communication between interstitial cells of cajal and gastrointestinal muscle
TRANSCRIPT
Communication between interstitial cells of Cajal and
gastrointestinal muscle
E. E. DANIEL
Department of Pharmacology, University of Alberta, Edmonton, AB, Canada
Abstract Interstitial cells of Cajal (ICC) pace gastro-
intestinal muscle by initiating slow waves in both
muscle layers and appear to be preferred sites for
reception of neurotransmitters. ICC of the myenteric
plexus (ICC-MP) pace stomach and small intestine,
while intramuscular ICC (ICC-IM) receive nerve
messages. Recently, ICC-IM have been found to pro-
vide regenerative responses to and amplification of
pacing messages from ICC-MP, at least in some sys-
tems. This review will examine the assumption that
gap junctions provide low-resistance contacts for pa-
cing. Structural and functional evidence will be eval-
uated. Structural, theoretical and experimental
difficulties with the gap junctions hypothesis for pa-
cing will be considered. So far little direct evidence
about the role of gap junctions in neurotransmission
exists, although a structural basis exists. Alternate
possibilities for transmission of ICC pacing and neural
messages will be examined and suggestions for future
research made.
Keywords field coupling, gap junctions, immuno-
cytochemistry, slow waves, ultrastructure.
IS THERE A STRUCTURAL BASIS FOR GAPJUNCTIONS IN PACING ANDNEUROTRANSMISSION?
That ICC-MP play a crucial role in pacing slow waves
and contractions1 and ICC-IM probably play such a role
to receive neural messages1,2 is not in dispute. What is
unclear is how pacing messages get to muscle and
neural message get from ICC to muscle. Gap junctions
have been found between ICC-MP in stomach, intes-
tine and colon,3,4 perhaps allowing them to function as
a network. Few or no gap junctions, none in mouse
intestine and very rare ones in canine intestine exist
between ICC-MP and either longitudinal (LM) or
circular muscle (CM).3,5 In mouse intestine, from
which much evidence of the role of ICC in pacing
was derived, neither ultrastructural nor immunochem-
ical evidence for such gap junctions exists (5, Cho &
Daniel unpublished). In canine intestine, gap junctions
between ICC-MP and muscle are very rare.4 In the rat
intestine gap junctions have been described between
ICC-MP and muscle.6 Structural evidence exists in
circular muscle of various tissues that gap junctions
couple ICC-IM to muscle.2,3 See Table 1 for a summary
of some of this evidence.
Absence of structural gap junctions has not
deterred assumptions about their role in pacing.
Reliance is placed on the assumed existence of gap
junctions too small to be observed by electron
microscopy (EM) or immunochemistry. Gap junctions
consisting of one or two connections, 9–20 nm across
in sections, will probably be invisible in EM sections
of �100 nm. It is assumed that they are also invisible
to immunochemical study. No evidence shows that
current sufficient to pace the functional syncytium of
the CM or the LM passively can pass a few small
junctions. However, this possibility provides an
excuse to avoid considering the problem or alterna-
tives. To achieve certainty that gap junctions (seen
by EM or identified by immunocytochemistry) are
present or absent requires major technical advances,
so far unforseen.
THEORETICAL PROBLEMS FOR THE GAPJUNCTION HYPOTHESIS IN PACING
In studies of the canine colon CM, paced primarily by
an ICC network in the submuscular plexus just
beneath the CM,3 currents appeared to spread passively
from the ICC to the CM.7 Passive spread of sufficient
current from ICC-MP to muscle to activate the CM
syncytium was assumed in other systems. If so, all the
Address for correspondence:E. E. Daniel, Room 9–10 MSB, Department of Pharmacology,University of Alberta, Edmonton, AB, T6G 2H7, Canada.Tel.: (780) 492 2105; e-mail: [email protected]
Neurogastroenterol Motil (2004) 16 (Suppl. 1), 118–122
118 � 2004 Blackwell Publishing Ltd
current to charge the capacity of the CM functional
syncytium and depolarize its membranes would have
to be supplied from and pass through a few, small low-
resistance contacts between ICC-MP and CM. This
seems implausible. Although LM in most intestinal
tissues appears3 to be electrically coupled (space con-
stants measured using Abe-Tomita-type bath longer
than cell length, but the validity of this as a measure of
space constants is unclear-see later), no gap junctions
have been found between LM cells.8 Again the
assumption is made that small undetectable gap
junctions account for this. However, passive spread of
current from ICC-MP to LM becomes even more
difficult to accept; i.e. currents would have to utilize
these sparse connections to reach the LM and spread
passively through it.
