GASTROENTEROLOGY

1992;103:496-505

Mucin and Protein Release in the Rabbit Jejunum: Effects of Bethanechol and Vagal Nerve Stimulation BEVERLY GREENWOOD and MICHfiLE MANTLE Department of Pharmacology and Toxicology, Gastrointestinal Research Grouo, Departments Calgary, Calgary, Alberta, Canada _

Medical College of Wisconsin, Milwaukee, Wisconsin; and of Medical Biochemistry and Pediatrics, University of

The role of the vagus nerve and cholinergic mechanisms in the control of rabbit jejunal mucin and protein release was investigated in vivo. In anesthetized animals, a lo-cm segment of the jejunum was cannulated and perfused with saline. Perfusate was collected and analyzed for mucin (by immunoassay) and protein. Bilateral cervical vagotomy had no effect on basal mucin or protein output, suggesting that the vagus nerve does not exert a tonic control on jejunal macromolecule secretion. Electrical stimulation of the vagi did not alter mucin release, even in the presence of muscarinic cholinergic (scopolamine) or adrenergic (propranolol and phentolamine) blockade. In contrast, protein output increased significantly after vagal stimulation, an effect inhibited by scopolamine. In both vagotomized and vagally intact rabbits, the cholinergic agonist bethanechol (ZOOpg/kg intraperitoneally) induced a scopolamine-sensitive increase in both mucin and protein output. Predominantly serum proteins were released into intestinal perfusates after vagal or cholinergic stimulation. It is concluded that the extrinsic vagus nerve does not regulate rabbit jejunal mucin secretion in vivo and that cholinergic control of intestinal goblet cells is implemented entirely by the intrinsic enteric nervous system. In addition, cholinergic or vagal stimulation increases intestinal vascular and epithelial permeability, resulting in the passage of serum proteins into the lumen, possibly by opening tight junctions and paracellular pathways.

G

oblet cell mucin protects and lubricates the intestinal tract by forming a viscoelastic gel over the epithelium that physically separates the underlying tissue from potentially harmful luminal contents (such as digestive enzymes, bacteria, and fecal material).l Despite its importance in host defense, regulation of intestinal mucin production by extrinsic and intrinsic enteric nerves is still incompletely understood.

Cholinergic agents are known to increase secretion from intestinal and colonic goblet cells. Ultrastructural analyses have indicated that exocytosis of mucin after cholinergic stimulation is restricted to crypt goblet cells in the small intestine and colon, both in vivo and in organ culture.“3 In contrast, villus and surface goblet cells only secrete in the presence of luminal irritants.3*4 Other potential neurotransmitters (such as adrenergic agonists, vasoactive intestinal peptide, substance P, and serotonin) have no effect on either crypt or villus goblet cells.’ Studies on isolated intestinal epithelia have suggested that cholinomimetics induce mucin release by acting directly on crypt goblet cells,4 a finding supported by experiments on mucus-producing colonic cell lines.5s” It has therefore been proposed that systemic control of intestinal mucin secretion is exercised through cholinergic stimulation of crypt goblet cells whereas local, immediate control occurs by luminal stimuli acting on villus goblet cells.24 Because pharmacological doses of cholinomimetits were used in all of the above experiments, the actual role of acetylcholine in the regulation of intestinal mucin secretion in vivo can only be inferred. However, the hypothesis is supported by ultrastructural studies on rat intestinal and colonic tissue subjected to electrical field stimulation in vitro.’ This work showed that cholinergic (in the ileum and colon) and noncholinergic (in the colon) intrinsic nerves may modulate mucin secretion from crypt (but not villus or surface) goblet cells.7 Although qualitative ultrastructural studies clearly show the location of goblet cells responding to cholinergic stimulation, the actual magnitude of that secretory response has not been established. In addition, it is not known whether cholinergic regulation of mucin release involves extrinsic (as well as intrinsic) enteric nerves. Early observations by Wright et al. suggested that stimulation of the vagus nerve in0 1992 by

