Defect in colonic smooth muscle contraction in patients with ulcerative colitis WILLIAM J. SNAPE, JR., R. WILLIAMS, AND P. E. HYMAN Department of Medicine and Surgery and University of California, Los Angeles, Inflammatory Bowel Disease Center, Harbor- UCLA Medical Center, Torrance, California 90502
SNAPE, WILLIAM J., JR., R. WILLIAMS, AND P. E. HYMAN. Defect in colonic smooth muscle contraction in patients with ulcerative colitis. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G987-G991, 1991.-Patients with ulcerative colitis have decreased postprandial colonic contractions. The purpose of this study was to determine whether the smooth muscle from patients with ulcerative colitis responds abnormally in vitro to different stimuli. Circular colonic smooth muscle strips from patients with ulcerative colitis, acute diverticular disease, or adenocarcinoma were stretched to the optimal length and stimulated with electrical field stimulation (EFS), bethanechol, or increased concentrations of extracellular K’. The EFS-stimulated on-contraction was similar in each group, but the offcontraction was decreased in patients with colitis compared with patients with cancer (P < 0.02) or diverticular disease (P < 0.01). Bethanechol stimulated a dose-dependent colonic contraction, which was less in the strips from patients with colitis compared with cancer (P < 0.02) or diverticular disease (P < 0.05). The response to increased extracellular K+ was less in muscle from patients with colitis (P < 0.01) than in the other tissues. Muscle from diverticular disease developed greater stress to K+ stimulation than did muscle from cancer (P < 0.05). These studies suggest that there is a decrease in the force of muscle contraction in colonic muscle obtained from patients with colitis compared with normal muscle resected from patients with cancer or with muscle associated with diverticular disease of the colon. The similar relatively low amplitude of the on-contraction in each group suggests the physiological release of an inhibitory neurotransmitter. mucosal inflammation; inflammatory bowel disease; electrical field stimulation; cholinergic; isometric tension
THE FORCE of colonic contractions after different stimuli
is decreased in patients with ulcerative colitis compared with healthy controls or with patients with diverticular disease of the colon (3, 9, 15, 19, 23). Diarrhea, which is occasionally associated with incontinence, is a major symptom in ulcerative colitis. The decrease in segmenting contractions can accentuate the diarrhea in colitis by allowing rapid forward movement of the colonic contents (2, 4). The defect in smooth muscle contraction may be generalized, since the force of contractions also is decreased in the small intestine, which is not histologically involved in the disease process (16). The present study was designed to evaluate the mechanism of the decreased colonic smooth muscle contraction in patients with ulcerative colitis by comparing the in vitro contraction of colonic muscle from ulcerative colitis with muscle from 0193-1857/91
$1.50
Copyright
patients with adenocarcinoma or diverticular
disease of
the colon. METHODS
Tissue preparation. Colonic tissue distal to the splenic flexure was removed during colectomy from 10 patients with an adenocarcinoma, 5 patients with diverticular diseaseof the colon, and 8 patients with ulcerative colitis. Table 1 shows the clinical characteristics of these patients with ulcerative colitis. Patients with globally severe symptoms of ulcerative colitis had severe diarrhea (6 or more bowel movements for 24 h) with macroscopic blood in the stools, tachycardia, fever (>37.8”C), and anemia (hemoglobin ~75%) (7). These patients required frequent hospitalizations. There were no patients with mild disease, since colectomy was a prerequisite for the study. Patients with moderate disease had symptoms estimated to be intermediate between severe and mild. These patients required continued medical observation, but could function at home or work on a part-time basis. The two patients who had a colectomy for moderate symptoms complained of significant fecal incontinence. The degree of mucosal inflammation was approximated by visual observation at colonoscopy (17): 3+ inflammation consisted of confluent ulcers and spontaneous bleeding; 2+ inflammation consisted of marked erythema, absent vascular pattern, and increased friability with erosions; l+ inflammation consisted of erythemadecreased vascular pattern and mild friability. The patients had a mean age of 41.8 t 5.7 years compared with patients with cancer (52.6 t 4.3 years) or diverticular disease (58.3 t 4.8 years). The patients with ulcerative colitis had chronic disease (7.4 t 2.7 years). These studies were approved by the human subjects committee at Harbor-UCLA Medical Center on March 5, 1985. All muscle contraction studies were performed on the tissue from each patient. The colonic muscle used for control studies was taken from an area free of carcinoma. The colonic muscle, from the colitis and diverticular patients, was taken from areas with mucosal inflammation. Tissue was not used from areas that were necrotic. The vascular supply of the tissues (l-2 cm ring) was ligated just before removal. The excised tissue was removed immediately and placed in 22OCoxygenated Krebs solution with the following composition (in mM): 115.5 NaCl, 4.6 KCl, 2.5 CaCIZ, 2.1 MgCIZ, 21.9 NaHC03, 1.2 NaHP04, and 15.5 glucose. The tissue, placed in a spe-
0 1991 the American
Physiological
Society
G987
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G988
DEFECTIVE
COLONIC
CONTRACTION
IN
COLITIS
1. Summary of patients with ulcerative colitis
TABLE Patient
1 2 3 4 5 6 7 8
Age, Yr
31 58 60 24 39 27 32 62
Duration Colitis, Yr
2 15 1.5 2 22 4 11 2
of
Severity of Symptoms, hematochezia
Severe Severe Moderate Severe Severe Severe Severe Moderate
Inflammation 2OV, 4+ 4+ l+ 4+ 4+ 3+ 4+ 3+
16H2,O.g
ms
T=s 1. A chart
recording of the response of human distal muscle after electrical field stimulation (EFS; 20 V, 16 Hz, EFS pulse is shown in the bottom tracing. FIG.