Another problem for the assumption of passive
spread of pacing currents through gap junctions exists:
how could stable pacing activity be maintained when
so much current is spread through low-resistance
contacts? Even if it could be achieved, the expectation
is that the pacing current frequency and amplitude
would be affected by the properties of the whole
system including the muscle, assuming that sufficient
low resistance contacts exist to pass the necessary
currents. Isolated ICC should be unable to mimic the
activity of the intact system.
EXPERIMENTAL EVIDENCE
The presence of functional gap junctions is demon-
strated by injecting a low molecular weight dye
(lucifer yellow or neurobiotin) into a cell and obser-
ving its spread into another. Study of dye spread in
pacing requires identifying the ICC-MP (with antibod-
ies to c-kit or by shape), injecting the dye and
observing its spread between cells. Another method
involves insertion of electrodes into two cells connec-
ted by gap junctions and showing that current injec-
tion into one directly affects the other without major
attenuation or rectification (see Evans & Boitano
20019 and references therein). A putative inhibitor of
gap junction coupling should inhibit dye or current
spread. These techniques are often applied between
cell pairs or, in the case of dye coupling, in a
functional syncytium. Application of either technique
to ICC-MP and to ICC with smooth muscle is difficult
and requires extensive dissection. Few attempts have
been made: one showed limited dye spread in the
canine colon between LM and CM apparently via ICC-
MP.10 Another showed very limited dye spread
between ICC-MP and LM of mouse intestine.11 David
Hirst reported dye spread between ICC-MP of an-
trum12 and Hanani et al. reported it between ICC-MP
of human ileum.13 In these two cases, no spread to
muscle was observed. Of course it is possible, even
likely, that conditions for the spread of dye and �ionic
current� may not be identical.
Several agents are known to inhibit dye spread:
aliphatic alcohols such as octanol, anaesthetics such as
halothane, 18a-glycyrrhetinic acid and its hemi-succi-
nate, carbenoxolone, fatty acids such as arachidonic
acid, tumour promoters such as phorbol esters, and
others.9 None is highly selective for inhibition of gap
junctions conductance of dyes. Perhaps the most
selective are connexin mimetic peptides such as Gap
27, a peptide sequence found next to the second and
fourth transmembrane domains of connexin 43, a
major component of smooth muscle and ICC gap
junctions.9 It has not yet been applied to ICC coupling.
Not all these inhibitors affect electrical coupling,
especially when applied to complex systems. 18a-
Glycyrrhetinic acid and carbenoxolone have been
shown not to inhibit ICC-MP pacing in canine or
mouse intestine and submuscular ICC pacing of canine
colon.14,15 Octanol affected but did not abolish spread
of slow waves into canine intestine CM.4 Thus,
available functional evidence does not support that
gap junctions are required for electrical pacing of
smooth muscle.
Table 1 Distribution of gap junctions in mouse and dog intestine
Location Mouse intestine Canine intestine
ICC-MP to ICC-MP Gap junctions present Gap junctions presentICC-MP to LM No gap junctions Gap junctions rare or absentICC-MP to CM No gap junctions Gap junctions rare or absentICC-DMP to ICC-DMP Gap junctions present Gap junctions presentICC-DMP to outer CM Gap junctions present Gap junctions presentICC-DMP to inner CM Gap junctions absent Gap junctions absentICC-IM to CM No or very few ICC-IM found Gap junctions present
See 3–5,33.
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Volume 16, Supplement 1, April 2004 How do ICC talk to muscle?
