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Am J Physiol Gastrointest Liver Physiol 292: G734-G745, 2007. First published November 22, 2006; doi:10.1152/ajpgi.00428.2006
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NEUROREGULATION AND MOTILITY

Do gap junctions play a role in nerve transmissions as well as pacing in mouse intestine?

E. E. Daniel, Ahmed El Yazbi, Marco Mannarino, Gary Galante, Geoffrey Boddy, Jonathan Livergant, and Tahereh Eteraf Oskouei

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada

Submitted 15 September 2006 ; accepted in final form 11 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Varicosities of nitrergic and other nerves end on deep muscular plexus interstitial cells of Cajal or on CD34-positive, c-kit-negative fibroblast-like cells. Both cell types connect to outer circular muscle by gap junctions, which may transmit nerve messages to muscle. We tested the hypotheses that gap junctions transmit pacing messages from interstitial cells of Cajal of the myenteric plexus. Effects of inhibitors of gap junction conductance were studied on paced contractions and nerve transmissions in small segments of circular muscle of mouse intestine. Using electrical field stimulation parameters (50 V/cm, 5 pps, and 0.5 ms) which evoke near maximal responses to nitrergic, cholinergic, and apamin-sensitive nerve stimulation, we isolated inhibitory responses to nitrergic nerves, inhibitory responses to apamin-sensitive nerves and excitatory responses to cholinergic nerves. 18beta-Glycyrrhetinic acid (10, 30, and 100 µM), octanol (0.1, 0.3, and 1 mM) and gap peptides (300 µM of 40Gap27, 43Gap26, 37,43Gap27) all failed to abolish neurotransmission. 18beta-Glycyrrhetinic acid inhibited frequencies of paced contractions, likely owing to inhibition of L-type Ca2+ channels in smooth muscle, but octanol or gap peptides did not. 18beta-Glycyrrhetinic acid and octanol, but not gap peptides, reduced the amplitudes of spontaneous and nerve-induced contractions. These reductions paralleled reductions in contractions to exogenous carbachol. Additional experiments with gap peptides in both longitudinal and circular muscle segments after NG-nitro-L-arginine and TTX revealed no effects on pacing frequencies. We conclude that gap junction coupling may not be necessary for pacing or nerve transmission to the circular muscle of the mouse intestine.

nitric oxide; adenosine 5'-triphosphate; inhibitory nerve transmission; cholinergic nerve transmission; interstitial cells of Cajal; pacing of intestinal muscle

IN THE MOUSE INTESTINE, structural evidence suggests that few or no gap junctions connect the interstitial cells of Cajal (ICC) of the myenteric plexus (ICC-MP) with either the circular (CM) or the longitudinal (LM) muscles (11, 12, 17, 49, 50, 59). The ICC-MP are considered responsible for pacing the muscle slow waves (23, 37, 38). They are coupled to one another by gap junctions, as are the CM cells, but none have been found coupling LM cells to one another (1, 11, 12, 17, 32, 36, 49, 50, 53, 59, 60–62). Nevertheless, gap junctions are assumed to be essential for coupling between slow waves in ICC-MP and both muscle layers. Gap junctions are also assumed to be required for electrical coupling within the muscle layers (14, 22, 32, 37, 38) and other components of the pacing and nerve transmission systems in the gastrointestinal system (13, 21, 22, 47).

There is another network of ICC in the mouse intestine, located at the deep muscular plexus (DMP; ICC-DMP). These ICC are more closely associated with enteric nerve endings than are smooth muscle cells (11, 12, 15, 17, 24, 36, 38, 49, 50, 58, 60–62). Evidence has been presented that neurokinin 1 receptors on these ICC are activated or downregulated by enteric nerve stimulation (14, 15, 24, 25, 34, 36, 61, 62). Other receptors for neurotransmitters are also present on these cells (14). Because they are connected to outer CM by gap junctions (11, 12, 17, 36, 49, 50, 61, 62), the question arises; are these gap junctions necessary for neurotransmission to smooth muscle from enteric nerves? In addition to cholinergic nerves, which also contain substance P, there are nitrergic nerves and nerves that release ATP and other mediators in the DMP (36, 38, 60–62). ATP acts mainly to activate SK3 (small conductance Ca2+ activated K+) channels (4, 27, 52, 53, 60–62). However, the target of innervation of these nerves does not appear to be the ICC-DMP, but rather fibroblast-like cells, which are SK3, CD34 positive, but c-kit (marker for ICC) negative (52, 53). They, too, are connected to outer CM cells by gap junctions. So the question arises, is coupling by gap junctions necessary for spread of nerve signals from enteric nerves in the DMP to CM?

