AMERICAN JOURNAL OF PHYSIOLOGY Vo!. 230, No. 1, January 1976. Prinld
in
U.S.A.
Correlation of contractions in rat small intestine
and transit
LARRY D. SCOTT AND ROBERT W. SUMMERS Division of Gastroenterology, Department of Internal Iowa City, Iowa 52242
SCOTT, LARRY D., AND&BERT W. SUMMERS. CorreZation of contractions and transit in rat small intestine. Am. J. Physiol. 230(l): 132-137. 1976. -A single strain gauge was implanted chronically in the proximal jejunum of the rat to record contractions. Fasted pentobarbital-anesthetized animals received glucagon, caerulein, or isotonic saline control infusions intravenously while contractions were recorded. In control animals, the distribution of contractions in time showed clusters, with peaks at intervals of 14.24 -+ 5.6 (SD) min. Glucagon produced dose-related inhibition of contractions. Caerulein at two lower doses produced a more uniform distribution of contractions in time; a higher dose caused inhibition. In other fasted rats, an isotopically labeled bolus was given through a chronically implanted duodenal catheter, and its distribution along the small intestine was examined in animals receiving the same doses of glucagon, caerulein, and saline. Glucagon caused a dose-related delay in transit. Those doses of caerulein that produced a uniform distribution of contractions accelerated transit; the dose that inhibited contractions delayed transit. Doses of glucagon and caerulein known to affect both motility and transit did not significantly affect water movement. The fasted rat resembles the fasted dog in respect to the temporal distributions of jejunal contractions at a single point. Both quantitative and qualitative changes in contractions induce changes in transit. motility;
glucagon;
caerulein
FORCES accomplishing small bowel flow are generated in the wall of the gut. The responsible contractions may also provide much of the resistance to flow. Neural and hormonal control mechanisms have been considered important in the distribution and incidence of contractions (6). Records of contractions themselves, or of the small bowel electromyogram, give no information about flow. The patterns of contractions and flow are very much altered by fasting in the dog (7, 17). The quite uniform distribution of contractions in time, characteristic of the fed state, gives way to a very ordered distribution in fasting in which contractions occur in clusters with peaks at quite regular intervals in time. This ordered distribution, called the “interdigestive migratory myoelectric complex” in the dog, seems to be the constant in ruminants, irrespective of the state of feeding (10). This study was designed to define the pattern of contractions, to measure net transit in the rat smal1 intestine under fasting conditions, and to examine the effects
THE PROPULSIVE
Medicine, University
of Iowa,
of glucagon and caerulein, agents known to affect small intestinal muscle, on contractions, water movement, and net flow. MATERIALS
AND METHODS
Studies of contractions. Male albino Sprague-Dawley rats, 10&250 g, were anesthetized with intraperitoneal pentobarbital sodium (45 mg/kg) for implantation of strain gauges on the proximal jejunum. One small unbacked semiconductor strain gauge transducer (BLH Electronics, Waltham, Mass.) was sewn to the serosa in each rat within 1 cm of the ligament of Treitz (method of K. Hubel, personal communication). The gauges were oriented so that the active element was perpendicular to the long axis of the intestine. The wires were led subcutaneously to the back of the neck and connected to a plug, embedded in a dental acrylic button sewn to the paraspinous muscles. The wounds were closed and., after 5 days, the rats were anesthetized as before for experimental study. For this study, they had been given access to water but not food for the preceding 18-22 h. A cannula was placed in a femoral or jugular vein and the transducer output was led, through the cervical plug, to an ink-writing polygraph (type 411 Dynograph recorder with type 9853A coupler modified for use with a Beckman Instrument). The electrical transducer signal, representing contractions, was recorded for approximately 1 h. Then intravenous infusions were made for at least a successive hour of recording, after which the cannula was removed and the animal allowed to recover. Most animals underwent three to four trials with at, least a 4day interval. Glucagon was infused (Eli Lilly and Company, Indianapolis, Ind.) in 18 trials, caerulein (Societa Farmaceutici Italia, Milan, Italy) in 18 trials, and 0.9% NaCl, the solvent for the peptides, was infused in six trials. Glucagon was infused at 1.0, 10.0, and 100.0 lug/kg per min, each ani ma1 receiving only one dose with-six animals in each dosage group. Caerulein was given at 0.001, 0.01, and 1.0 pg/kg per min, the doses being allocated among the animal .s as described for glucagon. All infusi ons were given at 0.04 ml/min by a constantinfusion syringe pump (Harvard Apparatus Co., Inc., Millis, Me.). Records of transducer output showed contractions occurring in clusters or bursts of five to a dozen or more contractions with intervening rest periods lasting sev-
1 32 Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 13, 2018.
