Idiopathic chronic constipation is associated with decreased colonic vasoactive intestinal peptide

GASTROENTEROLOGY

1988;94:300-10

Idiopathic Chronic Constipation Is Associated With Decreased Colonic Vasoactive Intestinal Peptide TIMOTHY R. KOCH, J. AIDAN CARNEY, LISA GO, and VAY LIANG W. GO Gastroenterology Minnesota

IJnit and Ikpartment

of Pathology.

Mayo

Clinic. and Foundation,

Kochester.

To investigate the reported association between idiopathic chronic constipation and morphologic abnormalities of enteric nerves, we measured the concentrations of six neuropeptides, vasoactive intestinal peptide, peptide histidine-methionine, substance P, methionine5-enkephalin, neuropeptide Y, and the bombesinlike intestinal peptides, in descending colon from 4 patients with idiopathic chronic constipation. Decreased concentrations of vasoactive intestinal peptide (707 f 112 rig/g wet tissue) and peptide histidine-methionine (543 f 58 rig/g))were found in the muscularis externa obtained from constipated patients compared with normal concentrations (40 patients) of vasoactive intestinal peptide (1199 + 47 rig/g)) and peptide histidinemethionine (815 f 45 ng/g). Vasoactive intestinal peptide was identified by immunocytochemistry in nerve fibers within the circular smooth muscle layer of descending colon obtained from 6 control patients, but not in nerve fibers within the circular smooth muscle of descending colon obtained from 3 patients with idiopathic chronic constipation. By contrast, the distribution of immunoreactive met5enkephalin was similar in normal descending colon and in descending colon obtained from patients with idiopathic chronic constipation. Decreased coionic concentrations of vasoactive intestinal peptide (a candidate nonadrenergic, noncholinergic inhibitory neurotransmitter) may be associated with diminution of inhibitory innervation of colonic circular smooth muscle in some patients with idiopathic chronic constipation.

bolic or hormonal disorders (such as hypokalemia and hypothyroidism), and toxins or drugs (such as narcotic agents and ganglionic blocking agents). In some patients, the cause of chronic constipation remains unknown (idiopathic). Multiple mechanisms have been proposed to explain the etiology of idiopathic chronic constipation. Studies using radiopaque markers have demonstrated a colonic motility disorder associated with delayed colonic transit (1-3)in some patients with idiopathic constipation. Anorectal manometric studies, electromyography of the pelvic floor, and studies utilizing balloon proctograms have provided inconclusive results in other cases. These studies have suggested that an outlet obstruction, caused by failure of pelvic floor muscle relaxation (3-6),loss of inhibition of the external anal sphincter (7,~)). or alteration of the anorectal inhibitory reflex (4,~). could be the cause of constipation in some patients. III the past decade, light microscopic (I O-I 3) and ultrastructural (14) immunocytochemistry have demonstrated that a group of peptides, including vasoactive intestinal peptide (VIP), pcptide histidine-methionine (PHM), substanc:c P, mcthionine”enkephalin (met”-ENK), neuropeptide Y (NPY), anti the bombesinlike intestinal peptides (HI,IPs), are present within enteric nerves. Irnmurloc:hemit:al methods have allowed examination of the distribution and quantity of these neuropeptides in several human colonic diseases including ulcerative colitis. Crohn’s colitis, and Hirschsprung’s disease (lFi.lti). In this study, we determined the concentrations of

T

Abbreviafions used in this paper: BLIP, bombesinlike intestinal peptide; ENK, enkephalin; met”-ENK, methionine5-enkephalin; NANC, nonadrenergic, noncholinergic; NPY. neuropeptide Y; PHM, peptide histidine-methionine. $7 1988 by the American Gastroenterological Association 0016-5085/88/$3.50

he known etiologies of constipation include neuropathic disorders (Hirschsprung’s disease and Chagas’ disease), myopathic disorders (such as scleroderma and amyloidosis), mechanical obstruction (such as adenocarcinoma of the rectum), meta-

these six neuropeptides, VIP, PHM, substance P, enkephalin (ENK), NPY, and BLIP, in the muscularis externa and in the mucosal-submucosal layer of descending colon from 4 patients with idiopathic chronic constipation using specific radioimmunoassays. We also examined the distributions of VIP and ENK within the muscularis externa of descending colon obtained from these patients using immunocytochemistry. Idiopathic chronic constipation was associated with decreased concentrations of VIP and PHM in the muscularis externa of descending colon, and with diminished staining of VIP-containing nerves in the colonic circular smooth muscle layer.

