pattern of variation was 18°C in steps of about 2”C, and then raised similarly to seen.
E434
DE
1
4 Stirnub 1.
sotI t\
. 1 c\ I \I
2.5
T
‘- 2’5
SZABO,
AND CHRISTENSEN
S8C
Signal
Ofkesponses in a single transverse strip of smooth muscle from middle part of body of esophagus are shown at 37°C (A) and 26% (B). In both panels, bottom traces show train of pulses. Two FIG.
CARLE,
I
I
I
30
35
40
Temperature
(“Cl
-FIG. 2. Latency of off-response is plotted against temperature. Each point represents mean 2 1 SE of latency of 8 strips at each .@nperature. Solid line represents function y = aebr where y is latency in seconds, a = 57.57, e = 2.72, and b = -0.13. Points fit this curve with an r value of 0.99.
The itime from injection of acetylcholine to the onset of co&ra&iun due to acetylcholine was measured in four ,a$ various tern-peratures and was .the same at all .strips . fensperrrtures. .Th,is ;time reflects a combination of the kk& &~kbe drug to pass from the injection site to the .h -and &he time required for activation of the muscle. &$ect of tempe&ture QJLoff-respoflse amplitude. Al=~ature caused a*&ange in amplitude of the .a~.. The :maximum amplitude was at 35OC.
arrows in upper traces of both panels indicate points used to measure latency of twitch. It can be seen that latency has more than doubled and that amplitude has diminished at lower temperature.
Lowering of temperature caused a linear fall in offresponse amplitude (Fig. 3), with a slope of 6.37, an x intercept of 19.59, and an r value 0.98. Raising the temperature caused a dramatic fall in off-response amplitude with a slope of -15.13, an x intercept of 42.27, and an r value of 0.96. There was no change in basal tension over the range of temperatures tested. The amplitude of contraction after acetylcholine did not change at low temperatures, whether measured with a single moderate dose of acetylcholine or with a complete doseresponse curve. As can be seen in Fig. 1, the duration of the off-response increased at low temperatures. This was not quantitated and .it was not observed with temperatures between 37 and 40°C. Effect of temperature on LES relaxation. The magnitude of the relaxation in the junctional strips during electrical field stimulation remained approximately constant at temperatures from 25 to 37OC. Between 25 and ZO”C, the basal tension in the strips rose, giving an apparent rise in relaxation amplitude. Above 37OC the depth of the relaxation diminished in a linear fashion over a very short range without change in basal tension. The slope of this decline was -12.79, thex intercept was 42.0, and the r value was 0.97. Below 20°C the basal tension in the strips fell slightly, and the magnitude of the relaxation diminished in a linear fashion over a short range. The slope was 15.88, the x intercept was 14.21, and the r value was 0.93 (Fig. 4). The inhibitory response to isopropylnorepinephrine was unchanged by reducing temperature from 37 to 23.l”C. DISCUSSION
These studies show that the operation of the circular layer of smooth muscle in the esophageal body is sensitive to changes in temperature. Changes occur both in the amplitude and in the latency of contraction after nerve stimulation. The changes in amplitude may reflect alterations in rates of transmitter release or dispersion which h,ave been described in other smooth muscle
ESOPHAGEAL
SMOOTH
E435
MUSCLE
8 70 8 2 k
FIG. 3. Amplitude of off-response is plotted against temperature. Solid line on left has a slope of 6.37 and an x intercept of 19.59. Points below 35°C fit this with an R value of 0.98. Line on right has a slope of -15.13 and an x intercept of 42.27. Points above 35°C fit this with anR value of 0.96. Each point represents mean of from 4 to 12 observations. Standard errors ranged from 3.3 to 12.6 but are not shown in order to improve clarity of illustration.
60
10 18
20
22
24
26
28
3Q
Temperature
32
34
loo-
8
90
-
80
-
70l
s *s 5 e k \ 0 % .s 3 E T
0 0
0 0
60
-
0 0 0
5oc
40-
30
-
20
0
10
0
-t/ -14
18
22
26
30
34
36
38
40
42
(OC)
38’
$2
Temperature (“C) FIG. 4. Amplitude of LES basal tension inhibition is plotted against temperature. Solid line on left has a slope of 15.88, and an x intercept of 14.21, and points 20°C and below fit with an R value of 0.93. Line on right has a slope of -12.79. and an x intercept of 42. Points 37°C and above fit with an R value of 0.97. Each point represents mean of from 3 to 8 observations. Standard errors ranged from 2.7 to 15.3 but are not shown in order to improve cfarity of illustration.
