Muscularis mucosae contraction evokes colonic secretion via prostaglandin synthesis and nerve stimulation

W. H. Percy, T. H. Fromm, and C. E. Wangsness

Division of Basic Biomedical Sciences, School of Medicine, The University of South Dakota, Vermillion, South Dakota 57069-2390


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This in vitro study tested the hypothesis that muscularis mucosae contractile activity contributes to rabbit colonic mucosal function by mechanisms other than simple mechanical deformation of the epithelium. Experiments were performed by using a technique that allows simultaneous recording of muscle activity and transmucosal potential difference, a measure of epithelial ion transport. ATP, bradykinin, histamine, PGE2, PGF1alpha , and PGF2alpha elicited muscularis mucosae contractions that were resistant to atropine and TTX. Only ATP-induced contractions were indomethacin sensitive, and only those to dimethylphenylpiperazinium iodide (DMPP) were reduced by atropine. All agonist-evoked increases in transmucosal potential difference were atropine resistant, and, with the exception of those to PGE2, PGF2alpha , and VIP, they were also TTX sensitive. Mucosal responses to ATP, bradykinin, and histamine were indomethacin sensitive, whereas those to DMPP, the prostaglandins, and VIP were not. When cyclooxygenase activity or the mucosal innervation was compromised, even maximal muscularis mucosae contractions did not produce large secretory responses. It is concluded that contraction-related prostaglandin synthesis and noncholinergic secretomotor neuron stimulation represent the physiological transduction mechanism through which muscularis mucosae motor activity is translated into mucosal secretion.

motility; mucosa; submucosal plexus; transduction pathway; integrated function; rabbit


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RELATIONSHIP BETWEEN INTESTINAL motility and mucosal absorption and secretion has been given only scant attention over the last three decades, and almost without exception the focus has been on the effects of muscularis propria motor activity on mucosal function (15-19, 37). More recently, attention has shifted to the influence of muscularis mucosae contractions on mucosal activity. This was based on the observations that this muscle layer is physically attached to the mucosa, both are innervated via the submucosal plexus (30), and the muscularis mucosae can respond to specific changes in the luminal environment via a neurally mediated afferent-efferent link (27, 30). In the rabbit colon, the latter pathway appears to be associated with mucosal protection, as evidenced by the potentially harmful stimuli required to elicit this type of response (30).

In an early Ussing chamber study, morphological analysis of rat colonic mucosa immediately following stimulation with bradykinin revealed significant changes in mucosal architecture associated with anion secretion (3). The authors felt that this was unrelated to muscularis mucosae contraction because bradykinin caused relaxation of the colonic muscularis propria (13); therefore, they assumed it would have the same effect on the third muscle layer. Subsequent work on the muscularis mucosae has shown this belief to be incorrect in any region of the rat colon (29), and thus a significant contribution by this muscle layer to mucosal events cannot be excluded based solely on those experiments.

The mechanisms by which mechanical events occurring in the muscularis mucosae are translated into secretion by the mucosa remain unknown. In the stomach, for example, it was long believed that, on the basis of its anatomic relationship to the mucosa, this muscle layer would promote gastric acid secretion by "squeezing" the gastric glands (39). Recent studies (35) in the rabbit stomach have shown that this assumption may not be correct, because the muscularis mucosae in this region receives a predominantly inhibitory intrinsic innervation, is not contracted by secretagogues, and may thus actually facilitate acid secretion by relaxing and opening the gastric glands.

The importance of the muscularis mucosae to mucosal activity is further emphasized by a recent in vitro investigation of their integrated function in rabbit distal colon, where it was noted that, in the presence of TTX, large agonist-induced muscle contractions persisted but the corresponding mucosal responses were attenuated (30). This led to the suggestion that the functional relationship between these structures might be more complex than epithelial secretion simply being augmented via muscularis mucosae contraction-induced squeezing of the mucosa. On the basis of this proposition, the aim of the present in vitro study was to test the hypothesis that in rabbit distal colon mucosal compression is not the primary mechanism through which muscularis mucosae motor activity is linked to epithelial secretion.

