Division of Basic Biomedical Sciences, School of Medicine, University of South Dakota, Vermillion, South Dakota 57069-2390
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ABSTRACT |
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This in vitro study investigated the relationship between rabbit colonic muscularis mucosae motor activity and changes in transmucosal potential difference. Spontaneous muscle contractions and potential difference oscillations occurred independently and were not neurally driven. ACh and histamine directly stimulated the muscularis mucosae, but their mucosal effects were largely indirect, suggesting that muscularis mucosae contractions promote epithelial secretion. 1,1-Dimethyl-4-phenyl-piperazinium iodide and vasoactive intestinal polypeptide induced large potential difference changes but small muscularis mucosae contractions, demonstrating mucosal secretion without significant muscle activity. Lowered intraluminal pH directly stimulated the muscle, whereas a bile salt-lipid mixture evoked TTX- and atropine-sensitive increases in its contractile activity. Increased intraluminal pressure and hypertonic luminal perfusion did not elicit muscularis mucosae excitation. Thus under basal conditions muscle and mucosal activities are independent, but evoked muscularis mucosae contractions can stimulate epithelial secretion. In response to specific luminal stimuli, muscularis mucosae motor activity is increased via the activation of cholinergic nerves. These data suggest that muscularis mucosae and mucosal functions are physiologically linked and that their activities can be coordinated by multiple mechanisms.
motility and secretion; luminal stimuli; submucosal plexus; integrated function; afferent-efferent pathway
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INTRODUCTION |
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PREVIOUS STUDIES (27) have demonstrated an association between intestinal muscularis propria contraction and changes in transmural potential difference, a measure of mucosal absorption and secretion. There is good evidence to support the belief that alterations in potential difference associated with spontaneous longitudinal and circular muscle activity are at least partly neural in origin (12) and that this process is modulated by the extrinsic innervation of the gut (9, 11, 13, 14). In the guinea pig small intestine, several types of histologically identifiable afferent fibers project from the mucosa to the myenteric plexus (35) and a variety of mucosal stimuli elicit excitation of myenteric neurons (2, 33), associated with neurally mediated changes in the electrical activity of the circular muscle layer (32, 34, 39). However, although the myenteric plexus may contribute to these events, there is also evidence to suggest that muscularis propria motor activity and mucosal function can be coordinated via the submucosal plexus (30), which is known to provide innervation to both the circular muscle (29) and the mucosa itself (3).
One unknown factor arising from this information, therefore, is the potential contribution of the muscularis mucosae to these events, because this muscle is physically attached to the mucosa and both receive innervation via the submucosal plexus (7). In a series of in situ experiments (18, 38), it was noted that canine small intestinal villous motility could be selectively stimulated by the mucosal application of certain amino acids, lipids, and bile salts, but not by an increased osmotic load or altered pH. As it is now known that mucosal application of bile salts can activate intestinal afferent fibers (17, 36), the discovery of an atropine-sensitive villous response to a luminally applied mixture of taurocholic and oleic acid (18) may be considered the first empirical evidence for a mucosal-to-muscularis mucosae afferent-efferent pathway.
Despite this discovery, the functional relationship between the muscularis mucosae and the corresponding mucosa throughout the gastrointestinal tract remains poorly understood. Based on indirect evidence, it has been suggested that this muscle may be involved in functions as diverse as maintaining structural integrity (16), modulating blood flow (26), assisting acid expulsion from the gastric pits (25, 31, 37), and promoting epithelial secretion (20). However, much of this is mere speculation, inferred from data obtained in experiments that were not specifically designed to assess the function of the muscularis mucosae or the nature of its relationship to the mucosa at any given location. The paucity of such experiments has, in large part, been due to the lack of a preparation in which the function of both systems could be measured concurrently. Because of this shortfall, the aims of the present study were to 1) develop an in vitro technique to simultaneously record colonic muscularis mucosae contraction and relaxation and transmucosal potential difference and 2) use this preparation to assess the nature of the relationship between muscularis mucosae motor activity and mucosal function under basal and stimulated conditions.