TRANSIT
IN
RAT
133
INTESTINE
era1 minutes. Net activity was measured in 1-min units of time with a map mileage marker that yielded a number indicating the length of the line written by the polygraph pen, This measurement was made in one direction only, from the base line. This number, representing an integration of force and frequency, is referred to as the motility index. With the DC amplifier gain such that the biggest excursions yielded full-scale deflections (2.5 cm), deflections less than 3 mm were considered to be noise and were not included in this measurement. Figure 1 shows a correlation of an actual record and the calculated motility index in 1-min intervals, Records were also read for the frequency of contractions within contraction bursts and for the lengths of the periods between the termination of bursts. Studies of transit. Other rats, like those described above, were anesthetized as described above. In each, a PE-50 polyethylene tube was passed subcutaneously from the back of the neck to and through the abdominal wall and into the forestomach through an incision, to terminate in the duodenum about 3 cm beyond the pylorus. The proximal end of the tube was attached to a 24-gauge hypodermic needle hub that was sutured to the paraspinous muscles, and the incisions were closed (15). Animals so prepared gained weight and showed no obvious signs of impaired gastrointestinal function. Each animal was allowed free access to food and water for 5 days when, after an 18- to 22-h fast, it was anesthetized as before. A femoral or jugular vein was cannulated with PE-10 polyethylene tubing and an intravenous infusion was begun with one of the solutions described above. Twelve animals received isotonic saline while each rat in each of three dosage groups of six animals received one of the same three doses of caerulein or glucagon. After 10 min of intravenous infusion, a O.&ml bolus of a solution of mannitol (300 mosM) containing 50 &i of “lCr as sodium chromate (Na,“VrO,, AmershamlSearle Corp., Arlington Heights, Ill.) was injected into the duodenal cannula and flushed with a volume of isotonic saline equal to the dead space of the cannula. The cannula was plugged and intravenous infusion was continued for 15 min, The animal was then killed by intracardiac injection of pentobarbital sodium (25 mg) and atropine (0.4 mg). The abdomen was opened and ligatures were tied about the small intestine at 3- to 5-cm intervals all along its length, the first being at the pylorus and the last at the ileocecal junction. The stomach, small intestine, and cecum were removed en bloc and placed on a 122 x 7.5 cm platform. This platform was chain-driven by an electric motor so that it could be
I FIG.
1. Actual
motility
record
moved in its long axis under a shielded collimated scintillation detector at 6.5 cm/min. The output of the detector counter was connected to a Packard 271 dual-channel linear-log analyzer and to a Honeywell ElectroniK 194 strip-chart recorder equipped with a series 200 DISC integrator. The record of radioactivity could thus be plotted as a function of distance along the gut. The radioisotope detected distal to the cannula was termed small intestinal radioactivity. The distance from the cannula tip to that point at which 50% of the small intestinal radioactivity had traveled was measured. This distance, expressed as percent of the total length of the small intestine from cannula tip to the ileocecal junction, was termed DA”2, A sample record so analyzed is shown in Fig. 2. The DA”,2 was determined in each animal and the mean of each treatment group was compared with that of the control group by Duncan’s multiple-range test (11). As shown in the example (Fig. 2), the portion of the graph corresponding to the distance from the cannula tip to the ileocecal junction was divided into 10 equal segments. The proportion of isotope in each segment was expressed as percent of total small intestinal radioactivity. Proportions in each segment were averaged within treatment and control groups, plotted and compared graphically. Studies of water movement. Net water movement was studied by a recirculation technique. The conditions of the studies of contractions and transit were duplicated as nearly as possible. Rats were fasted overnight and anesthetized with pentobarbital, 45 mg/kg. Through a midline incision, a ligature was placed around the esophagus. The biliary and pancreatic ducts were left intact and open. Through an incision in the nonglandular portion of the stomach a polyethylene tube was manipulated into the pylorus and secured with a ligature in order to keep the pylorus open. Inlet and outlet cannulas were then secured with ligatures in the same gastric incision and at the distal end of the upper one-half of the small intestine. The femoral or jugular veins were cannulated with PE-IO tubing. Net movement of water was studied during two successive 15-min periods in each rat after a 30-min equilibration period. The intravenous infusion was started at the end of the 30-min equilibration perfusion and continued through the experiment at 0.04 ml/min with a constantinfusion syringe pump. The infusion was saline (8 rats), glucagon at 100 pg/kg per min (7 rats), or caerulein at 1 luglkg per min (7 rats). The perfusion solution was isotonic with rat plasma and consisted of mannitol, 300 mosM, plus labeled polyethylene glycol ([l”C]PEG, New
I showing
minute-by-minute
analysis.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 13, 2018.
o,‘h h!M.