The pharmacological agents used to test this hypothesis were selected on the basis of their respective abilities to 1) directly stimulate either the muscularis mucosae (30) or the mucosa (1, 14), 2) initiate prostaglandin production (1, 2, 8, 20, 23, 25), 3) reproduce the actions of endogenous eicosanoid mediators, 4) stimulate ganglionic nicotinic receptors on submucosal neurons, and 5) block the synthesis or the effects of agents with actions on the muscle or the mucosa and/or their respective innervation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of the muscularis mucosae/mucosa cylinder. The technique used in this study was that recently described by Percy et al. (30) for the simultaneous measurement of rabbit distal colonic transmucosal potential difference and muscularis mucosae motor activity in vitro.

Male New Zealand White rabbits (2-3 kg) were euthanized by pentobarbital overdose. After laparotomy, a segment of distal colon ~4 cm in length situated immediately proximal to the pelvic brim was removed and transferred to a dish coated with Sylgard (Dow Corning, Midland, MI) and filled with oxygenated Krebs' solution. Under a low-power dissecting microscope, the longitudinal and circular muscle layers were removed en bloc by sharp dissection, leaving a cylinder composed of submucosa, submucosal plexus, muscularis mucosae, and mucosa. Throughout this procedure, the lumen was regularly perfused with fresh oxygenated Krebs' solution to ensure epithelial viability. The ability to maintain colonic mucosal integrity during extended periods of luminal perfusion with an oxygenated physiological solution has previously been noted (17, 30). A polyethylene catheter with a flared end connected to a peristaltic pump was then tied securely into the proximal end of the cylinder.

The whole muscularis mucosae/mucosa cylinder was next transferred to a 50-ml organ bath where the distal end was attached to one arm of a "T" connector fixed in the base of the bath. The second arm of this connector was used as a luminal outflow. An agar salt bridge electrode was inserted into the third arm of the T piece, bringing it into electrical contact with the luminal perfusion solution of oxygenated Krebs' solution at 37 ± 0.5°C at a flow rate of 12 ml/min, delivered via a Masterflex peristaltic pump (model 7520-35; Cole-Parmer, Chicago, IL). A second agar salt bridge was then placed in contact with the bathing medium. Great care was taken to ensure that no air bubbles entered the luminal perfusion line, because these could cause regions of electrical discontinuity, preventing accurate reading of the voltage difference between the two electrodes. Potential difference between the cylinder lumen and the bathing medium was measured after amplification of the signal via a Grass polygraph preamplifier and amplifier. Increases in transmucosal potential difference recorded in this fashion represent an increase in luminal negativity resulting from chloride ion secretion (30).

Finally, the oral end of the preparation was connected to a force-displacement transducer (model FTO3C; Grass Instruments, Quincy, MA). The entire preparation was maintained under a tension equivalent to a 2.0-g load (19.6 mN). The tissue was then allowed to warm up to a maintained temperature of 37 ± 0.5°C and to equilibrate for at least 30 min before the start of each experiment. All responses were recorded on a Grass model 7D polygraph. Under these recording conditions, movement artifacts were excluded from the potential difference recordings because neither electrode was in contact with the preparation itself.

Effects of pharmacological agents on the cylinder preparation. All drugs were added to the serosal bathing medium in amounts that did not exceed 10% of the total chamber volume, and they were removed by draining and refilling the bath. Acetylcholine (10-9-10-3 M) was added to the serosal bathing solution to quantitate excitatory changes and to confirm tissue viability. Concentration-response curves for each agonist were constructed by using noncumulative addition protocols, and the ensuing muscle and mucosal responses were expressed as a percentage of their respective maximum responses to acetylcholine. To avoid tissue fatigue and to eliminate errors associated with tissue deterioration over time, responses to pharmacological agents in the presence of atropine, indomethacin, and TTX were each obtained from separate preparations and compared with untreated controls. TTX was added 5 min before beginning pharmacological manipulation. Atropine and indomethacin exposure began 30 min in advance of the addition of the agent under study. When their effects on agonist-induced responses were being studied, TTX, atropine, and indomethacin were present continuously throughout the entire drug addition sequence to ensure that their concentration in the serosal bathing medium remained constant and that their effects did not diminish over time.