the American Gastroenterological 0016-5065/92/$3.00

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August 1992

RABBIT JEJUNAL MLJCIN AND PROTEIN RELEASE 497

duced “sticky” or mucoid juice from the stomach and duodenum whereas stimulation of the pelvic nerve caused mucoid secretion from the co10n.8*gIn the small intestine, vagal stimulation (particularly after sympathetic denervation) resulted in the discharge of viscous fluid.’ These experiments implied that extrinsic nerves play a role in the regulation of mucus production in the gastrointestinal tract. Thus, the present study aimed to examine whether the vagus nerve is capable of affecting mutin output in the rabbit jejunum in vivo. In addition, we quantitated the increase in mucin secretion after administration of a low dose of the cholinergic agonist bethanechol. Because previous studies have reported that the permeability of the small intestine to macromolecules is increased during accelerated mutin secretion induced by cholinomimetics,” we also measured protein output after either cholinergic or vagal nerve stimulation. Our results show (a) that the vagus nerve does not regulate jejunal goblet cell activity, thereby suggesting that control of mucin secretion (by cholinergic mechanisms) operates entirely within the enteric nervous system in vivo, and (b) that both cholinergic or vagal nerve stimulation induce release of serum proteins into the intestinal lumen, possibly by opening paracellular secretory pathways. Materials and Methods Preparation

of Animals

After an overnight fast, male New Zealand White rabbits (n = 61) weighing 985 + 26 g (mean f SEM) were anesthetized with a single intraperitoneal (IP) injection of urethane (0.2 mL/lOO g body wt of a 50% solution). A tracheal catheter was inserted to provide a clear airway, and the left jugular vein was cannulated for the subsequent administration of drugs. Throughout the experiment, blood pressure and heart rate were monitored via a catheter inserted into the left carotid artery and body temperature was maintained at 37°C via a rectal probe connected to a homeothermic blanket (Harvard Instruments, Ealing, England). After a midline incision, a IO-cm segment of the jejunum was isolated from the remainder of the gut with its blood supply intact. The segment was cannulated at both ends with polyethylene tubing (ID, 2 mm; OD, 4 mm). The laparotomy was then sutured closed to prevent desiccation and heat loss. For the duration of the experiment, the intestinal segment was perfused with prewarmed (3Y’C), isotonic saline at a rate of 0.7 mL/min using a Harvard syringe pump (Harvard Instruments). This perfusion rate was selected because preliminary experiments indicated that it did not cause distension of the jejunum or tissue damage.

Experimental

Procedures

Basal controls. After a 30-60-minute equilibration period after surgery, jejunal perfusate from the distal can-

nula was collected on ice at lo-minute intervals for a total of 160 minutes. Proteolytic inhibitors (1.5 mmol/L phenylmethylsulfonyl fluoride, 1.5 mmol/L N-ethylmaleimide, and 0.1 mmol/L Na, ethylene diaminetetraacetic acid (EDTA), final concentrations) were added to all samples, which were then lyophilized and stored at -8O’C until analyzed. In some cases, tissue from the middle of the perfused intestinal segment was taken at the end of the experiment for morphological examination. Sections of Carnoy’s-fixed tissue (5 pm) were stained with periodic acid-Schiff and hematoxylin to visualize goblet cells and nuclei, respectively. Vagotomy and vagal nerve stimulation. After initial equilibration, two lo-minute basal control samples were collected. Bilateral cervical vagotomy was then performed (as described previously,“.‘* and the system was allowed to re-equilibrate for a further 30 minutes. Subsequently, perfusate was collected and treated as described above. In one series of experiments, the peripheral cut ends of both vagi were placed over platinum or tungsten carbide bipolar electrodes and were electrically stimulated (square wave pulses with a duration of 0.5 milliseconds, 10 Hz, 5-mA constant current, and 30 seconds on/30 seconds off for 30 minutes or continuously for 30 minutes) starting immediately after collection of the third postvagotomy sample. Because it has been reported that periodic and continuous electrical stimulation of nerves may have different effects on secreting target tissues,13 the vagi were subjected to both forms of stimulation. Tissue was again taken from some animals 10 minutes after the start of nerve stimulation for morphological analysis as described above. In another series of experiments, either scopolamine (bolus dose of 5-10 mg/kg body wt in sterile saline delivered intravenously (IV) followed by 100 pg. kg-.’ min-’ continuous IV infusion; Sigma Chemical Co., St. Louis, MO) or phentolamine and propranolol (bolus of 1 mg/kg each intra-arterially; Sigma) were administered to vagotomized animals, and, after 20-30 minutes, the cervical vagi were again stimulated for 30 minutes. Intestinal perfusate was collected throughout the above procedures as described for basal controls. Bethanechol stimulation. Bethanechol (200 pg/kg; Sigma) was administered IP by slow (2-minute) infusion to either vagally intact or vagotomized animals (30 minutes after sectioning the vagi), and jejunal perfusate was collected for 40-50 minutes. In some cases, the experiment was terminated 10 minutes after bethanechol administration, and tissue from the perfused intestinal segment was taken for morphological analysis. In a final series of experiments, animals (vagally intact and vagotomized) received scopolamine (5-10 mg/kg bolus followed by a continuous IV infusion of 100 )*g - kg-’ - min-‘) 30 minutes before administration of bethanechol. Again, perfusate was collected for 40-50 minutes and treated as described for basal controls. The above experimental procedures have been used extensively in neurophysiological investigations on vagal and cholinergic regulation of intestinal motility and fluid and electrolyte transport.“*‘2~14 Approval to perform these studies was granted by the animal welfare committees of