Materials

and Methods

(Grosslyand histologically normal specimens of descending colon were obtained at surgery from 40 patients with nonobstructing colonic carcinoma, solitary colonic polyp, or colonic angiodysplasia. Permission for human studies was granted by the Mayo Clinic Institutional Review Board ou December 6, 1984. The mean patient age was 62 yr (range 19-87 yr). Idiopathic

Chronic

Constipation

Uuse I. This patient is a 53-yr-old woman with a 15-qr history of intractable constipation. Past medical treatment included prune juice, docusate sodium, bran fiber, and psyllium hydrophilic mucilloid. Partial colectomy with an ileosigmoidostomy had been previously performed. Histologic review (J. A. C.) of this resected colon revealed no diagnostic abnormality. Severe constipation persisted, and she was evaluated at the Mayo Clinic. Serum electrolytes, fasting blood glucose, and total thyroxine were normal. Proctoscopic examination was normal. and a barium colon x-ray study revealed a freely functioning ileocolostomy. Anorectal manometry demonstrated a normal resting pressure of 55 mmHg with a mean squeeze pressure of 50 mmHg. However, the rectoanal inhibit#Dry reflex was inconsistently demonstrated. A coionic transit study showed that the first radiopaque markers reached the sigmoid colon 3 days after ingestion, and that radiopaque markers remained in the sigmoid colon 6 days after ingestion. This study was interpreted as showing an increased colonic transit time. The patient underwent surgical resection of a dilated loop of sigmoid colon with formation of an ileorectostomy. Routine histologic examination of sigmoid colon was normal. (Ius(: 2. This patient is a 63-yr-old woman with a 7-yr histor!, of daily rectal urgency who was able to pass fecal material per rectum only once every 4-5 days. Past medical treatment included lactulose syrup. psyllium plantagcl hydrocolloid, and enemas. LJpon evaluation at the Mayo Clinic:, serum electrolytes. fasting blood glucose, and total thyroxine were normal. Colonoscopy was normal. Neither anorectal manometry nor a colonic transit studv was obtained preoperatively. An elongated, dilated

colon was identified at surgery, and resection of ascending, transverse, and descending colon with formation of an ileosigmoidostomg was performed. Routine histologic examination of descending colon was normal. Cnse 3. This patient is a 3%yr-old woman with an 8-yr history of intractable constipation. Past medical treatment included a phenolthalein cathartic, prune juice, magnesium salts, and disimpaction enemas. Two years before evaluation at the Mayo Clinic, a partial colectomy with an ascending-sigmoid colostomy had been performed. Histologic review (J. A. C.) of this resected colon revealed melanosis coli, but no diagnostic abnormality. Intractable constipation persisted, and she was evaluated at the Mayo Clinic. Serum electrolytes. fasting blood glucose, and total thyroxine were normal. Proctoscopic examination was normal, and a barium colon x-ray study revealed a freely functioning ascending-sigmoid colostomy. Anorectal manometry demonstrated a normal rectoanal inhibitory reflex, but the patient was not able to detect rectal pressure with the rectal balloon distended to 50 ml. A colonic transit study wac‘ not performed preoperativelg. Surgical resection of the cecum and sigmoid colon Rouwith formation of an ileorectostomy was performed. tine histologic examination of sigmoid colon showed hypertrophy of the circular ~n~w:le layer, but a normal myenteric plexus. Cuse 4. This patient is a 42-yr-old man with a 7-yr history of constipation. The patient required 2-3 h of prolonged defecation straining for passage of a small volume of stool, and noted a residual sensation of incomplete evacuation. Past medical treatment included a benzodiazepine-class drug, a cholinergic receptor blocking agent, docusate sodium, psyllium hydrophilic: mucilloid, and enemas. Before evaluation at the Mayo Clinic, the patient underwent resection of sigmoid colon with formation of a colocolostomy. Histologic re\+erv (J. ,L C.) of this resected colon revealed hypertrophy of the circular smooth muscle layer, but no abnormality of the myenteric plexus. Because of persistent constipation, the patient was evaluated at the Mayo Clinic. Serum electrolytes, fasting blood glucose, and total thyroxine were normal. Proctoscopic examination was normal, and a barium colon x-ra) study revealed a freely functioning colocolostomv. Anorectal manometry revealed an elevated mean restitlg pressure of 92 mmHg and an elevated mean squeeze pressure of 180 mmHg, but the rectoanal inhibitory reflex was present. A colonic transit study showed that no radiopaque markers remained in the colon on the fourth day after ingestion, and this was interpreted as a normal study. The patient underwent surgical resection of the cecum and the ascending, transverse. and descending colon with formation of a side-to-end ileorectostomy. Routine histologic examination of the descending colon was normal.