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METHODS |
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Preparation of Muscularis Mucosae-Mucosa Cylinder
The technique used in this study was based on an early preparation for the investigation of muscularis mucosae contractile activity in vitro (15) and is also a modified version of a technique originally described by Goldhill and Percy (9) for the simultaneous measurement of rabbit distal colonic transmural potential difference and both longitudinal and circular muscle 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 maintain epithelial viability. The ability to maintain colonic mucosal integrity during extended periods of luminal perfusion with an oxygenated physiological solution has previously been noted (12). 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 preparation was next transferred to a 50-ml organ bath as
described previously by Goldhill and Percy (9). The distal
end of the muscularis mucosae-mucosa cylinder was next 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. For control experiments, this solution was oxygenated
Krebs solution at 37 ± 0.5°C, at a flow rate of 12 ml/min,
delivered via a Masterflex peristaltic pump (Cole-Parmer, Chicago, IL;
model 7520-35). A second agar-salt bridge was then placed in contact
with the bathing medium. In subsequent procedures, the various luminal
perfusion solutions utilized in attempts to stimulate the preparation
were introduced by switching between reservoirs via a three-way
stopcock (Fig. 1). 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. The potential difference between the cylinder lumen and the
bathing medium was measured after amplification of the signal via a
Grass polygraph preamplifier and amplifier. Finally, the oral end of
the preparation was connected to a Grass FTO3C force-displacement
transducer (Quincy, MA) (Fig. 1). The entire preparation was maintained
under a tension equivalent to a 2-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. By convention, increases in muscle tone and transmucosal potential difference (increased luminal negativity) were denoted by upward movements of the chart recorder pens.
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Muscle Strip Preparation
Strips of muscularis mucosae with mucosa attached were prepared using the "sutured edge" technique originally described by Percy and Christensen (21). Briefly, strips of mucosa, muscularis mucosae, and submucosa 3 cm in length were tied in the middle with 5-0 surgical thread and folded, with mucosal surfaces facing inward, so that they were one-half their original length. The oral and aboral ends, now side by side, were tied together to form a loop. Because curling of the tissue tends to expose the mucosal surface rather than the submucosal aspect, the vertical edges of the preparation were sutured at four points with 7-0 surgical thread. This holds the preparation flat and ensures that the submucosa and muscularis mucosae are fully exposed to the bathing medium but does not compromise its ability to contract.Strips prepared in this way were mounted in 2-ml organ baths at 37 ± 0.5°C, and one end of the tissue was connected to a stationary mounting point on the bottom of the bath. The other end was connected to a Grass FTO3D force-displacement transducer under a tension equivalent to a 1-g load (9.8 mN). Based on preliminary experiments, a load of this magnitude stretched this muscle to approximately the optimal length for the generation of active tension.
Ussing Chamber Studies
As previously described (10), segments of distal colon were opened along their mesenteric border and rinsed in Krebs solution to remove residual fecal material. The tissue was next pinned, mucosal surface down, in a petri dish in oxygenated Krebs solution. The muscularis propria was removed by sharp dissection, leaving a preparation consisting of mucosa, lamina propria, muscularis mucosae, and submucosal plexus. This was then mounted between two halves of an Ussing chamber. Both halves of the chamber were circulated with oxygenated Krebs solution. The potential difference was monitored using one pair of agar-salt bridges connected to a voltage-current clamp (DVC 1000, WPI, Sarasota, FL). A pair of current-passing electrodes was connected to this apparatus and short-circuit current automatically determined. Compensation for fluid resistance and potential difference arising from electrode asymmetry was made before mounting tissues.Effects of Pharmacological Agents on Cylinder Preparation
ACh (10Statistical Analysis
Statistical differences were assessed using either a paired Student's t-test or, in the case of multiple group comparisons, ANOVA followed by an appropriate parametric or nonparametric post hoc test. P < 0.05 was considered to be significantly different. In all cases, n represents 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 NaHCO3, and 11 glucose. ACh chloride (Sigma, St. Louis, MO) was dissolved in 5% sodium phosphate and diluted in Krebs solution acidified to pH 4 with 0.1 N HCl. Atropine sulfate, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and histamine (all from Sigma) were dissolved in and diluted with a modified Krebs solution (composition in mM: 143 NaCl, 4.75 KCl, and 2.54 CaCl2). TTX (Sigma) was dissolved in a citrate buffer (composed of 50 mM citric acid and 48 mM NaH2PO4). Vasoactive intestinal polypeptide (VIP; Sigma) was dissolved in and diluted with distilled water. VIP stock solutions were frozen at ![]() |
RESULTS |
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Baseline Activity
Under resting conditions, the preparations exhibited atropine- (10
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Responses to Pharmacological Stimuli
ACh.