Statistical analysis. Statistical differences were assessed using a nonpaired Student's t-test or, in the case of multiple group comparisons, ANOVA with Bonferroni correction for parametric data or a Kruskal-Wallis ANOVA with a Dunn's multiple comparison post hoc test for nonparametric data. Statistical analyses were performed using GraphPad Instat version 3 (GraphPad Software, San Diego, CA), and a P value of <0.05 was considered to represent a significant difference. In all cases, n values represent one preparation from one animal.

Drugs and solutions. Experiments were performed using a Krebs' solution of the following composition (in mM): 118.5 NaCl, 4.75 KCl, 2.54 CaCl2, 1.19 NaH2PO4, 1.19 MgSO4, 25.0 NaHCO3, and 11.0 glucose. Acetylcholine chloride (Sigma, St. Louis, MO) was dissolved in 5% sodium phosphate and diluted in Krebs' solution acidified to pH 4.0 with 0.1 N HCl. Atropine sulfate, dimethylphenylpiperazinium iodide (DMPP), famotidine, histamine diphosphate, pyrilamine maleate, PGE2, PGF1alpha , and PGF2alpha (all Sigma) were dissolved in, and diluted with, a modified Krebs' solution (in mM: 143 NaCl, 4.75 KCl, 2.54 CaCl2). TTX (Sigma) was dissolved in a citrate buffer (50 mM citric acid, 48 mM NaH2PO4). ATP, bradykinin, and VIP (all Sigma) were dissolved in, and diluted with, distilled water. ATP, bradykinin, PGE2, PGF1alpha , PGF2alpha , and VIP stock solutions were frozen at -5°C when not in use, and fresh dilutions were made daily. Indomethacin (Sigma) was initially dissolved in 10 ml of a 54-mM Na2CO3 solution and subsequently diluted with distilled water.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Histamine. The muscularis mucosae and the mucosa responded to histamine (10-9-10-3 M) with increases in tone and in transmucosal potential difference that reached, respectively, 105.7 ± 3.7 and 75.6 ± 10.0% of the acetylcholine maximum (Fig. 1). These responses were significantly reduced by a 30-min pretreatment with the histamine H1 receptor antagonist pyrilamine (10-6 M) to 65.4 ± 4.1 and 21.2 ± 3.8% (both P < 0.001; n = 6 each) but not by the H2 receptor antagonist famotidine (10-6 M; not shown). This concentration of pyrilamine did not alter the responses of the muscularis mucosae or the mucosa to acetylcholine (not shown).


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Fig. 1.   Effect of serosally applied atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to histamine. Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the effects of histamine on the mucosa but not the muscularis mucosae were attenuated by indomethacin and TTX but not by atropine. Data are means ± SE of the number of observations indicated. Ach, acetylcholine. ***P < 0.001, significantly smaller than the largest histamine-induced response.

Muscularis mucosae responses to histamine were unaffected by a 30-min pretreatment with the muscarinic antagonist atropine (10-6 M). However, although the mucosal effects of lower concentrations of this agent were enhanced, the largest histamine responses did not differ significantly from controls under these conditions (Fig. 1). This concentration of atropine reduced the largest acetylcholine-induced responses of both the muscularis mucosae and the mucosa to 54.5 ± 8.5 and 57.4 ± 12.5% (n >=  6), respectively.

Neither TTX (10-6 M) nor indomethacin (10-6 M) altered the muscularis mucosae responses to histamine. In contrast, both procedures significantly attenuated the largest responses of the mucosa under the same experimental conditions (Fig. 1).

Bradykinin. As with histamine, bradykinin (4 × 10-13-4 × 10-6 M) elicited contractions and increases in transmucosal potential difference whose respective largest responses (96.5 ± 4.4 and 137.5 ± 9.8%; n = 12) were unaffected by atropine (10-6 M), although the linear portion of the mucosal bradykinin concentration-response curve was shifted to the left under these conditions (Fig. 2). TTX (10-6 M) significantly attenuated the mucosal responses to bradykinin, whereas the contractile responses of the muscularis mucosae were unchanged by this procedure (Fig. 2). Indomethacin (10-6 M) caused a rightward shift in the bradykinin concentration-response curves for both the muscularis mucosae and the mucosa, but the largest responses of each tissue were not significantly reduced in either case (Fig. 2).