Recently, it was reported that a gap junction uncoupler, 18beta-glycyrrhetinic acid at 10–30 µM, inhibited the coupling of Ca2+ oscillations and slow waves between ICC-MP and LM and inhibited dye coupling in ICC (32). However, this study did not rule out the possibility that 18beta-glycyrrhetinic acid acted by other mechanisms, not involving gap junctions. On the other hand, many references evaluating dye or electrical coupling support the ability of 18beta-glycyrrhetinic acid and related agents to inhibit gap junctions, including dye coupling; a few are noted here (2, 3, 7, 19, 28, 31, 63).

However, 18beta-glycyrrhetinic acid is known to have numerous non-gap junction-related effects and these may have contributed to the observed uncoupling (48). At 10 µM, it markedly inhibits inward currents through L-type Ca2+ channels in isolated mouse jejunal smooth muscle and at 50 µM abolishes them. It also inhibits contractions of jejunal smooth muscle strips at 10 µM, presumably by inhibiting L-type Ca2+ channels, but it does not, at 30 µM, abolish or reduce the frequency of slow waves even though it reduces their amplitudes. In the presence of apamin, it increases tetraethyl-ammonium-sensitive currents through large-conductance Ca2+-activated K+ channels. At 10 µM, it also decreases delayed-rectifier K+ current, especially at positive membrane potentials and possibly also inhibits A-type outward currents.

Earlier, we showed that a gap junction uncoupler, carbenoxolone, did not affect pacing in the mouse or canine intestine except modestly at a concentration that had other actions on non-gap junction-mediated contractions (39). Carbenoxolone is the water-soluble hemisuccinate of 18{alpha}-glycyrrhetinic acid, chemically related to 18beta-glycyrrhetinic acid.

Other widely used inhibitors of gap junctions are the alcohols heptanol and octanol. Octanol has been widely used and has been shown to block electrical and dye coupling in various cell types (1, 5, 9, 16, 28, 31, 33, 35), but it failed to abolish slow waves in canine intestine CM (11). Like the glycyrrhetinic acid derivatives, octanol also affects ion channels, including Ca2+, Na+, and Cl– channels (9, 35).

The most selective inhibitors of gap junction coupling are peptide analogs of epitopes in the extracellular domain of connexins involved in gap junctions (6, 7, 17, 26). In smooth muscle of the intestine, connexins 43 and 40 have been found (8, 12, 59), and in other smooth muscles connexin 37 is also present (20). Therefore we used peptide analogs which recognize the extracellular domains of each of these connexins. These have also been shown to inhibit dye and electrical coupling through gap junctions (6, 7, 17, 26) and to inhibit the coupling between endothelial cells and arterial smooth muscle (6, 17, 26).

To answer the question "Is coupling by gap junctions necessary for spread of pacing from ICC-MP and nerve signals from enteric nerves in the DMP to CM?," we evaluated the effects of these three gap junction uncouplers on ICC-paced contractions and nerve responses in CM of the mouse intestine. We developed approaches to determine whether effects on neurotransmission were due to block of gap junction coupling or to another action, described later.

Three main nerve mediators are released from nerves in the DMP: nitric oxide (NO), acetylcholine, and ATP (4, 38, 52, 53, 61, 62). Peptide mediators such as substance P, pituitary adenylate cyclase-activating polypeptide, and VIP (10, 25, 29, 30, 51) are released at higher frequencies of electrical field stimulation (EFS) of enteric and autonomic nerves. We chose EFS stimulus parameters (50 V/cm, 0.5 ms, 5 pps) that minimize the release of peptide mediators. We also set up three protocols so that a single one of the three major mediators was acting: acetylcholine, after block of NO productions with L-NNA and ATP action with apamin; NO, after block of acetylcholine action with atropine and ATP action with apamin; and ATP, after block of action of acetylcholine with atropine and block of NO release with NG-nitro-L-arginine (L-NNA). Thus our aim was to establish whether the actions of any or all of these three mediators as well as ICC-mediated pacing depended on gap junction coupling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experiments involving the use of animals were conducted according to a laboratory animal protocol approved by our institutional Animal Policy and Welfare Committee.