Both the muscularis mucosae and the mucosa responded to ACh
(109-10
3 M) with, respectively,
concentration-dependent increases in tension and potential difference.
There was a clear distinction between basal and stimulated events, and
the largest ACh-induced mucosal responses were up to 20-30 times
larger than ongoing spontaneous potential difference oscillations (Fig.
4). Basal transmucosal potential
difference in the rabbit distal colon is known to be ~50% amiloride
sensitive, reflecting a significant contribution from Na+
absorption (8). However, stimulus-evoked increases in
short-circuit current in this tissue have been shown to be due to
Cl
secretion (1), and in these experiments,
agonist-induced increases in potential difference are considered to
reflect luminally directed anion movement. It was also noted that
although the time taken for the development of maximal transmucosal
potential difference changes was similar to the time for peak muscle
contraction to be achieved, mucosal responses persisted long after the
muscle had returned to baseline tone following removal of agonists from the bathing medium. In many instances, 20 min and several additional washes were required for full mucosal recovery (Fig. 4).
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Histamine.
The muscularis mucosae responded to histamine
(109-10
3 M) with
concentration-dependent contractions that reached 113.8 ± 4.8% (n = 10) of the corresponding ACh maximum (Fig.
6A). Histamine-induced changes
in transmucosal potential difference, however, plateaued at
10
5 M histamine and at 10
3 M were only
39.9 ± 13.7% (n = 10) of the ACh maximum (Fig.
6A). Under these conditions, the largest histamine-induced
muscularis mucosae contractions were significantly larger than the
corresponding maximal potential difference changes (P < 0.001; n = 10) when each was expressed as a
percentage of their respective maximum responses to ACh.
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DMPP and VIP.
The ganglion-stimulating agent DMPP
(109-10
3 M) elicited
concentration-dependent muscularis mucosae contractions and increases in transmucosal potential difference (Fig.
7A). In contrast to the
effects of histamine, the largest DMPP-induced change in transmucosal potential difference (88.7 ± 7.1%) was significantly larger than the corresponding maximal muscularis contraction (56.5 ± 5.2) (P < 0.001; n = 19), when each was
expressed as a percentage of the tissues' maximum responses to ACh.
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Responses to Luminal Stimuli
Increased osmotic load. With the use of the second luminal perfusion reservoir and the three-way stopcock (Fig. 1), the luminal perfusion solution was changed from normal Krebs solution to a Krebs solution containing 140 mM mannitol. Throughout a 30-min luminal perfusion with this solution, there was no measurable change in muscularis mucosae tone or transmucosal potential difference (n = 8).
Increased intraluminal pressure.
Increased intraluminal pressure was achieved by occluding the
perfusion outflow (Fig. 1) for a period of 10 min so that the inflow remained at 12 ml/min, but the outflow was reduced to 6 ml/min.
Under these recording conditions, preparations were seen to
"balloon" as the volume of fluid in the system increased, but no
coordinated elevation of muscularis mucosae motor activity was noted in
response to the change in luminal diameter. Distension was,
however, associated with a small increase in muscularis mucosae resting
tension (7 of 14 preparations) coupled to an attenuation or total loss
of ongoing spontaneous motor activity (14 of 14 preparations) (Fig.
