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Am J Physiol Gastrointest Liver Physiol 281: G533-G543, 2001;
0193-1857/01 $5.00
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Vol. 281, Issue 2, G533-G543, August 2001

Gap junctions in gastrointestinal muscle contain multiple connexins

Y. F. Wang and E. E. Daniel

Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the canine gastrointestinal tract, the roles that gap junctions play in pacemaking and neurotransmission are unclear. Using antibodies to connexin (Cx)43, Cx45, and Cx40, we determined the distribution of these connexins. Cx43 was present in all locations where structural gap junctions occur. Cx40 was also widely distributed in the circular muscle of the lower esophageal sphincter (LES), stomach, and ileum. Cx45 was sparsely distributed in circular muscle of the LES. In the interstitial cells of Cajal (ICC) networks of myenteric plexus, in the deep muscular and submuscular plexuses, sparse Cx45 and Cx40 immunoreactivity was present. In colon, immunoreactivity was found only in the myenteric and submuscular plexus and nearby circular muscle cells. No immunoreactivity was found in sites lacking structural gap junctions (longitudinal muscle, inner circular muscle of the intestine, and most circular muscle of the colon). Studies of colocalization of connexins suggested that in the ICC networks, some colocalization of Cx43 with Cx40 and/or Cx45 occurred. Thus gap junctions in canine intestine may be heterotypic or heteromeric and have different conductance properties in different regions based on different connexin compositions.

gap junction composition; coupling; slow waves; interstitial cells of Cajal as pacemakers; inhibitory neurotransmission


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN CANINE INTESTINE, GAP JUNCTIONS have been observed with electron microscopy between circular muscle (CM) cells, except cells of the inner CM (iCM) of intestine and most cells of CM of colon, between interstitial cells of Cajal (ICC) in the myenteric plexus everywhere, the deep muscular plexus (DMP) of intestine, and the submuscular plexus of colon (1-5, 9-17, 20). Good electrical coupling has been observed or inferred between CM cells (3, 8-11, 13-17, 19, 20, 22, 29, 31, 32,) and assumed but not established experimentally between ICC and between ICC and CM. Even though longitudinal muscle cells have no gap junctions visible by electron microscopy except near the myenteric plexus of colon (5, 8, 9, 11, 13, 15-17), some electrical coupling has been observed and deduced between them because they have regular slow waves coupled to those in CM in intestine (8, 9, 15, 19), and in several species, space constants longer than the cell length have been observed (see Refs. 9, 15).

Recently, strong evidence has accumulated that slow waves throughout the gastrointestinal tract are paced by the networks of ICC in the myenteric plexus of stomach and intestine and in the submuscular plexus of colon (9, 23, 29, 30, 42), as originally proposed by Thuneberg (35). In the intestine, a network of ICC in the DMP plays a subsidiary role (7, 22) as does the ICC network in the myenteric plexus in the colon (31, 32). However, gap junctions visible with electron microscopy between ICC in the myenteric plexus and CM are rare and small (5, 15, 16) and, except in the colon, nonexistent between ICC and longitudinal muscle (5). In contrast, there are numerous gap junctions between the ICC of the submuscular plexus of colon and the adjacent CM (2, 4) and between the DMP and adjacent outer CM (oCM) (9, 16, 17, 20). Recently, accumulated evidence has been interpreted to imply that slow waves on gastrointestinal muscle are driven passively by current flow from the ICC networks in the myenteric plexus or submuscular plexus of colon (19, 29-30).

Furthermore, evidence has accumulated that the intramuscular ICC play an essential role in inhibitory neurotransmission (6, 30, 43). This was originally suggested because of the regular occurrence of nerve endings very close to intramuscular ICC, which are in gap junction contact with CM (12). Additional observations in canine gastrointestinal CM have shown similar structural relationships (1-5, 10, 13-16, 20). How the intramuscular ICC may amplify and transmit neural inhibitory information to the muscle is unclear, but the gap junctions connecting them are considered likely to be essential (15).

These observations raise several structural paradoxes. 1) How can the rare (to CM) or nonexistent (to longitudinal muscle) gap junctions between the myenteric plexus ICC of the myenteric plexus of the stomach and small intestine pass sufficient current to fill the large capacities and drive slow waves of these muscle layers that appear to be syncytia? 2) How can the numerous gap junctions between ICC networks of the submuscular plexus of colon and DMP of intestine provide current to drive slow waves in the adjacent syncytial CMs and still maintain independent pacemaking activities? One possibility is that these different gap junctions have different current-passing properties, including possible rectification of current flow, because they may contain different connexins, may be heteromeric with more than one connexin in each connexon, or may be heterotypic with a different connexin comprising each connexon to form a channel (44).

