Effect of Cholinergic Agonists on Muscle From Rodent Proximal and Distal Small Intestine

GASTROENTEROLOGY 1985;88:1118-25

Effect of Cholinergic Agonists on Muscle From Rodent Proximal and Distal Small Intestine THOMAS V. NOWAK and BONNIE HARRINGTON

Department of Medicine, University of Rochester, St. Mary's Hospital, Rochester. New York

Proximal and distal rat small intestine was cut into strips measuring 6.0 x 10.0 mm. Strips cut along the oral-caudal axis were called longitudinal strips, whereas those cut 90° to that axis were called circular strips. Stress in circular and longitudinal muscle strips was measured continuously as they were superfused with acetylcholine, carbamylcholine, methacholine, bethanecho1, or physostigmine. Resting stress during stretch, acetylcholine-stimulated active stress, and total stress were determined. Proximal circular muscle was five times as sensitive to acetylcholine as distal circular muscle (p < 0.05); proxima110ngitudina1 muscle was 2.8 times as sensitive to bethanecho1 as distal muscle (p < 0.05). Resting, active, and total stress were similar in proximal and distal muscle, but circular muscle showed nearly twice the resting stress of longitudinal muscle at either proximal or distal sites (p < 0.05). Physostigmine (10- 6 M) increased acetylcholine-stimulated active stress in proximal and distal circular muscle by 29% and 70%, respectively (p < 0.05), but not in longitudinal muscle (p > 0.05). This difference between proximal and distal circular muscle (41%) was also significant (p < 0.05). Thus, the proximal and distal muscle of the rat small intestine differs in its sensitivity to various cholinergic agonists, but not in its length-stress properties. Both muscarinic agonists and cholinesterase inhibitors enhance contractions of the small intestine (1). It is clear that cholinergic influence is essential for Received April 19, 1984. Accepted November 13, 1984. Address requests for reprints to: Thomas V. Nowak, M.D., Department of Medicine, St. Mary's Hospital, 89 Genesee Street, Rochester, New York 14611-9981. This study was supported by Biomedical Research Grant 2S07RR05403 from the Public Health Service, National Institutes of Health and the St. Mary's Hospital Clinical Research Fund. The authors thank Dawn Foley for secretarial assistance, Dr. John Kalbfleisch for statistical analysis, and Dr. William Y. Chey for advice and encouragement. © 1985 by the American Gastroenterological Association 0016-5085/85/$3.30

propulsive enteric motility, as the peristaltic reflex can be inhibited by blockage of muscarinic receptors with atropine (2). Despite the fact that the net movement of luminal contents is in an aboral direction, there has been little comparative study of cholinergic influence on muscle from the proximal and distal small intestine. Our purpose was to evaluate the responses of muscle from the proximal and distal small intestine to various cholinergic agonists.

Materials and Methods Adult retired male breeder rats, weighing 500-700 g, were used. Pentobarbitol (50 mg/kg body wt) was injected intra peritoneally to anesthetize the animals. The abdomen was opened and two segments of intestine, each about 10 cm long, were removed just distal to the pylorus and just proximal to the ileocecal valve. Both segments were opened along their respective mesenteric insertions and the mucosas were rinsed with warm Krebs' solution. The Krebs' solution had the following composition (mM): NaCl, 115.48; KCI, 4.63; NaH 2 P0 4 , 1.16; NaHC0 3 , 21.91; CaCl 2 , 2.47; MgS0 4 , 1.16; and glucose, 11.5. Both segments were then pinned on a wax block, mucosal side up, in a bath of Krebs' solution gassed with 95% O 2 -5% CO 2 at 36.5°-37.5°C. The mucosa was removed. Both segments were cut into strips 6 mm wide with an assembly of razor blades held in a clamp. Each strip was about 0.9-1.1 cm long. Those strips cut parallel to the oral-caudal axis of the segment were called longitudinal strips. The others, cut perpendicular to the oral-caudal axis of the segment, were called circular strips. The strips were tied at each end with silk thread. One end was fixed and the other fastened to a force-displacement transducer (Grass Instruments, Quincy, Mass.). Responses were recorded on a dynograph recorder (Beckman Instruments, Schiller Park, Ill.). Krebs' solution, equilibrated with 95% O 2-5% CO 2 at 36S37.5°C, flowed over the strips at a rate of 5.6 mllmin in an organ bath as previously described (3). The bath consisted of a block of plexiglass (11.5 x 2.5 x 2.5 cm) with a central Abbreviations used in this paper: Fa, active stress; Fro resting stress; Flo total stress; Lj, initial length; Loo length at which a maximal active tension response occurs.