preparations (g-10). The change in the latency of the offresponse is unexplainable at present because the mechanism of the latency itself is not known. It is possible that this change is also related to an altered role of transmitter release or dispersion. It has been suggested (7) that the latency of the off-response is related to the rate of passive partial depolarization after hyperpolarization during stimulation of inhibitory nerves. Repolarization is dependent, at least in part, on membrane conductance, and our findings are consistent with the hypothesis that temperature changes alter off-response latency by changing smooth-muscle membrane conductance. It has been reported, however (9), that temperature changes cause little alteration in membrane conductance in the taenia coli. In view of the fact that muscle from the esophageal body was able to respond normally to an exogenous excitatory chemical stimulus over a wide range of temperatures, it seems unlikely that the changes seen in the off-response are due to nonspecific depression of muscle ebatractility . The’ findings in the smooth muscle from the esophagogastric junction are different from those in the esophagea1 body, in that the response to field stimulation, relaxation, remains relatively constant in amplitude over a wide range of temratures and then diminishes sharply at both ends of that range. The nerve-muscle combination is rela%iveIy ins‘ensi%ive to temperature changes and. thus differs from the esophageal body in some physical characteri&ic. In junctional strips the response to an exogenuas inMl$to~y chemical stimulus was unc*hanged over a range of WwYatures, suggesting that &tinges seeh with temperattite were not due to nonspecific alteration in the ability of the muscle to relax. It is generally assumed that the al!Ur&Ws in esophageal function caused by inge&ioti of co&! Merials in normal persons (1-1) and in p%ien%s with emhageal
E436 spasm are mediated by afferent nerves activated by thermoreceptors. This study was prompted by the idea that a second possibility exists as well, a direct effect of temperature on local neuromuscular mechanisms in the esophagus itself. The alterations reported here with temperature are, however, difficult to relate to those seen in patients with esophageal spasm. We did not observe that a temperature reduction increased contraction amplitude or led to repetitive off-responses, the two things that might have been expected from the assumption that the off-response, as seen in vitro, can be related to swallowing-induced peristalsis in the smoothmuscle esophagus (6). These experiments, then, fail to reveal any effect of temperature on the local neuromuscular mechanisms in esophageal smooth muscle that can be clearly related to
DE CARLE,
SZABO,
AND
CHRISTENSEN
the peculiar alterations in esophageal function in vivo produced by esophageal cooling. What they do reveal, however, is that the off-response is graded (both in amplitude and latency) over a wide range of temperatures whereas the relaxation of the junctional segment is not. These observations are consistent with the view that the off-response represents a “rebound” contraction, a passive response of the muscle after some change induced by the preceding hyperpolarization induced by stimulation of an inhibitory nerve. If this rebound contraction follows passive entry of ions allowed by a permeability change in the muscle cell membrane, for example, such a gradation with temperature might be expected. Received
for
publication
14 July
1976.
REFERENCES
1.
W. W.-L., AND N. E. DIAMANT. Electrical off response of cat esophageal smooth muscle: an analog simulation. Am. J. Physiol. 230: 233-238, 1976. 2. CHRISTENSEN, J., J. L. CONKLIN, AND B. W. FREEMAN. Physiologic specialization at the esophagogastric junction in three species. Am. J. Physiol. 225: 1265-1270, 1973. 3. CHRISTENSEN, J., B. W. FREEMAN, AND J. K. MILLER. Some physiological characteristics of the esophagogastric junction in the opossum. Gastroenterology 64: 1119-1125, 1973. 4. CHRISTENSEN, J., AND G. F. LUND. Esophageal responses to distension and electrical stimulation. J. Clin. Invest. 48: 40% 419, 1969. 5. DE CARLE, D. J., AND J. CHRISTENSEN. A dopamine receptor in esophageal smooth muscle of the opossum. Gastroenterology 70: 216-219, 1976. 6. HUBBARD, J. I., S. F. JONES, AND E. M. LANDAU. The effect of temperature change upon transmitter release, facilitation and
post-tetanic
CHAN,
potentiation. J. Physiol., London 216: 591-609, 1971. L. P., AND A. DEN HERTOG. Effect of temperature on the transmitter release from the ‘purinergic’ nerves in the guineapig taenia coli. European J. PharmacoZ. 29: 201-205, 1974. MUKHOPADHYAY, A. K., AND N. W. WEISBRODT. Neural organization of esophageal peristalsis: role of vagus nerve. GastroenteroZogy 68: 444-447, 1975. TUCH, A., AND S. COHEN. Lower esophageal sphincter relaxation: studies on the neurogenic inhibitory mechanism. J. CZin. Invest. 52: 14-20, 1973. WEISBRODT, N. W., AND J. CHRISTENSEN. Gradients of contractions in the opossum esophagus. Gastroenterology 62: 1159-1166, 1972. WINSHIP, D. H., S. R. VIEGAS DE ANDRADE, AND F. F. ZBORAL~KE. Influence of bolus temperature on human esophageal motor function. J. CZin. Znuest. 49: 243-250, 1970.
7. JAGER, 8.
9.
10.
11.