L. D. SCOTT
(31.1%)
12.6%,
6.5%1Z9.5%
4 Opening of Canwla in Duodenum FIG.
analysis
2. Example described
~34.1%,15.9%
J.4%
l 0.0% t 0.0% ,O.O%,O.O%
Small Intestine Divided Into Ten Equa I Segments
of actual in text..
transit
record
demonstrating
Ileocecal Junction
method
of
England Nuclear Corp., Boston, Mass.), 25 &i/liter. A tapered centrifuge tube served as a reservoir. The initial volume of the perfusing solution was 15.4 ml and it was perfused at a rate of 1.9 mllmin with a peristaltic pump (Process and Instruments, Brooklyn, N.Y.), Duplicate O.&ml aliquots were removed from the reservoir initially, after the 30-min equilibration period, and 15 and 30 min after the infusion of saline, glucagon, or caerulein. The radioactivity in each sample was estimated with a beta scintillation counter (model LS-250, Beckman Instruments, Inc., Fullerton, Calif.). At the end of the perfusion the entire small intestine from pylorus to ileocecal junction was stripped from the mesentery, opened longitudinally, wiped and blotted, and the proximal and distal halves were weighed. The following equations were used to calculate water flux:
AND
R. W.
SUMMERS
The infusion of glucagon at 1.0 pg/kg per min had no effect on the basal control pattern detected in the control period. The larger doses inhibited contractions, the inhibition being apparently complete at a dose of 100 pg/ kg per min (Fig. 4). Caerulein altered the pattern of contractions from one of periodic bursts to one of a more uniform distribution of contractions in time at the two lower doses, and it strongly inhibited contractions at 1.0 pg/kg per min (Fig. 5). Studies of transit. The Dp for the distribution of the isotope during the isotonic saline infusion in the 12 control animals was 33 t 7%. Glucagon did not signiflcantly affect the DA 112 or the distribution (Fig. 6) at doses of 1.0 pg/kg per min, but the two larger doses inhibited transit, significantly (P < 0.05) reducing the Dp and
of motility
1. Characteristics
TABLE Rat
Interval*,
1 2 3 4 5 6
min
9.88 23.90 13.14
+ 2.12 k 3.27 k 2.82
18.93
k 7.97 + 2.38 2 5.51
13.24 14.50
in fasted rat Frequency+,
31.56 32.21 28.41 27.61
29.25 27.65
* Mean interval + SD in minutes tMean number phase of each cycle. SD per minute.
per min k 1.47 -t- 0.63 + 2.01
f 1.53 k 1.38 2 1.48
between onset of quiescent of rhythmic contractions +
I
+
0.9% NaCl N=6(6)
L
I
V ?t = w n-1 - 0.4)(e)] AV = V, - V,-1 where V is the volume in milliliters of the luminal fluid, n refers to the end of the equilibration period or 15 or 30-min periods after the infusion of glucagon or caerulein, ~2 - I refers to the end of the previous period (equivalent to the beginning of the same period), PEG is the counts per minute per milliliter from [14C]PEG, and AV from any period is the volume change in milliliters of the luminal fluid. The changes in volume 15 and 30 min after infusion of saline, glucagon, or caerulein were analyzed statistically by analysis of variance with a split plot design (13).