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hollow core, 0.4 cm in diameter. Agonist drugs were dissolved in Krebs' solution daily and serial dilutions were made. The drugs were superfused from the reservoir feeding the organ baths.

Preliminary Studies Preliminary experiments were performed on 6 rats to determine the optimal muscle strip length (L) that provides a maximal response. Distance between the two ligatures on the muscle strip was carefully measured with a caliper. The initial length (Ld, the length at which muscle stress (F) is still at zero but ready to increase with any further increase in muscle length, was determined. Maximal amplitude of contraction occurred at 150%170% of Li for both circular and longitudinal strips from proximal and distal intestine.

Dose-Response Studies To examine the responses to various cholinergic agents, the muscle strips were stretched to 150%-170% of their initial length in several small increments over 30 min. This stress was maintained as the baseline stress throughout the experiment. The following drugs were used: acetylcholine chloride, carbamylcholine chloride, methacholine chloride, bethanechol chloride, and physostigmine. Each agent was dissolved in Krebs' solution. In determining the dose-response curve for each agent, the lowest concentration used was 10- 10 M. Each agent superfused the strips until the tissue response to that agent had stabilized. The strips were then washed free of the agent with Krebs' solution. They were then exposed to a 2.5-fold to fivefold higher concentration of the agent and this sequence was repeated until a maximal response was achieved. At least 20 min was allowed to elapse between doses of the agonist.

Length-Stress Studies To determine the length-stress properties, the Li was determined for each strip. The strips were allowed to stabilize in the bath for 30 min and were then maximally stimulated with a 5 x 10- 2 M solution of acetylcholine chloride dissolved in Krebs' solution. According to the dose-response properties of acetylcholine chloride, the strips are maximally contracted at this concentration. Acetylcholine flowed over the strips for 5 min during which time a stable response was obtained. The stress developed was the total stress (Ftl. Resting stress (Fr) was that recorded before administration of acetylcholine. The difference between the total stress (Ftl and resting stress (Fr) was called the active stress (Fa). After administration of the acetylcholine, the muscle was washed free of the agent and allowed to return to a level of stress that was stable. The muscle was then progressively stretched by 1mm increments. After the muscle had stabilized at each level of stretch, the resting stress and length of each muscle strip was measured and the strips were again stimulated with acetylcholine. At least 20 min were al-

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lowed to elapse between each exposure to acetylcholine. Each strip was stretched to at least 200% of its initial length and an active stress response was no longer generated. At the end of each experiment each muscle strip was gently blotted and weighed.

Effect of Cholinesterase Inhibition To determine the effect of cholinesterase inhibition, circular and longitudinal strips from proximal and distal intestine were superfused with Krebs' solution at Lo, the length of each strip at which a maximal active tension response occurs, for 30 min. The strips were then superfused with acetylcholine (5 x 10- 2 M) until a stable response was obtained, after which they were washed with Krebs' solution for at least 20 min. The strips were then superfused with physostigmine (10- 6 M) for 5 min, washed with Krebs' solution for 20 min, and then again superfused with acetylcholine chloride (5 x 10- 2 M). After the experiment the strips were gently blotted with tissue paper and weighed.

