In vivo signal-transduction pathways to stimulate phasic contractions in normal and inflamed ileum

Sushil K. Sarna

Departments of Surgery and Physiology, Medical College of Wisconsin, and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated the in vivo signal-transduction pathways to stimulate phasic contractions in normal and inflamed ileum by close intra-arterial infusions of test substances. Methacholine stimulated phasic contractions dose dependently. This response was suppressed during inflammation. Verapamil inhibited the response to methacholine dose dependently in both normal and inflamed ileum. Neomycin inhibited the response partially in normal ileum and almost completely in inflamed ileum. H-7 and chelerythrine partially inhibited the methacholine response in normal ileum but had no significant effect in inflamed ileum. Ryanodine stimulated phasic contractions that were blocked by TTX, hexamethonium, atropine, or ruthenium red. Ruthenium red, however, had no significant effect on the contractile response to methacholine. Conclusions: 1) Ca2+ influx through the L-type channels may be the primary source of Ca2+ to stimulate in vivo phasic contractions. 2) Phosphatidylinositol hydrolysis enhances the stimulation of in vivo phasic contractions in the normal ileum. In the inflamed ileum, phosphatidylinositol hydrolysis may be essential to stimulate phasic contractions. 3) Inflammation may downregulate the protein kinase C pathway. 4) Ryanodine stimulates phasic contractions by the release of ACh.

gastrointestinal motility; smooth muscle; calcium; protein kinase C; ryanodine; ruthenium red; neomycin; H-7; W-7; chelerythrine; inflammation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AN INCREASE in intracellular Ca2+ concentration ([Ca2+]i) is an essential step for smooth muscle cells to contract. The increase in [Ca2+]i is part of a series of signal-transduction steps that result in the phosphorylation of contractile proteins. The two sources of Ca2+ to increase cytosolic concentration are the extracellular medium and the rapidly exchanging intracellular stores.

The gut smooth muscle generates at least three different types of contractions to perform the complex motility functions of mixing and propulsion: 1) rhythmic phasic contractions, 2) giant migrating contractions (GMCs), and 3) tone. The rhythmic phasic contractions produce mixing and net slow distal propulsion of chyme in the postprandial state and of residual food and secretions in the fasting state (31). The GMCs produce mass movements (29). The precise role of the generation of tone in circular muscle cells is not known, but the resulting decrease in the diameter of the gut may enhance the efficiency of the phasic contractions in mixing and propulsion. The time course, frequency, and force generated by each of the above three types of contractions are significantly different from each other. The phasic contractions occur rhythmically at a few cycles per minute in different species, last for about 3-5 s, and generate a moderate force (~75-100 g), and their occurrence in time and space is controlled by slow waves (30, 37). The GMCs occur infrequently (about once or twice a day), last for about 20 s, and generate a very strong force (>150 g), and these contractions are independent of slow waves (29). The tone is normally of small to moderate force; it is independent of slow waves and it can last for prolonged periods of time (several minutes to hours). It is remarkable that, using a limited number of second messengers, the same smooth muscle cells can generate so many different types of contractions. Our hypothesis is that the signal transduction in smooth muscle cells to stimulate different types of contractions is different. A significant amount of work has been done to identify the signal-transduction pathways to generate tone in dispersed small intestinal smooth muscle cells (4, 5, 13, 16, 20, 23, 24). However, very little is known about the signal-transduction pathways for the stimulation of in vivo phasic contractions and GMCs.

The two major effects of inflammation on small intestinal motility are the suppression of phasic contractions and the stimulation of GMCs (1, 2, 17). The suppression of phasic contractions compromises the mixing and slow orderly distal propulsion of the ingested meal, whereas the stimulation of GMCs produces frequent mass movements that are associated with diarrhea and abdominal cramping (2, 17, 34). The inflammatory response has been shown previously to alter the enteric neuronal function (6, 10) as well as the characteristics of slow waves in smooth muscle cells (21). The effects of inflammation on signal transduction in small intestinal circular smooth muscle cells are not known.