498

Table

GASTROENTEROLOGY

GREENWOOD AND MANTLE

1. Basal (Unstimulated)

Release of Intestinal

Mucin

and Protein Mean + SEM @g/l0 min) Mucin output

27.7 f 2.7 349.5 f 30.6 7.9 + 0.1

Protein output Volume

NOTE. Rabbits were anesthetized with urethane, and a IO-cm segment of the jejunum was perfused (0.7 mL/min) with isotonic saline at 37°C. After a 30-minute equilibration period, perfusate was collected for two lo-minute intervals and analyzed for mucin and protein. Basal output for each animal was calculated by averaging the results from the two collections. Data shown represent the mean k SEM for a total of 42 rabbits.

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160 minutes (Figure 1A). Similarly, total protein output showed interanimal variability, but each individual rabbit had constant levels of release during prolonged perfusion of the jejunum (Table 1;Figure 1B). The volume of perfusate collected showed little variability among rabbits and did not change over time (Table 1; Figure 1C). The DNA content of perfusate samples from all rabbits was well below the minimum detectable by the fluorimetric assay (

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Vol. 103, No. 2

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Figure 1. Mucin and protein release in the rabbit jejunum in vivo. Rabbits were prepared as described in Table 1. After a so-minute equilibration, intestinal perfusate was collected at lo-minute intervals and analyzed for (A) mucin and (B) protein. The volume of perfusate collected at each time interval is shown in (C). Data indicate the values for an individual, representative animal.

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Figure 2. Effects of vagotomy and vagal stimulation on rabbit jejunal mucin secretion in vivo. (A) Mucin secretion after bilateral cervical vagotomy (closed arrow). (B) Mucin secretion after vagotomy (closed arrow) and electrical stimulation of the vagi (open arrow) either periodically (10 Hz, 5 mA, 30 seconds on/30 seconds off for 30 min; 0) or continuously (10 Hz, 5 mA, for 30 minutes; 0). (C) Mucin secretion after vagotomy (closed arrow), administration of scopolamine (3-10 mg/kg body wt IV, dotted arrow), and vagal stimulation (open arrow). (D) Mucin secretion after vagotomy (closed arrow), administration of propranolol and phentolamine (1 mg/kg body wt each intra-arterially, doffed arrow), and vagal stimulation (open arrow). Data shown represent the mean rt SEM for six rabbits in each experimental group.