8). During this time, spontaneous
transmucosal potential difference oscillations were lost (9 of 14 preparations), and the potential difference itself gradually declined
(9 of 14 preparations) (Fig. 8). Preparations in which one of these
mucosal phenomena was unchanged either lacked potential difference
oscillations or had a small basal potential difference before the
occlusion procedure. The characteristics of the occlusion-induced
muscle and mucosal responses were not altered by pretreating a separate group of preparations with serosal TTX (106 M; 5-min
exposure; n = 9; Fig. 8).
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Bile salt-lipid mixture.
When the luminal perfusion solution was changed to Krebs containing
taurocholic acid (15 mM), oleic acid (10 mM), and monoolein (5 mM),
there was an increase in muscularis mucosae resting tone and an
increase in the amplitude of spontaneous contractions (18 of 21 preparations). The increase in muscle tone was small relative to the
amplitude of spontaneous contractions (Fig.
9). This was associated with an initial
concurrent increase in transmucosal potential difference (16 of 21 preparations) that declined over the ensuing 20 min. In 2 of 21 preparations, the increase in potential difference was sustained, and
in an additional 3 of 21 preparations, the decline in potential
difference was not preceded by an increase. The muscle response was
attenuated by a 5-min pretreatment with serosal TTX (106
M; 5-min exposure; n = 6; Fig. 9). The responses of the
muscularis mucosae to luminal perfusion with this bile salt-lipid
mixture were also attenuated after a 30-min pretreatment with serosal atropine (10
6 M; 30-min exposure; n = 6;
not shown). The characteristics of the mucosal response to the bile
salt-lipid mixture were unchanged by either TTX or atropine at these
concentrations.
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Decreased intraluminal pH.
When the pH of the luminal perfusion solution was reduced to 4 by the
addition of acetic acid, the transmucosal potential difference began a
decline to zero immediately on its entry to the lumen (6 of 21 preparations); in the remaining 15 preparations, the reduction in
potential difference was interrupted by a transient increase before the
decline was complete (Fig. 10).
However, there was a delay of 1-2 min before the muscularis
mucosae exhibited a contractile response to this stimulus (19 of 21 preparations) (Fig. 10). In an additional two preparations, the onset
of the muscle and mucosal responses appeared to be concurrent. In
contrast to the effects of the bile salt-lipid perfusion, acetic
acid-induced responses of the muscularis mucosae were large compared
with spontaneous motor activity. Neither the muscle nor the mucosal
responses were modified by serosal pretreatment with TTX
(106 M; 5-min exposure; n = 16; Fig. 10),
atropine (10
6 M; 30-min exposure; n = 6),
indomethacin (10
6 M; 30-min exposure, n = 6), or pyrilamine (10
6 M; 30-min exposure;
n = 5).
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DISCUSSION |
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The results of the present study demonstrate that the cylinder preparation utilized in this investigation provides a viable method for the concurrent recording and analysis of the relationship between muscularis mucosae motor activity and transmucosal potential difference in vitro. Under basal conditions, it was possible to discriminate muscular events as small as 4.9 mN (0.5 g) and transmucosal potential difference changes of 0.5 mV. In response to pharmacological stimuli, muscular contractions in excess of 100 mN and transmucosal potential differences of 10-20 mV were seen routinely. Thus the recording system is capable of measuring responses that vary by between one and two orders of magnitude, while maintaining a large signal-to-noise ratio across this range. The data obtained under these experimental conditions also show that the system is capable of distinguishing serosally initiated responses from those that are mucosal in origin. Furthermore, with the appropriate pharmacological tools, this preparation can be used to determine the relative contributions of neural, muscular, and epithelial events to the overall responses obtained after different stimuli.