circular 0.9 ms).
cifically designed carrier containing continuously aerated A -A DIVERTICULITIS 4501 (95% O&% COZ) Krebs solution, was brought quickly to the laboratory. 400 1 The tissue was placed in oxygenated Krebs solution at 350 22°C for dissection. The mucosa was removed without c-d E 300 stretching or damaging the underlying muscle. The mus0 cle segments were cut into strips measuring 1 cm X 1 \z 250 \5 mm. These full-thickness strips were oriented in the axis g 200 of the circular muscle. The thickness of circular muscle E and weight of the strip was similar for each disease $j 150 category. 100 Isometric tension measurements. Each strip was placed 50 in a l-ml well filled with oxygenated Krebs solution 0 maintained at a temperature of 37°C (22). One or two 0 10 20 30 40 50 60 70 80 90 100 strips of circular muscle were used from each patient. One end of the muscle was attached to a stationary Hz clamp. The other end was attached via an inelastic wire to an isometric force transducer (model FT-03C, Grass, O-O colitis Quincy, MA). The muscle length was adjusted by a 0-O cancer AA diverticulitis micrometer. Initial length and the change in length were measured directly in the bath using a caliper. Each recording was graphed on a rectilinear ink-writing poly800 graph. The muscle tissues were stabilized in the bath for 1 h. After stabilization, the tissues were stretched, by increments of 1 mm, until muscle tension was ready to increase with any further increase in muscle length. The length was measured and noted as the initial length (Li). From this initial length, the muscle strip was progressively stretched by 2-mm increments. At each increment of stretch, the muscle was contracted using lOA M bethanechol. Bethanechol was washed from the bath and the tissue was allowed to 50 60 70 80 90 100 0 10 20 30 40 return to baseline before the next stretch. The optimal Hz length of the muscle (L,), at which peak active tension FIG. 2. On-contraction (A) and off-contraction (B) to increasing developed, was determined for each strip. At L,, the resting stress (no stimulation) was 328 t 15 in cancer, frequency of EFS in circular muscle from the distal colon. Parameters of EFS were 20 V, 0.9-ms pulse duration, and 10-s train duration. 375 t 18 in diverticulosis, and 329 t 7 mN/cm2 in colitis Results are expressed as means k SE. tissue (P > 0.05). The passive stress [no stimulation plus 2 mM ethylene glycol-bis (P-aminoethyl ether)-&& were placed parallel to the muscle strip (18). The elecN’,N’-tetraacetic acid (EGTA)] at L, was similar for trodes were placed 1 mm from the strip. Square-wave strips from colitis, diverticulitis, and cancer patients (73- pulses were delivered to the tissue. The frequency of the 75 mN/cm2) (P > 0.05). The bethanechol-stimulated stimulus was varied between 1.0 and 60 Hz, the pulse tension (active tension) was measured from the stable duration was 0.9 ms, and the voltage was supramaximal level of resting tension to the peak tension recorded after at 20 V. The voltage actually delivered to the tissue was bethanechol administration. Tension was expressed as measured from an oscilloscope (Tektronix T9112). The stress (force normalized to cross-sectional area) (13, 22). duration of the train of electrical pulses was 10 s. Increasing doses of extracellular K+ concentration iMuscle stimulation. Electrical field stimulation (EFS) (lK+l,. 4.5-120 mM) or bethanechol (Merck Sharp & was nerformed using Zmm-wide silver electrodes that 0
f
I
I
I
I
1
1
1
1
1
1
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
DEFECTIVE
O-O 0-O
A-
A
COLONIC colitis cancer diverticuli
CONTRACTION
IN
COLITIS
G989
The dose-response curves were compared using an analysis of variance (6). All data are shown as means t SE.
tis
RESULTS
0
I -8
-6
-7
-5 BETHANECHOL
-4
-3
LOG(M)
3. Contractile response to bethanechol in circular the distal colon. Results are expressed as means k SE. FIG.
O-O 0-O
A-
0
20
40
60
[K+l,
-2
80
A
muscle
from
colitis cancer diverticulitis
100
120
140
mM
FIG. 4. Contractile response to increased extracellular tion of K’ ([K’],) in circular muscle from the distal colon. expressed as means _t SE.
concentraResults are
Dohme, West Point, PA) (10B8to 10B3M) stimulated the muscle. As [K+10 increased, osmolality was maintained by reducing [Na+lO. Tetrodotoxin (TTX, low6 M) was added to the buffer in studies using [K’10 as the agonist. This concentration of TTX blocked EFS stimulation of the colonic enteric plexus in preliminary studies. Each drug was diluted in Krebs solution and added to the organ bath to provide the desired molar concentration. After exposure to each agent until a stable response occurred (-5 min), the muscle was washed free of the agent and tension was allowed to return to its previous resting level. At least 15 min elapsed between doses. Cumulative dose-response curves were not used. At the end of all experiments, the muscle strips were blotted gently and weighed. Statistical analysis. Statistical analysis was performed using unpaired Student’s t test where appropriate. The concentration of bethanechol required to elicit the halfmaximal response (ED& of bethanechol was calculated using a nonlinear, least-squares computer program (6).