By microdissection, the muscularis externa and the mucosal-submucosal layer of fresh descending colon obtained from controls and from patients with idiopathic chronic constipation were separated. aud neuropeptides

302 KOCH ET AL.

GASTKOENTRKOLOGY

were extracted as previously described (15). Specific radioimmunoassays were used to measure tissue concentrations of VIP (17), PHM (18),substance P (19),ENK (20). and BLIPS (21). A sensitive and specific radioimmunoassay for NPY has been recently developed in our laboratory (22). This radioimmunoassay utilizes a rabbit antibody (antibody 221) raised against synthetic porcine NPY. Antibody 221 is used at a final dilution of 1:12,800, and synthetic human NPY is used as standard and for preparing ‘?-NPY. Separation utilizes goat-antirabbit serum (Cambridge Medical Diagnostics, Billerica, Mass.) with 6% PEG 8000. The assay does not cross-react with VIP, PHM, or human pancreatic polypeptide, but does cross-react 0.6% with porcine peptide YY. In dilutions of tissue extracts, parallelism compared with the standard curve has been demonstrated. The assay is sensitive to 20 pgltube. The intraassay variation is 7% and the interassay variation is 12%. The immunoreactive species measured by these radioimmunoassays in extracts of human colon have been previously characterized by gel chromatography (15). After separation of extracts of normal human colon on a Sephadex G-50 superfine column, radioimmunoassay identified one peak of immunoreactive VIP that appeared to coelute with porcine VIP (15). After separation of extracts of normal human colon on a Sephadex G-25 superfine column, radioimmunoassay for substance P identified one immunoreactive peak that appeared to coelute with porcine substance P; radioimmunoassay for met’-ENK identified one major immunoreactive peak that appeared to coelute with synthetic met”-ENK; and radioimmunoassay for BLIP identified three peaks of immunoreactive material (one peak appeared to coelute with porcine gastrin-releasing peptide, the second peak appeared to coelute with bombesin-decapeptide, and a third minor peak appeared to be of smaller molecular weight than bombesin-10) (I 5). In previous work (151, we lyophilized histologically normal colon and colon obtained from patients with diverticulitis, ulcerative colitis, and Crohn’s colitis to determine colonic water content. There were no significant differences in water content (range 79%-81%) among these different groups (15). In this study, we therefore chose to express the results as nanograms of immunoreactive peptide per gram wet tissue. Population mean values and standard errors of the mean were calculated. Significant differences between mean neuropeptide concentrations were tested by two-tailed unpaired Student’s t-tests.

Tissue

Fixation

and

Immunohistochemistry

Transmural sections of descending colon from 6 control patients and from 3 patients with idiopathic chronic constipation were incubated for 20 h in Zamboni’s fixative (23) at 4’C. Tissues were washed in multiple changes of 0.01 M phosphate-buffered saline with 3% sucrose (pH 7.4) for 48 h, and were embedded in paraplast, followed by sectioning at 5 pm. The indirect immunoperoxidase technique of Nakane (24,25) was used for visualization of immunoreactive VIP and ENK. For localization of immunoreactive VIP, rabbit antise-

Vol.

94. No. z

rum 4823 raised against porcine VIP was used at a final dilution of 1:300. Goat-antirabbit immunoglobulin Ghorseradish peroxidase conjugate (Tago Inc., Burlingame, Calif.) at a dilution of 1:100 was then applied. Duplicate sections from each tissue block were immunostained, and examined in a blinded method to determine the distribution of immunoreactive VIP. As controls, nonimmune rabbit serum at a dilution of 1: 300 or phosphate-buffered saline was substituted for the primary rabbit antiserum 4823, or phosphate-buffered saline was substituted for the secondary conjugated goat antiserum. In adsorption experiments, immunoreactivity was extinguished by preincubation of the primary antisera at a dilution of 1:300 with VIP (at 500 ngiml antiserum) or with peptide histidinemethionine, a structurally similar peptide present in the preproVIP molecule (26), (at 5 pg/ml antiserum) for 24 h at 4°C. Immunoreactivity was not extinguished by preincubation of the primary antiserum (1: 300 dilution) with pancreatic giucagon at up to 75 pg/ml antiserum for 24 h at 4°C. For localization of immunoreactive ENK. rabbit antiserum N382 raised against synthetic met”-ENK was used at a final dilution of 1: 200. Duplicate sections from each tissue block were immunostained and examined (blinded) to determine the distribution of immunoreactive ENK. As controls, nonimmune rabbit serum at a dilution of 1:200 or phosphate-buffered saline was substituted for the primary antiserum. In adsorption experiments, immunoreactivity was extinguished by preincubation of the primary antiserum N382 (1:200 dilution) with met’-ENK (500 ngiml antiserum) at 4°C for 20 h. Immunoreactivity was not extinguished by preincubation of the primary antiserum N382 (1: 200 dilution) with leucine”-ENK, dynorphin, or p-endorphin [each 500 ng/ml antiserum).