The objective of this study was to use immunocytochemistry to evaluate whether any gap junctions of canine gastrointestinal tissues are composed exclusively of Cx43, as is sometimes implied (24, 26), or whether they contain other connexins such as Cx45, which has recently been reported in canine DMP (28), or Cx40, which has been reported in other smooth muscles (25). Although Cx37 has recently been reported to be present in airway smooth muscle (27) and sparsely present in vascular smooth muscle (39), it was not studied. Immunohistochemistry with a light microscope lacks sufficient resolution to determine if the presence of more than one connexin in a given region implies that gap junctions there are heteromeric or heterotypic, but it does allow their localization to different sites where gap junctions occur.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Four mongrel dogs of either sex were euthanized with an overdose of pentobarbital sodium (100 mg/kg) in accordance with a protocol approved by the McMaster University Animal Care Committee and following the guidelines of the Canadian Council on Animal Care. The abdomen was opened along the midline; segments of lower esophagus, gastric antrum, ileum, and colon were excised and immediately put into oxygenated Krebs Ringer solution containing (in mM) 115.0 NaCl2, 4.6 KCl, 1.2 MgSO4, 22.0 NaHCO3, 2.5 CaCl2, and 11.0 glucose. Tissues were opened along the gastroesophageal junction and mesenteric border and pinned on a piece of Sylgard silicon rubber (mucosa side facing down) and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) overnight at 4°C. The fixed tissues were rinsed several times with PB, were then placed in PB containing 30% sucrose as a cryoprotection agent for 24 h, and were stored at -70°C until used.

Immunofluorescent labeling. Frozen sections of 8-µm thickness were cut using a Leitz 1720 digital cryostat, mounted on glass slides coated with gelatin, and dried at room temperature overnight. The steps for immunoreaction with the antibodies (shown in Table 1) were performed as follows. Blocking was carried out for 2 h with 3% BSA and 10% normal goat serum in 0.1 M PB at room temperature. Specimens were separately incubated with the mouse monoclonal antibody against gap junction protein Cx43 and the rabbit polyclonal antibody against gap junction protein Cx40 and Cx45 (Chemicon International) overnight at 4°C; antibodies were diluted 1:400 (Cx43), 1:300 (Cx40), and 1:100 (Cx45). The sections were washed three times with PB and then incubated for 60-120 min with fluorescein-cyanine-3 (Cy3)-labeled goat anti-rabbit (for Cx40 and Cx45) or anti-mouse (for Cx43) IgG (BIO/CAN Scientific) diluted 1:80. After being washed with PB, the specimens were then mounted in 80% glycerol in PB and viewed with a Leitz microscope equipped with a fluorescence epi-illuminator. Kodak T-MAX 400 film was used for black and white photography.

                              
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  		Table 1.   Antibodies used for immunoreaction

For double labeling of Cx43 and Cx40 or Cx45, the sections were incubated with a solution containing a mixture of the two primary antibodies. The secondary antibodies, Cy3-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG, were also applied in a mixed solution.

For peptide inhibition experiments, the antibodies and antigen were incubated together at 1:4 (1 part antibody to 4 parts peptide) for 24 h at 4°C in a cold room with agitation and were then spun using an air centrifuge for 20 min before application to the tissue sections for replacement of the primary antibodies.

Double-immunolabeled sections were examined by laser scanning confocal microscopy with a Carl Zeiss LSM instrument equipped with an argon/henel laser and fitted with the appropriate filter blocks for detection of FITC-Cy3. The images recorded and saved were projections on optima focus.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Distribution of Cx43 immunoreactivity. Indirect fluorescein immunohistochemistry on cryosections of canine gastrointestinal tract smooth muscle demonstrated the widespread expression of Cx43. The monoclonal mouse antibody against Cx43-specific peptide revealed strong punctate labeling in the CM of the lower esophageal sphincter (LES) and gastric antrum (Fig. 1, a and c). The punctate labels in stomach appeared smaller than those in LES. Pucntate labeling was also found in oCM of ileum but not in longitudinal muscle (Fig. 2, a and b). In the myenteric plexus of ileum between circular and longitudinal muscle layers and in the DMP between the inner and outer CM layers, immunoreactivity appeared to be associated with structures within the plexus, likely ICC (Fig. 2, a and b). Moreover, there was intense staining of the oCM but not the iCM. In the myenteric plexus and submuscular plexus of colon (Fig. 2, c and d), occasional immunoreactive sites were found associated with the plexuses and, likely, ICC or adjacent muscle. Labeling by this antibody and by a polyclonal antibody to Cx43 (data not shown) was similar at all sites. Very rare labeling (Fig. 2c) of colon longitudinal muscle and of CM of colon was found near the myenteric plexus. Near the submuscular plexus, within CM, very rare labeling was also seen. Preincubation of the antibody with the peptide antigen used to raise the Cx45 antibody reduced, whereas the peptide used to raise the Cx43 antibody abolished, immunostaining against the Cx43 antibody (Fig. 1, b and d).