EFS initiates an on- and off-contraction of distal circular colonic smooth muscle (Fig. 1). Before the onset of the on-contraction there is a 680-ms latency period. The off-contraction is larger than the on-contraction. Figure 2 compares the effect of EFS on distal circular smooth muscle from patients with colitis, diverticular disease, and cancer. The on-contraction in muscle from colitis is similar to the response in muscle from patients with cancer or diverticular disease (P > 0.05) (Fig. 24). The off-contraction increases as the frequency of EFS increases up to 60 Hz (Fig. ZB), where the response reaches a plateau. Less stress is generated by muscle from colitis patients compared with cancer patients (P < 0.01) or diverticular patients (P < 0.01). There is no significant difference between the response of tissue from patients with cancer or diverticular disease. We evaluated tissue contraction in response to bethanechol to determine whether there were any differences in response to a cholinergic agonist in tissue from colitis patients. Bethanechol stimulated a dose-dependent increase in stress in colonic muscle from each condition (Fig. 3). Bethanechol stimulated a larger maximum contraction in muscle from patients with cancer (1,438 t 95 mN/cm2) and diverticular disease (1,411 t 108 mN/cm2) than in muscle from patients with colitis (1,020 t 119 mN/cm2) (P < 0.05). The potency of bethanechol stimulation of circular colonic muscle was similar for cancer (ED = 2.6 + 0.3 X 10D5M), diverticular disease (1.9 t 0.3 :lO-” My, and colitis (2.5 t 0.5 X lo-” M) muscle. To bypass the need for receptor activation and to assesswhether the observed decrease in efficacy was due to a decreased number of muscarinic receptors or to a postreceptor event, we stimulated the colon muscle strips with increasing concentrations of K+, which stimulates the smooth muscle directly by depolarizing the cell membrane (Fig. 4). Increasing concentrations of K+ stimulated an increase in stress in each tissue. However, the increase in stress was less in muscle from colitis compared with muscle from patients with cancer or diverticular disease (P < 0.01). The contraction was greater in diverticular muscle than in muscle from cancer patients (P < 0.05). DISCUSSION
The present studies demonstrate that the decrease in in vivo colonic contraction (3, 19, 23) in patients with ulcerative colitis persists in vitro. Although the decrease in the force of contraction could be due to a decrease in the release of neurotransmitters, the present study suggests that it may be intrinsic to the smooth muscle cell. The studies were all done in the distal circular colonic muscle and avoided regional differences in response (20). EFS releases both excitatory and inhibitory neurotransmitters from the intrinsic colonic nerve plexus, first when electrical current is on and then when the current is turned off (18). It is probable that altering the param-
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G990
DEFECTIVE
COLONIC
CONTRACTION
eters of EFS alters the character of the neurotransmitter release (10). The major putative stimulatory neurotransmitters released by EFS are acetylcholine and substance P (12, 18). Vasoactive intestinal polypeptide (VIP) and catecholamines inhibit colonic contractions (1,24). However, the data characterizing neurotransmitters is incomplete in humans compared with other animals. The on-contraction after EFS has a similar amplitude in colitis muscle and tissue from cancer or diverticular patients, despite decreased force development of colitis muscle after bethanechol or increased extracellular K+. One explanation for the similarity in the on-contraction in the different tissues is the release of an inhibitory neurotransmitter during the electrical pulse that decreases active force development in colitis as well as in muscle from cancer or diverticular disease. The low amplitude of the on-contraction compared with the offcontraction supports this hypothesis. In the rabbit during the electrical stimulation there is a consistent relaxation, which is due in part to VIP (25). Concentrations of VIP and catecholamine in the myenteric plexus are similar in ulcerative colitis and carcinoma (11, 14). EFS could inhibit contraction similarly by releasing large quantities of VIP in each group. However, there could be a different ratio of excitatory to inhibitory neurotransmitters released from colitis tissue compared with cancer or divertitular disease. The decrease in the off-contraction in muscle from ulcerative colitis could be secondary to a defect in neurotransmitter release or an intrinsic defect in smooth muscle contraction. The decreased contraction by K+ suggests an intrinsic defect in colitis. The similar potency for bethanechol stimulation of the colonic muscle in the different groups suggests that the mucosal inflammation in ulcerative colitis does not affect the affinity of muscarinic receptor binding. The decreased contraction after K+ and the similar potency of bethanechol suggest that the defect causing decreased colitic smooth muscle contraction lies beyond the membrane receptors on the cell. It is possible that the decreased contraction could be due to altered Ca’+-, calmodulin-dependent regulation of actin-myosin crossbridge formation (8, 13). Increased intracellular Ca2’ complexes with calmodulin and activates myosin light chain kinase, which phosphorylates the myosin light chain, allowing myosin-actin cross-bridge formation (5). Preliminary studies showed that the potency of Ca2+ to stimulate isometric force development is similar in a permeabilized strip from rabbits with experimental colitis compared with control animals, despite a decrease in force development in the tissue from colitis animals (21). Therefore, the defect appears to be distal to the regulation of intracellular Ca2’ concentration. Future studies should examine a disturbance in actin, myosin, or the cross-bridge formation. The authors thank Nancy Ghattas and Douglas Root for technical assistance and Connie Madrigal for secretarial assistance. We also thank John Parker for construction of the electronic equipment used in this study. This work was supported by research grants from the National Institutes of Health (ROl-DK-3114 and R32-HD-22912), the Inflammatory Bowel Disease Center (5P30-DK-36200), and the National
IN
Foundation Address ical Center, Received
COLITIS
for Ileitis and Colitis. for reprint requests: W. J. Snape, Jr., Harbor-UCLA 1124 W. Carson St., Torrance, CA 90502. 16 July
1990; accepted
in final
form
8 July
Med-
1991.