riodic electrical stimulation of the vagi had no effect on mucin output (Figure 2C and D). The total protein content of the perfusate was also unaffected after bilateral cervical vagotomy, but periodic or continuous stimulation of the vagi caused a statistically significant increase in output during the first 10 minutes of stimulation (Figure 3A). Treatment with scopolamine (but not propranolol and phentolamine) prevented the increase in protein release induced by vagal stimulation (Figure 3B and C). Throughout no significant changes were these experiments, noted in the volume of perfusate collected at each lo-minute interval, and the DNA content of perfusate samples remained at basal (prevagotomy) levels. To confirm that vagal nerve stimulation had not caused mucin secretion or epithelial breakdown, tissue was examined by light microscopy. Crypt and villus goblet cells were clearly visualized (darkly stained) by periodic acid-Schiff reagent and did not appear depleted of mucin (Figure 4). There was no sign of trapped mucin in the crypts. These observations support the concept that mucin secretion had not occurred after nerve stimulation. Furthermore, the epithelium was not apparently damaged. Thus,

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both DNA and light microscopic analyses provided no evidence of tissue degradation, suggesting that the increase in protein release after vagal stimulation was not caused by nonspecific breakdown of the epithelial barrier. In all the above experiments, the appropriate cardiovascular responses to vagal stimulation were observed.“~‘2 Resting blood pressure and heart rate were 178/56 t 12/10 (mean -t SEM) mm Hg and 293 -t 30 beats/min, respectively (average of basal and postvagotomy periods for 20 animals); these values decreased to 50/20 * 10/6 mm Hg and 159 + 22 beats/min (n = 14) during electrical stimulation of the sectioned nerves. Treatment with scopolamine abolished the cardiovascular effects induced by stimulation of the vagi. After administration of phentolamine and propranolol to vagotomized animals (n = 6) blood pressure and heart rate decreased to 70,’ 50 * 11/7 mm Hg and 230 + 20 beats/min, respectively, and subsequent nerve stimulation caused a further decrease in both parameters (blood pressure, 50/30 + 10/5 mm Hg; heart rate, 123 + 10 beats/ min; n = 6). In view of these cardiovascular responses and the observed changes in protein release into the intestine during the above experiments, it seems unlikely that inadequate neurotransmission

30 Time

60 (min)

Figure 3. Effects of vagotomy and vagal stimulation on rabbit jejunal protein release in vivo. (A) Protein release after bilateral cervical vagotomy (closed arrow) and electrical stimulation of the vagi (open arrow; 10 Hz, 5 mA, 30 seconds on/30 seconds off for 30 min). (B) Protein release after vagotomy (closed arrow), administration of scopolamine (5-10 mg/kg body wt IV, dotted arrow), and vagal stimulation (open arrow). (C) Protein release after vagotomy (closed arrow), administration of propranolol and phentolamine (1 mg/kg body wt each intra-arterially, dotted arrow), and vagal stimulation (open arrow). Data shown represent the mean + SEM for six rabbits in each experimental group. *P < 0.05,

500 GREENWOOD

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caused by postsurgical damage to the vagi was responsible for the lack of change in mucin secretion following electrical nerve stimulation. Bethanechol

Stimulation

To ensure that increased mucin secretion could occur in our animals in response to an appropriate stimulus, we assessed the effects of the cholinergic muscarinic agonist bethanechol on jejunal mutin release. Because cholinergic agonists are known

Figure 4. Rabbit jejunal tissue after vagal nerve stimulation. After bilateral cervical vagotomy, jejunal tissue was removed for histology before and after electrical stimulation of the vagi for 10 minutes. Sections (5 pm) were stained with periodic acidSchiff and hematoxylin. (A and C) Villus and crypt regions, respectively, from tissue before nerve stimulation. (B and D) Villus and crypt regions, respectively, after vagal nerve stimulation. Black arrows indicate villus goblet cells; white arrows show crypt goblet cells. Goblet cells remain clearly visible and do not appear depleted after nerve stimulation (bar = 100 pm).