Results Neuropeptide Colon

Concentrations

in Descending

Measurement of tissue concentrations of neuropeptides demonstrated that in muscularis externa of descending colon, the mean t SEM concentration of immunoreactive VIP was significantly decreased in patients with idiopathic chronic constipation (707 i- 112 ngig wet tissue) compared with controls (1199 +- 47 rig/g)) (Figure 1). The mean concentration of immunoreactive PHM was similarly decreased in the muscularis externa in patients with idiopathic chronic constipation (543 t 58 ngig) compared with controls (815 k 45 ngig) (Figure 1). By contrast, in the mucosal-submucosal layer, the mean concentrations of VIP and PHM in colon from patients with idiopathic chronic constipation were similar to controls (Figure 1). There were no significant differences in the mean concentrations of substance P, ENK, or BLIP in colon obtained from patients with idiopathic chronic constipation compared with normal colon (Figure 2). In

\‘lP IN IDIOPATHIC

CHRONIC

CONSTIPATION

303

1600

*

I 0 0 0

0

800

\”

600

F

8 400

*

200

?? Chronic

p<.o1

Constipation

oMucosa-

Muscularis Externa

Submucasa

MucosaSubmucosa

VIP Figure

PHM

1. Concentrations of vasoactive intestinal polypeptide (VIP) and peptide histidine-methionine (PHhll in descending colon from 40 control patients and 4 patients with idiopathic chronic constipation. Horizontal bars indicate mean concentrations. with the standard error of the mean represented by the vertical bars. There were significantly decreased concentrations of VIP and PHM in the muscularis externa of colon from patients with idiopathic chronic constipation compared with control colon (unpaired Student’s t-tests, p < 0.01)

lu

z k

Mucosa -

Submucoso

Muscularis Externo

SUBSTANCE

Figure

Muscularis Ex tern0

P

Muscularis Externo

MET5ENKEPHALIN

8 0 6

Musculoris Externo

BLIP

.2. Concentrations of substance P. met”-ENK, and bombesinlike intestinal peptides (BLIP) in descending colon from 40 controls and 4 patients with idiopathic chronic constipation. Horizontal bars indicate mean concentrations, with the standard error of the mean represented by the vertical bars. There were no significant differences in the colonic concentrations of these three peptidcs between these two groups of patients.

C;AS’I‘KOI~:N’I‘I:KOl,O~Y

both groups of patients, concentrations of ENK and BLIP in the mucosal-submucosal layer were present at the lower detection limit of each radioimmunoassay, 4 and 1.5 ngig wet tissue, respectively. As shown in Figure 3, there was a trend toward increased concentrations of NPY (p = 0.07) in muscularis externa of descending colon obtained from patients with idiopathic chronic constipation corn-pared with control colon. Similar to other neuropeptides in this study, the mean concentrations of NPY in the mucosa-submucosa layer from control colon and descending colon obtained from patients with idiopathic chronic constipation were not significantly different. Among the 4 cases of idiopathic chronic constipation, case 1 was found to have the lowest concentrations of VIP (512 ngig wet tissue], ENK (27 ngig), and externa. Case 3 was BLIP (1.9 llgig) in the muscularis found to have the lowest concentration of substance P (17 rig/g wet tissue) in the muscularis externa. Case 4 was found to have the lowest concentrations of PHM (368 ngig wet tissue), but the highest concen-

Figure

100

Vol. ‘31. No. 2

r

60

*

p=.o7

El Contra/s ?? Chronic Constipation Muscularis Externo

Mucoso -

Submucoso

NPY Concentrations ot neuropeptidc Y (NPYJ in descending colon from 40 controls and 4 patients tvith idiopathic: chronic: constipation Horizontclf bclrs indicate mean concentrations. with the standard error of the mean reprewntrd by the vrrticcll burs. There was a trend t~~ward inurastrd [:onc.r,iltratiolls of NPY in the muscuI~rris cxtrrna.

4. Immunoreactive VIP in the myenteric: plexus of control descending colon visualized technique. Cytoplasmic. staining of a nerve cell body is indicated by tho Iurge ur~.o~.heud. of a VIP-containing nerve fiber. is indicated t)y the smtrll WTOLV Original magnification

by an indirect immunoperoxidasrt A linear ,wray of granulrs. indicative XWO.

Figure

,i. Immunoreactive VIP in the circular smooth muscle of control tec:hnique. Linear array of granules, indicative of VIP-containing circ:ular smooth muscle layer. Original magnification ~400.

trations of NPY (83 ngig) muscularis externa.