REFERENCES 1. BIANCANI, P., J. H. WALSH, AND J. BEHAR. Vasoactive intestinal polypeptide. A neurotransmitter for lower esophageal sphincter relaxation. J. Clin. Invest. 73: 963-967, 1984. 2. CHAN, S. S., S. N. REDDY, J. VILLANUEVA, I. MENA, K. AKASHI, G. BAZZOCCHI, AND W. J. SNAPE, JR. Colonic motility and transit in intraluminal contents in ulcerative colitis (Abstract). Gastroenterology 96: A81, 1989. 3. CHAUDHARY, N. A., AND S. C. TRUELOVE. Human colonic motility: a comparative study of normal subjects, patients with ulcerative colitis, and patients with the irritable colon syndrome. Gastroenterology 40: l-36, 1961. 4. CONNELL, A. M. The motility of the pelvic colon. II. Paradoxical motility in diarrhea and constipation. Gut 3: 342-348, 1962. 5. DILLON, P. F., M. 0. AKSOY, S. P. DRISKA, AND R. A. MURPHY. Myosin phosphorylation and the crossbridge cycle in arterial smooth muscle. Science Wash. DC 211: 495-497, 1981. 6. DIXON, W. J. BMDP StatisticaL Software. Berkeley: Univ. of California Press, 1985. 7. EDWARDS, F., AND S. C. TRUELOVE. The course and prognosis of ulcerative colitis. Gut 4: 299-315, 1983. 8. HARTSHORNE, D. J. Biochemistry of the contractile process in smooth muscle. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 423-482. 9. KERN, F., JR., T. P. ALMY, F. K. ABBOT, AND M. D. BOGDONOFF. The motility of the distal colon in non-specific ulcerative colitis. Gastroenterology 19: 492-503, 1951. 10. KILBINGER, H., AND I. I. WESSLER. The variation of acetylcholine release from myenteric neurons with stimulation frequency and train length. Role of presynaptic muscarinic receptors. NaunynSchmiedeberg’s Arch. Pharmacol. 324: 130-133, 1983. 11. KOCH, T. R., D. R. CAVE, H. FORD, AND J. KIRSNER. Histofluorescent and radioenzymatic analysis of colonic catecholamines in man. J. Auton. Neru. Syst. 11: 383-391, 1984. 12. KOELBEL, C. B., A. PATEL, H. M. ENNES, W. J. SNAPE, JR., AND E. A. MAYER. Evidence for the involvement of substance P in noncholinergic excitation of rabbit colonic muscle. Am. J. Physiol. 256 (Gastrointest. Liver PhysioZ. 19): G246-G253, 1989. 13. MURPHY, R. A. Mechanics of vascular smooth muscle. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Sot., 1980, sect. 2, vol. II, chapt. 13, p. 325-351. 14. O’MORAIN, C., A. E. BISHOP, G. P. MCGREGO, A. J. LEVI, S. R. BLOOM, J. M. POLAK, AND T. J. PETERS. Vasoactive intestinal peptide concentrations and immunocytochemical studies in rectal biopsies from patients with inflammatory bowel disease. Gut 25: 57-61,1984. G. M. ARDRAN, AND M. TUCKEY. 15. PAINTER, N. S., S. C. TRUELOVE, Segmentation and the localization of intraluminal pressure in the human colon, with special reference to the pathogenesis of colonic diverticula. Gastroenterology 49: 169-178, 1965. 16. RITCHIE, J. A., AND S. N. SALEM. Upper intestinal motility in ulcerative colitis, idiopathic steatorrhea, and the irritable colon syndrome. Gut 6: 325-337, 1965. 17. SCHROEDER, K. W., W. J. TREMAINE, AND P. M. ILSTRUP. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. N. Engl. J. Med. 317: 1625-1629, 1987. 18. SNAPE, W. J., JR., B. H. KIM, R. WILLENBUCHER, C. KOELBEL, E. A. MAYER, AND J. H. WALSH. The responses of proximal and distal rabbit colonic muscle after electrical field stimulation. Gastroenterology 96: 321-326, 1989. 19. SNAPE, W. J., JR., S. A. MATARAZZO, AND S. COHEN. Abnormal gastrocolonic response in patients with ulcerative colitis. Gut 21: 392-396,198O. 20. SNAPE, W. J., JR., E. A. MAYER, C. M. KOELBEL, P. E. HYMAN, R. WILLIAMS, AND D. ROOT. Responses of smooth muscle from proximal and distal human colon. J. Gastrointest. MotiZ. 1: 29-34, 1989. 31 YI. SNAPE, W. J., JR., Y. XIE, S. N. REDDY, V. E. EYSSELEIN, AND F.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
DEFECTIVE Effect of contraction. J. 22. SNAPE, W. J., JR., AND colonic smooth muscle 235: 690-695, 1985. 23. SPRIGGS, E. A., C. F. Motility of the pelvic patients with ulcerative COMINELLI.
muscle
COLONIC
mucosal inflammation on colonic smooth Gastrointest. Motil. 1: 69, 1989. S. YOO. Effect of amino acids on isolated from the rabbit. J. Pharmacol. Exp. Ther. J. A. BARGEN, AND R. K. CURTISS. colon and rectum of normal persons and colitis. Gustroenterology 19: 480-491, 1951.