Cross-Sectional Area Analysis To determine the relative thickness of the longitudinal and circular muscle layers from proximal and distal intestine, 6 rats were killed with pentobarbital sodium and transverse sections were taken from intact segments of duodenum and ileum. These specimens were fixed in formalin under zero tension, imbedded in paraffin blocks, and cut transversely across the intestinal lumen into 6-pm sections. Each section was mounted on a microscope slide and stained with hematoxylin and eosin. The thickness of the circular and longitudinal muscle layers was measured in four quadrants through a microscope (American Optical, Buffalo, N.Y.) with a stage micrometer (E. Leitz, Rockleish, N.J.). These values were then averaged to obtain the mean thickness of each layer. To calculate the cross-sectional area of each layer, the following equation was used: cross section (mm 2 )

=

tissue weight (mg)/Lo (mm) x (1.05 mg/mm3),

where 1.05 mg/mm 3 is the assumed density of smooth muscle. This equation is similar to that used by Herlihy and Murphy (4), Keatinge (5), and Brown (6). Assuming that most of the tissue weight is composed of longitudinal (L) and circular (C) muscle, L plus C equals the total tissue weight. Referring to our histologic studies (Figure 1), 60% of the total tissue weight is due to circular muscle and 40% of the total tissue weight is due to longitudinal muscle. The weight for the longitudinal layer to be used in the calculation would therefore be 40% of the total tissue weight; and that for the circular layer would be 60% of the total tissue weight.

Statistical Analysis The recordings were analyzed visually to determine the amplitude of response. In determining the dose-

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NOWAK AND HARRINGTON

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LONGITUDINAL & CIRCULAR

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Figure 1. Thickness of longitudinal and circular muscle layers in the duodenum and ileum. Values are means ± SE from 6 rats.

response for each agent, the amplitude of each response was normalized. The maximal response was designated 100%, and the other responses were calculated as a percentage of the maximal response. The mean response at each dose was compared for each set of strips. As a first step in the calculation of the ED 50 , the response of each muscle strip was fit with a least-squares logistic regression curve. From the curves an ED50 (the dose at which the muscle would give a 50% response) and the interquartile range (the difference between the ED75 and the ED 25 ) were calculated. The ED50 provided an estimate of the location of the response curve and the interquartile range provided an estimate of the rate of change of response (7). Mean values for ED50 and interquartile ranges were tested with the paired t-test and Student's t-test. A probability level of p ~ 0.05 was used to indicate statistical significance. Relative sensitivity of proximal and distal muscle was assayed by the method described by Ashton (8). Unless otherwise stated, data are shown as the mean ±SE for observations made in a minimum of eight muscle strips from the same number of animals.

Results Responses to Cholinergic Agonists All agents produced dose-dependent tonic contraction in longitudinal muscle and phasic contraction in circular muscle (Figure 2). Table 1 shows the ED50 of the cholinergic agonists in circular and longitudinal muscle of the rat duodenum and ileum. Table 2 shows the interquartile ranges. Figure 3 shows the dose-response curves.

ED50 values or the interquartile ranges. Proximal muscle was significantly more sensitive to bethanechol chloride than distal muscle at concentrations of 10- 6 through 5 x 10- 5 M (Figure 3). Potency ratio (±95% confidence interval) of the ED50 values showed that proximal longitudinal muscle was 2.8 (1.6-4.7) times as sensitive to bethanechol as distal muscle. No significant difference was noted in the interquartile ranges. Carbamylcholine chloride, methacholine chloride, and physostigmine showed no difference in potency between proximal and distal intestine. No significant differences in ED50 values or interquartile ranges were seen. Physostigmine also had no effect on acetylcholine-produced contraction in longitudinal muscle (Figure 4). In proximal strips acetylcholine generated a maximum stress of 25.9 x 10 4 ± 1.9 dyn/cm 2 and 24.2 x 10 4 ± 1.7 dyn/cm 2 before and after superfusion with physostigmine, respectively. Likewise, in distal strips acetylcholine produced a maximum stress of 28.8 x 10 4 ± 4.7 dyn/cm 2 and 30.4 x 10 4 ± 5.7 dyn/cm 2 before and after superfusion with physostigmine, respectively. Circular Muscle Proximal circular muscle was significantly more sensitive to acetylcholine than distal muscle at concentratins of 5 x 10- 4 M and 10- 3 M. Potency ratio (±95% confidence interval) of the ED 50 values showed that proximal circular muscle was 5.0 (1.418.3) times as sensitive to acetylcholine as distal circular muscle. No significant difference was found between the interquartile ranges. The ED50 values and interquartile ranges for bethanechol chloride, carbamylcholine chloride, methacholine chloride, and physostigmine were similar in proximal and distal intestine. Differences were noted CIRCULAR