ImmunocptochemicaI Vasnactive Intestinal

and

ENK (65 ngig) in the

Localization Peptide

of

In the muscularis externa of control colon, immunoreactive VIP was localized within the cytoplasm of nerve cell bodies and within nerve fibers (arrays of granules] in the myenteric plexus (Figure 4). Immunoreactivc VIP-containing nerve fibers (arrays of granules) were oriented parallel to the long axis of the circular smooth muscle layer in control colon (Figure 5); immunoreactive VIP was rarely identified in the longitudinal smooth muscle layer. III descending colon obtained from patients with idiop(athic chronic constipation, immunoreactive VIP was also localized within the cytoplasm of nerve cell bodies and within nerve fibers in the myenteric plexus (Figure 6). Within the circular smooth muscle layer, rare granules of VIP but no VIP-containing nerve fibers were identified in descending colon

descending colon visualized by an indirect immunoperoxidase nerve fibers (arrow), is oriented parallel to the long axis of the

obtained stipation

from patients (Figure 7).

with

Immunocytochemicc~I Methionine5-Enkepholin

idiopathic

Localizution

chronic

con-

of

In the muscularis externa of control colon, immunoreactive ENK was rarely identified within the cytoplasm of nerve cell bodies in the myenteric plexus or within the circular smooth muscle layer. No immunoreactive ENK was present in the longitudinal smooth muscle layer. In the myenteric plexus, immunoreactive ENK was diffusely present in arrays of granules (presumably nerve fibers), as shown in Figure 8. Varicosities of immunoreactive ENK often were seen surrounding nonstaining nerve cell bodies (Figure 8). In the muscularis externa of descending colon obtained from patients with idiopathic chronic constipat/on, the distribution of immunoreactive ENK was identical to that observed in control colon. Immunoreactive ENK was identified in nerve fibers

306

Figure

KOCH ET AL.

GASTROENTEROLOGY

Vol. 94. No. 2

6. Immunoreactive VIP in the myenteric plexus of descending colon obtained from a patient with idiopathic chronic constipation visualized by an indirect immunoperoxidase technique. Cytoplasmic staining of a nerve cell body is indicated by the large arrowhead. An array of granules, indicative of a VIP-containing nerve fiber, is indicated by the smull orrow Original magnification X400.

in the myenteric plexus, ing nerve cell bodies.

often surrounding

nonstain-

Discussion Our examination of colonic neuropeptides has demonstrated decreased concentrations of VIP and PHM in the muscularis externa of descending colon obtained from 4 patients with idiopathic chronic constipation compared with concentrations of these neuropeptides in control descending colon. Immunoreactive VIP-containing nerve fibers were not identified in the circular smooth muscle layer of descending colon from 3 patients with idiopathic chronic constipation. Normally, VIP-containing nerve fibers are present in the circular smooth muscle layer of descending colon (ll), as confirmed by our immunocytochemical examination of VIP in control descending colon. Our results extend previous morphologic studies that have implicated neural abnormalities including hypoganglionosis (reduced number of ganglion cells

by histologic examination) of the myenteric plexus (27), and reduced numbers of both neurons and axons in the myenteric plexus (determined by silver staining) as possible causes of idiopathic constipation (28-30). It was uncertain from these previous studies whether morphologic changes revealed by silver staining of the myenteric plexus were primary in origin (28) or secondary, for example, to anthraquinone cathartic use (29). None of the patients with idiopathic chronic constipation in our study had used these substances. There is presently no means of directly determining whether the decreased concentrations of VIP and PHM in the muscularis externa of descending colon obtained from patients with idiopathic chronic constipation were the cause of or the result of constipation. Hypertrophy of the circular smooth muscle layer in 2 patients with idiopathic chronic constipation and elongation with dilatation of the colon in 2 patients with idiopathic chronic constipation probably resulted from chronic, partial colonic obstruction. In animal models of intestinal obstruction.

VIP IN IDIOPATHIC

Figure

7. In the circular muscle of descending colon obtained from a patient immunoperoxidase technique has been used to localize immunoreactive in the circular smooth muscle layer in patients with idiopathic chronic

morphologic studies have shown increased numbers of ganglion cells in the myenteric plexus of the intestine proximal to the obstructed segment compared with control intestine (31,323. If these results may be extrapolated to patients with idiopathic chronic constipation, one might have expected increased colonic concentrations of VIP and PHM, rather than the decreased colonic concentrations of these neuropeptides that were found. Among the gut neuropeptides, VIP has been the most extensively studied. It produces relaxation in vitro of human colonic circular muscle (33). This observation and other findings demonstrating that projections of VIP-containing intestinal nerves correspond to expected projections of enteric inhibitory nerves (34) have led to the hypothesis that VIP is a nonadrenergic, noncholinergic (NANC) inhibitory neurotransmitter (35). In agreement with this hypothesis, there is recent evidence that VIP is a mediator of descending colonic relaxation in guinea pig and rat colon (36). In aganglionic colon obtained from patients with Hirschsprung’s disease, deficiency of NANC inhibi-