20 FIG. 3. Graphic travenous infusion fasted rat. Number sponded in manner
40 TIME
120
representation of typical effect of continuous of 0.9% NaCl on basal pattern of contractility in parentheses is number of animals that displayed. 1
GLUCAGON
1.0 ta/kq/min N=6 (6)
15
+
10
l-l
l-l
1 GLUCAGON
15t lo-
inin re-
1
+
RESULTS
Studies of contractions (under basal conditions). Contractions occurred in bursts separated by long intervals of quiescence in the uninfused control animals (Fig. 1). Each cluster, in general, tended to begin gradually and to end abruptly. The interval between the ends of the clusters was 14.2 2 5.6 (SD) min (Table I). The frequency of contractions within bursts was 29.63 t 2.28 (SD) contractions/min (Table 1). One-way analysis of variance to compare animal-to-animal variation in cluster intervals to within-animal variation in cluster intervals among saline-infused animals yielded F, 35= 11.64, P < 0.001 (ll), The infusion of isotonic salihe did not influence the pattern of activity (Fig. 3),
100
60 80 (minutes)
N
q6
10.0
(6)
pg/kg/min
]
+
-
5OTT1
20
40
60
1 80
I 100
I 120
J
140
TlME (minutes) FIG. 4. Graphic representation of typical effect of continuous intravenous infusion of varying doses of glucagon on contractility. Numbers in parentheses are numbers of animals within each group that responded in manner displayed.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 13, 2018.
TRANSIT
IN
RAT
135
INTESTINE
5
0
20
40
60
80
100
120
TIME (minutes) FIG. 5. Graphic representation of typical effect of continuous intravenous infusion of varying doses of caerulein on contractility. Numbers in parentheses are numbers of animals within each group that responded in manner displayed,
apparently changing the distribution. The D, 112values for the two larger doses differed (P < 0.05) as well, indicating a dose-related effect. Caerulein accelerated transit (P < 0.05) at the two smaller dosesand retarded it at the highest dose, as evidenced by the comparison of D, 112 values and distributions (Fig. 7). The D, 112 values of the two smaller dosesdid not differ (P > 0.05) but both differed from that of the highest dose (P < OlX$ Studies of water movement. Table 2 shows the results of the studies of net water flux 15 and 30 min after the intravenous infusion of saline, glucagon (100 pg/kg per min) or caerulein (1 pg/kg per min). Similar segments of intestine were perfused. There were no significant differences between groups or between times and no group-by-time interaction.
through hypothermia. Other investigators, however, have reported frequencies (either for contractions or slow waves) in the rat duodenum of ZP32/min (I). We have independently observed a slow-wave frequency of 28 8 -+ 0.90 cycles/min in rat jejunum (unpublished observations). A potential criticism of the technique of measuring transit is that the method of sacrifice or removal of the gut might alter the distribution of isotope and the conclusions about transit. Experiments in our laboratory that preceded this study demonstrated that sacrifice by either inhalation, decapitation, intracardiac barbiturate, or barbiturate plus atropine or antemortem removal of the gut under intraperitoneal barbiturate anesthesia made no significant difference in the distribution of the isotope of DA112 even though a few agonal contractions were often observed as the abdomen was opened (unpublished observations) + In other experiments, a 2.0-ml bolus of isotope was injected into each of several previously resected small intestines and the distribution of isotope was recorded as described in METHODS. Ligatures were then secured at 3-cm intervals in some specimens and around the midportion of the bolus in others and the scanning was repeated. There was no significant change in the segmental distribution or D, 112from the untied ti the tied specimens. The method of sacrifice and manipulation of the gut was the same for treated and control animals; thus the bias is the same for both groups and not responsible for the differences observed. The observed effects of the two peptides is consonant with observations of others. Glucagon inhibited contractions in rat jejunal muscle strips in vitro (9) and in the human small bowel in vivo (8). Caerulein, sharing the same COOH-terminal pentapeptide as gastrin and cho-
DISCUSSION
These observations indicate that contractions in fasted anesthetized rats are temporally distributed much as they are in the unanesthetized fasted dog (7), with clusters of contractions occurring at long intervals in an otherwise quiet gut. Although we recorded only at a single point, it seems reasonable to suppose that the burst of activity moves slowly caudad as it does in the fasted dog (10, 16). The high value for the F statistic indicates that variations in the period of the burst arise mainly from variations among animals rather than within individuals; indeed, the cluster pattern seemed to occur quite regularly, the largest variation being in rat 4 (Table 1) in which this large value appeared to represent a skipped cycle. Only five cycles occurred in that animal, the largest being 32,93 min, about twice the mean of the four other cycles, 15.11 min. If one ignores that long cycle, the SD for the other four is reduced to 1.76 min, a value close to that of the other five animals. The frequency of rhythmic contractions within clusters in our animals is less than that reported by Cannon (4) for the rat duodenum. This may be attributed either to the position of the transducer beyond the duodenum, where the frequency should be lower, or to an effect of the anesthetic on freauencv either dire&v or indirectlv
"p
in
U.S.A.