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  		Fig. 1.   Distribution of connexin (Cx)43 in circular muscle (CM) of canine lower esophageal sphincter (LES) and stomach (Sto) antrum. a and c: Note dense distribution of immunoreactivity in CM. b: Ability of the peptide antigen used to raise the Cx45 antibody (PeCx45) to reduce staining against the Cx43 antibody is shown (compare a and b, same experiment). d: Specificity of the Cx43 antibody is shown as a result of preabsorption with the peptide antigen against which it was raised (PeCx43). Sph, sphincter. Bar, 12.5 µm for a-d.



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  		Fig. 2.   Distribution of Cx43 immunoreactivity in plexuses [interstitial cells of Cajal (ICC) networks] of ileum (ILe) and colon (Col). a: Immunoreactivity in ICC network (arrows) of myenteric plexus (MyP) between longitudinal muscle (LM) and CM of ILe shows presence of staining in CM but not LM. b: Immunoreactivity in the ICC network in the deep muscular plexus (DMP; arrowheads) between outer CM (oCM) with many gap junctions and inner CM (iCM) with none. SM, submucosa. c: Immunoreactivity in ICC network (arrows) of Auerbach's plexus between LM and CM of Col shows sparse immunoreactivity in ICC network and CM (arrowhead) and its absence in LM. d: Immunoreactivity in the ICC network in submuscular plexus at the border of CM and SM (arrowheads) in Col shows the virtual absence of staining (arrow) in CM. Bar, 12.5 µm for a-d.

Distribution of Cx40 immunoreactivity. Dense immunoreactivity to Cx40, like that to Cx43, was observed in the CM of the LES, gastric antrum, and oCM of ileum near the DMP (Fig. 3, a, c, and f). This was not affected by preabsorption with peptide antigen from Cx45 (Fig. 3b). Sparse immunoreactivity to Cx40 was also observed in myenteric plexus between longitudinal muscle and CM and in the DMP in ileum (Fig. 3, e and f) as well as in the submuscular plexus of colon (Fig. 3d). These may represent gap junctions on ICC networks located there. There was an occasional occurrence of immunoreactivity to Cx40 in the CM of colon (Fig. 3d). In the antral myenteric plexus, Cx40 antibody also occasionally labeled putative ICC (see sections on colocalization studies).


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  		Fig. 3.   Distribution of Cx40 immunoreactivity in gastrointestinal tract. a and b: Dense immunoreactivity to Cx40 in LES, which is not affected by preabsorption with peptide antigen from Cx45 (PeCx45). c: Dense immunoreactivity to Cx40 in Sto (antrum CM). d: Immunoreactivity to Cx40 in Col at submuscular plexus (arrowheads) between CM and SM. Some sparse immunoreactivity can be seen in CM (arrows). e: Immunoreactivity to Cx40 in ICC network (arrows) of Auerbach's plexus between LM and CM in ILe. Note sparse immunoreactivity in LM and CM. f: Immunoreactivity at the ICC network in the DMP (arrowheads) between oCM (with many gap junctions; dense staining) and iCM (with none; no staining). Bar, 12.5 µm for a-f.

Distribution of Cx45 immunoreactivity. In contrast to Cx43 and Cx40, very sparse or no immunostaining of Cx45 was found in the CM of the LES, antrum, and ileum both near the myenteric plexus and near the DMP (Fig. 4, a-d). Rare Cx45 immunoreactivity was found (Fig. 4, b and c) around the periphery of antral and ileal myenteric plexuses (for colon, see colocalization studies), near the DMP of ileum, and in the submuscular plexus of colon (Fig. 4, d and e). The immunoreactivity to Cx45 was abolished by preabsorption of the Cx45 antibody with its specific peptide (Fig. 4f).