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Longitudinal Muscle Proximal and distal longitudinal muscle showed no significant difference in sensitivity for acetylcholine at any of the concentrations studied. Likewise, there was no significant difference in the

lACETYLCHOLlNE 10-3 M

Figure 2. Effect of acetylcholine on one circular and one longitudinal muscle strip from a single rat duodenum. Acetylcholine produces multiple phasic contractions in the circular strip and a sustained tonic contraction in the longitudinal strip.

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Table 1. ED50 Values of Cholinergic Agonists in the Rat Small Intestine Distal

Proximal

Acetylcholine Carbamylcholine Bethanechol Methacholine Physostigmine o

Longitudinal

Circular

Drug 6.70 2.53 8.26 Q.02 1.05

x x x x x

p < 0.05 (circular vs. longitudinal).

10- 4 Mo. b 10- 7 M 10-(; M 10 6 M 10 oM b

3.79·X 4.23 x 6.05 x 4.31 x 1.51 x

10- 5 M 10 7M 10 [j Me 10 "M 10 6M

Longitudinal

Circular 3.35 7.67 1.74 1.58 8.01

4.03 3.38 1.69 8.66 1.27

X 10- 5 M" x 10- 7 M X 10- 5 M x 10- 0 M x 10- 7 M

x 10 -0 M X 10- 7 M x 10- 0 Me X 10- 6 M x 1O- 6 M

p < 0.05 (proximal circular vs. distal circular). 'p < 0.01 (longitudinal vs. longitudinal).

only at concentrations of 5 x 10- 4 M for methacholine and 7.75 x 10- 6 M for physostigmine. Physostigmine significantly increased acetylcholine-produced contraction in circular muscle (Figure 4). Before superfusion with physostigmine, proximal and distal strips produced maximum tensions of 8.9 x 10 4 ± 1.4 dyn/cm 2 and 8.3 x 10 4 ± 2.4 dyn/cm2, respectively. After physostigmine these tensions increased to 11.5 x 10 4 ± 1.5 dyn/cm 2 and 13.7 x 10 4 ± ~.O dyn/cm 2 , respectively. These changes are statistically significant at both sites. Viewed in percent, physostigmine increased acetylcholine-produced contraction in proximal muscle by 29% and in distal muscle by 70%. This difference between proximal and distal circular muscle is statistically significant. Circular Versus Longitudinal Muscle At either site in the intestine longitudinal muscle was more sensitive to acetylcholine than circular muscle. Potency ratio (±95% confidence intervals) of the ED50 values showed longitudinal muscle was 17.8 (1.2-256.6) times as sensitive to acetylcholine as circular muscle in proximal intestine, whereas distally longitudinal muscle was 83.1 (24.9-277.6) times as sensitive. Bethanechol chloride, carbamylcholine chloride, methacholine chloride, and physostigmine produced similar amplitudes of response, ED50 values, and interquartile ranges in both muscle layers at either locus in the intestine.

Length-Stress Properties Length-stress properties were determined for each set of strips (Table 3). As each muscle strip was stretched, there was an appreciable increase in active stress development up to the optimal length (Lo), after which active stress development declined (Figure 5). No significant differences in total, active, or resting stress were noted between proximal and distal circular muscle or between proximal and distal longitudinal muscle. When circular muscle was compared with longitudinal muscle at either site in the intestine, no significant differences were noted in total stress development. At either locus in the intestine, however, circular muscle showed significantly greater resting stress and lower active stress than longitudinal muscle.