Preparation of the tissue. Male 6- to 8-wk-old BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were killed by cervical dislocation. After the abdominal wall was opened, the gastrointestinal tract, from the stomach to the rectum, was removed from the mouse and immediately placed into a beaker of Krebs-Ringer solution containing (in mM) 115.5 NaCl, 21.9 NaHCO3, 11.1 D-glucose, 4.6 KCl, 1.16 MgSO4, 1.16 NaH2PO4, and 2.5 CaCl2, at room temperature (21–22°C), and preequilibrated with carbogen (95% O2 and 5% CO2). In a dissection dish filled with Krebs-Ringer solution and continuously bubbled with carbogen, small intestinal tissue was isolated and cut into ~0.5 cm CM segments. To study the CM contractile activity, the open side of a thin metal triangle was slid through the lumen of the tissue segment. The triangle was then hooked together. A stainless steel rod attached to the bottom of the electrode holder was inserted into the lumen of the tissue under the metal triangle. Silk suture thread, attached to the apex of the triangle opposite to the tissue, was tied to a force transducer (Grass FT-03). Two thin platinum rods, situated on either side of and parallel to the tissue, were used for the electrical stimulation of the tissue. The muscle preparations were placed in muscle baths filled with Krebs-Ringer solution, continuously bubbled throughout the experiment with carbogen, and maintained at a temperature of 37°C. The tension on the tissue was increased or decreased slowly until the tension which produced the maximum phasic activity was reached (about 0.5 g). Tissue contractile activities were recorded on a Grass model 7D polygraph.

In some experiments, involving both segments of LM and CM, enteric nerves of the tissue preparations were stimulated using EFS from a Grass S88 (Grass Instruments, Quincy, MA) stimulator set at 50 V/cm, 5 pulses/s, and 0.5-ms pulse duration to observe the characteristic contractile response of the tissue. L-NNA (10–4 M), an inhibitor of NO synthase, was added to each bath to prevent NO-mediated inhibitory nerve activity. The L-NNA was allowed to incubate with the tissue for 5 min. Immediately following incubation of the tissue with L-NNA 10–6 M TTX was added to each bath to block all enteric nerve activity. EFS was performed 5 min after addition of TTX: a lack of response to a second EFS was interpreted as being due to total blockade of enteric nervous system activity and signified the fact that the muscle was entirely under intrinsic control. Preparation of CM segments was described above. Longitudinal segments were prepared as previously described (39). In brief, segments 1 to 1.5 cm long were prepared by tying off both ends of the segments and attaching one end to the electrode holder and the other to the strain gauge. It should be noted that in LM segments, as the ends were tied, peptides were not exposed to intestinal mucosa and their peptidases.

Experimental protocols. All tissues were allowed to recover for 30 min. After recovery, some tissues were equilibrated in the organ bath with a combination of L-NNA (10–4 M) and apamin (10–6 M) for 15 min to study effects on cholinergic neurotransmission. To study the responses mediated by NO, other tissues were equilibrated in the organ bath with a combination of atropine (10–7 M) and apamin (10–6 M) for 15 min. To study the responses mediated by ATP, additional tissues were equilibrated in the organ bath with a combination of L-NNA (10–4 M) and atropine (10–7 M) for 15 min. EFS for a duration of 10 s (parameters: 50 V/cm and 0.5-ms pulse duration at 5 pps) was carried out. Figure 1 shows the responses typical of each mediator, pure excitation for acetylcholine (Fig. 1A), pure inhibition for NO (Fig. 1B), and initial inhibition followed by some recovery during the EFS for ATP (Fig. 1C). In some cases, all three agents (atropine, L-NNA, and apamin) were added to ensure that there was no significant response to EFS (data not shown). In other cases, two of the three were added, EFS was tested, and then the third was added to show that the response was inhibited (data not shown).


Figure 1
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  		Fig. 1. Representative tracings of 10 s of electrical field stimulation (EFS) at 50 V/cm, 5 pps, with square waves of 0.5-ms duration. A: after 10–4 M NG-nitro-L-arginine (L-NNA) and 10–6 M apamin, leaving primarily acetylcholine-mediated neurotransmission. B: after 10–7 M atropine and apamin, leaving primarily nitric oxide (NO)-mediated neurotransmission. C: after atropine and L-NNA, leaving primarily ATP-mediated neurotransmission at the deep muscular plexus. With acetylcholine as mediator there was pure excitation in A. NO as mediator produced pure and sustained inhibition in B. ATP as mediator produced initial inhibition that was not sustained in C. Responses in A were inhibited by atropine, those in B by L-NNA, and those in C by apamin (data not shown).

 
The gap junction uncouplers octanol and 18beta-glycyrrhetinic acid were given at three different concentrations, each to a separate segment of CM. For octanol the concentrations were 0.1, 0.3, and 1 mM. For 18beta-glycyrrhetinic acid the concentrations were 10, 30, and 100 µM. Responses to EFS were tested before addition of inhibitor and at the end of the exposure period. In experiments in which acetylcholine was acting, carbachol at a just maximal concentration of 10–5 M was applied at the end or at the beginning and at the end of the experiment. When atropine was used in experiments to evaluate actions on NO- or ATP-mediated contractions, 60 mM KCl was applied instead of carbachol.