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  		Fig. 4.   Distribution of immunoreactivity to different antibodies to Cx45 in gastrointestinal tract. Monoclonal antibody (MAb) 3100 and MAb 3101, which gave similar staining in all regions, are represented in a-d. a: Immunoreactivity vs. Cx45 in LES shows sparse (compared with Cx43) immunoreactivity. Some larger immunoreactive sites may represent intramuscular ICC, which have gap junctions to adjacent CM cells. b: Immunoreactivity at Sto antrum MyP reveals sparse scattered staining at edges of MyP. c and d: Immunoreactivity to Cx45 antibody in small intestine. c: staining of ICC in MyP between LM and CM, as in ILe, consisted of sparse staining around periphery of MyP. Note sparse and/or absent staining in CM and LM. d: sparse staining of ICC in the deep MyP (arrowheads) between inner and outer CM. There was very little or no staining in oCM or iCM. e: Staining of ICC or special smooth muscle in Col submuscular plexus between CM and submucosa. f: Peptide antigen saturated the antibody and abolished staining. Bar, 12.5 µm for a-f.

Immunoreactivity in regions without structural gap junctions. No immunoreactivity to gap junctions 43, 40, or 45 was consistently found in longitudinal muscle throughout the digestive tract or in iCM in ileum. As illustrated above, very sparse immunoreactivity was found in colon CM, consistent with the ultrastructural findings (6, 8). It was not possible to clearly distinguish immunoreactivity within CM of the various tissues associated with smooth muscle from that associated with intramuscular ICC, but some larger cells, possibly ICC, had Cx43 and Cx40.

Colocalization: Cx43 and Cx40. In these studies (Figs. 5-7), Cx43 antibodies were labeled with secondary antibodies conjugated to FITC (green), and Cx40 antibodies were labeled with secondary antibodies conjugated to Cy3 (red). Thus colocalization is indicated by yellow. In CM throughout the gastrointestinal tract, immunoreactivity to Cx43 and Cx40 was closely colocalized in the LES (Fig. 5a).


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  		Fig. 5.   Colocalization of Cx43 and Cx40 (a) and Cx43 and Cx45 (b) in LES. Cx43 was labeled with FITC (green), and Cx41 and Cx45 were each labeled with cyanine-3 (Cy3; red). a: Cx43 and Cx40 were colocalized at most sites, including sites of aggregated immunoreactivity (arrows, possible intramuscular ICC), all of which show yellow. Bar, 10 µm. b: Sites of Cx43 (green) and Cx45 (arrowheads) immunoreactivity were present along with sites of colocalization when immunoreactivities were aggregated (arrows, possible intramuscular ICC). Bar, 20 µm.



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  		Fig. 6.   Colocalization (yellow) of Cx43 (green) and Cx40 (red) in various gastrointestinal muscles. a: Low-power view of MyP region of Sto antrum in which most labeling occurs in CM. Note colocalization of the 2 antigens. Endothelia of blood vessels (bv) were predominantly labeled by antibodies to Cx40. b: Higher-power view of antral muscle in which most sites have colocalization and are labeled by both antibodies. Some sites are labeled only by antibodies to Cx43 (solid arrowheads) or to Cx40 (open arrowheads). c: LM was unlabeled, and there was extensive colocalization (arrows) of Cx43 and Cx40 in CM in the MyP region of the ILe. In addition, there were sites at which only Cx40 was present (open arrowheads), often sites of aggregated immunoreactivity. Also, sites with only Cx43 were present (solid arrowheads). d: oCM but not iCM was densely labeled, usually by colocalized antibodies to both Cx43 and Cx40 in the region of the DMP. At a few sites in the DMP, the labeling was only for Cx40 (arrowheads). e: Sparse labeling of the colon MyP region, mostly by antibodies to Cx43 and occasionally by colocalized antibodies to Cx43 and Cx40 (arrows) is shown. Note very sparse labeling of both LM and CM. f: Labeling in the region of the submuscular plexus between SM and CM shows that at some sites, antibodies to Cx43 and Cx40 were colocalized (arrows). At others, only Cx43 (solid arrowhead) or Cx40 (open arrowhead) was recognized.



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  		Fig. 7.   Colocalization (yellow) of Cx43 (green) and Cx45 (red) in various gastrointestinal muscles. a: Sto antrum showing extensive labeling by Cx43 antibodies in CM (solid arrowheads), occasional labeling by Cx45 antibodies in CM (open arrowheads), and in the region between CM and LM, presumably MyP (unlabeled) is shown. In this region, some colocalization of antibodies to Cx43 and Cx45 occurred (arrows). b: Deeper in CM, near the MyP, labeling by antibodies to Cx43 (unlabeled) was much more extensive than to Cx45 (arrowheads). Occasional colocalization of antibodies occurred near the SM (arrows). c: Labeling near the MyP of ileum demonstrates that no labeling occurred in LM. At the periphery of MyP, as in CM, most labels were to Cx43 (solid arrowheads), but there were occasional sites at which both antigens were recognized (arrows). Within CM, occasional sites had only Cx45 present (open arrowheads). d: Labeling near the DMP, mostly by the antibody to Cx43 in oCM, revealed no labeling, as found in iCM. Occasional sites labeled by the antibody to Cx45 were present in CM (arrowheads). Very rare sites of colocalization of connexins 43 and 45 were also found (arrow). e: MyP region of Col shows almost no labeling of CM or LM except near the plexus, and this was mostly to Cx45 (arrowheads). Occasional sites of colocalization of Cx43 and Cx45 were also present (arrows). f: Almost no labeling was present in CM or SM near the submuscular plexus of Col. Most label was found in the submuscular plexus sites and appeared to show colocalization of Cx43 and Cx45 (arrows), including some where labels were aggregated. Occasional sites were labeled only by antibody to Cx45 (arrowheads).