CODE,
CONTRACTION
IN
COLITIS
G991
24. WIENBECK, M., AND J. CHRISTENSEN. Effects of some drugs on electrical activity of the isolated colon of the cat. Gastroenterology 61: 470-478, 1971. 25. WILLENBUCHER, R. F., V. E. EYSSELEIN, J. H. WALSH, N. GHATTAS, P. E. HYMAN, AND W. J. SNAPE, JR. Flash photolysis of caged CAMP reproduce the effect of vasoactive intestinal peptide release during electric field stimulation of the rabbit colon (Abstract). Gastroenterology 98: A532, 1990.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
SNAPE, WILLIAM J., JR., R. WILLIAMS, AND P. E. HYMAN. Defect in colonic smooth muscle contraction in patients with ulcerative colitis. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G987-G991, 1991.-Patients with ulcerative colitis have decreased postprandial colonic contractions. The purpose of this study was to determine whether the smooth muscle from patients with ulcerative colitis responds abnormally in vitro to different stimuli. Circular colonic smooth muscle strips from patients with ulcerative colitis, acute diverticular disease, or adenocarcinoma were stretched to the optimal length and stimulated with electrical field stimulation (EFS), bethanechol, or increased concentrations of extracellular K’. The EFS-stimulated on-contraction was similar in each group, but the offcontraction was decreased in patients with colitis compared with patients with cancer (P < 0.02) or diverticular disease (P < 0.01). Bethanechol stimulated a dose-dependent colonic contraction, which was less in the strips from patients with colitis compared with cancer (P < 0.02) or diverticular disease (P < 0.05). The response to increased extracellular K+ was less in muscle from patients with colitis (P < 0.01) than in the other tissues. Muscle from diverticular disease developed greater stress to K+ stimulation than did muscle from cancer (P < 0.05). These studies suggest that there is a decrease in the force of muscle contraction in colonic muscle obtained from patients with colitis compared with normal muscle resected from patients with cancer or with muscle associated with diverticular disease of the colon. The similar relatively low amplitude of the on-contraction in each group suggests the physiological release of an inhibitory neurotransmitter. mucosal inflammation; inflammatory bowel disease; electrical field stimulation; cholinergic; isometric tension
THE FORCE of colonic contractions after different stimuli
is decreased in patients with ulcerative colitis compared with healthy controls or with patients with diverticular disease of the colon (3, 9, 15, 19, 23). Diarrhea, which is occasionally associated with incontinence, is a major symptom in ulcerative colitis. The decrease in segmenting contractions can accentuate the diarrhea in colitis by allowing rapid forward movement of the colonic contents (2, 4). The defect in smooth muscle contraction may be generalized, since the force of contractions also is decreased in the small intestine, which is not histologically involved in the disease process (16). The present study was designed to evaluate the mechanism of the decreased colonic smooth muscle contraction in patients with ulcerative colitis by comparing the in vitro contraction of colonic muscle from ulcerative colitis with muscle from 0193-1857/91
$1.50
Copyright
patients with adenocarcinoma or diverticular
disease of
the colon. METHODS
Tissue preparation. Colonic tissue distal to the splenic flexure was removed during colectomy from 10 patients with an adenocarcinoma, 5 patients with diverticular diseaseof the colon, and 8 patients with ulcerative colitis. Table 1 shows the clinical characteristics of these patients with ulcerative colitis. Patients with globally severe symptoms of ulcerative colitis had severe diarrhea (6 or more bowel movements for 24 h) with macroscopic blood in the stools, tachycardia, fever (>37.8”C), and anemia (hemoglobin ~75%) (7). These patients required frequent hospitalizations. There were no patients with mild disease, since colectomy was a prerequisite for the study. Patients with moderate disease had symptoms estimated to be intermediate between severe and mild. These patients required continued medical observation, but could function at home or work on a part-time basis. The two patients who had a colectomy for moderate symptoms complained of significant fecal incontinence. The degree of mucosal inflammation was approximated by visual observation at colonoscopy (17): 3+ inflammation consisted of confluent ulcers and spontaneous bleeding; 2+ inflammation consisted of marked erythema, absent vascular pattern, and increased friability with erosions; l+ inflammation consisted of erythemadecreased vascular pattern and mild friability. The patients had a mean age of 41.8 t 5.7 years compared with patients with cancer (52.6 t 4.3 years) or diverticular disease (58.3 t 4.8 years). The patients with ulcerative colitis had chronic disease (7.4 t 2.7 years). These studies were approved by the human subjects committee at Harbor-UCLA Medical Center on March 5, 1985. All muscle contraction studies were performed on the tissue from each patient. The colonic muscle used for control studies was taken from an area free of carcinoma. The colonic muscle, from the colitis and diverticular patients, was taken from areas with mucosal inflammation. Tissue was not used from areas that were necrotic. The vascular supply of the tissues (l-2 cm ring) was ligated just before removal. The excised tissue was removed immediately and placed in 22OCoxygenated Krebs solution with the following composition (in mM): 115.5 NaCl, 4.6 KCl, 2.5 CaCIZ, 2.1 MgCIZ, 21.9 NaHC03, 1.2 NaHP04, and 15.5 glucose. The tissue, placed in a spe-
0 1991 the American
Physiological
Society
G987
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G988
DEFECTIVE
COLONIC
CONTRACTION
IN
COLITIS
1. Summary of patients with ulcerative colitis
TABLE Patient
1 2 3 4 5 6 7 8
Age, Yr
31 58 60 24 39 27 32 62
Duration Colitis, Yr
2 15 1.5 2 22 4 11 2
of
Severity of Symptoms, hematochezia
Severe Severe Moderate Severe Severe Severe Severe Moderate
Inflammation 2OV, 4+ 4+ l+ 4+ 4+ 3+ 4+ 3+
16H2,O.g
ms
T=s 1. A chart
recording of the response of human distal muscle after electrical field stimulation (EFS; 20 V, 16 Hz, EFS pulse is shown in the bottom tracing. FIG.
circular 0.9 ms).