In experiments with mimetic peptides, three (see Materials below) were given together, each at a concentration of 300 mM. In some experiments, a scrambled version of the peptide corresponding to loop 1 of connexin 43 was applied ion addition to the 900 µM combination of three peptides corresponding to loop 2 of connexin 40 and loops 2 of connexins 43 and 37. After recovery, CM segments were exposed to 10–7 M atropine for 15 min and responses to EFS were tested. After atropine, the responses to EFS were always simple inhibition. Then, in the presence of atropine, segments were incubated with either a combination of peptides (see Materials) or a corresponding volume of DMSO for 2 h, with responses to EFS, contraction amplitudes and frequencies measured every 15 or 30 min for the duration of the experiments. In each experiment, two CM segments were mounted simultaneously, one of each tissue type serving as a time control, while the other was treated with the peptides. In some experiments, LM as well as CM segments were used. They were treated similarly but received TTX and L-NNA before exposure to peptides.

At the end of each experiment, all tissues were washed twice with 10 ml Ca2+-free Krebs-Ringer solution with 1.0 mM EGTA to relax the tissues to basal passive tension and abolish spontaneous contractions and tone. All contraction or tone amplitude measurements were made relative to the basal passive tension, which was determined for each individual tissue.

Data analysis. Frequencies of paced contractions were measured after the block of all but one mediator had occurred and again after each application of gap junction uncouplers. Frequencies were measured over at least 20 s. Amplitudes of spontaneous contractions and the response to EFS were similarly measured as the values above the passive tension determined at the end of the experiments. They were measured as the mean of at least 10 individual contractions, except that during evaluation of acetylcholine-mediated responses the contractions that occurred during EFS (6 to 8) were averaged. When NO- or apamin-mediated responses were studied, the relaxations of spontaneous tone (not including spontaneous contractions) obtained relative to the passive tension were measured. Changes in contraction amplitude were normalized to both the initial and the final amplitudes of spontaneous contractions or left without being normalized. The measurements were entered into GraphPad Instat 3 and statistically tested with either ANOVA with Bonferroni post hoc tests or paired or unpaired t-test as appropriate. When small samples were studied, nonparametric analysis was applied. A P value <0.05 was considered to be statistically significant. The n values represent the number of mice whose intestine provided segments for study.

Materials. Stock solutions of 10–2 M L-NNA and 10–3 M TTX were premade and kept refrigerated and frozen, respectively. TTX was obtained from Alomone Laboratories (Jerusalem, Israel). Atropine sulfate, apamin, L-NNA, 18beta-glycyrrhetinic acid, and octanol were purchased from Sigma (Oakville, ON, Canada). Dimethyl sulfoxide and EGTA were purchased from Caledon Laboratories (Georgetown, ON, Canada). Apart from apamin, 18beta-glycyrrhetinic acid, and octanol, double-distilled water was used to dissolve the drugs used in experiments in this study. Apamin was dissolved in 0.05 M acetic acid. 18beta-Glycyrrhetinic acid was dissolved in DMSO and diluted before use so that the final concentration of DMSO was less that 0.1%. Octanol was dissolved in ethanol and diluted before use so that the final concentration of ethanol was less than 0.1%. DMSO, ethanol, and 0.05 M acetic acid did not affect the functional activity of the tissue in the amounts added.

Four peptides were employed in this study: 1) A peptide corresponding to extracellular loop 2 of connexin 40. This peptide, 40Gap27, had the sequence SRPTEKNVFIV, with a corresponding molecular weight of 1,289.5 Da. 2) A peptide corresponding to extracellular loop 2 of connexins 37 and 43. This peptide, 37,43Gap27, had the sequence SRPTEKTIFII, with a corresponding molecular weight of 1,304.6 Da. 3) A peptide corresponding to extracellular loop 1 of connexin 43. This peptide, 43Gap26, had the sequence VCYDKSFPISHVR, with a corresponding molecular weight of 1,549.8 Da. 4) A scrambled version of peptide 3 with the sequence KVDSCYSPFIVHR. This peptide had a molecular weight identical to that of peptide 3.