In antrum, too (Fig. 6, a and b), there was extensive colocalization of Cx43 and Cx40, but note the predominance of Cx40 staining in blood vessel endothelium near the myenteric plexus and the occurrence of Cx40 staining with and without Cx43 in deeper CM (Fig. 6b). Staining of Cx43 independently of Cx40 was rare. In ileum (Fig. 6, c and d), there was extensive colocalization of Cx40 and Cx43, but Cx40 also occurred free of Cx43 staining, notably near the plexuses, and, rarely, Cx43 immunoreactivity occurred independently of Cx40. Occasionally, clustered colocalized staining occurred. Comparison of Fig. 6, c and d, suggests that Cx40 was predominant in CM near the myenteric plexus but not near the DMP. In the portion of the oCM near the DMP, the immunoreactivity to both Cx43 and Cx40 was strong, and they were nearly completely colocalized except for a few sites that appeared to contain only Cx40 (Fig. 6d).

Figure 6, e and f, shows that immunolabeling of neither Cx43 nor Cx40 was prominent in circular or longitudinal muscle in colon. There was some colocalized staining near the myenteric plexus (Fig. 6e) and near the submuscular plexus (Fig. 6f). In the latter case, occasional sites had either Cx40 or Cx43 alone.

Colocalization of Cx43 and Cx45. Again, Cx43 antibodies were labeled with secondary antibodies conjugated to FITC, and Cx45 antibodies were labeled with secondary antibodies conjugated to Cy3. In LES (Fig. 5b), there was clear labeling of Cx45 as well as Cx43 sites. Some colocalization occurred, predominantly in sites where immunoreactivity was aggregated. These may be intramuscular ICC. These were similar to sites at which immunoreactivity to Cx43 and Cx40 was aggregated.

In antrum (Fig. 7, a and b), there was sparse labeling in the CM with Cx45 compared with Cx43. The exception was within the myenteric plexus, which was predominantly labeled by antibodies to Cx45. Colocalization, when it occurred, was in or near the myenteric plexus.

In ileum (Fig. 7, c and d), myenteric plexus, and oCM, sparse labeling with Cx45 was found compared with that of Cx43, but occasional colocalization occurred on both inner and outer aspects of the myenteric plexus. In the muscle, rare sites of Cx45 labeling as well as numerous sites of Cx43 labeling were seen. Note that in the DMP and oCM (Fig. 6d), the labeling of Cx45 was very sparse compared with that of Cx43, and colocalization was very rare.

In colon (Fig. 7e), myenteric plexus, and submuscular plexus (Fig. 7f), there was sparse labeling with Cx45, which was partially colocalized with Cx43. Near the submuscular plexus, some sites of colocalization appeared to be aggregated. Note the absence of staining to either Cx43 or 45 in most colon circular or longitudinal muscles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that canine gastrointestinal smooth muscle and the associated ICC have gap junctions that are frequently composed of multiple connexins. Cx43 and Cx40 are most prominent in CM of the LES, antrum, and ileum. Their immunoreactivities were present but more sparse in regions where ICC are located, such as the myenteric plexus of antrum, intestine, and colon and in the deep muscular and submuscular plexuses of ileum and colon, respectively. Immunoreactivity to Cx45 was sparse everywhere but present in LES muscle and all plexuses. There was no special concentration in the DMP of ileum, in contrast to a previous report (28). However, regions containing all ICC networks had some immunoreactivity from Cx40, Cx45, and Cx43, partly but not fully colocalized. Table 2 summarizes our findings.

                              
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  		Table 2.   Summary of locations of connexins in canine intestine

Another general observation was that immunoreactivity to these connexins was never found in regions that lack structural gap junctions as observed by electron microscopy (1-5, 9-17, 20). Thus longitudunal smooth muscle, iCM of intestine, and the longitudinal and CMs of colon, except near ICC networks, were nearly devoid of immunoreactivity.