Moreover, there is no need for low-resistance con-
tacts in electrical coupling. Numerous publications
have shown that the production of an electrical field
potential in narrow clefts between cells is sufficient to
allow electrical coupling.16–23 When cells make peg
and socket connections as do LM and CM to ICC-MP
of mouse intestine and K+ accumulates in these narrow
clefts, electrical coupling without gap junctions is
facilitated.21,23
Functions of ICC-IM. ICC-IM connect to smooth
muscle by gap junctions.2,3 Evidence from mice lack-
ing ICC-IM suggests that they are sites for the primary
actions of NO and acetylcholine in LES, pylorus and
gastric fundus.2 Only one study3 tried to evaluate if gap
junction function is required, but 18a-glycyrrhetinic
acid had no effect on NO-mediated relaxations of
canine LES. Thus the role of gap junctions in trans-
mitting neural information from ICC-IM to muscle
remains unclear. Recently ICC-IM in mouse and
guinea-pig antrum have been found to participate in
amplification of pacing signals from ICC-MP.24–27
They do so by giving regenerative responses to depo-
larizing currents from ICC-MP, external electrodes or
exposure to acetylcholine. ICC-IM are not connected
directly to ICC-MP. No direct evidence shows how the
message is received and transmitted, but gap junctions
are assumed to be involved. In small CM muscle
bundles lacking ICC-MP slow waves did occur, con-
sisting of a pacemaker and regenerative component,
both of which were composed of elementary events
called spontaneous transient depolarizations (STDs),
associated with local Ca2+ transients.28 Some evidence
associated the Ca2+ transients and STDs with ICC-IM
in the muscle bundles. Slow waves emerged when
STDs became entrained like relaxation oxcillators.
This study found that 18a-glycyrrhetinic acid perfused
across the centre of the strips caused slow waves at
each end of the strip to run independently. Gap
junctions may be involved in synchronizing and
entraining slow waves.
In canine small intestine which has ICC-IM as well
as a network of ICC in the deep muscular plexus (ICC-
DMP), slow waves of different configuration were
present in CM after removal of ICC-MP.29 These may
arise from interaction of ICC-IM with ICC-DMP but
this needs further study, for example, of the effect of
gap junction uncouplers on pacing in the absence of
ICC-MP. In canine intestine CM, long (50–100 ms)
pulses from electrical stimulation (EFS) could induce
slow waves if the ICC-MP was present but not if it
was absent.30 This does not fit the hypothesis that
only ICC-IM or ICC-DMP respond to depolarizing
stimuli.
ARE GAP JUNCTIONS BETWEEN ICC-IMAND CM CELLS REQUIRED TO EXPLAINCOUPLING?
Not all gastrointestinal muscles contain ICC-IM. For
example, the mouse intestine contains few or none (5,
unpublished observations, personal communication
from H. Mikklesen). In this tissue, gap junction
inhibitors minimally influence pacing frequencies
and do not prevent responses to EFS pacing by 50–
100 ms pulses.15 Even in intestine of W/WV mice, CM
responded to these pulses (Daniel & Bodie unpub-
lished). There must be an excitable system indepen-
dent of ICC-MP or ICC-IM in the intestine.
Moreover, in ongoing experiments using PSpice
simulation of action potentials in a planar network
of 25 smooth muscle cells (five parallel chains (A–E) of
five cells each) connected to one ICC-IM), it was
found that gap junctions between the ICC cell and the
last cell (E5) of the network were not necessary for the
ICC-IM cell to excite the entire 25 cells of the
network (Sperelakis & Daniel unpublished). Adding
many gap junctions at this junction (e.g. 1000 or
10 000/junction) simply made the voltage change in
the E5 cell follow closely the voltage change of the
action potential in the ICC cell; the E5 cell then
initiated an slow wave in E4 cell (via the depolarizing
negative cleft potential), which propagated excitation
throughout the network. In this model the smooth
muscle cells were not connected by gap junctions
tunnels, and transmission of excitation occurred via
the electric field generated in the junctional clefts
when the prejunctional membrane fired an action
potential. Excitation jumped to parallel chains by a
similar mechanism.
FUTURE DIRECTIONS
Any necessary role of low-resistance contacts for
coupling of ICC-MP and ICC-IM to muscle is doubtful.
Technical advances to rule in or out the presence of
small gap junctions between ICC-MP and muscle are
needed. How ICC-MP may be coupled to ICC-IM needs
elucidation. Also, the properties of gap junctions,
which are usually heteromeric, including in intes-
tine,31,32 depend on their connexin composition.31 The
roles of electrical field potentials in peg and socket
clefts between muscle and ICC-MP and of mechano-
sensitive ion channels operated by connections be-
tween muscle and ICC33 need further evaluation.
Finally, the question of whether intestinal muscle is
electrically excitable needs to be re-evaluated because
Prosser & Sperelakis34 found that CM cells from cat
120 � 2004 Blackwell Publishing Ltd
E. E. Daniel Neurogastroenterology and Motility
intestine did respond reproducibly to depolarizing
stimuli.
ACKNOWLEDGMENTS
The work from my laboratory was supported by the
Canadian Insititutes of Health Research. The author
gratefully acknowledges advice and critical comments
from Professor Nicholas Sperelakis.
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