GASTROENTEROLOGY

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to increase intestinal and colonic goblet cell secretion,z-6 we chose to administer only one dose of the cholinomimetic IP for these experiments. The dose selected (200 pg/kg) was significantly lower than that used in previous studies on mucin secretior? but was similar to that used in physiological experiments on intestinal fluid and electrolyte transport and motility.1**‘4 Bethanechol was administered IP to avoid changes in cardiovascular function (as occurs after systemic administration) and, hence, alterations in intestinal blood flow that may indirectly affect mucin secretion and protein release. Finally, bethanechol was chosen as the cholinergic agonist because (unlike carbachol) it acts directly on postganglionic (end-organ) muscarinic receptors and does not have neural effects. In vagally intact rabbits, bethanechol caused a rapid and significant (-J-fold) increase in mucin re20 minlease that was sustained for approximately utes after administration of the drug (Figure 5A). The total protein content of the perfusate also increased after bethanechol stimulation (Figure 5B). DNA output remained below minimum detectable limits. The volume of perfusate collected for the lo-minute interval immediately following bethanechol treatment increased slightly (from 7.8 * 0.2 to 9.2 * 0.7 mL), but the change was not statistically significant. The bethanechol-induced increase in mucin, protein, and fluid output was completely inhibited by pretreatment with scopolamine (Figure 5C). Vagotomized rabbits showed similar responses to bethanechol stimulation (Figure 6A), which were also inhibited by scopolamine (Figure 6B). Morphological assessment of tissue from the perfused intestinal segment after bethanechol treatment showed no evidence of damage to the epithelium or mucosa and no trapping of mucin in the crypts. Crypt goblet cells in bethanechol-stimulated tissue stained very poorly (pale pink) with periodic acid-Schiff reagent and could barely be detected, suggesting depletion of mucin. Although villus goblet cells remained heavily stained (dark pink) after bethanechol, some cells did appear to be extruding mucin from their apical surface, suggesting that they may have been stimulated to secrete by the cholinomimetic. Therefore, the perfusate mucin detected in these experiments was probably derived from crypt goblet cells, but some may have come from villus goblet cells. Because tissue appeared undamaged by light microscopy and because the DNA content of the perfusate remained extremely low, we conclude that mucin release was the result of true secretion from goblet cells and that protein release was not caused by epithelial breakdown. Bethanechol administered via the IP route caused a slight but nonsignificant decrease in heart rate and

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Time (min) Figure 5. Effects of betbanecbol on rabbit jejunal mucin and protein release in vivo. (A) Mucin and(B) protein release into the perfusate after administration of bethanechol (ZOO&kg IP, open arrow). (C) Mucin (0) and protein (H) release after administration of scopolamine (5-10 mg/kg body wt IV, doffed arrow) and bethanechol(200 pg/kg IP, open arrow). Data shown represent the mean + SEM for four rabbits in each experimental group. *P < 0.05

JEJLJNAL MUCIN

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501

release in the upper small intestine, we established an in vivo jejunal perfusion system in the anesthetized rabbit. Under nonstimulated baseline conditions, mucin and protein output occurred at a continuous rate (27.7 + 2.7 and 349.5 + 30.6 pg/lO min, respectively) that remained stable for over 2 hours. The vagus nerve does not seem to exert a tonic (ongoing) control on basal macromolecule secretion in the jejunum, because bilateral cervical vagotomy had no effect on either mucin or protein release. Although electrical stimulation of both cervical vagi (under conditions previously shown to affect intestinal motility and fluid and electrolyte secretion”~‘2~14)did not alter mucin output or cause goblet cell depletion, it did cause a significant increase in protein release during the first 10 minutes of stimulation. Because serum proteins were detected in the perfusate in the absence of morphological damage to the epithelium, it appears that vagal stimulation induced a transient increase in epithelial permeability, thereby allowing these macromolecules to enter the jejunal lumen. These findings agree with previous reports indicating that transport of serum protein across the intestinal epithelium may be affected by neural mechanisms.” Because the cervical vagus nerve may contain sympathetic (as well as parasympathetic) fibers, the lack of effect of nerve excitation on mucin secretion may

blood pressure in both normal and vagotomized rabbits. However, profuse salivation and lacrimation were observed. Gel Electrophoresis