cifically designed carrier containing continuously aerated A -A DIVERTICULITIS 4501 (95% O&% COZ) Krebs solution, was brought quickly to the laboratory. 400 1 The tissue was placed in oxygenated Krebs solution at 350 22°C for dissection. The mucosa was removed without c-d E 300 stretching or damaging the underlying muscle. The mus0 cle segments were cut into strips measuring 1 cm X 1 \z 250 \5 mm. These full-thickness strips were oriented in the axis g 200 of the circular muscle. The thickness of circular muscle E and weight of the strip was similar for each disease $j 150 category. 100 Isometric tension measurements. Each strip was placed 50 in a l-ml well filled with oxygenated Krebs solution 0 maintained at a temperature of 37°C (22). One or two 0 10 20 30 40 50 60 70 80 90 100 strips of circular muscle were used from each patient. One end of the muscle was attached to a stationary Hz clamp. The other end was attached via an inelastic wire to an isometric force transducer (model FT-03C, Grass, O-O colitis Quincy, MA). The muscle length was adjusted by a 0-O cancer AA diverticulitis micrometer. Initial length and the change in length were measured directly in the bath using a caliper. Each recording was graphed on a rectilinear ink-writing poly800 graph. The muscle tissues were stabilized in the bath for 1 h. After stabilization, the tissues were stretched, by increments of 1 mm, until muscle tension was ready to increase with any further increase in muscle length. The length was measured and noted as the initial length (Li). From this initial length, the muscle strip was progressively stretched by 2-mm increments. At each increment of stretch, the muscle was contracted using lOA M bethanechol. Bethanechol was washed from the bath and the tissue was allowed to 50 60 70 80 90 100 0 10 20 30 40 return to baseline before the next stretch. The optimal Hz length of the muscle (L,), at which peak active tension FIG. 2. On-contraction (A) and off-contraction (B) to increasing developed, was determined for each strip. At L,, the resting stress (no stimulation) was 328 t 15 in cancer, frequency of EFS in circular muscle from the distal colon. Parameters of EFS were 20 V, 0.9-ms pulse duration, and 10-s train duration. 375 t 18 in diverticulosis, and 329 t 7 mN/cm2 in colitis Results are expressed as means k SE. tissue (P > 0.05). The passive stress [no stimulation plus 2 mM ethylene glycol-bis (P-aminoethyl ether)-&& were placed parallel to the muscle strip (18). The elecN’,N’-tetraacetic acid (EGTA)] at L, was similar for trodes were placed 1 mm from the strip. Square-wave strips from colitis, diverticulitis, and cancer patients (73- pulses were delivered to the tissue. The frequency of the 75 mN/cm2) (P > 0.05). The bethanechol-stimulated stimulus was varied between 1.0 and 60 Hz, the pulse tension (active tension) was measured from the stable duration was 0.9 ms, and the voltage was supramaximal level of resting tension to the peak tension recorded after at 20 V. The voltage actually delivered to the tissue was bethanechol administration. Tension was expressed as measured from an oscilloscope (Tektronix T9112). The stress (force normalized to cross-sectional area) (13, 22). duration of the train of electrical pulses was 10 s. Increasing doses of extracellular K+ concentration iMuscle stimulation. Electrical field stimulation (EFS) (lK+l,. 4.5-120 mM) or bethanechol (Merck Sharp & was nerformed using Zmm-wide silver electrodes that 0
f
I
I
I
I
1
1
1
1
1
1
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
DEFECTIVE
O-O 0-O
A-
A
COLONIC colitis cancer diverticuli
CONTRACTION
IN
COLITIS
G989
The dose-response curves were compared using an analysis of variance (6). All data are shown as means t SE.
tis
RESULTS
0
I -8
-6
-7
-5 BETHANECHOL
-4
-3
LOG(M)
3. Contractile response to bethanechol in circular the distal colon. Results are expressed as means k SE. FIG.
O-O 0-O
A-
0
20
40
60
[K+l,
-2
80
A
muscle
from
colitis cancer diverticulitis
100
120
140
mM
FIG. 4. Contractile response to increased extracellular tion of K’ ([K’],) in circular muscle from the distal colon. expressed as means _t SE.
concentraResults are
Dohme, West Point, PA) (10B8to 10B3M) stimulated the muscle. As [K+10 increased, osmolality was maintained by reducing [Na+lO. Tetrodotoxin (TTX, low6 M) was added to the buffer in studies using [K’10 as the agonist. This concentration of TTX blocked EFS stimulation of the colonic enteric plexus in preliminary studies. Each drug was diluted in Krebs solution and added to the organ bath to provide the desired molar concentration. After exposure to each agent until a stable response occurred (-5 min), the muscle was washed free of the agent and tension was allowed to return to its previous resting level. At least 15 min elapsed between doses. Cumulative dose-response curves were not used. At the end of all experiments, the muscle strips were blotted gently and weighed. Statistical analysis. Statistical analysis was performed using unpaired Student’s t test where appropriate. The concentration of bethanechol required to elicit the halfmaximal response (ED& of bethanechol was calculated using a nonlinear, least-squares computer program (6).