Discussion There is evidence to suggest that differences in propulsive ability exist between proximal and distal intestine. Nonabsorbable markers traverse the initial 50% of rat intestine in ~20 min, whereas >1 h is needed to cover the next 30% of bowel length (9). Differences in work performance also exist. Isolated, perfused segments of cat ileum have the ability to transport fluid aborally and against a significant pressure gradient, whereas duodenal and jejunal segments lack this property (10). Despite these observe~ variations in contractile performance be-

Table 2. Interquartile Ranges of Drugs in Circular and Longitudinal Muscle of Proximal and Distal Rat Small Intestine Distal

Proximal Drug Acetylcholine Ca~bamylcholine

Bethanechol Metha(;holine Physostigmine

Circular -1.1736759 -1.3675215 -0.7345799 -1.3355255 -0.8011791

0

Longitudinal

Circular

Longitudinal

-2.7039605 -1.0331319 -0.8755517 -1.2658292 -0.7333188

-1.1666553 -0.6519410 -0.1733499 -1.0448406 -0.7685097

0

-2.0426423 -1.0099828 -0.8755517 -1.2863127 -0.8084405

Each interquartile range represents the mean of the differences between the ED zo and the ED" for each set of strips. Each entry is expressed as the logarithm base ten of dose units. 0 p < 0.05 (circular vs. longitudinal).

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Figure 4. Effect of acetylcholine (5 x 10 2 M) on circular and longitudinal muscie from rat intestine before and after treatment with physostigmine (10- 5 M). Physostigmine caused an increase in the amplitude of contraction in circular muscle, especially in the distal intestine. No effect was noted in iongitudimil muscle. Values are expressed as means ± SE in at least Brats.

tween proximal qnd distal intestine, there is very little information regartling the factors that determine these regional differences. , The current Iltudy shows that proximai anti distal intestinal muscle differ in their sensitivit~ to some cholinergic agoriists. Proximal circular muscle is five times as sen~itive to acetylcholine and proximal longitudinal muscle is nearly threefold ~s sensitive to bethanechol as respective dIstal muscle. In contrast to other studies [11, 12), we found no difference in sensitivity betweert longitudinal and circular muscle fdt either carbamylcholine or methacholine. Because one of the differences between acetylcholine and the other choline esters is resistance to Table 3. Lengtp-Stress Properties of Muscle From Rat Small Intestine Proximal Circular

Longitudinal

Distal Circular

Longitudinal

Total stress 29.0 ± 3.4 35.9 ± 3.5 32.4 ± 5.6 33.9 ± 4.8 Active stress 14.9 ± 2.8" 29.2 ± 3.7 13.4 ± 3.6" 26.1 ± 3.4 Resting stress 14.1 ± 2.4" 6.7 ± 1.5 19.0 ± 4.4" 7.8 ± 2.2 Values are means ± SE, expressed as clyn x 10 4 /cm 2 , obtained at La. " p < 0.05 (circular vs. longitudinal layers).

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DISTAL LONGITUDINAL MUSCLE

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LlLo Figure 5. Length-stress curves for circular and longitudinal muscle from proximal and distal rat small intestine. On the horizontal axis muscle length (LlLo) is standardized to the optimal length, La. On the vertical axis stress (F/F q ) is standardized for the optimal stress (Fa) developed at La. No significant difference was noted among tptal stress for any of the four sets of strips. In both prQximal and distal intestine, however, circular muscle had significantly higher resting stress and lower active stress than longitudinal muscle. Values are expressed as means ± SE in at least 8 rats.

hydrolysis by cholinesterase, differences in cholinesterase activity between the two muscle layers at various loci in the intestine might explain variations in sensitivity to cholinergic agonists. Histologic studies have shown that the circular and longitudinal muscle layers of the cat ileum vary in their cholinesterase content (13) and in their respective