of Perfusates

To determine the nature of the proteins present in perfusate samples, aliquots were subjected to SDS-PAGE. In basal and postvagotomy samples, the most prominent protein band detected on stained gels had a molecular weight of -66 kilodaltons, equivalent to that of serum albumin (Figure 7). After vagal stimulation, the staining intensity of the 66-kilodalton band increased, and a variety of other proteins became apparent on the gels. The same changes (although even more marked) were observed in perfusate samples collected after bethanechol treatment (Figure 7). To determine whether the perfusate proteins were derived from serum, Western blots were probed with antibodies against either rabbit whole serum or rabbit albumin (Figure 7). These experiments confirmed that the 66-kilodalton band present in all of the perfusates was albumin and that vagal or cholinergic stimulation resulted in the passage of serum proteins into the jejunal lumen. Discussion

To investigate the role of the vagus nerve in modulating goblet cell mucin secretion and protein

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Figure 6. Effects of bethanechol on jejunal mucin secretion in vagotomized rabbits in vivo. Rabbits were prepared as described in Table 1, and bilateral cervical vagotomy was then performed (closed arrow). (A) Mucin release after administration of bethanechol(200 pg/kg IP, open arrow).(B) Mucin release after administration of scopolamine (5-10 mg/kg body wt IV, dotted arrow) and bethanechol(200 pg/kg IP, open arrow). Data shown represent the mean f SEM for six rabbits in each experimental group. *P i 0.05.

GASTROENTEROLOGY Vol. 103, No. 2

502 GREENWOOD AND MANTLE

ABC

DE

FG

HI

J

K

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Figure 7. SDS-PAGE and Western blots of rabbit jejunal perfusates. Jejunal perfusates were subjected to SDS-PAGE under reducing conditions using 7.5% separating gels with 5.7% stacking gels. Gels were either stained with Coomassie blue or transferred onto nitrocellulose sheets that were then probed with goat anti-rabbit albumin or goat anti-rabbit whole serum antibodies followed by horseradish peroxidase-conjugated rabbit anti-goat IgG and peroxidase substrate. (A-G)Coomassie bluestained gels: (A)basal perfusate, (B) perfusate collected after vagotomy, (C) perfusate collected after vagal stimulation, (D) basal perfusate, (E)perfusate collected after bethanechol treatment, (F) rabbit albumin (40 pg), and (G) rabbit serum (5 pL). (H-K) Western blots probed with anti-rabbit albumin antibodies: (H) basal perfusate, (I) perfusate after vagal stimulation, (I) rabbit albumin, and (K) rabbit serum. (L-O) Western blots probed with anti-rabbit whole serum antibodies: (L) basal perfusate, (M) perfusate collected after bethanechol treatment, (N) rabbit albumin, and (0) rabbit serum.

have been caused by the activation of both stimulatory and inhibitory pathways. Vagotomized animals were therefore treated with either parasympathetic (scopolamine) or sympathetic (phentolamine and propranolol) antagonists before nerve stimulation. Neither parasympathetic nor sympathetic blockers had any effect on basal mucin output, indicating the absence of either cholinergic or adrenergic tonic control. Furthermore, vagal stimulation in the presence of either type of antagonist did not affect mucin secretion. These findings differ from those of Florey et al.* who reported an increase in intestinal mucin secretion after sympathetic denervation and vagal stimulation, implying that goblet cells were subject to sympathetic inhibitory input and were capable of responding to the vagus nerve. However, the conclusions from these early experiments were based on changes in the volume and visual consistency of intestinal secretions. Because vagal stimulation increases intestinal fluid and electrolyte secretion”~‘2 and protein release (the current study), it is likely that early data reflected fluid and protein (rather