These peptides are identical in sequence to the peptides that have been successfully employed to block gap junctional intercellular communication individually and collectively in other published studies (6, 7, 17, 26). Solutions of peptides were made up in DMSO, aliquoted, and frozen until before use. All peptides were obtained from Dalton Chemical Laboratories (Toronto, ON).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Actions of 18beta-glycyrrhetinic acid. Three of the four CM segments were exposed to 18beta-glycyrrhetinic acid for 5 min, at 10, 30 or 100 µM. The additional segment served as a time control. After 15 min, EFS was applied again and then either 10–5 M carbachol or 60 mM KCl was applied. Finally, all tissues were washed twice in Ca2+-free Krebs-Ringer solution with 1 mM EGTA, to determine the passive tension. Measurements of frequencies and amplitudes (relative to the passive tension) of contraction and responses to EFS were made after blocking agents and after the actions of 18beta-glycyrrhetinic acid were complete. Contractile responses to carbachol and KCl were also determined.

Representative traces of all experiments showing the effects of 10 and 100 µM 18beta-glycyrrhetinic acid on pacing and nerve transmission are shown in Fig. 2. Note the decrease in frequencies and amplitudes of paced contractions by both concentrations of 18beta-glycyrrhetinic acid, greater at 100 than at 10 µM. However, in all cases relaxations to nitrergic and apamin-sensitive mediators and contractions to cholinergic mediation persisted. In the case of relaxations, the level of tone achieved did not change. In the case of cholinergic contractions, the amplitudes were reduced but they were not abolished.


Figure 2
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  		Fig. 2. Response to EFS in control segments before and 15 min after exposure to 10 or 100 µM 18beta-glycyrrhetinic acid (18betaGR). Top: effects of this agent when acetylcholine was the mediator. Note that, although paced contractions were small or absent, EFS still activated contractions in the presence of both concentrations of 18betaGR. Middle: when NO was the mediator, relaxation still occurred at both concentrations of 18betaGR, even though there were no paced contractions. Bottom: when ATP was the main mediator, initial relaxations still occurred after both concentrations of 18betaGR, even though there were no paced contractions. EFS durations were ~10 s, initiated at the triangles.

 
A summary of the effects of 18beta-glycyrrhetinic acid on frequencies of contractions with all combinations of agents is shown in Fig. 3. In controls, all mean frequencies were in the range of 26–31 cpm, typical of jejunum. With all three combinations of blocking agents, 18beta-glycyrrhetinic acid significantly inhibited frequencies of contractions concentration dependently, except when atropine and apamin were used. Then all concentrations had similar effects. When atropine and L-NNA were used together, 100 µM 18beta-glycyrrhetinic acid abolished paced contractions. P values from these experiments are summarized in Table 1.


Figure 3
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  		Fig. 3. Frequencies in time controls at 0 and 15 min (T0 and T15, respectively), were similar and unchanged in all 3 experimental conditions. A: frequency changes from 18betaGR in apamin and atropine. B: frequency changes from 18betaGR in L-NNA and atropine. C: frequency changes from 18betaGR in L-NNA and apamin. However, 18betaGR concentration dependently decreased them in all 3 conditions. Note that all 3 concentrations of 18betaGR can abolish or severely diminish activity of L-type Ca2+ channels. In this and subsequent figures, *, **, ***, and **** refer to P values <0.05, <0.01, <0.001, and <0.0001, respectively.

 

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  		Table 1. Significance of effects of 18beta-glycyrrhetinic acid on frequencies of contractions

 
When atropine and apamin blocked all but NO-mediated responses, EFS produced sustained relaxation often followed by an "off" response. 18beta-Glycyrrhetinic acid has no effect at any concentration on the proportion of spontaneous tone eliminated during EFS as summarized in Fig. 4. It reduced resting tone significantly (P = 0.014) only at 30 µM concentration.


Figure 4
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  		Fig. 4. Effects of 18betaGR on spontaneous tone in response to release of NO by EFS in the presence of atropine and apamin. Top: spontaneous tone during EFS was not significantly affected by 18betaGR compared with the time control, as also illustrated in Fig. 2. Moreover, the percentage reduction in spontaneous tone during EFS was not reduced by any concentration of 18betaGR except 30 µM, as shown in the middle panel. Bottom: responses to 60 mM KCl were not significantly reduced, even though L-type Ca2+ channels are known to be inhibited by 18betaGR. The release of peptide mediators by depolarization of nerve endings was not prevented and may account for these responses to KCl.

 
When L-NNA and atropine blocked all but responses to apamin-sensitive mediators, EFS produced inhibition of phasic and tonic contractions, but the inhibition was not maintained during EFS. As when NO was the mediator, 18beta-glycyrrhetinic acid did not inhibit the relaxation measured as the proportion of spontaneous tone eliminated during EFS as summarized in Fig. 5. It reduced the resting tone insignificantly at 100 µM of 18beta-glycyrrhetinic acid. Responses to KCl (Fig. 5) were reduced significantly only by 100 µM 18beta-glycyrrhetinic acid (P = 0.0313). High concentrations of 18beta-glycyrrhetinic acid failed to abolish contractions to depolarization, probably because KCl released peptides such as substance P from nerves to act on mechanisms other than L-type Ca2+ channels.