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NOWAK AND HARRINGTON

concentrations of specific and nonspecific cholinesterase. Specific cholinesterase localizes to the inner layer of the circular muscle. Nonspecific cholinesterase concentrates in longitudinal muscle and is found at very low concentrations in circular muscle. These cholinesterases differ mainly in their affinity for acetylcholine and in their sensitivity to cholinesterase inhibitors. Our experiments with physostigmine sugges~ that proximal and distal circular muscle might differ in their cholinesterase concentrations as well. Conceivably, variations in muscarinic receptor density in tissues might explain differences in sensitivity to cholinergic agonists. One study examined the binding of [3H]quinuclidinyl benzilate to characterize the distribution of muscarinic receptors in feline esophageal smooth muscle (14). Specific binding of [3H]quinuclidinyl benzilate paralleled the relative amount of smooth muscle at any given level in the esophagus. This corresponds with the recognized regional variation in response of cat esophageal muscle to stimulation by acetylcholine, carbachol, and methacholine (14,15). Using similar methods another study found that the longitudinal muscle layet of the guinea pig ileum contains 90% of the muscarinic receptors found within the ileal wall (16). This supports our observation and that of others (11) that longitudinal muscle is significantly more sensitive to cholinergic agonists than circular muscle. Conflicting evidence exists regarding the distributiori of muscarinic receptors along the length of the small intestine. One study showed .that muscarinic receptor density increased from duodenum to ileum (17), whereas another concluded that receptors were similarly distributed at proximal and distal sites (14). Neither study, however, examined the relative density of muscarinic receptors in respective longitudinal and circular muscle layers. Previous studies of the length-tension properties of intestinal smooth muscle have dealt only with circular muscle and are confounded by the fact that different methods were used for stimulation and for the control of spontaneous activity. Meiss and Prosser (18) studied circular muscle strips from cat duodenum with AC stimulation at 30°C. They found a peak active tension of 0.42 kg/cm 2 . Scott et ill. (19) stimulated circular muscle strips from rat intestine with carbachol at 25°C and found maximal tensions of 4.10,4.82, and 6.64 x 10 5 Nlm 2 in the duodenum, jejunum, and ileum, respectively. In that study determination of cross-sectional area was based on dry weight of tissue. Our data show no significant difference in the length-stress properties of proximal and distal intestinal muscle. Significant differences, however, were

GASTROENTEROLOGY Vol. 88. No.5. Part 1

noted between the two muscle layers at either site in the intestine. The total stress generated by the two muscle layers was similar, but active stress generated by longitudinal muscle at Lo was nearly twice that shown by circular muscle. Conversely, circular muscle had more than twice the resting stress at Lo than did longitudinal muscle. The values obtained in the present study are comparable to values cited for other smooth muscles: guinea pig taenia coli (20), canine trachealis (21), and rabbit uterus (22). High resting stress at Lo has also been described in other smooth muscles such as guinea pig taenia coli (23), proximal rabbit colon (24), rabbit taenia coli (25), and others (18,26, 27). The relative positions of the active and passive tension curves appear to depend upon the amount of connective tissue present in the muscle, which explains the high resting tension seen in taenia coli (25). Our studies of the length-tension properties expand the known physiologic differenceS between the two muscle layers of the intestine. Some species variation must also occur, as other investigators using opossums and guinea pigs have found a difference. in sensitivity between the two layers for the synthetic choline esters, whereas we did not. We found muscle from proximal and distal intestine to differ in two main respects. Proximal muscle is more sensitive to some cholinergic agonists. Second, cholinesterase activity seems to differ between proximal and distal intestinal muscle and this difference appears to be restricted to the circular muscle layer. The import of these observations on the aboral movement of intraluminal contents remains to be determined.