than mucin) secretion. From our results, we concluded that mucin release from rabbit jejunal goblet cells is not under tonic cholinergic or adrenergic control and is not influenced by the vagus nerve. Thus, the mechanisms that regulate intestinal mucin secretion in vivo are not the same as those that modulate fluid/electrolyte transport and motility. In addition, control of mucin release in the small intestine differs substantially from that in the respiratory tract where secretion increases significantly after electrical stimulation of the vagus nerve using the same conditions as those applied in the present studies.” Experiments with parasympathetic and sympathetic antagonists also provided some insight into the mechanisms mediating protein secretion in the rabbit jejunum. The increase in protein output after vagal stimulation seems to involve cholinergic nerves, because it was inhibited by parasympathetic (but not sympathetic) blockers. The effects of vagal stimulation probably result from activation of efferent fibers; however, the possible contribution from antidromic firing of afferent fibers cannot yet be eliminated. In the trachea of several rodent species, vagal stimulation induces vasodilation, a rapid (within 1-2 minutes) increase in vascular permeability and extravasation of plasma protein into the tissue (neurogenie inflammation). This response has been attributed to antidromic firing of substance P-containing afferent C fibers.21-24 Large molecules (66 and 156 kilodaltons) have also been shown to pass into the airway lumen through transient openings between epithelial cells that promptly close after transudation.24-27 An atropine-sensitive cholinergic component has been implicated in control of epithelial permeability.25~27 A similar mechanism may occur in the intestine. Further studies on chronic capsaicintreated rabbits are required to investigate the contribution of afferent sensory fibers to the present experimental results. In contrast to our findings, earlier studies on guinea pigs reported that protein extravasation into intestinal tissue does not occur in response to vagal nerve stimulation.21~22 This may be caused by differences between species in their sensitivity to stimuli causing intestinal protein extravasation23*24 or, alternatively, differences in experimental design, because the conditions of nerve stimulation used in the present studies were not the same as those used earlier and we examined intestinal perfusates not tissues.“*” It is possible that proteins extrasavated in the guinea pig intestine were not seen in the tissue because they were rapidly cleared into the gut lumen. Because our experiments provided no evidence of vagal, cholinergic, or adrenergic tonic control of basal protein release, it is not yet clear why serum pro-

August 1992

teins (particularly albumin) were present in basal perfusates. Nonetheless, serum proteins have been detected in the lumen of the normal, nonstimulated intestine previously.28 In preliminary studies on rats and ferrets using the same protocol as described herein for rabbits, we also found significant amounts of protein in intestinal perfusates collected under basal conditions (133 + 26 and 206 + 20 l&l0 min, respectively). Again, most of the protein appeared to be serum albumin (Greenwood and Mantle, unpublished observations). Whether these proteins passed through transient leaks in the epithelium or were transported by other mechanisms remains to be elucidated. Of additional interest, our early experiments on ferrets also suggested that protein release into the intestinal lumen increased after vagal nerve stimulation, thus supporting our observations on the rabbit. Cholinergic agents have been reported to increase intestinal and colonic mucin secretion from crypt (but not villus and surface) goblet cells.2-4 Large doses of carbachol induce mucin release in the rat ileum and colon by direct stimulation of muscarinic receptors on goblet cells, whereas in the duodenum and jejunum, the response involves both a direct effect on goblet cells and an indirect neural (ganglionic) component because both muscarinic and nicotinic receptor antagonists are required to inhibit mucin secretion.24 In the present studies, a low dose of bethanechol(200 pg/kg) caused a significant (-4 fold) increase in mucin output in both vagally intact or vagotomized rabbits. Scopolamine completely inhibited the bethanechol response, thus supporting the hypothesis that direct muscarinic receptor occupation on goblet cells plays a role in regulating jejunal mucin secretion in vivo. Light microscopic examination of tissue after bethanechol treatment suggested that the secreted mucin came from crypt goblet cells, the reported site of action of cholinomimetics,2-4 but some villus goblet cells also appeared to be secreting. These observations are in agreement with recent studies by Kemper and Specian2’ showing that villus goblet cells can respond to cholinomimetics. Because stimulation of the vagus nerve is not sufficient to affect jejunal mucin output, it seems likely that cholinergic control of goblet cells is exercised entirely within the intrinsic enteric nervous system. Local stimulation of this system by chemical or mechanical means and the subsequent release of acetylcholine is possibly the major regulatory pathway for controlling jejunal mucin secretion. From studies on isolated intestinal epithelia4 and on mucus-secreting cell lines,5s6 it appears that acetylcholine then acts directly on goblet cells. In the rat ileum, carbachol has previously been shown to induce leakage of circulating horseradish