Figure 5
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  		Fig. 5. Effects of 18betaGR on responses to release, primarily, of ATP in the presence of atropine and L-NNA. Top: spontaneous tone was not significantly changed compared with the time control by any concentration of 18betaGR. Middle: percentage reduction in spontaneous tone during EFS was not significantly reduced by any concentration of 18betaGR compared with the time control. Bottom: 18betaGR concentration dependently reduced the responses to KCl. These may involve the release of peptide mediators as well as depolarization and opening of voltage-dependent Ca2+ channels by KCl.

 
When L-NNA and apamin blocked all but responses to acetylcholine, EFS increased tone and phasic contractions. 18beta-Glycyrrhetinic acid concentration dependently reduced the amplitudes of EFS-induced contractions whether measured in millimeters of chart paper (data not shown) or after amplitudes were normalized to the amplitudes of initial spontaneous contractions (Fig. 6). However, when the responses to EFS were normalized to the amplitudes of spontaneous contractions just before application of EFS, responses were less only at 100 µM 18beta-glycyrrhetinic acid. This was the same concentration that significantly reduced responses to 10–5 M carbachol (Fig. 6).


Figure 6
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  		Fig. 6. Effects of 18betaGR on responses to acetylcholine released to EFS in the presence of L-NNA and apamin. A: 18betaGR reduced, but did not abolish, the contractions to EFS at all concentrations of 18betaGR. When these data were normalized to the concurrent amplitude of spontaneous contractions (B), the responses to EFS were enhanced in controls and inhibited by 18betaGR only at 100 µM. C: responses to 10–6 M carbachol were concentration dependently inhibited by 18betaGR and, like the results in bottom left, the reductions were significant at 100 µM.

 
Actions of octanol. Octanol had no effect on frequencies of paced contractions at 0.1 and 0.3 mM. At 1 mM, octanol abolished paced contractions in most cases. It concentration dependently reduced the amplitudes of contraction before abolishing them (Fig. 7).


Figure 7
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  		Fig. 7. A: octanol had no effects on the frequencies of spontaneous contractions at 0.1 and 0.3 mM concentrations, although it concentration dependently reduced their amplitudes (not shown). At 1 mM concentration, octanol abolished spontaneous contractions in some cases and their frequencies could not reliably be measured. B: changes in amplitudes in response to various concentrations of octanol. Note that the values for amplitudes at 1 mM octanol reflect primarily the residual spontaneous tone rather than spontaneous contractions.

 
When exposure to atropine and apamin left responses to EFS predominantly due to NO, octanol at all concentrations had no effect on the proportion of spontaneous tone lost (Fig. 8), even though it inhibited paced contractions, as shown in Fig. 7. It did not abolish spontaneous tone even at 1 mM. Similarly, when atropine and L-NNA left responses to EFS primarily by ATP, octanol at all concentrations had no effect on the proportion of spontaneous tone lost (Fig. 9), even though it inhibited paced contractions.


Figure 8
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  		Fig. 8. A: increasing concentrations of octanol had no significant inhibition of residual tone during EFS releasing primarily NO when expressed as a percentage of the initial residual tone during EFS. B: effects of various concentrations of octanol on the amplitudes of NO-mediated relaxation to EFS. Note that there was no significant change, although at 1 mM the amplitudes were reduced.

 

Figure 9
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  		Fig. 9. A: increasing concentrations of octanol had no significant inhibition of residual tone during EFS releasing primarily ATP when expressed as a percentage of the initial residual tone during EFS. B: effects of various concentrations of octanol on the amplitudes of NO-mediated relaxation to EFS. Note that there was no significant change, although at 1 mM the amplitudes were reduced.

 
When L-NNA and apamin had abolished EFS-induced release of NO and responses to ATP, cholinergic responses were concentration dependently reduced but not abolished (Fig. 10A). However, when responses were normalized to the level of paced contractions or tone (at 1 mM octanol), there were no significant changes (Fig. 10C). Moreover, responses to carbachol, a proxy for the responses to acetylcholine, were also concentration dependently reduced by octanol (Fig. 10, B and D). Thus the changes in EFS-induced cholinergic responses were largely or completely due to inhibition of the response to cholinergic agonists.