Correlation of contractions in rat small intestine
and transit
LARRY D. SCOTT AND ROBERT W. SUMMERS Division of Gastroenterology, Department of Internal Iowa City, Iowa 52242
SCOTT, LARRY D., AND&BERT W. SUMMERS. CorreZation of contractions and transit in rat small intestine. Am. J. Physiol. 230(l): 132-137. 1976. -A single strain gauge was implanted chronically in the proximal jejunum of the rat to record contractions. Fasted pentobarbital-anesthetized animals received glucagon, caerulein, or isotonic saline control infusions intravenously while contractions were recorded. In control animals, the distribution of contractions in time showed clusters, with peaks at intervals of 14.24 -+ 5.6 (SD) min. Glucagon produced dose-related inhibition of contractions. Caerulein at two lower doses produced a more uniform distribution of contractions in time; a higher dose caused inhibition. In other fasted rats, an isotopically labeled bolus was given through a chronically implanted duodenal catheter, and its distribution along the small intestine was examined in animals receiving the same doses of glucagon, caerulein, and saline. Glucagon caused a dose-related delay in transit. Those doses of caerulein that produced a uniform distribution of contractions accelerated transit; the dose that inhibited contractions delayed transit. Doses of glucagon and caerulein known to affect both motility and transit did not significantly affect water movement. The fasted rat resembles the fasted dog in respect to the temporal distributions of jejunal contractions at a single point. Both quantitative and qualitative changes in contractions induce changes in transit. motility;
glucagon;
caerulein
FORCES accomplishing small bowel flow are generated in the wall of the gut. The responsible contractions may also provide much of the resistance to flow. Neural and hormonal control mechanisms have been considered important in the distribution and incidence of contractions (6). Records of contractions themselves, or of the small bowel electromyogram, give no information about flow. The patterns of contractions and flow are very much altered by fasting in the dog (7, 17). The quite uniform distribution of contractions in time, characteristic of the fed state, gives way to a very ordered distribution in fasting in which contractions occur in clusters with peaks at quite regular intervals in time. This ordered distribution, called the “interdigestive migratory myoelectric complex” in the dog, seems to be the constant in ruminants, irrespective of the state of feeding (10). This study was designed to define the pattern of contractions, to measure net transit in the rat smal1 intestine under fasting conditions, and to examine the effects
THE PROPULSIVE
Medicine, University
of Iowa,
of glucagon and caerulein, agents known to affect small intestinal muscle, on contractions, water movement, and net flow. MATERIALS
AND METHODS
Studies of contractions. Male albino Sprague-Dawley rats, 10&250 g, were anesthetized with intraperitoneal pentobarbital sodium (45 mg/kg) for implantation of strain gauges on the proximal jejunum. One small unbacked semiconductor strain gauge transducer (BLH Electronics, Waltham, Mass.) was sewn to the serosa in each rat within 1 cm of the ligament of Treitz (method of K. Hubel, personal communication). The gauges were oriented so that the active element was perpendicular to the long axis of the intestine. The wires were led subcutaneously to the back of the neck and connected to a plug, embedded in a dental acrylic button sewn to the paraspinous muscles. The wounds were closed and., after 5 days, the rats were anesthetized as before for experimental study. For this study, they had been given access to water but not food for the preceding 18-22 h. A cannula was placed in a femoral or jugular vein and the transducer output was led, through the cervical plug, to an ink-writing polygraph (type 411 Dynograph recorder with type 9853A coupler modified for use with a Beckman Instrument). The electrical transducer signal, representing contractions, was recorded for approximately 1 h. Then intravenous infusions were made for at least a successive hour of recording, after which the cannula was removed and the animal allowed to recover. Most animals underwent three to four trials with at, least a 4day interval. Glucagon was infused (Eli Lilly and Company, Indianapolis, Ind.) in 18 trials, caerulein (Societa Farmaceutici Italia, Milan, Italy) in 18 trials, and 0.9% NaCl, the solvent for the peptides, was infused in six trials. Glucagon was infused at 1.0, 10.0, and 100.0 lug/kg per min, each ani ma1 receiving only one dose with-six animals in each dosage group. Caerulein was given at 0.001, 0.01, and 1.0 pg/kg per min, the doses being allocated among the animal .s as described for glucagon. All infusi ons were given at 0.04 ml/min by a constantinfusion syringe pump (Harvard Apparatus Co., Inc., Millis, Me.). Records of transducer output showed contractions occurring in clusters or bursts of five to a dozen or more contractions with intervening rest periods lasting sev-
1 32 Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 13, 2018.