CHRONIC

CONSTIPATION

SO7

with idiopathic chronic consiipation. an indirect VIP. No VIP-containing nerve fibers were identified constipation. Original magnification x-100.

tory neural input is associated with absence of colonic circular muscle relaxation (3 7). Deficiency of colonic NANC inhibitory neural input might therefore be expected to cause constipation by diminution of circular smooth muscle relaxation. As a pathophysiologic correlate, patients with idiopathic constipation in several studies have demonstrated diminished rectoanal inhibitory reflex (4,9). This result suggests that there has been alteration of the descending, inhibitory pathway. These studies permit speculation that it should be possible to identify alteration of NANC inhibitory innervation in some patients with idiopathic chronic constipation. In our study, the patient with the lowest colonic concentration of VIP (case 1) did have an in vivo physiologic abnormality as suggested by an increased colonic transit time, and an inconsistent demonstration of the rectoanal inhibitory reflex. Although the identity of the NANC inhibitory neurotransmitter in human colonic smooth muscle remains unproven, VIP remains a candidate for this function. In case 4, the possible relationship between prolonged defecation straining with elevated mean rest-

308

Figure

KOCH ET AI,.

(;ASTKO~~N’I’~~KOI,O(;Y

Vol. 94, No. z

8. Immunoreactive met”-ENK in the myenteric plexus of control deswnding colon visualized by an indirect irnmunoI)eroxidast! technique. Arrays of granules (trrrowhrods), indicative of ENK-c;ontaining nerve fibers, were diffusely present throughout the myenteric plexus, whereas nerve wll bodies containing ENK were rare. An array of ENK varic:ositit:h surrounding a nonstaining nerve cell body is shown by an c~rrorvhec~tl. Original magnific:ation ~400.

ing pressure, as determined by anorectal manometry, and alteration of neuropeptide concentrations is presently unclear. In these 4 cases of idiopathic chronic constipation, it was difficult to identify other associations between alteration of neuropeptide concentrations and abnormal in vivo rectocolonic functions. Other colonic disorders in which decreased tissue concentrations of VIP have previously been reported include Chagas’ disease (38) and Hirschsprung’s disease (16,391. In a comparison of colonic diseases in which there is abnormal motility, we found that the mean concentrations of VIP and PHM in the muscularis externa from the aganglionic: colonic segment in Hirschsprung’s disease were 414 and 281 ngig wet tissue, respectively (16). These concentrations were about 33’5, of normal concentrations of VIP and PHM; in descending colon obtained from patients with idiopathic chronic: constipation, the concentrations of VIP and PHM in our present study were about 600/o of normal concentrations. Immunohistochemical localization of VIP in Hirschsprung’s disease demonstrated an absence of VIPcontaining nerve endings and nerve cell bodies in the aganglionic segment of colon (40). I3y contrast,

our present study of the distribution of VIP in colon obtained from patients with idiopathic chronic constipation has demonstrated diminished staining of VIP-containing nerve fibers, a result that is confined to the circular smooth muscle layer. In contrast to Hirschsprung’s disease, none of the patients with idiopathic chronic constipation in our study demonstrated constriction of a segment of colon. This difference was presumably due to the presence of VIP that was not visualized by immunohistoc:hernistry in circular smooth muscle, but that was measurable by radioimmunoassay using extracts of the colonic muscularis cxterna. Electrophysiologic studies using smooth muscle from human colon have suggested that ENKs may inhibit the release of the NANC inhibitory neurotransmitter (41). The hypothesis that alteration of c:nkephalinergit: nerves might be involved in the pathophysiology of chronic constipation has been previously examined in a clinical study by Kreek and associates (42). 111 their study (42), naloxone therapy increased fecal passage in patients with idiopathic chronic: constipation. However, in our study, we found no immunohistochemical alteration of the distribution of, or the tissue conc:entration of,

Februar\

VII’ IN IDIOl’ATHl(:

lY88

met”-ENK in 3 patients with idiopathic chronic constipation. In guinea pig colon, NPY appears to produce smooth muscle relaxation by inhibition of acetylcholine rel,ease (43). Immunohistochemical studies using rat gut have shown that a proportion of NPY is present within extrinsic sympathetic nerve fibers (44). In our present study, there was no statistically significant alteration of NPY concentrations in patients with idiopathic chronic constipation. This result does not support the possibility that there has been degeneration of extrinsic nerve fibers containing the candidate inhibitory neuropeptide NPY. Norrnal concentrations of six neuropeptides were found in the mucosal-submucosal layer of descending colon obtained from patients with idiopathic chronic constipation. We did not identify a neuropeptide abnormality that could be examined in coionic mucosal biopsy specimens obtained from other patients with chronic constipation. The etiology of idiopathic chronic constipation is unlikely to be related to an alteration of enteric nerves in all patients presenting with this symptom. However, our results do suggest that an abnormality of VIP-containing nerves in colonic circular smooth muscle should be considered to be associated with the development of idiopathic chronic constipation in some patients.