If no additional gap junctions exist, it is unclear that gap junctions provide for pacemaking in the gastrointestinal tract. Modes of electrical coupling alternative to gap junctions do exist, and theoretical modeling suggests that they may be effective; field coupling between cell membranes closely apposed (33) can occur, and has its ability to transmit electrical events enhanced if there in an intrusion of a projection of one cell into another (40) or if potassium accumulates in the cleft between cells (33, 41). A special type of intrusion, the peg and socket joint, has recently been described in detail in mouse intestine (36-38, 41) and shown to vary with the incidence of physiological events. Peg and socket connections are postulated to function as stretch sensors, leading to activation of stretch-sensitive channels during segmenting and sleeve contractions (38).

It seems unlikely that field coupling of ICC networks to muscle layers could provide sufficient currents to drive slow waves passively throughout the muscularis externae. However, stretch coupling during shortening by ICC or muscle might transmit pacemaking activity throughout the muscle layer by inducing coupled smooth muscle cells to initiate their own currents, which then spread to other smooth cells by a similar mechanism. Moreover, the unidirectional relation between peg and socket joints between ICC and adjacent smooth muscle, the ICC that receive the peg and socket projection from smooth muscle cells, suggests that smooth muscle cells may have activity initiated by stretch during contraction (38).

If pacemaking currents flow through gap junctions, there are two paradoxes. One is that it is difficult to accept that the very low number of small gap junctions coupling ICC of the myenteric plexus to CM and the negligible number coupling them to longitudinal muscle could pass sufficient current to drive these smooth muscle syncytia passively. This study does not help resolve that paradox because no additional large gap junctions formed by connexins connecting ICC to muscle were found. The possibility that other modes of coupling contribute to the pacemaking by ICC networks in the myenteric plexus needs careful evaluation.

The other paradox is that the primary pacemaking region of the colon, the ICC network of the submuscular plexus, has numerous gap junctions to adjacent CM as does the secondary pacemaking area of the intestine, the ICC network of the DMP (4, 15, 17). Yet these pacemaking regions appear to be able to function independently of their coupling to large syncytia of muscle cells. It has been shown, for example, that isolated ICC cells produce pacemaking currents whether connected to smooth muscle or not (23, 30). The findings of this study may help explain those observations by showing that gap junctions in both these regions may contain both Cx45 and Cx40 as well as Cx43, all of which appear to be colocalized in part. We were unable to determine whether these gap junctions were heterotypic or heteromeric. However, it has been shown by expression studies that channels that are heterotypic for Cx43 and Cx40, as well as for Cx43 and Cx45, have altered conductance, greater sensitivity to transjunctional voltage differences, and altered pH sensitivity (18, 21, 34). Thus these channels may allow rectification of currents, allowing current passing primarily from the ICC network to smooth muscle.

Some caveats to our findings and suggested interpretations must be noted. Our findings, in addition to previous electron microscopy studies (9, 11, 13, 15-17, 24, 35), suggest that gap junctions are unlikely to mediate pacing by ICC or coupling between longitudinal muscle cells of the canine gastrointestinal tract or CM cells of colon. However, the ability of electron microscopy to resolve small gap junctions is limited by the thickness of the thin sections (~100 nm), whereas a small gap junction may be only 9- to 18-nm thick and fail to be resolved (15). It is unclear whether a strong fluorescent signal from such a small gap junction would be observable or could be captured on film. Nevertheless, at this time, no evidence supports the existence of small gap junctions in the above-mentioned regions of the canine gastrointestinal tract, but occurrence of very small gap junctions undetected by electron microscopy or by confocal immunofluorescence cannot be excluded. If such gap junctions exist, it is uncertain whether they can account for coupling between ICC and muscle.

In summary, this study showed that connexins in addition to Cx43 exist in the canine gastrointestinal tract and sometimes appear to be colocalized. It failed to reveal the presence of gap junctions in regions where structural gap junctions have not been found by electron microscopy. Therefore, understanding of coupling in the muscularis externae of the canine gastrointestinal tract may require evaluation of alternate modes of coupling in addition to gap junctions, modes such as stretch coupling by peg and socket joints.


    ACKNOWLEDGEMENTS

This research was supported by the Medical Research Council of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: Address for reprint requests and other correspondence: E. E. Daniel, Dept. of Pharmacology, Univ. of Alberta, 9-70 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada (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.

Received 6 December 2000; accepted in final form 19 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allescher, HD, Berezin I, Jury J, and Daniel EE. Characteristics of canine lower esophageal sphincter: a new electrophysiological tool. Am J Physiol Gastrointest Liver Physiol 255: G441-G453, 1988[Abstract/Free Full Text].