TRANSIT
IN
RAT
133
INTESTINE
era1 minutes. Net activity was measured in 1-min units of time with a map mileage marker that yielded a number indicating the length of the line written by the polygraph pen, This measurement was made in one direction only, from the base line. This number, representing an integration of force and frequency, is referred to as the motility index. With the DC amplifier gain such that the biggest excursions yielded full-scale deflections (2.5 cm), deflections less than 3 mm were considered to be noise and were not included in this measurement. Figure 1 shows a correlation of an actual record and the calculated motility index in 1-min intervals, Records were also read for the frequency of contractions within contraction bursts and for the lengths of the periods between the termination of bursts. Studies of transit. Other rats, like those described above, were anesthetized as described above. In each, a PE-50 polyethylene tube was passed subcutaneously from the back of the neck to and through the abdominal wall and into the forestomach through an incision, to terminate in the duodenum about 3 cm beyond the pylorus. The proximal end of the tube was attached to a 24-gauge hypodermic needle hub that was sutured to the paraspinous muscles, and the incisions were closed (15). Animals so prepared gained weight and showed no obvious signs of impaired gastrointestinal function. Each animal was allowed free access to food and water for 5 days when, after an 18- to 22-h fast, it was anesthetized as before. A femoral or jugular vein was cannulated with PE-10 polyethylene tubing and an intravenous infusion was begun with one of the solutions described above. Twelve animals received isotonic saline while each rat in each of three dosage groups of six animals received one of the same three doses of caerulein or glucagon. After 10 min of intravenous infusion, a O.&ml bolus of a solution of mannitol (300 mosM) containing 50 &i of “lCr as sodium chromate (Na,“VrO,, AmershamlSearle Corp., Arlington Heights, Ill.) was injected into the duodenal cannula and flushed with a volume of isotonic saline equal to the dead space of the cannula. The cannula was plugged and intravenous infusion was continued for 15 min, The animal was then killed by intracardiac injection of pentobarbital sodium (25 mg) and atropine (0.4 mg). The abdomen was opened and ligatures were tied about the small intestine at 3- to 5-cm intervals all along its length, the first being at the pylorus and the last at the ileocecal junction. The stomach, small intestine, and cecum were removed en bloc and placed on a 122 x 7.5 cm platform. This platform was chain-driven by an electric motor so that it could be
I FIG.
1. Actual
motility
record
moved in its long axis under a shielded collimated scintillation detector at 6.5 cm/min. The output of the detector counter was connected to a Packard 271 dual-channel linear-log analyzer and to a Honeywell ElectroniK 194 strip-chart recorder equipped with a series 200 DISC integrator. The record of radioactivity could thus be plotted as a function of distance along the gut. The radioisotope detected distal to the cannula was termed small intestinal radioactivity. The distance from the cannula tip to that point at which 50% of the small intestinal radioactivity had traveled was measured. This distance, expressed as percent of the total length of the small intestine from cannula tip to the ileocecal junction, was termed DA”2, A sample record so analyzed is shown in Fig. 2. The DA”,2 was determined in each animal and the mean of each treatment group was compared with that of the control group by Duncan’s multiple-range test (11). As shown in the example (Fig. 2), the portion of the graph corresponding to the distance from the cannula tip to the ileocecal junction was divided into 10 equal segments. The proportion of isotope in each segment was expressed as percent of total small intestinal radioactivity. Proportions in each segment were averaged within treatment and control groups, plotted and compared graphically. Studies of water movement. Net water movement was studied by a recirculation technique. The conditions of the studies of contractions and transit were duplicated as nearly as possible. Rats were fasted overnight and anesthetized with pentobarbital, 45 mg/kg. Through a midline incision, a ligature was placed around the esophagus. The biliary and pancreatic ducts were left intact and open. Through an incision in the nonglandular portion of the stomach a polyethylene tube was manipulated into the pylorus and secured with a ligature in order to keep the pylorus open. Inlet and outlet cannulas were then secured with ligatures in the same gastric incision and at the distal end of the upper one-half of the small intestine. The femoral or jugular veins were cannulated with PE-IO tubing. Net movement of water was studied during two successive 15-min periods in each rat after a 30-min equilibration period. The intravenous infusion was started at the end of the 30-min equilibration perfusion and continued through the experiment at 0.04 ml/min with a constantinfusion syringe pump. The infusion was saline (8 rats), glucagon at 100 pg/kg per min (7 rats), or caerulein at 1 luglkg per min (7 rats). The perfusion solution was isotonic with rat plasma and consisted of mannitol, 300 mosM, plus labeled polyethylene glycol ([l”C]PEG, New
I showing
minute-by-minute
analysis.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 13, 2018.
o,‘h h!M.