Figure 10
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  		Fig. 10. A: cholinergic responses to EFS in the presence of L-NNA and apamin were reduced but not abolished by increasing concentrations of octanol. Inset: representative example of responses before and after 1 mM octanol. C: when the responses to cholinergic stimulation were normalized to the concurrent amplitudes of contraction, there were no significant changes in response. Moreover, as shown in B, responses to 10–6 M carbachol were also concentration dependently reduced by increasing concentrations of octanol. Inset in B: representative example of responses to carbachol before and after 1 mM octanol. D: normalization to the concurrent amplitudes of contraction reduced the apparent inhibitory effects of increasing concentrations of octanol on responses to carbachol.

 
Effects of gap peptides. The gap peptides, epitopes from extracellular loops of connexins 43, 40, and 37, each at 300 µM, were applied for 2 h to CM segments treated with atropine. After atropine, response to EFS resembled those after atropine and apamin (Fig. 1) but involved release of both NO- and apamin-sensitive mediators. Figure 11, top left, shows that 2 h of exposure to these peptides did not affect the frequency of paced contractions. Neither 1- nor 2-h exposure to these peptides affected the relaxations produced by EFS compared with time controls in DMSO (Fig. 11, bottom). The decrease in the amplitudes of paced contractions were similar in segments exposed to the peptides and to DMSO (Fig. 11, top right).


Figure 11
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  		Fig. 11. Effects of exposure of CM segments to 300 µM of each of 3 gap peptides for up to 2 h. Top left: there was no decrease in frequencies of paced contractions; indeed there was a significant increase in pacing frequency. Top right: there were similar decreases (~40%) in amplitudes of paced contractions in DMSO controls and in segments exposed to gap peptides. Bottom: at 1 h (left) and at 2 h (right), the percentage decreases in amplitude of contractions during EFS in the presence of atropine was similar in the presence of DMSO and in the presence of the 3 gap peptides.

 
After block of nerve function and NO synthase, the 900 mM combination of three peptides each at 300 mM produced results identical to those obtained without block of nerves and NO synthase on LM as well as CM segments. There were no significant changes in contraction frequency after 1 h in either peptide-treated or control tissues (Fig. 12). Although some tissues did show a drop in contraction amplitude over time, there were no significant differences in amplitude between the treated and control tissues (Fig. 12). In addition, the 1,200 mM combination of three active peptides, and a scrambled peptide, each at 300 mM produced results consistent with those obtained three peptides. No significant changes in frequency were noted over time, and contraction amplitudes in treated and control tissues were not significantly different from each other (Fig. 13).


Figure 12
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  		Fig. 12. A: a 900 µM combination of 3 peptides has no significant effect on the contraction frequency of circular (CM) and longitudinal (LM) smooth muscle segments when incubated with the tissues over a period of 60 min (CM and LM treated, respectively). Corresponding DMSO controls are shown and also demonstrate no significant change in contraction frequency over time; n = 11 for all groups. B: a 900 µM combination of 3 peptides including a scrambled peptide has no significant effect on the contraction frequency of CM and LM treated segments. The 3 peptides used were peptides corresponding to extracellular loop 2 of connexins 40 and 37/43, and a scrambled version of the peptide corresponding to extracellular loop 1 of connexin 43. Corresponding DMSO controls are shown and also demonstrate no significant change in contraction frequency over time; n = 7 for all groups.

 

Figure 13
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  		Fig. 13. A: effects of the 900 µM combination of 3 peptides (described previously) on the contraction amplitudes of CM and LM treated segments. Corresponding DMSO controls are also shown. In this case, neither the peptides nor DMSO had any significant effect on the contraction amplitude of LM segments. In contrast to the results shown in Fig. 6, both control and treated CM tissues showed a significant decline in amplitude after 30 and 60 min of incubation; n = 10 for all groups. B: effects of the 900 µM combination of 2 peptides plus a scrambled peptide on the contraction amplitudes of circular and longitudinal muscle segments (CM and LM treated, respectively). Corresponding DMSO controls are also shown. Neither the peptides nor DMSO had any significant effect on the contraction amplitude of LM segments. Both control and treated CM tissues showed a significant decline in amplitude after 30 and 60 min of incubation. In this case, there was also a significant difference between the contraction amplitude of the CM control segments at 30 and 60 min.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of gap junctions in pacing and in neurotransmission of the intestine has been controversial for many years (12). Structural evidence described in the Introduction suggests that few or no gap junctions connect ICC-MP to the CM but that gap junctions do connect ICC-DMP to outer CM. The limitations of structural evidence include the possibility of overlooking very small gap junctions and the fact that the presence of gap junctions does not guarantee that they are required (12, 41). However, from the structural information, we expected to find that inhibition of gap junction coupling did not affect pacing of contractions, but since nerve varicosities in the intestinal DMP (11, 12, 17, 49, 50, 60–62) and elsewhere in the gastrointestinal tract (12, 21, 47) appear to end on ICC that connect to CM by gap junctions, we expected to find that such inhibition interfered with nerve transmission.