2.   Berezin, I, Allescher HD, and Daniel EE. Ultra-structural localization of VIP-immunoreactivity in canine distal oesophagus. J Neurocytol 16: 749-757, 1987[ISI][Medline].

3.   Berezin, I, Daniel EE, and Huizinga JD. Ultrastructure of interstitial cells of Cajal in the canine distal esophagus. Can J Physiol Pharmacol 72: 1049-1059, 1994[ISI][Medline].

4.   Berezin, I, Huizinga JD, and Daniel EE. Interstitial cells of Cajal in canine colon: a special communication network at the inner border of the circular muscle. J Comp Neurol 273: 42-51, 1988[ISI][Medline].

5.   Berezin, I, Huizinga JD, and Daniel EE. Structural characterization and interconnections of interstitial cells of Cajal in myenteric plexus and circular muscle of canine colon. Can J Physiol Pharmacol 68: 1419-1431, 1990[ISI][Medline].

6.   Burns, AJ, Lomax AE, Torihashi S, Sanders KM, and Ward SM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach. Proc Natl Acad Sci USA 93: 12008-12013, 1996[Abstract/Free Full Text].

7.   Cayabyab, FS, Jimenez M, Vergara P, de Bruin H, and Daniel EE. Influence of nitric oxide and vasoactive intestinal peptide on the spontaneous and triggered electrical and mechanical activities of the canine ileum. Can J Physiol Pharmacol 75: 383-397, 1997[ISI][Medline].

8.   Cheung, DW, and Daniel EE. Comparative study of the smooth muscle layers of the rabbit duodenum. J Physiol (Lond) 309: 13-27, 1980[Abstract/Free Full Text].

9.   Daniel, EE, and Berezin I. Interstitial cells of Cajal: are they major players in control of gastrointestinal motility? J Gastrointest Motil 4: 1-24, 1992.

10.   Daniel, EE, Berezin I, Allescher HD, Manaka H, and Posey-Daniel V. Morphology of the canine pyloric sphincter in relation to function. Can J Physiol Pharmacol 67: 1560-1573, 1989[ISI][Medline].

11.   Daniel, EE, Daniel VP, Duchon G, Garfield RE, Nichols M, Malhotra SK, and Oki M. Is the nexus necessary for cell-to-cell coupling in smooth muscle? J Membr Biol 28: 207-239, 1976[ISI][Medline].

12.   Daniel, EE, and Posey-Daniel V. Neuromuscular structures in opossum esophagus: role of interstitial cells of Cajal. Am J Physiol Gastrointest Liver Physiol 246: G305-G315, 1984[Abstract/Free Full Text].

13.   Daniel, EE, Robinson K, Duchon G, and Henderson RM. The possible role of close contacts (nexuses) in the propagation of control electrical activity in the stomach and small intestine. Dig Dis 16: 611-622, 1971.

14.   Daniel, EE, Sakai Y, Fox JET, and Posey-Daniel V. Structural bases for function of circular muscle of canine corpus. Can J Physiol Pharmacol 62: 1304-1314, 1984[ISI][Medline].

15.   Daniel, EE, and Wang YF. Gap junctions in intestinal smooth muscle and interstitial cells of Cajal. Microsc Res Tech 47: 309-320, 1999[ISI][Medline].

16.   Daniel, EE, Wang YF, and Cayabyab F. Role of gap junctions in structural arrangements of interstitial cells of Cajal and canine ileal smooth muscle. Am J Physiol Gastrointest Liver Physiol 274: G1125-G1141, 1998[Abstract/Free Full Text].

17.   Duchon, G, Henderson R, and Daniel EE. Circular muscle layers in the small intestine. In: Proceedings of the Fourth International Symposium on GI Motility, edited by Daniel EE.. Vancouver, Canada: Mitchell, 1974, p. 635-646.

18.   Gu, H, Ek-Vitorin JF, Taffet SM, and Delmar M. Coexpression of connexins 40 and 43 enhances the pH sensitivity of gap junctions: a model for synergistic interactions among connexins. Circ Res 86: E98-E103, 2000.

19.   Hara, Y, Kubota M, and Szurszeweski JH. Electrophysiology of smooth muscle on the small intestine of some mammals. J Physiol (Lond) 372: 510-520, 1986.

20.   Henderson, RM, Duchon G, and Daniel EE. Cell contacts in duodenal smooth muscle layers. Am J Physiol 221: 564-574, 1971.

21.   Hermans, MM, Kortekaas P, Jongsma HJ, and Rook MB. pH sensitivity of the cardiac gap junction proteins, connexin 45 and 43. Pflügers Arch 431: 138-140, 1995[ISI][Medline].