L. D. SCOTT
(31.1%)
12.6%,
6.5%1Z9.5%
4 Opening of Canwla in Duodenum FIG.
analysis
2. Example described
~34.1%,15.9%
J.4%
l 0.0% t 0.0% ,O.O%,O.O%
Small Intestine Divided Into Ten Equa I Segments
of actual in text..
transit
record
demonstrating
Ileocecal Junction
method
of
England Nuclear Corp., Boston, Mass.), 25 &i/liter. A tapered centrifuge tube served as a reservoir. The initial volume of the perfusing solution was 15.4 ml and it was perfused at a rate of 1.9 mllmin with a peristaltic pump (Process and Instruments, Brooklyn, N.Y.), Duplicate O.&ml aliquots were removed from the reservoir initially, after the 30-min equilibration period, and 15 and 30 min after the infusion of saline, glucagon, or caerulein. The radioactivity in each sample was estimated with a beta scintillation counter (model LS-250, Beckman Instruments, Inc., Fullerton, Calif.). At the end of the perfusion the entire small intestine from pylorus to ileocecal junction was stripped from the mesentery, opened longitudinally, wiped and blotted, and the proximal and distal halves were weighed. The following equations were used to calculate water flux:
AND
R. W.
SUMMERS
The infusion of glucagon at 1.0 pg/kg per min had no effect on the basal control pattern detected in the control period. The larger doses inhibited contractions, the inhibition being apparently complete at a dose of 100 pg/ kg per min (Fig. 4). Caerulein altered the pattern of contractions from one of periodic bursts to one of a more uniform distribution of contractions in time at the two lower doses, and it strongly inhibited contractions at 1.0 pg/kg per min (Fig. 5). Studies of transit. The Dp for the distribution of the isotope during the isotonic saline infusion in the 12 control animals was 33 t 7%. Glucagon did not signiflcantly affect the DA 112 or the distribution (Fig. 6) at doses of 1.0 pg/kg per min, but the two larger doses inhibited transit, significantly (P < 0.05) reducing the Dp and
of motility
1. Characteristics
TABLE Rat
Interval*,
1 2 3 4 5 6
min
9.88 23.90 13.14
+ 2.12 k 3.27 k 2.82
18.93
k 7.97 + 2.38 2 5.51
13.24 14.50
in fasted rat Frequency+,
31.56 32.21 28.41 27.61
29.25 27.65
* Mean interval + SD in minutes tMean number phase of each cycle. SD per minute.
per min k 1.47 -t- 0.63 + 2.01
f 1.53 k 1.38 2 1.48
between onset of quiescent of rhythmic contractions +
I
+
0.9% NaCl N=6(6)
L
I
V ?t = w n-1 - 0.4)(e)] AV = V, - V,-1 where V is the volume in milliliters of the luminal fluid, n refers to the end of the equilibration period or 15 or 30-min periods after the infusion of glucagon or caerulein, ~2 - I refers to the end of the previous period (equivalent to the beginning of the same period), PEG is the counts per minute per milliliter from [14C]PEG, and AV from any period is the volume change in milliliters of the luminal fluid. The changes in volume 15 and 30 min after infusion of saline, glucagon, or caerulein were analyzed statistically by analysis of variance with a split plot design (13).