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Fig. 2.   Effect of serosally applied atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to bradykinin. Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the effects of bradykinin on the muscularis mucosae and mucosa were atropine resistant but indomethacin sensitive. Only the mucosal responses were affected by TTX. Data are means ± SE of the number of observations indicated. ***P < 0.001, significantly smaller than the largest bradykinin-induced response.

ATP. Muscularis mucosae and mucosal responses to ATP (10-9-10-3 M) were concentration related. Paradoxically, although the largest ATP-induced responses of the muscularis mucosae were small (63.0 ± 4.4%; n = 7), the corresponding mucosal responses were large (120.5 ± 15.3%; n = 7; Fig. 3). In contrast to all other agents studied, muscularis mucosae responses to ATP were significantly reduced by indomethacin (10-6 M) but were resistant to both atropine (10-6 M) and TTX (10-6 M). Mucosal responses to ATP were atropine resistant, but they were significantly attenuated by both indomethacin (10-6 M) and TTX (10-6 M; Fig. 3).


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Fig. 3.   Effect of serosally applied atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to ATP. Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the effects of ATP on the muscularis mucosae and mucosa were atropine resistant but indomethacin sensitive. Only the mucosal responses were attenuated by TTX. Data are means ± SE of the number of observations indicated. **P < 0.01, significantly smaller than the largest ATP-induced response.

DMPP. The ganglion-stimulating agent DMPP (10-9-10-3 M) elicited muscularis mucosae contractions that reached 54.6 ± 6.6% (n = 10) of the acetylcholine maximum. These responses were unaffected by indomethacin (10-6 M) but were significantly attenuated in the presence of atropine (10-6 M; P < 0.001; Fig. 4). Mucosal responses to DMPP reached 84.7 ± 12.9% (n = 10) of the acetylcholine maximum and were also resistant to indomethacin (10-6 M). In the presence of atropine (10-6 M), the mucosal concentration-response curve for DMPP was shifted to the right, but, although the largest response to this agent was then numerically smaller than the control (65.6 ± 11.4%; n = 8), this was not a statistically significant difference (Fig. 4).


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Fig. 4.   Effect of serosally applied atropine (10-6 M) and indomethacin (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to the ganglion-stimulating agent dimethylphenylpiperazinium (DMPP). Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the effects of DMPP on both structures were indomethacin resistant. Muscularis mucosae contractions were reduced by atropine, and, although the largest mucosal response was unchanged by this procedure, the DMPP concentration response curve was shifted to the right and responses to lower concentrations of this agent were attenuated. Data are means ± SE of the number of observations indicated. ***P < 0.01, significantly smaller than the largest DMPP-induced response.

Prostaglandins. Muscularis mucosae responses to PGE2, PGF1alpha , and PGF2alpha (2.8 × 10-11-2.8 × 10-6 M) were resistant to atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M). The largest muscularis mucosae responses were elicited by PGE2 (68.5 ± 4.6%; n = 6; not shown) and PGF2alpha (64.4 ± 6.1; n = 10; not shown); the smallest were elicited by PGF1alpha (27.4 ± 5.6; n = 6; Fig. 5). Similarly, mucosal responses to these prostaglandins occurred in the following order: PGE2 (119.4 ± 9.0%; n = 10), PGF2alpha (83.1 ± 13.4%; n = 10), and PGF1alpha (61.7 ± 12.8%; n = 6). The potential difference changes elicited by PGE2 and PGF2alpha were resistant to atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M; not shown). In contrast, the largest mucosal response to PGF1alpha was significantly reduced by TTX (10-6 M), but, as with the other eicosanoids, it was unaffected by either atropine (10-6 M) or indomethacin (10-6 M; Fig. 5).


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Fig. 5.   Effect of serosally applied atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to PGF1alpha . Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the effects of PGF1alpha on the muscularis mucosae and mucosa were atropine and indomethacin resistant. The mucosal response was TTX sensitive; the muscularis mucosae response was not. Data are means ± SE of the number of observations indicated. *P < 0.01, significantly smaller than the largest PGF1alpha -induced response.