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peroxidase across the crypt (but not the villus) epithelium and into the crypt lumen.*’ The enzyme seemed to take a paracellular pathway to reach the lumen, passing through occluding junctions between crypt epithelial cells. In agreement with those studies, we also noted that bethanechol increased serum protein release into the jejunum. Although not investigated, we assume that bethanechol opened occluding junctions between crypt cells.‘o Because the effect was inhibited by scopolamine, it appears that direct stimulation of muscarinic receptors on the epithelium can cause an increase in paracellular permeability. This concept is supported by experiments showing the presence of muscarinic receptors on intestinal and colonic epithelial cells.30r31 Neurotransmittors and inflammatory mediators have been observed to increase epithelial permeability to large macromolecules in a number of tissues, 10,21-27*32,33 suggesting that this may be a common epithelial response to stimulation and may possibly be linked to host mucosal defense. As yet, control of epithelial permeability is poorly understood, but physiological signals (activation of nutrient transporters) and the cytoskeleton are thought to be important in regulating tight junctions.34*35 However, it does not seem that increased permeability and exocytotic secretion are coincident in the intestine, because (a) carbachol-induced mucin secretion was not always accompanied by increased epithelial permeabilitylO and (b) our studies show that enhanced intestinal permeability occurred after vagal nerve stimulation without changing mucin secretion, Because blood pressure and heart rate were significantly affected during some of our experimental procedures, vascular changes may have influenced our results. However, it seems unlikely that serum protein release resulted purely from an increase in hydrostatic pressure in the capillaries and interstitium, because both vagal nerve stimulation and bethanechol treatment would be expected to induce vasodilation and, hence, a decrease in pressure. Thus, at least part of the protein response in the intestine must involve a direct action on microvascular and epithelial permeability. In addition, because mucin secretion only increased in response to bethanechol (but not to nerve stimulation), it seems that changes in blood flow per se were not able to affect goblet cell activity. In summary, therefore, the current study indicates that mucin and protein release in the anesthetized rabbit jejunum under basal conditions is not subject to tonic control either by the vagus nerve or by cholinergic or adrenergic mechanisms. Thus, if macromolecule secretion in the jejunum is under any tonic neural control in this model, it must be mediated by as yet unidentified noncholinergic, nonadrenergic

504 GREENWOOD AND MANTLE

neurotransmitters. In addition, the vagus nerve does not regulate rabbit jejunal goblet cell activity, even under stimulated conditions. This contrasts with the known influence of this nerve on other aspects of gut function, including motility, fluid and electrolyte transport,“*‘2~14 and protein output (current study). Because the vagus nerve mediates central nervous system modulation of gastrointestinal function,36s37 including mucus production in the stomach,gv38 its inability to affect intestinal mucin secretion suggests that jejunal goblet cells are unlikely to be subject to central nervous system regulation by this pathway. Finally, our studies show that muscarinic receptor stimulation with bethanechol significantly increases jejunal mucin secretion and protein release. Although it now seems clear that goblet cells are entirely controlled by cholinergic elements of the intrinsic enteric nervous system, further studies are required to define the pathways regulating protein transport into the intestinal tract.

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Received March 15,1991. Accepted February 13, 1992. Address requests for reprints to: M. Mantle, Ph.D., Department of Medical Biochemistry, Health Sciences Centre, 3336 Hospital Drive North West, Calgary, Alberta, Canada T2N 4Nl. Supported by the Canadian Foundation for Ileitis and Colitis and Proctor & Gamble Co., Cincinnati, Ohio. M.M. is the recipient of a Scholarship Award from the Alberta Heritage Foundation for Medical Research. The authors thank M. Eckert (Medical College of Wisconsin), E. Thakore (University of Calgary), and Dr. G. Cope (St. James’ Hospital, Leeds, England) for technical assistance and A. Bailey (University of Calgary) for secretarial help. Dr. Greenwood’s present address: Eli Lilly&Co., Lilly Corporate Center 28/l, Indianapolis, Indiana 46285.