We investigated the hypothesis that the influx of Ca2+ through L-type channels is the primary source for the stimulation of in vivo ileal phasic circular muscle contractions by muscarinic receptor activation. The hydrolysis of phosphatidylinositol (PI) by muscarinic receptor activation enhances these contractions. Our second hypothesis is that inflammation modulates signal-transduction pathways to suppress the phasic contractions in ileal circular smooth muscle cells. These hypotheses were tested in conscious dogs by close intra-arterial infusions of test substances in short segments of the ileum. We chose methacholine, a stable analog of ACh, as the agonist to stimulate phasic contractions. ACh is the physiological neurotransmitter of spontaneous small intestinal phasic contractions at the neuroeffector junction. Atropine, a nonspecific muscarinic receptor antagonist, completely blocks spontaneous phasic contractions in the conscious state (25, 32).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Surgical procedure. The experiments were performed on 11 healthy conscious dogs of either sex. Each dog weighed 20-29 kg (mean wt 24 ± 1.7 kg). The dogs were anesthetized with 30 mg/kg pentobarbital sodium (Abbott Laboratories, Chicago, IL). Access to the abdominal cavity was obtained by a midline ventral laparotomy. An intraluminal catheter (2.6 mm ID, 4.9 mm OD) was implanted 158 ± 4 cm proximal to the ileocolonic junction to infuse ethanol and acetic acid for the induction of inflammation, as described below. A stainless steel fistula was implanted 20 cm proximal to the ileocolonic junction to drain ethanol and acetic acid and prevent them from reaching the colon.

Two mesenteric arteries were identified in the segment between the intraluminal catheter and the fistula. The arteries were freed completely from the mesentery, preserving the nerves. A Silastic catheter (0.75 mm ID, 1.63 mm OD) was inserted in the centripetal direction in a branch artery so that its tip rested 1-2 mm from the junction of the branch artery and the main artery. The boundaries of the perfused segment were identified by infusing saline at 15-20 ml/min for 10-15 s. The segment refilled with blood within 2-3 s after the end of the infusion. Infusion of saline at 1 ml/min for up to 10 min produced no apparent change in color of the segment and did not stimulate any contractions. The length of the infused segment was limited to 5-6 cm by ligating the secondary branch arteries, if necessary. The catheters were secured by ties to the branch artery and the mesentery.

One electrode-strain-gauge pair and two strain-gauge transducers were attached to the seromuscular layer in the infused segment by 3-O Surgilon sutures (Davis & Geck, Danbury, CT). An additional strain-gauge transducer was attached to the seromuscular layer 10 cm distal to each infused segment. All transducers were oriented to record circular muscle contractions.

The intraluminal and intra-arterial catheters were exteriorized subcutaneously in the subscapular region. The catheters were housed in jackets that the dogs wore at all times. Each intra-arterial catheter was flushed twice daily with 2,000 IU of heparin. The dogs were allowed 5 days to recover from surgery.

Experimental protocol. All experiments were performed in the conscious state after an overnight fast. At least one phase III activity was recorded to establish the fasting state. The contractile and electrical signals were recorded on a 12-channel Grass recorder (model 7D; Grass Instruments, Quincy, MA), with lower and upper cut-off frequencies set at direct current and 15, 0.1, and 15 Hz, respectively.

All test substances were infused at 1 ml/min during phase I or a quiescent period during phase II activity of the migrating myoelectric/motor complex cycle. Preliminary experiments were done to establish the duration at which infusion of each test substance was maximally effective. The agonist, methacholine, was infused for 1 min. The antagonists were infused for 1-, 5-, or 10-min periods. When an antagonist was infused for 1 min, methacholine was infused about 2 min after the end of the antagonist infusion. When an antagonist was infused for 5- or 10-min periods, the 1-min infusion of methacholine started 2 or 5 min after the beginning of the antagonist infusion, respectively. A waiting period of at least 30 min was allowed between successive infusions. Preliminary experiments indicated that the responses to repeated infusions after this interval were the same. All experiments were done first in the normal state and then during inflammation.

Induction of inflammation. Ileal inflammation was induced by mucosal exposure to ethanol and acetic acid, as described previously (17). Briefly, 75 ml of 95% ethanol were infused intraluminally on day 1. The same amount of ethanol was infused on days 3 and 5, followed 1 h later by infusions of 50 ml of 20% acetic acid. Mucosal exposure to ethanol and acetic acid induces an inflammatory response that lasts for about 10 days (17). Myeloperoxidase activity and neutrophil infiltration are increased in both the mucosa and the muscle layers (17). The experiments in the inflamed state were done on days 3 and 4 and days 6-9 after the first exposure to ethanol. Shi and Sarna (34) reported recently that there is no difference in response to methacholine on days 3 and 6 of inflammation. The performance of experiments with different antagonists was distributed randomly during the period of inflammation on days 3 and 4 and days 6-9.