To answer the questions about gap junction involvement in coupling, most investigators use uncouplers of gap junction functions as pharmacological tools. Interpretation of findings with these tools has always been complicated by their multiple other actions on ion channels and contractile function. Thus those who postulate a role for gap junctions in pacing or neurotransmission interpret positive findings with these agents as establishing their role (32). They reject negative findings (e.g., 39) in which uncoupling did not appear to inhibit pacing or neurotransmission by pointing to the inevitable side effects which are produced. With the introduction of the gap peptides, which have few side effects, the argument against findings with negative outcomes has become that the peptides did not penetrate to the sites of gap junctions.

In this study, we used three different gap junction uncouplers, each of which has been shown to be an effective uncoupler. One of our main findings is that no gap junction uncoupler inhibited frequencies of paced contractions except at concentrations that markedly inhibited the contractions themselves. In the case of both 18beta-glycyrrhetinic acid and octanol, this inhibition of contractions occurred at concentrations which have been shown to inhibit currents through L-type Ca2+ channels (48). Activation of these channels is necessary for intestinal muscle contractions (32, 37). Thus the decrease in frequency and amplitude of contractions with all concentrations of 18beta-glycyrrhetinic acid could be due either to block of gap junctions or to block of voltage-dependent Ca2+ channels. In the case of the gap peptides, there was no inhibition of contraction amplitudes beyond that in the time controls with DMSO and no inhibition of paced contraction frequency. This was true in experiments with and without nerve block and in LM segments with a closed lumen, in which peptides could not access mucosal peptidases, as well as in CM segments in which the lumen was open.

We think it likely, but cannot prove, that after 1 or 2 h the gap peptides had penetrated the relatively thin LM layer and reached the LM and ICC-MP. However, concentrations of octanol known to block gap junctions, about which there is no doubt of their ability to penetrate tissue and produce side effect on CM activity, also failed to inhibit the frequency of paced contractions. Moreover, the inhibition of paced contraction by 18beta-glycyrrhetinic acid occurred at concentrations known to inhibit L-type Ca2+ channels and contractions of mouse intestinal CM (47). We conclude that the balance of evidence fails to support the hypothesis that functioning gap junctions are required for pacing.

Gap junctions, even though present structurally, also do not seem to be required for transmission of nitrergic, cholinergic, or purinergic nerve messages from the DMP to the outer CM. No concentration of any gap junction uncoupler abolished neurotransmission, even though 18beta-glycyrrhetinic acid and octanol at higher concentrations inhibited both the amplitudes of paced contractions and responses to carbachol and KCl. In the case of the gap peptides, the possibility exists that they failed to penetrate the mucosa of the intestine or were degraded by intestinal peptidases. However, as with paced contractions, 18beta-glycyrrhetinic acid and octanol clearly reached the outer CM, where they exerted side effects, but failed to inhibit nerve transmission. This was a surprising result and a contradiction to our expectations. However, our findings are consistent with those in a recent study of electrical coupling in the mouse colon (40). Inhibitory junction potentials, both fast and slow, were not blocked by carbenoxolone, a glycyrrhetinic acid derivative, even though octanol and heptanol did abolish them, presumably by nonspecific actions. There appears to be no doubt that any gap junction electrical coupling function in the DMP was blocked with the concentrations of 18beta-glycyrrhetinic acid and octanol we applied. Thus these gap junctions may play another role in neurotransmission, acting to ensure proximity of either ICC or fibroblast to outer CM cells and/or transferring a stretch or other physical or chemical effect from one cell to the other.

These results raise the question, by what mechanisms are pacing and nerve messages transmitted? At this time there are no experimental answers, but few studies have looked for them. Some possibilities exist, e.g., field coupling can provide for electrical coupling (41–45, 54), including the possibilities provided by special structural relations among smooth muscle cells like peg and socket connections (50, 54, 56, 57), which activate stretch channels for electrical and mechanical coupling (50). The results of this study may provide an incentive for experimental evaluation of these and other possibilities (18, 42–46, 52).


   ACKNOWLEDGMENTS
 
Present address for T. E. Oskouei: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. E. Daniel, Rm. 9-10, Medical Sciences Bldg., Dept. of Pharmacology, Faculty of Medicine and Dentistry, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7 (e-mail: edaniel{at}ualberta.ca)

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.


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