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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
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.
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RESULTS |
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.
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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.
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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.
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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).
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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.
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DISCUSSION |
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.
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.
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ACKNOWLEDGEMENTS |
This research was supported by the Medical Research Council of Canada.
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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.
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Am J Physiol Gastrointest Liver Physiol 281(2):G533-G543
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