Test substances. The following substances were used: methacholine, verapamil, neomycin, ruthenium red, TTX, and hexamethonium, all dissolved in 0.9% saline; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and chelerythrine, dissolved in sterile water and diluted in 0.9% saline; N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), dissolved in DMSO and diluted in sterile water; and ryanodine, dissolved in ethanol and diluted in 0.9% saline. All of these substances were purchased from Sigma Chemical (St. Louis, MO). Atropine sulfate was purchased from Lymphomed (Deerfield, IL) and dissolved in 0.9% saline. The infusion of vehicle alone had no effect on the contractile activity.

Data analysis. The contractile response was quantified as the area under contractions (WINDAQ/EX program; DATAQ Instruments, Akron, OH). The area under contractions was measured from the beginning of the first contraction after the start of infusion to the point at which the tracing returned to the baseline and contractions ceased to occur.

All data are means ± SE. The n value represents the number of dogs. Statistical analysis was done by analysis of variance with repeated measures. Multiple comparisons were done by Student-Newman-Keul's test. P <=  0.05 was considered statistically significant. This study was approved by the Animal Studies Subcommittee at the Zablocki Veterans Affairs Medical Center.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Inflammatory modulation of the ileal phasic contractile response to methacholine. Close intra-arterial infusions of methacholine (0.05-8 nmol · ml-1 · min-1 for 1 min) stimulated a series of phasic contractions in a dose-dependent fashion. Inflammation significantly reduced the response to methacholine (Fig. 1A; F = 17.2, degrees of freedom = 69, P < 0.001). The half-maximal effective dose during inflammation, 1.7 ± 0.33 nmol, was significantly greater than that in the normal state, 0.55 ± 0.26 nmol. The dose of methacholine (4 nmol · ml-1 · min-1 for 1 min) that produced nearly maximum response was used in the following experiments.


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Fig. 1.   A: methacholine dose dependently increased the phasic contractile response in both normal and inflamed ileum. Response in inflamed ileum was significantly less than that in normal ileum. B: contractile response to methacholine was not affected significantly by prior close intra-arterial infusions of TTX and hexamethonium but was blocked completely by atropine in both normal and inflamed ileum.

The contractile response to methacholine was not affected significantly by prior close intra-arterial infusions of TTX (75 nmol · ml-1 · min-1 for 1 min) or hexamethonium (70 µmol · ml-1 · min-1 for 1 min), but it was blocked completely by prior infusion of atropine (150 nmol · ml-1 · min-1 for 1 min) (Fig. 1B; n = 5). These doses of TTX, hexamethonium, and atropine have been reported previously to effectively block Na+ channel enteric neural conduction, nicotinic receptors, and muscarinic receptors, respectively (11, 32). Thus methacholine, given close intra-arterially, acted primarily on muscarinic receptors on circular smooth muscle cells in the normal and the inflamed ileum to stimulate phasic contractions.

Role of Ca2+ influx through L-type channels and PI hydrolysis in stimulating the phasic contractile response to methacholine in normal and inflamed ileum. Close intra-arterial infusions of verapamil, an L-type Ca2+ channel blocker (0.1-800 nmol · ml-1 · min-1 for 5 min, n = 6; Fig. 2), and of neomycin, an antagonist of phospholipase C (PLC; 1-12 µmol · ml-1 · min-1 for 10 min, n = 5; Fig. 3), dose dependently inhibited the phasic contractile response to methacholine in both the normal and the inflamed ileum. In each case, the response was expressed as a percentage of the control response to methacholine in the normal or the inflamed ileum. The absolute value of the control response in the inflamed ileum, however, was less than that in the normal ileum (Fig. 1A). At the maximum dose, verapamil blocked the response almost completely in both the normal and the inflamed ileum. There was no significant difference between the half-maximal inhibitory dose values of verapamil between the normal and the inflamed states (42.4 ± 20.2 and 23.0 ± 16 nmol · ml-1 · min-1 for 5 min, respectively). In normal ileum, the highest dose of neomycin inhibited the response by ~50%. In the inflamed ileum, the same dose of neomycin completely inhibited the contractile response to methacholine (Fig. 3).


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Fig. 2.   Close intra-arterial infusions of verapamil dose dependently inhibited the phasic contractile response to methacholine in normal and inflamed ileum. In both states, response to methacholine with saline infusion was taken as 100%. There was no significant difference between the two inhibitory dose-response curves.