22.   Jimenez, M, Cayabyab FS, Vergara P, and Daniel EE. Heterogeneity in electrical activity of the canine ileal circular muscle: interaction of two pacemakers. Neurogastroenterol Motil 8: 339-350, 1996[ISI][Medline].

23.   Koh, SD, Sanders KM, and Ward SM. Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol (Lond) 513: 203-213, 1998[Abstract/Free Full Text].

24.   Li, Z, Zhou Z, and Daniel EE. Expression of gap junction connexin 43 and connexin 43 mRNA in different regional tissues of intestine in dog. Am J Physiol Gastrointest Liver Physiol 265: G911-G916, 1993[Abstract/Free Full Text].

25.   Little, TL, Beyer EC, and Duling BR. Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo. Am J Physiol Heart Circ Physiol 268: H729-H739, 1995[Abstract/Free Full Text].

26.   Mikkelsen, HB, Huizinga JD, Thuneberg L, and Rumessen JJ. Immunohistochemical localization of a gap junction protein (connexin 43) in the muscularis externa of murine, canine, and human intestine. Cell Tissue Res 274: 249-256, 1993[ISI][Medline].

27.   Nakamura, K, Inai T, Nakamura K, and Shibata Y. Distribution of gap junction protein connexin 37 in smooth muscle cells of the rat trachea and pulmonary artery. Arch Histol Cytol 62: 27-37, 1999[ISI][Medline].

28.   Nakamura, K, Kuraoka A, Kawabuchi M, and Shibata Y. Specific localization of gap junction protein, connexin 45, in the deep muscular plexus of dog and rat small intestine. Cell Tissue Res 292: 487-494, 1998[ISI][Medline].

29.   Sanders, KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111: 492-515, 1996[ISI][Medline].

30.   Sanders, KM, Ordog T, Koh SD, Torihashi S, and Ward SM. Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil 11: 311-338, 1999[ISI][Medline].

31.   Smith, TK, Reed JB, and Sanders KM. Origin and propagation of electrical slow waves in circular muscle of canine colon. Am J Physiol Cell Physiol 252: C215-C224, 1987[Abstract/Free Full Text].

32.   Smith, TK, and Sanders KM. Interaction of two electrical pacemakers in muscularis of canine proximal colon. Am J Physiol Cell Physiol 252: C290-C299, 1987[Abstract/Free Full Text].

33.   Sperelakis, N, LoBracco B, Jr, Mann JE, and Marschall R. Potassium accumulation in intercellular junctions combined with electric field stimulation for propagation in cardiac muscle. Innov Tech Biol Meth 6: 25-43, 1985.

34.   Steiner, E, and Ebihara L. Functional characterization of canine connexin 45. J Membr Biol 150: 153-161, 1996[ISI][Medline].

35.   Thuneberg, L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryol Cell Biol 71: 1-130, 1982[Medline].

36.   Thuneberg, L, and Peters S. Toward a concept of stretch-coupling in smooth muscle. I. Anatomy of intestinal segmentation and sleeve contraction. Anat Rec 262: 110-124, 2001[Medline].

37.   Thuneberg, L, and Peters S. Formation of peg and socket junctions (Abstract). Neurogastroenterol Motil 12: 294, 2000.

38.   Thuneberg, L, and Peters S. Peg-and-socket junctions: accumulating evidence of a role in intestinal mechanoreception (Abstract). Neurogastroenterol Motil 10: 479, 1998.

39.   Van Kempen, MJ, and Jongsma HJ. Distribution of connexin 37, connexin 40 and connexin 43 in the aorta and coronary artery of several mammals. Histochem Cell Biol 112: 479-486, 1999[ISI][Medline].

40.   Vigmond, EJ, and Bardakjian BL. The effect of morphological interdigitation on field coupling between smooth muscle cells. IEEE Trans Biomed Eng 42: 162-171, 1995[ISI][Medline].

41.   Vigmond, EJ, Bardakjian BL, Thuneberg L, and Huizinga JD. Intercellular coupling mediated by potassium accumulation in peg-and-socket junctions. IEEE Trans Biomed Eng 47: 1576-1583, 2000[ISI][Medline].

42.   Ward, SM, Burns AJ, Torihashi S, and Sanders KM. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol (Lond) 480: 91-97, 1994[ISI][Medline].

43.   Ward, SM, Morris G, Reese L, Wang XY, and Sanders KM. Interstitial cells of Cajal mediate inhibitory neurotransmission in the lower esophageal and pyloric sphincters. Gastroenterology 115: 314-328, 1998[ISI][Medline].

44.   White, TW, and Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61: 283-310, 1999[ISI][Medline].

Am J Physiol Gastrointest Liver Physiol 281(2):G533-G543
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society


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