EFS initiates an on- and off-contraction of distal circular colonic smooth muscle (Fig. 1). Before the onset of the on-contraction there is a 680-ms latency period. The off-contraction is larger than the on-contraction. Figure 2 compares the effect of EFS on distal circular smooth muscle from patients with colitis, diverticular disease, and cancer. The on-contraction in muscle from colitis is similar to the response in muscle from patients with cancer or diverticular disease (P > 0.05) (Fig. 24). The off-contraction increases as the frequency of EFS increases up to 60 Hz (Fig. ZB), where the response reaches a plateau. Less stress is generated by muscle from colitis patients compared with cancer patients (P < 0.01) or diverticular patients (P < 0.01). There is no significant difference between the response of tissue from patients with cancer or diverticular disease. We evaluated tissue contraction in response to bethanechol to determine whether there were any differences in response to a cholinergic agonist in tissue from colitis patients. Bethanechol stimulated a dose-dependent increase in stress in colonic muscle from each condition (Fig. 3). Bethanechol stimulated a larger maximum contraction in muscle from patients with cancer (1,438 t 95 mN/cm2) and diverticular disease (1,411 t 108 mN/cm2) than in muscle from patients with colitis (1,020 t 119 mN/cm2) (P < 0.05). The potency of bethanechol stimulation of circular colonic muscle was similar for cancer (ED = 2.6 + 0.3 X 10D5M), diverticular disease (1.9 t 0.3 :lO-” My, and colitis (2.5 t 0.5 X lo-” M) muscle. To bypass the need for receptor activation and to assesswhether the observed decrease in efficacy was due to a decreased number of muscarinic receptors or to a postreceptor event, we stimulated the colon muscle strips with increasing concentrations of K+, which stimulates the smooth muscle directly by depolarizing the cell membrane (Fig. 4). Increasing concentrations of K+ stimulated an increase in stress in each tissue. However, the increase in stress was less in muscle from colitis compared with muscle from patients with cancer or diverticular disease (P < 0.01). The contraction was greater in diverticular muscle than in muscle from cancer patients (P < 0.05). DISCUSSION
The present studies demonstrate that the decrease in in vivo colonic contraction (3, 19, 23) in patients with ulcerative colitis persists in vitro. Although the decrease in the force of contraction could be due to a decrease in the release of neurotransmitters, the present study suggests that it may be intrinsic to the smooth muscle cell. The studies were all done in the distal circular colonic muscle and avoided regional differences in response (20). EFS releases both excitatory and inhibitory neurotransmitters from the intrinsic colonic nerve plexus, first when electrical current is on and then when the current is turned off (18). It is probable that altering the param-
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
G990
DEFECTIVE
COLONIC
CONTRACTION
eters of EFS alters the character of the neurotransmitter release (10). The major putative stimulatory neurotransmitters released by EFS are acetylcholine and substance P (12, 18). Vasoactive intestinal polypeptide (VIP) and catecholamines inhibit colonic contractions (1,24). However, the data characterizing neurotransmitters is incomplete in humans compared with other animals. The on-contraction after EFS has a similar amplitude in colitis muscle and tissue from cancer or diverticular patients, despite decreased force development of colitis muscle after bethanechol or increased extracellular K+. One explanation for the similarity in the on-contraction in the different tissues is the release of an inhibitory neurotransmitter during the electrical pulse that decreases active force development in colitis as well as in muscle from cancer or diverticular disease. The low amplitude of the on-contraction compared with the offcontraction supports this hypothesis. In the rabbit during the electrical stimulation there is a consistent relaxation, which is due in part to VIP (25). Concentrations of VIP and catecholamine in the myenteric plexus are similar in ulcerative colitis and carcinoma (11, 14). EFS could inhibit contraction similarly by releasing large quantities of VIP in each group. However, there could be a different ratio of excitatory to inhibitory neurotransmitters released from colitis tissue compared with cancer or divertitular disease. The decrease in the off-contraction in muscle from ulcerative colitis could be secondary to a defect in neurotransmitter release or an intrinsic defect in smooth muscle contraction. The decreased contraction by K+ suggests an intrinsic defect in colitis. The similar potency for bethanechol stimulation of the colonic muscle in the different groups suggests that the mucosal inflammation in ulcerative colitis does not affect the affinity of muscarinic receptor binding. The decreased contraction after K+ and the similar potency of bethanechol suggest that the defect causing decreased colitic smooth muscle contraction lies beyond the membrane receptors on the cell. It is possible that the decreased contraction could be due to altered Ca’+-, calmodulin-dependent regulation of actin-myosin crossbridge formation (8, 13). Increased intracellular Ca2’ complexes with calmodulin and activates myosin light chain kinase, which phosphorylates the myosin light chain, allowing myosin-actin cross-bridge formation (5). Preliminary studies showed that the potency of Ca2+ to stimulate isometric force development is similar in a permeabilized strip from rabbits with experimental colitis compared with control animals, despite a decrease in force development in the tissue from colitis animals (21). Therefore, the defect appears to be distal to the regulation of intracellular Ca2’ concentration. Future studies should examine a disturbance in actin, myosin, or the cross-bridge formation. The authors thank Nancy Ghattas and Douglas Root for technical assistance and Connie Madrigal for secretarial assistance. We also thank John Parker for construction of the electronic equipment used in this study. This work was supported by research grants from the National Institutes of Health (ROl-DK-3114 and R32-HD-22912), the Inflammatory Bowel Disease Center (5P30-DK-36200), and the National
IN
Foundation Address ical Center, Received
COLITIS
for Ileitis and Colitis. for reprint requests: W. J. Snape, Jr., Harbor-UCLA 1124 W. Carson St., Torrance, CA 90502. 16 July
1990; accepted
in final
form
8 July
Med-
1991.