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Fig. 3.   Close intra-arterial infusion of neomycin dose dependently inhibited the contractile response to methacholine in normal and inflamed ileum. Inhibition at maximum dose was partial in normal ileum but complete in inflamed ileum. Inhibition was significantly greater in inflamed than in normal ileum (F = 4.37, degrees of freedom = 29, P = 0.0075). Control response to methacholine in normal and inflamed ileum was taken as 100%.

The infusion of H-7 (5-200 nmol · ml-1 · min-1 for 5 min) or chelerythrine (5-400 nmol · ml-1 · min-1 for 5 min), inhibitors of protein kinase C (PKC), dose dependently inhibited the phasic contractile response to methacholine in the normal ileum (Fig. 4). The inhibition at the maximum dose was about 40% of the control response. In the inflamed state, H-7 and chelerythrine in the same dose range had no significant effect on the contractile response to methacholine (Fig. 4).


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Fig. 4.   H-7 ( A) and chelerythrine ( B) dose dependently inhibited the contractile response to methacholine in normal ileum. In inflamed ileum, they had no significant effect. Control response to methacholine in normal and inflamed ileum was taken as 100%.

The role of ryanodine-sensitive intracellular Ca2+ stores and calmodulin in the contractile response to methacholine in normal and inflamed ileum. Close intra-arterial infusion of ryanodine at 20 nmol · ml-1 · min-1 for 5 min stimulated a series of phasic contractions in the normal ileum (n = 4). The mean maximum amplitude of these contractions was 23 ± 4% of the maximum amplitude of phasic contractions stimulated by methacholine. However, the total duration of occurrence of ryanodine-induced contractions (405 ± 120 s) was about threefold longer than that of methacholine-induced contractions (128 ± 13 s). As a result, the total area under ryanodine-induced contractions was not significantly different from that under methacholine-induced contractions (Fig. 5A). The response to ryanodine began 265 ± 60 s after the start of infusion, as opposed to 24 ± 4 s for methacholine (P < 0.05). The contractile response to ryanodine was blocked completely by a 5-min concurrent infusion of 1.0 nmol · ml-1 · min-1 ruthenium red, an antagonist of ryanodine receptors. However, the 5-min infusion of ruthenium red in the dose range of 0.5-1.5 nmol · ml-1 · min-1 had no significant effect on the contractile response to methacholine (Fig. 5B). The contractile response to ryanodine was also blocked by TTX, atropine, and hexamethonium infusions (data not shown). Ryanodine did not stimulate phasic contractions in the inflamed ileum (Fig. 5A).


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Fig. 5.   A: close intra-arterial infusion of ryanodine (hatched bars) stimulated a phasic contractile response in normal ileum that was not significantly different from response to methacholine (open bars; P > 0.05). Response to methacholine was suppressed significantly in inflamed ileum. However, ryanodine hardly stimulated any contractions in inflamed ileum. Response to combined infusions of ryanodine and methacholine (stippled bars) was slightly greater than that to methacholine alone in both normal and inflamed ileum, but they were not significantly different (P > 0.05). However, response to combined infusions in inflamed ileum was significantly less than that in normal ileum (* P < 0.05). B: ruthenium red had no significant effect on contractile response to methacholine. Control response to methacholine in normal and inflamed ileum was taken as 100%.

The contractile response to close intra-arterial infusion of methacholine during a concurrent infusion of ryanodine in normal and inflamed ileum exhibited a slight increase, but it was not statistically significant (Fig. 5A).

The infusion of W-7, a calmodulin antagonist, at 0.4 or 1.6 µmol · ml-1 · min-1 for 5 min had no significant effect on the phasic contractile response to methacholine in the normal or the inflamed ileum (data not shown). Infusion of W-7 at 1.6 µmol · ml-1 · min-1 for 5 min has been reported previously to inhibit the contractile response to 5-hydroxytryptamine (11).

Relationship between myoelectrical activity and phasic contractions in normal and inflamed ileum. Each spontaneous phasic contraction in the ileum was associated with a slow wave and a spike burst (Fig. 6B). Only one spike burst occurred in each slow wave cycle. No significant contractile activity was recorded when spike bursts were absent (Fig. 6A; n = 6). The methacholine-induced phasic contractions were related similarly to the spike bursts and slow wave cycles (Fig. 6C). This 1:1:1 relationship among phasic contractions, spike bursts, and slow wave cycles did not change during inflammation. Inflammation had no significant effect on the frequency of slow waves (15.7 ± 0.2 vs. 15.9 ± 0.2 cycles/min in normal and inflamed ileum, respectively; n = 6, P > 0.05).