In the absence of an external stimulus, there were measurable spontaneous contractions of the muscularis mucosae that occurred in association with clearly defined oscillations in transmucosal potential difference. The higher rate of spontaneous muscle activity in individual preparations may be related to the observation that the time taken for mucosal events to occur is greater than that required for similar degrees of muscle contraction and relaxation. This was particularly obvious during large agonist-induced responses. The resistance of spontaneous muscularis mucosae and mucosal events to both atropine and TTX suggests that neither is neurally driven nor dependent on spontaneous ACh release. In contrast, sutured edge muscularis mucosae preparations have been found to contract spontaneously at a higher frequency that can be increased or decreased by treatment with eserine or atropine, respectively (24). This implies that cutting strips of muscularis mucosae elicits continuous ACh release from submucosal neurons impinging on the muscle, whereas in the cylinder preparation the submucosal plexus is better preserved and this does not occur. Although spontaneous muscularis mucosae contractions occurred at a lower frequency under these experimental conditions, the rate of spontaneous potential difference oscillations lay within a range of values previously obtained (9) utilizing Ussing chamber techniques and also a cylinder preparation including the muscularis propria. This suggests that, as for the muscularis mucosae, a tonic release of ACh does not modulate spontaneous mucosal events in the cylinder preparation and is consistent with the observation that a significant component of the innervation of this colonic mucosa is noncholinergic (1).
The data obtained with ACh and histamine illustrate several important
points. First, although both agents elicited muscle contraction by a
direct action, their effects on the mucosa were predominantly indirect.
This was demonstrated by the TTX sensitivity of the mucosal response to
ACh and also by the lack of mucosal sensitivity to histamine in the
Ussing chamber studies. It is tempting to infer from these data that
changes in potential difference elicited by muscularis mucosae
contraction may be partly neurally in origin. This is based on the
observations that 50% maximal histamine-induced muscle contractions
elicited
40% maximal increases in potential difference, but in the
presence of TTX, 70% maximal ACh-induced contractions produced only
15% maximal increase in potential difference. This hypothesis is
further supported by our (22) recent discovery that,
under the same experimental conditions, maximal bradykinin-induced
changes in transmucosal potential difference are reduced by ~80% in
the presence of TTX, although contractions of the muscularis mucosae
are unchanged in amplitude by this procedure. These data support the
belief that the in vivo motor activity of the muscularis mucosae does modulate mucosal function and further suggests that this is, in part,
coordinated via the submucosal plexus.
Although histamine elicited large muscularis mucosae contractions but relatively small changes in transmucosal potential difference, DMPP-induced mucosal responses were significantly larger than those of the muscle, when both were expressed as a percentage of their respective maximal responses to ACh. This is consistent with the observations that this mucosa has a substantial excitatory (secretomotor) innervation (1), whereas neurally driven muscularis mucosae excitation is small and compounded by a concurrent inhibition (24). Similarly, VIP evoked large increases in transmucosal potential difference but only trivial responses from the muscularis mucosae. Again, this is consistent with the known actions of VIP on the mucosa (1) and the muscularis mucosae (24). Thus under certain conditions large changes in transmucosal potential difference can be achieved in the absence of maximal muscularis mucosae contractions.
From these observations, it can be seen that the properties of the combined muscularis mucosae-mucosa cylinder closely resemble the known behavior of the individual components of this preparation. Therefore, this strongly suggests that in terms of serosal stimuli the responses seen in the present study are a true reflection of the nature of the relationship between the muscle and the mucosa and their modulation by the submucosal plexus in the intact organ in vivo.
In dog small intestine, the mucosal application of a bile salt-lipid mixture elicits an atropine-sensitive increase in villous motor activity (18), which cannot be reproduced with saline or acidified predigested food (38). In the present study, a similar atropine- and TTX-sensitive, muscularis mucosae motor response to the presence of a bile salt-lipid mixture in the colonic lumen was found. Although luminally applied bile salts stimulate colonic afferent fibers (17, 36), the muscularis mucosae responses obtained previously (18) and in the present study were atropine sensitive, implying that the increase in motor activity was not the result of the release of substance P from sensory neurons. Release of this peptide from afferent fibers has previously (28) been suggested to contribute to the excitatory responses of opossum esophageal muscularis mucosae to electrical field stimulation. Therefore, the present data suggest the existence of a specific afferent-efferent pathway that ultimately leads to the activation of cholinergic submucosal motoneurons. Selective neural mediation of this motor phenomenon is further supported by the inhibitory actions of the same bile salt-lipid mixture when applied to the serosal aspect of the preparation. It should be noted, however, that luminally applied bile salts stimulate colonic mucosal generation of prostaglandins and nitric oxide (4), and the potential contribution of these to the responses seen in the present study warrants further investigation.