References 1. Pre:
JE. Severe chronic. constipation slow transit constipation.’

of Gut

1986:27:41s8.

2. Watier A, Devroede G, Duranceau A, et al. Constipation with colonic. inertia: a manifestation of systemic disease’! Dig Dis Sci lY83;28:1025&33. 3. Shouler P, Keighley MKH. Changes in colorectal function in severe idiopathic: chronic constipation. Gastroenterology 1986;90:414-20. 4. Martelli H. Devroede G, Arhan P, Duguay C. Mechanisms of idiopathic czonstipation: outlet obstruction. Gastroenterology 1978.75:62x31. .I I

5. Preston I)hl, Lennard-Jones jE, Thomas BM. The balloon proctogram. Br J Surg 1984;71:29-32. 6. Kuijpcrs HC. Bleijenberg G. The spastic pelvic floor syndromc:: it ~.ause of constipation. Dis Colon Kectum lY85:28: 66%72. 7. Pwston IIM. L.ennard-Jones JE. Anismus in chronic constipation. Dig Dis S(,i 1985;30:413-8. 8. Kead NLZ’. Timms JM. Barfield LJ, Donnelly TC, Bannister JJ. Imp;iirmr:nt of defecation in young women with severe constipatioil. C;astrof:nterology 1986;90:53-60. Y Dur.rottca P. Denis P, Galmiche J-P, et al. Motricitrr anorectalc dans I11 c.onstipation idiopathique. Etude 111: 200 patients c.onscc.utifs. Gastroenterol Clin Biol 1985:9:10-5. 10. Polak Jhl. Bloom SK. Sullivan SN, Facer P. Pearse AGE. linkt:pt’~llirl-likt: immunoreactivity in the human gastrointestinal trCxt. Lancrt 1977:i:Y72-4. 11. Frsrri (i-1,. Adrian TE, Ghatei Ml\, et al. Tissue localization im~l rclativc distribution of regulatory peptides in separated

CHKONIC: (:ONSTIP.~TION

309

layers from the human bowel. (;astr[)enterolog~ tY83;84:77786. 12 Bishop AE. Polak JM. Yiangou Y. Christofides h’l), Bloom SK. The distributions of PHI and VIP in porcine gut and their c.o-localisation to d proportion of intrinsic ganglion wlls. Pcptides lYN4;5:255SY. JM, Tcrenius L. Hokfelt T. (ioldstcin M. High levels 13 Lundberg of neuropeptide Y in peripheral Iloraclrcllergic. neurons in various mammals includin:: man. Ncurosc.i Lett 1983:42:167F 72. L, DeMey J, Polak JM. Ultrastruc:tural localization ot 14 Probert four different neuropeptidcs Lvithin scpardte populations of ptype nerves in the guinea pig ~01011. C;astroent~!rolog), 1983: 85:1094-104. 15 Koch ‘IX. (Barney IA. Go VI,%’ Distribution and quantitation of gut neuropeptides in normal intrastine and thtr intlammatory bowel diseasrts. Dig Dis Sci 1987::12:‘36Y-76. 16 Kubota M. Koch TK, Telander KL. Kaufman B. Go \‘LiY, Szurszewski JH. Inhibitor:,~ innervation of thr: aganglionic scg~lent in Hirschsprung‘s discase (abstr). Gastroenterolog~ 1985:88:1458. 17 Yak& ‘I‘L. hbay EO II. Go VLM’. Stutlies on thr location and release of cholecystokinin and vasoactivr: intestinal peptide in rat and cat spinal cord. Brain Kcs lY82:242:279-90. 18 Kaufman HH, Koch TK, Sxurszewski JH, (:o VLW. Distributlon and quantitation of iinmul~orr?;ic:ti~~~~ gut Ileuropeptides in pi&Id mice and their normal litt~~rmatw. J Surg Kes 1985: 38:47%83 19 Miwvych PE. Yaksh TL, Go \‘I,W. Opi~ttcl-mecliated inhibition of the release of choler ystokinin an11 sl~bstance P. but not neurotensin from cat hyywthalalni[: slirzs. Brain Kes lY82; 250:283-Y. 20 Jhamandas K, Yaksh ‘TL. Go VI.Lf’. Ac:utc: and chronic morphine modifies the in viva rclewse of methioninr: cnkephaliillike inimunoreacti\,ity from the: c.at spinal cord and brain Brain Kes 1984;297:91-103. 21 r\ngeI F, (;o \‘LM’, Szurw.:wski 111 lilnc~rvation of the musc:ularis mucosae of r.aninc’ proximal cn111rl. J Phpsiol (Land) 1984:2Y7:Yl-103. 22 Koddy DK. Koch TK. Keilly b\‘hl, Gar~~(:y )A. Go VLW. Distribution and quantitation of neuropeptidr Y in the human and c:anine (;I tracts (abstr). C:~lstro~!nterolo:;! 1987:Y2:1598. 23 Stefanini M, DeMartino C. Zamboni I,. l’ixatioll at ejaculated spermatozoa for electron microscopy. Nature 1967:226:17:34. 24 Nakanc: PK. Pierc.e GM Jr. Enzyme-l~bellrd antibodies: prep1 aration and application lor the loc.alrzation ot antigens. Histochem Cytochem 1966:14:!92~1- :11. 25 Nakant: PK, Pierw GM Jr. Enz?lne-l;~belletl antibodies for the light and electron microscopic localization of tissue antigens. 1Cell Biol 1967:33:307-18. Itnh N. Obata K-i, Yanaihtra N, Okalnoto H. Human prepro26 vasoac.tive intestinal polvpeptitie contailis a novel PHl-27like peptide. PHM-27. Nature 1983:304:i47-Y. 27 hriel 1. Hcrshlag A, LernI~lxus. Gastroenteroogy lY85:88:2&34.