References 1. Kilbinger H. Weihrauch TR. Drugs increasing gastrointestinal

motility. Pharmacology 1982;25:61-72. 2. Kosterlitz HW. Lees GM. Pharmacological analysis of intrinsic intestinal reflexes. Pharmac Rev 1964;16:301-39. 3. DeCarie DJ, Christensen J. A dopamine receptor in the esophageal smooth muscle of the opossum. Gastroenterology 1976; 70:216-9. 4. Herlihy JT. Murphy RA. Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 1973; 33:275-83. 5. Keatinge WR. Sodium flux and electrical activity of arterial smooth muscle. J Physiol (Lond) 1968;194:183-200. 6. Brown BP, Anuras S, Heistad DD. Responsiveness of longitudinal and circular muscle iayers of the portal vein. Am J PhysioI1982;242:G498-G503. 7. Ashton WD. The logit transformation. In: Stuart A, ed. London: Charles Griffin, 1972:14-22 .. 8. Ashton WD. The logit transformation. In: Stuart A, ed. London: Charles Griffin;1972:43-5. 9. Summers RW, Kent TH, Osborne JW. Effects of drugs, ileal obstruction, and irradiation on rat gastrointestinal propulsion. Gastroenterology 1970;59:731-9. 10. Weems WA. Seygal GE. Fluid propulsion in cat intestinal

May 1985

11. 12. 13. 14.

15. 16.

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segments under conditions requiring hydrostatic work. Am J Physiol 1981;240:G147-56. Anuras S, Faulk DL, Christensen J. Effects of some autonomic drugs on duodenal smooth muscle. Am J Physiol 1979; 236(1):E33-8. Brownlee G, Harry J. Some pharmacological properties of the circular and longitudinal muscle strips from the guinea-pig isolated ileum. Br J Pharmacol 1963;21:544-54. Koelle GB. The histochemical differentiation of types of cholinesterases and their localization in tissues of the cat. J Pharmacol Exp Ther 1950;100:158-79. Rimele TJ, Rogers WA, Gaginella TS. Characterization of muscarinic cholinergic receptors in the lower esophageal sphincter of the cat: binding of [3H]quinuclidinyl benzilate. Gastroenterology 1979;77:1225-34. Christensen I. Dons RF. Regional variations in response of cat esophageal muscle to stimulation with drugs. J Pharmacol Exp Therap 1968;161:55-8. Yamamura HI, Synder SH. Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H]quinuclidinyl benzilate. Mol Pharmacol 1974;10: 861-7. Morisset J, Geoffrion L, Larose L, Lanoe I. Poirier GG. Distribution of muscarinic receptors in the digestive tract organs. Pharmacology 1981;22:189-95. Meiss RA, Prosser CL. Mechanical properties of cat intestinal smooth muscle. Fed Proc 1969;28:712.

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19. Scott LD, DeFlora E, Weisbrodt NW, Lester R. Pregnancy, sex steroids and contractility of rat small intestine (abstr). Gastroenterology 1982;82:1174. 20. Aberg AK, Axelsson J. Some mechanical aspects of an intestinal smooth muscle. Acta Physiol Scand 1965;64:15-27. 21. Stephens NL, Kroeger E, Mehta JA. Force-velocity characteristics of respiratory airway smooth muscle. J Appl Physiol 1969;26:685-92. 22. Csapo A. Dependence of isometric tension and isotonic shortening of uterine muscle on temperature and strength of stimulation. Am J Physiol 1954;177:348-54. 23. Bulbring E, Kuriyama H. The effect of adrenaline on the smooth muscle of guinea-pig taenia coli in relation to the degree of stretch. J Physiol (Lond) 1969;198-212. 24. Tucker HI. Snape WI. Cohen S. Comparison of proximal and distal colonic muscle of the rabbit. Am J Physiol 1979; 237:E383-8. 25. Gordon AR, Siegman MJ. Mechanical properties of smooth muscle. I. Length-tension and force-velocity relations. Am J Physiol 1971;221:1243-9. 26. Gluck SR. Kao CY. Active-state in uterine muscle. Fed Proc 1962;21:319. 27. Winton FR. Influence of length on the responses of unstriated muscle to electrical and chemical stimulation and stretching. J Physiol (Lond) 1926;61:368-82.