VIP. The responses of the muscularis mucosae to VIP (3 × 10-12-3 × 10-6 M) were small, reaching only 6.8 ± 2.0% (n = 5) of the acetylcholine maximum. In contrast, the largest VIP-induced increase in potential difference was 126.0 ± 16.8% (n = 5) of the acetylcholine maximum. Atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) had no significant effects on the mucosal responses to this peptide (Fig. 6). Muscularis mucosae responses to VIP were too small for a meaningful determination of the effects of these agents to be made.


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Fig. 6.   Effect of serosally applied atropine (10-6 M), indomethacin (10-6 M), and TTX (10-6 M) on responses of rabbit distal colonic muscularis mucosae (A) and mucosa (B) to VIP. Control and posttreatment data are from separate preparations to ensure that tachyphylaxis was not a factor in these responses. Note that the mucosal responses to VIP were resistant to each of these treatments; muscularis mucosae responses were too small for a meaningful assessment of these effects to be made. Data are means ± SE of the number of observations indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data obtained in this study add to our understanding of the physiological role of the muscularis mucosae and demonstrate that in rabbit distal colon there is a complex relationship between the motor activity of this muscle and the corresponding mucosal function. It has previously been shown that the muscularis mucosae may be linked to epithelial protection via a neural pathway that causes muscular contraction in response to noxious agents in the lumen, thus reducing the mucosal surface area (27, 30). The present data now suggest that this muscle plays a second important role in integrated gut function, namely promotion of epithelial secretion. However, this is not simply the fortuitous result of mechanical deformation of the mucosa as a consequence of muscularis mucosae shortening.

The nature of the relationship between the muscularis mucosae and the mucosa in this colonic region is clearly illustrated by the effects of histamine. In earlier Ussing chamber studies it was found that responses of rabbit distal colonic mucosa to histamine were H1 receptor mediated, small, transient, TTX resistant (28), and sometimes absent altogether (28, 30). With the use of the cylinder preparation, mucosal responses to histamine were again found to be H1 receptor mediated, but under these conditions they were large, persistent, and sensitive both to indomethacin and to TTX. The only significant functional difference between these two recording methods is the ability of the muscularis mucosae to contract. This strongly suggests, therefore, that the motor activity of this muscle is the first step in a complex series of events that can ultimately lead to mucosal secretion.

In addition, because histamine is essentially without a direct effect on this epithelium, the approx 80% maximal increase in transmucosal potential difference elicited by this agent in the cylinder preparation represents a measure of the total amount by which muscularis mucosae contraction can influence secretion at this site. Agents such as ATP or bradykinin that can promote additional prostaglandin production or directly stimulate secretomotor neurons can exceed approx 80% maximal transmucosal potential difference increases by augmenting these components of the intrinsic response to contraction.

The relationship between muscularis mucosae contraction and epithelial secretion characterized in the present investigation may also provide an explanation for the variable responses of this mucosa to histamine seen in a variety of Ussing chamber studies. If, in the Ussing chamber, the mucosa was pinned tightly and the muscularis mucosae was truly held immobile, the mucosa would appear to be unresponsive to histamine (28, 30); in instances in which the preparation was stretched to a lesser extent and the muscularis mucosae was able to contract in response to histamine, the mucosal responses would be attenuated by indomethacin, pyrilamine, and TTX (e.g., Ref. 38). Thus, although appearing to be mucosal in origin, the latter responses would represent muscularis mucosae-initiated secretion via the pathways identified here.

It could be argued that, in the cylinder preparation, indirectly mediated mucosal responses to agents such as histamine or bradykinin did not arise secondary to muscularis mucosae contraction but rather via stimulation of intestinal subepithelial myofibroblasts (4). These cells lie adjacent to the mucosa, possess a variety of pharmacological receptors, and are a likely source of prostaglandin production in this region (4, 36). This explanation is unlikely, given that histamine has minimal mucosal effects in the Ussing chamber when muscularis mucosae contractions are curtailed (28, 30) but causes large TTX- and indomethacin-sensitive potential difference changes in the cylinder preparation where contractions are unimpeded.

On the basis of the pharmacological profile of muscularis mucosae-related transmucosal potential difference increases, it appears that they are produced indirectly via mechanically induced prostaglandin synthesis and the concurrent stimulation of noncholinergic secretomotor neurons. These conclusions are drawn from the following observations.