20 FIG. 3. Graphic travenous infusion fasted rat. Number sponded in manner
40 TIME
120
representation of typical effect of continuous of 0.9% NaCl on basal pattern of contractility in parentheses is number of animals that displayed. 1
GLUCAGON
1.0 ta/kq/min N=6 (6)
15
+
10
l-l
l-l
1 GLUCAGON
15t lo-
inin re-
1
+
RESULTS
Studies of contractions (under basal conditions). Contractions occurred in bursts separated by long intervals of quiescence in the uninfused control animals (Fig. 1). Each cluster, in general, tended to begin gradually and to end abruptly. The interval between the ends of the clusters was 14.2 2 5.6 (SD) min (Table I). The frequency of contractions within bursts was 29.63 t 2.28 (SD) contractions/min (Table 1). One-way analysis of variance to compare animal-to-animal variation in cluster intervals to within-animal variation in cluster intervals among saline-infused animals yielded F, 35= 11.64, P < 0.001 (ll), The infusion of isotonic salihe did not influence the pattern of activity (Fig. 3),
100
60 80 (minutes)
N
q6
10.0
(6)
pg/kg/min
]
+
-
5OTT1
20
40
60
1 80
I 100
I 120
J
140
TlME (minutes) FIG. 4. Graphic representation of typical effect of continuous intravenous infusion of varying doses of glucagon on contractility. Numbers in parentheses are numbers of animals within each group that responded in manner displayed.
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TRANSIT
IN
RAT
135
INTESTINE
5
0
20
40
60
80
100
120
TIME (minutes) FIG. 5. Graphic representation of typical effect of continuous intravenous infusion of varying doses of caerulein on contractility. Numbers in parentheses are numbers of animals within each group that responded in manner displayed,
apparently changing the distribution. The D, 112values for the two larger doses differed (P < 0.05) as well, indicating a dose-related effect. Caerulein accelerated transit (P < 0.05) at the two smaller dosesand retarded it at the highest dose, as evidenced by the comparison of D, 112 values and distributions (Fig. 7). The D, 112 values of the two smaller dosesdid not differ (P > 0.05) but both differed from that of the highest dose (P < OlX$ Studies of water movement. Table 2 shows the results of the studies of net water flux 15 and 30 min after the intravenous infusion of saline, glucagon (100 pg/kg per min) or caerulein (1 pg/kg per min). Similar segments of intestine were perfused. There were no significant differences between groups or between times and no group-by-time interaction.
through hypothermia. Other investigators, however, have reported frequencies (either for contractions or slow waves) in the rat duodenum of ZP32/min (I). We have independently observed a slow-wave frequency of 28 8 -+ 0.90 cycles/min in rat jejunum (unpublished observations). A potential criticism of the technique of measuring transit is that the method of sacrifice or removal of the gut might alter the distribution of isotope and the conclusions about transit. Experiments in our laboratory that preceded this study demonstrated that sacrifice by either inhalation, decapitation, intracardiac barbiturate, or barbiturate plus atropine or antemortem removal of the gut under intraperitoneal barbiturate anesthesia made no significant difference in the distribution of the isotope of DA112 even though a few agonal contractions were often observed as the abdomen was opened (unpublished observations) + In other experiments, a 2.0-ml bolus of isotope was injected into each of several previously resected small intestines and the distribution of isotope was recorded as described in METHODS. Ligatures were then secured at 3-cm intervals in some specimens and around the midportion of the bolus in others and the scanning was repeated. There was no significant change in the segmental distribution or D, 112from the untied ti the tied specimens. The method of sacrifice and manipulation of the gut was the same for treated and control animals; thus the bias is the same for both groups and not responsible for the differences observed. The observed effects of the two peptides is consonant with observations of others. Glucagon inhibited contractions in rat jejunal muscle strips in vitro (9) and in the human small bowel in vivo (8). Caerulein, sharing the same COOH-terminal pentapeptide as gastrin and cho-
DISCUSSION
These observations indicate that contractions in fasted anesthetized rats are temporally distributed much as they are in the unanesthetized fasted dog (7), with clusters of contractions occurring at long intervals in an otherwise quiet gut. Although we recorded only at a single point, it seems reasonable to suppose that the burst of activity moves slowly caudad as it does in the fasted dog (10, 16). The high value for the F statistic indicates that variations in the period of the burst arise mainly from variations among animals rather than within individuals; indeed, the cluster pattern seemed to occur quite regularly, the largest variation being in rat 4 (Table 1) in which this large value appeared to represent a skipped cycle. Only five cycles occurred in that animal, the largest being 32,93 min, about twice the mean of the four other cycles, 15.11 min. If one ignores that long cycle, the SD for the other four is reduced to 1.76 min, a value close to that of the other five animals. The frequency of rhythmic contractions within clusters in our animals is less than that reported by Cannon (4) for the rat duodenum. This may be attributed either to the position of the transducer beyond the duodenum, where the frequency should be lower, or to an effect of the anesthetic on freauencv either dire&v or indirectlv
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