310

KOCH ET AL.

31. Okada A, Okamoto E. Myenteric plexus in hypertrophied intestine. J Neuro-Vise Relat 1971;32:75-89. 32. Pickard LR, Santoro S, Wyllie KG, Hailer JA Jr. Histochemical studies of experimental fetal intestinal obstruction. J Pediatr Surg 1981;16:256-60. 33. Couture R, Mizrahi J, Regoli D. Devroede G. Peptides and the human colon: an in vitro pharmacological study. Can J Physiol Pharmacol 1981;59:957-64. 34. Furness JB, Costa M. Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons. Neurosci Lett 1979;15:199-204. 35. Fahrenkrug J. Emson PC. Vasoactive intestinal polypeptide: functional aspects. Br Med Bull 1982;38:265-70. 36. Grider JR, Makhlouf GM. Colonic peristaltic reflex: identification of vasoactive intestinal peptide as mediator of df:scending relaxation. Am J Physiol 1986:251:G40-5. 37. Kubota M. Ito Y, Ikeda K. Membrane properties and innervation of smooth muscle cells in Hirschsprung’s disease. Am J Physiol 1983;244:G406-15. 38. Long RG, Barnes AJ. O’Shaughnessy DJ, et al. Neural and hormonal peptides in rectal biopsy specimens from patients with Chagas’ disease and chronic autonomic failure. Lancet 198O;i:559-62. 39. DuPont C. Navarro J, Chenut B, Rosselin G Modifications of VIP intestinal content associated with abnormal nervous myenteric plexus: a biological feature of chronic intestinal obstruction. J Pediatr 1980;96:1037-9. 40. Tsuto T, Okamura H, Fukui K, et al. An immunohistochemical investigation of vasoactive intestinal pnlypeptide in the

(;ASTROE:N’~EKOI.O(;Y

Vol. 94, No. 2

colon of patients with Hirschsprung’s disease. Neurosci Lett 1982;34:57-62. 41. Hoyle CHV, Burnstock G, Jass J. Lennard-Jones JE. Enkephalins inhibit non-adrenergic, non-cholinergic neuromuscular transmission in the human colon. Eur J Pharmacol 1986;131: 159-60. 42. Kreek M-J, Hahn EF. Schaefer RA, Fishman J. Naloxone, a specific opioid antagonist. reverses chronic idiopathic constipation. Lancet 1983;i:261-2. 43 Wiley J, Owyang C. Neuropeptide Y inhibits cholinergic transmission in the isolated guinea pig colon: mediation through p-adrenergic receptors. Proc Nat1 Acad Sci IJSA 1987;84:2047-51. 44. Sundler F, Moghimzadeh E. Hakanson R, Ekelund M. Emson P. Nerve fibers in the gut and pancreas of the rat displaying neuropeptide-Y immunoreactivity. Cell Tissue Res 1983:230: 487-93.

Received March 9, 1987. Accepted September 7, 1987. Address requests for reprints to: Dr. Timothy Koch, Gastrointestinal Section, III-C, Zablocki Veterans Administration Medical Center. 5000 West National Avenue, Milwaukee, Wisconsin 53295. Dr. Koch is a recipient of a Career Development Award from the National Foundation for Ileitis and Colitis. The authors thank J. Bailey. Il. Lucas, S. Michener, and D. Roddy for performing radioimmunoassays and C Stanislav for typing the manuscript