REFERENCES 1. BIANCANI, P., J. H. WALSH, AND J. BEHAR. Vasoactive intestinal polypeptide. A neurotransmitter for lower esophageal sphincter relaxation. J. Clin. Invest. 73: 963-967, 1984. 2. CHAN, S. S., S. N. REDDY, J. VILLANUEVA, I. MENA, K. AKASHI, G. BAZZOCCHI, AND W. J. SNAPE, JR. Colonic motility and transit in intraluminal contents in ulcerative colitis (Abstract). Gastroenterology 96: A81, 1989. 3. CHAUDHARY, N. A., AND S. C. TRUELOVE. Human colonic motility: a comparative study of normal subjects, patients with ulcerative colitis, and patients with the irritable colon syndrome. Gastroenterology 40: l-36, 1961. 4. CONNELL, A. M. The motility of the pelvic colon. II. Paradoxical motility in diarrhea and constipation. Gut 3: 342-348, 1962. 5. DILLON, P. F., M. 0. AKSOY, S. P. DRISKA, AND R. A. MURPHY. Myosin phosphorylation and the crossbridge cycle in arterial smooth muscle. Science Wash. DC 211: 495-497, 1981. 6. DIXON, W. J. BMDP StatisticaL Software. Berkeley: Univ. of California Press, 1985. 7. EDWARDS, F., AND S. C. TRUELOVE. The course and prognosis of ulcerative colitis. Gut 4: 299-315, 1983. 8. HARTSHORNE, D. J. Biochemistry of the contractile process in smooth muscle. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 423-482. 9. KERN, F., JR., T. P. ALMY, F. K. ABBOT, AND M. D. BOGDONOFF. The motility of the distal colon in non-specific ulcerative colitis. Gastroenterology 19: 492-503, 1951. 10. KILBINGER, H., AND I. I. WESSLER. The variation of acetylcholine release from myenteric neurons with stimulation frequency and train length. Role of presynaptic muscarinic receptors. NaunynSchmiedeberg’s Arch. Pharmacol. 324: 130-133, 1983. 11. KOCH, T. R., D. R. CAVE, H. FORD, AND J. KIRSNER. Histofluorescent and radioenzymatic analysis of colonic catecholamines in man. J. Auton. Neru. Syst. 11: 383-391, 1984. 12. KOELBEL, C. B., A. PATEL, H. M. ENNES, W. J. SNAPE, JR., AND E. A. MAYER. Evidence for the involvement of substance P in noncholinergic excitation of rabbit colonic muscle. Am. J. Physiol. 256 (Gastrointest. Liver PhysioZ. 19): G246-G253, 1989. 13. MURPHY, R. A. Mechanics of vascular smooth muscle. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Sot., 1980, sect. 2, vol. II, chapt. 13, p. 325-351. 14. O’MORAIN, C., A. E. BISHOP, G. P. MCGREGO, A. J. LEVI, S. R. BLOOM, J. M. POLAK, AND T. J. PETERS. Vasoactive intestinal peptide concentrations and immunocytochemical studies in rectal biopsies from patients with inflammatory bowel disease. Gut 25: 57-61,1984. G. M. ARDRAN, AND M. TUCKEY. 15. PAINTER, N. S., S. C. TRUELOVE, Segmentation and the localization of intraluminal pressure in the human colon, with special reference to the pathogenesis of colonic diverticula. Gastroenterology 49: 169-178, 1965. 16. RITCHIE, J. A., AND S. N. SALEM. Upper intestinal motility in ulcerative colitis, idiopathic steatorrhea, and the irritable colon syndrome. Gut 6: 325-337, 1965. 17. SCHROEDER, K. W., W. J. TREMAINE, AND P. M. ILSTRUP. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. N. Engl. J. Med. 317: 1625-1629, 1987. 18. SNAPE, W. J., JR., B. H. KIM, R. WILLENBUCHER, C. KOELBEL, E. A. MAYER, AND J. H. WALSH. The responses of proximal and distal rabbit colonic muscle after electrical field stimulation. Gastroenterology 96: 321-326, 1989. 19. SNAPE, W. J., JR., S. A. MATARAZZO, AND S. COHEN. Abnormal gastrocolonic response in patients with ulcerative colitis. Gut 21: 392-396,198O. 20. SNAPE, W. J., JR., E. A. MAYER, C. M. KOELBEL, P. E. HYMAN, R. WILLIAMS, AND D. ROOT. Responses of smooth muscle from proximal and distal human colon. J. Gastrointest. MotiZ. 1: 29-34, 1989. 31 YI. SNAPE, W. J., JR., Y. XIE, S. N. REDDY, V. E. EYSSELEIN, AND F.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
DEFECTIVE Effect of contraction. J. 22. SNAPE, W. J., JR., AND colonic smooth muscle 235: 690-695, 1985. 23. SPRIGGS, E. A., C. F. Motility of the pelvic patients with ulcerative COMINELLI.
muscle
COLONIC
mucosal inflammation on colonic smooth Gastrointest. Motil. 1: 69, 1989. S. YOO. Effect of amino acids on isolated from the rabbit. J. Pharmacol. Exp. Ther. J. A. BARGEN, AND R. K. CURTISS. colon and rectum of normal persons and colitis. Gustroenterology 19: 480-491, 1951.
CODE,
CONTRACTION
IN
COLITIS
G991
24. WIENBECK, M., AND J. CHRISTENSEN. Effects of some drugs on electrical activity of the isolated colon of the cat. Gastroenterology 61: 470-478, 1971. 25. WILLENBUCHER, R. F., V. E. EYSSELEIN, J. H. WALSH, N. GHATTAS, P. E. HYMAN, AND W. J. SNAPE, JR. Flash photolysis of caged CAMP reproduce the effect of vasoactive intestinal peptide release during electric field stimulation of the rabbit colon (Abstract). Gastroenterology 98: A532, 1990.
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on August 28, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