First, even maximal muscularis mucosae contractions did not elicit large secretory responses if prostaglandin synthesis or the innervation of the mucosa was pharmacologically compromised. This demonstrates that epithelial secretion is not simply the result of mucosal compression produced via muscularis mucosae contraction.

Second, all muscularis mucosae-related, TTX-sensitive mucosal responses in the rabbit colon were atropine resistant, despite the fact that mucosal secretion in this region is governed by both cholinergic and noncholinergic enteric nerves (6, 30). This cannot be attributed to selective damage of cholinergic neurons during the tissue preparation process, because functional cholinergic innervation of the muscle and the mucosa was seen as the atropine-sensitive component of their responses to the ganglion-stimulating agent DMPP. From this it appears that cholinergic submucosal neurons are present but that they do not participate in muscularis mucosae-initiated mucosal secretion. This contrasts starkly with the rat colon, where agonist-induced submucosal prostaglandin synthesis stimulates both cholinergic and noncholinergic secretomotor neurons (10).

The lack of a cholinergic component linking muscularis mucosae contraction to secretion may be because rabbit colonic secretomotor neurons do not possess the appropriate eicosanoid receptors or because of the known ability of certain inhibitory prostaglandins to attenuate acetylcholine release from submucosal nerves (11). The latter explanation seems improbable given the large number of arachidonic acid metabolites produced in the lamina propria in response to agents such as bradykinin (22, 25). Furthermore, absence of a significant endogenous inhibitory prostaglandin production in this system was strongly suggested by the inability of indomethacin to alter mucosal responses to exogenous PGE2, PGF1alpha , and PGF2alpha . There is good evidence that indomethacin pretreatment can enhance mucosal secretion to applied prostaglandins in regions where endogenous inhibitory prostaglandins (such as PGD2) oppose these excitatory effects (21, 24), because this negative influence is removed when cyclooxygenase activity is inhibited (24).

Third, on the basis of the effects of histamine and the selective actions of pyrilamine, it can be deduced that muscularis mucosae contraction is not linked to mucosal secretion via mechanically induced release of this autacoid from mast cells in the lamina propria.

Fourth, in response to VIP, a directly acting secretagogue, large TTX- and indomethacin-resistant increases in transmucosal potential difference occurred in the absence of significant muscularis mucosae motor activity; in contrast, all agonist-induced contractile activity occurred in association with increases in transmucosal potential difference. This demonstrates that muscle activity and mucosal function are indirectly coupled, because the secretory apparatus can be directly activated in the absence of significant muscularis mucosae contractions, but contractions cannot occur without concurrent secretory responses.

The ability of atropine to enhance mucosal responses to lower concentrations of histamine, and to a lesser extent of bradykinin, remains unexplained. Because there was no corresponding change in the magnitude of muscularis mucosae contractions to either agent, the atropine-induced augmentation of their mucosal effects was not simply a mechanical artifact. It would have been predicted that, if bradykinin- or histamine-induced stimulation of the mucosa involved a cholinergic pathway, muscarinic receptor blockade should decrease the transmucosal potential difference changes they evoke. This would occur not only via a reduction in the magnitude of the overall stimulus itself but also possibly by an increase in apical K+ secretion (12). The net result would thus be a decrease in the arithmetic sum of the negative and positive charges moving into lumen. However, because cholinergic secretomotor neurons were not found to participate in this process, atropine should have been without effect on these mucosal responses. One possible explanation for the augmentation of the effects of bradykinin and histamine under these conditions is that a component of integrated muscularis mucosae/mucosal function involves the activation of inhibitory muscarinic receptors on noncholinergic secretomotor neurons. Muscarinic receptor blockade would thus remove an endogenous inhibitory influence on secretion. This locus of action is supported by the inability of atropine to alter the mucosal responses to secretagogues such as PGE2, PGF2alpha , and VIP, whose effects were TTX resistant.