In contrast to the actions of the bile salt-lipid mixture, other luminal stimuli failed to elicit neurally mediated motor responses from the muscularis mucosae. Increased intraluminal pressure attenuated the motor activity of the muscle and elicited a decline in the transmucosal potential difference in a TTX-insensitive manner. Furthermore, distension of the cylinder preparation by this procedure did not elicit any observable coordinated muscle activity analogous to a "peristaltic reflex," suggesting that luminal clearance is not a physiological function of the muscularis mucosae in this region of the gut.
The resistance of the acetic acid Krebs-induced muscle response to a variety of pharmacological agents demonstrates that it was not neural in origin and did not arise from prostaglandin production or the release of histamine from mast cells. Interestingly, although short-chain fatty acids have been reported (5) to decrease colonic motility in vivo via the release of polypeptide YY, their excitatory in vitro actions result from direct stimulation of intestinal smooth muscle, supporting the findings of the present study. The lag time between the onset of the mucosal response and muscularis mucosae contraction to acetic acid suggests that this is a measure of the time taken for it to diffuse across the mucosa (19) and act on the muscle. The effects of the various luminal stimuli on the transmucosal potential difference may be explained either by a transient, distension-induced decrease in mucosal integrity or the high concentration of bile salts and charged ions overwhelming the ionic gradient that forms the basis of the potential difference being measured.
On the basis of these observations, it may be concluded that the neural pathways stimulated by the presence of the bile salt-lipid mixture are highly selective. This would suggest that neurally coordinated muscularis mucosae motor activity and mucosal function occur in response to specific luminal stimuli and that other mechanisms, such as release of paracrine substances (22), may facilitate absorption/secretion at other times. Given the varied pharmacological properties of the muscularis mucosae throughout the intestinal tract (e.g., Refs. 23-25), the possibility exists that different luminal stimuli elicit coordinated muscularis mucosae and mucosal activity in sequential regions of the gut.
Although it could be argued that radial distension in the absence of structural support from the muscularis propria, a bile salt-lipid mixture, or acidified Krebs solution are not truly physiological stimuli in the distal colon, their use in this instance was not designed to mimic in vivo conditions. Rather, these were chosen as tools having the potential to reveal mucosal-to-muscularis mucosae pathways available to coordinate the activity of these two structures. Thus the demonstration of muscularis mucosae contraction in response to irritant or potentially damaging luminal contents suggests that this may be one component of a mucosal protective response, because muscularis mucosae contraction reduces the mucosal surface area on which such substances can act. The nature of the in vivo physiological stimuli that initiate activity within the pathways now shown to be present requires further investigation.
The results of these experiments clearly demonstrate that the muscularis mucosae-mucosa cylinder in vitro exhibits basal and stimulated responses that do not differ significantly from several of the known properties of the individual tissues that comprise this system. However, the data from the present study greatly extend these observations by allowing for an evaluation to be made of the temporal relationships between these events and for the quantitative assessment of the consequences of activity in one structure on the function of the other structure. This novel in vitro technique significantly advances our ability to determine the nature of the relationship between the muscularis mucosae, the mucosa, and the various mechanisms by which the activities of these companion structures are coordinated throughout the gastrointestinal tract. By using this technique in conjunction with organ bath and Ussing chamber technology, it will now be possible to begin a determination of the function of the muscularis mucosae and its relationship to the mucosa throughout the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49803, National Science Foundation Grant OSR-9452894, the South Dakota Futures Fund, and the South Dakota Health Research Foundation (W. H. Percy). J. T. Brunz was supported by a University of South Dakota School of Medicine summer research stipend.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. H. Percy, Division of Basic Biomedical Sciences, School of Medicine, 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.
Received 7 December 2000; accepted in final form 21 March 2001.
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