In contrast to its inhibitory actions on a variety of smooth muscles (7), ATP elicits muscularis mucosae contractions in rabbit distal colon (33). The present data show this to be because ATP elicits excitatory prostaglandin production, which in turn causes direct stimulation of the muscle. Prostaglandin generation following exposure to endogenous or exogenous purines has been demonstrated in a variety of intestinal and nonintestinal tissues (1, 2, 8, 20, 23), and its occurrence in the muscularis mucosae/mucosa of the rabbit distal colon adds to the list of sites where this pathway operates. Paradoxically, ATP exerts its largest motor effects on the muscularis mucosae in the rabbit proximal colon, where prostaglandins elicit minimal contractions (31, 33). This suggests that regional variations in ATP-mediated responses reflect the relative contributions of the direct vs. indirect effects of this purine and supports the belief that the relationship between the muscularis mucosae and mucosa may be governed by different physiological factors in successive regions of the gut.

ATP-induced muscularis mucosae contractions were both atropine and TTX resistant, implying that the prostaglandins generated directly stimulate the muscularis mucosae. Mucosal stimulation by ATP was similarly mediated via prostaglandin production, but this process also involved the activation of noncholinergic secretomotor neurons, because, although atropine resistant, it was significantly attenuated by TTX. Although the prostaglandin(s) produced following ATP administration were not specifically identified in the present study, only the mucosal responses to applied PGF1alpha were found to be TTX sensitive. It is thus possible that this particular eicosanoid is an important component of the mucosal response to ATP and to other agents, such as histamine and bradykinin, whose indirect effects are also prostaglandin mediated and TTX sensitive. The indirect effects of ATP on rabbit colonic mucosa differ from those reported in the rat colon, where ATP-induced epithelial secretion results from a direct effect on crypt cells (26) and on secretomotor neurons, in the absence of prostaglandin production (9). These observations suggest that there may be significant species differences in the interrelationship of colonic muscularis mucosae motor activity and mucosal secretion.

Although the involvement of prostaglandins and submucosal nerves in epithelial secretion in the colon has previously been described (e.g., Refs. 5 and 10), the physiological necessity for having this complex apparatus available has not been satisfactorily explained. Without evidence for a functional interaction, it appeared that these were simply duplicate systems, each of which was capable of eliciting the same biological response under different experimental conditions. However, it is now possible to suggest that, in this region of the rabbit colon, these components are present because they represent the transduction mechanism through which muscularis mucosae motor activity is translated into epithelial secretion. On the basis of the indomethacin and TTX sensitivity of this pathway, the physiological mechanisms underlying this interaction are muscularis mucosae contraction-related prostaglandin synthesis coupled to the concurrent stimulation of noncholinergic secretomotor neurons (Fig. 7). Thus, although mechanical movement of the muscularis mucosae may change mucosal surface area, additional processes are required for this activity to influence secretion. This leads to the conclusion that the relationship between these structures is more complex than previously thought and that muscularis mucosae contraction is actively linked to mucosal function. The pathophysiological consequence of using muscularis mucosae-induced prostaglandin synthesis in a pathway that stimulates the mucosa is that, in colitis, this system may be disproportionately disrupted (34) because the muscularis mucosae and mucosa rapidly become desensitized to eicosanoids (14, 32) and the colonic mucosal innervation is significantly compromised (14).


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Fig. 7.   Events linking muscularis mucosae contraction to mucosal secretion in rabbit distal colon. Contraction of the muscularis mucosae (1) initiates prostaglandin production (2). The prostaglandins stimulate (+) noncholinergic secretomotor neurons (3), which in turn elicit epithelial secretion (4). The prostaglandins also have the potential to stimulate both the muscularis mucosae and the mucosal secretory apparatus directly (5), but this is usually overshadowed by the direct effects of agonists on the muscle and/or the mucosa and also by the excitatory actions of the eicosanoid(s) on noncholinergic secretomotor neurons. If prostaglandin production is inhibited or if neural conduction is blocked, simple mechanical deformation of the mucosa by muscularis mucosae shortening results in only a small secretory response.


    ACKNOWLEDGEMENTS

This work was supported by the South Dakota Health Research Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: W. H. Percy, Division of Basic Biomedical Sciences, School of Medicine, The Univ. of South Dakota, Vermillion, SD 57069-2390 (E-mail: wpercy{at}usd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpgi.00179.2002

Received 14 May 2002; accepted in final form 15 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(2):G213-G220
0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society




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