EDITORIAL FOCUS
Focus on "Molecular markers expressed in cultured and
freshly isolated interstitial cells of Cajal"
J. R.
Grider
Departments of Physiology and Medicine, Medical College of Virginia
Campus of Virginia Commonwealth University, Richmond, Virginia
23298
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ARTICLE |
THE PATTERN OF CONTRACTILITY of the
muscularis propria of the gut is the result of the regulation of three
types of cells: smooth muscle cells (SMC), enteric neurons, and
interstitial cells of Cajal (ICC). Whereas the neurons and SMC of the
gut are similar in many ways to those in other organs, the ICC
represents a cell type unique to the gut, making its characterization
and the identification of its physiological role more difficult.
Anatomic, morphological, and electrophysiological examinations of ICC
have clearly demonstrated the presence of several subclasses of ICC in
the muscularis and suggest that these subclasses are likely to mediate
different physiological functions (2, 7, 10). The ICC located at the border of the outer
longitudinal and inner circular muscle layer in the vicinity of the
myenteric plexus (IC-MY) are found in most species and regions of the
gut and are electrical pacemaker cells responsible for originating and
propagating the rhythmic oscillations of membrane potential known as
electrical slow waves. In the canine colon, a similar set of ICC
(IC-SM) are located at the submucosal surface of the circular muscle,
where they appear to act also as electrical pacemaker cells. A distinct
set of ICC are located within the muscle layer, either interspersed
between SMC (IC-IM) or clustered in a specialized region of the
circular muscle layer containing a network of nerve terminals termed
the deep muscular plexus (IC-DMP). These latter two groups of ICC have
been postulated to be involved in neurotransmission.
The ability to identify ICC in tissue and to study their anatomic
characteristics has been greatly enhanced by the use of immunohistochemical techniques to label Kit. ICC are the only cells in
the muscularis, other than mast cells, to express c-kit, the
protooncogene encoding the receptor tyrosine kinase Kit. Kit activation
is essential: immunoneutralization or mutation of c-kit or
its ligand, stem cell factor (SCF), severely interferes with the
development of some classes of ICC and of electrical activity (4, 11). Developmental studies
(6, 12) suggest the presence of a common
mysenchymal precursor cell that becomes ICC if Kit is activated or SMC
if Kit is not activated. SMC and some populations of ICC also share
common ultrastructural characteristics in the adult such as caveolae,
myofilaments, smooth endoplasmic reticulum, and dense bodies. These
studies strongly suggest that smooth muscle and ICC are closely related
and may therefore express common markers.
Whereas ICC are easily identified in situ, the loss or low levels of
Kit expression in dispersed preparations of muscularis makes it
difficult to study ICC in isolation. Similarly, the use of cultures of
ICC is hampered by the close relationship between ICC and SMC and the
potential reversion to a common precursor cell that shares
characteristics of both ICC and SMC. In the study by Epperson et al.,
the current article in focus (Ref. 1, see p. CXXX in this issue), the
authors have described a method of labeling ICC in murine gut with Kit
antibody coupled to Alexa 488 fluorescent dye, followed by enzymatic
dispersion. This technique allowed the ICC to be identified in cell
suspensions and harvested as individual cells. RT-PCR was applied to
mRNA isolated from single cells to characterize differential
transcription of key genes in SMC and ICC from two locations (IC-MY of
small intestine and IC-IM of gastric fundus). The pattern of
transcription was also determined for cultures of ICC and SMC and
compared with that for the freshly dispersed cells. The results
demonstrated that freshly dispersed ICC express c-kit mRNA
but not smooth muscle myosin heavy chain mRNA; freshly dispersed SMC
express the opposite pattern. In contrast, cultured ICC express smooth
muscle myosin heavy chain mRNA. This is consistent with the reversion
of ICC in culture to a less differentiated phenotype that possesses
characteristics of both ICC and SMC and supports the notion of a common
developmental precursor cell. It is also consistent with recent reports
that ICC demonstrate spontaneous contractile activity in culture
(9). Further studies are necessary to determine how
important this attribute of ICC is in the normal state and in
pathological conditions. It is worth noting that there are significant
changes in ICC in several pathological conditions, and it has been
proposed that ICC might be a site of therapeutic intervention in the
future (5, 7). The present study also
characterized the ICC with respect to the presence of mRNA for membrane
receptors and ion channels. All freshly dispersed and cultured ICC and
SMC expressed mRNA for muscarinic receptors M2 and
M3 and neurokinin receptors NK1 and
NK3. This pattern is consistent with neuronal signaling through both ICC and SMC and suggests that the presence of
neurotransmitter receptor mRNAs may not be specific markers of either
cell type. In contrast, in the present study freshly dispersed ICC
expressed only vasoactive intestinal peptide receptor type 1 (VIP-1)
mRNA, and freshly dispersed SMC did not express any VIP receptor mRNA, although both ICC and SMC cultures expressed mRNA for VIP-1 and VIP-2
receptors. This is a surprising finding in light of the ability of VIP
to relax SMC isolated from a number of species and regions of the gut
and the presence of VIP-2 mRNA in mRNA extracted from SMC of rabbit and
guinea pig (3, 8). The exact nature of
receptor expression and of the role of ICC in neurotransmission will
require detailed physiological studies as well as determination of
expression of the actual receptor protein by ICC. All cells studied
expressed mRNA for the nonselective cation channels Trp4 and Trp6,
whereas only SMC expressed mRNA for Trp1. Although it is likely that
these channels play a role in mediating the response to excitatory
transmitters, the exact nature of their role in the physiology of ICC
and SMC will require more detailed examination. This study does,
however, provide a basic categorization of the types present in each
cell on which future studies can build. The final marker examined in
these cells was the ligand for the Kit receptor, SCF. Freshly dispersed
IC-IM, IC-MY, and SMC from small intestine expressed only the soluble form of SCF, whereas gastric SMC expressed the bound form necessary for
ICC development; cultured ICC and SMC expressed both forms. These
differential expression patterns of SCF may point the way to an
understanding of why ICC develop different phenotypes in different
regions. As with other markers, the expression of both bound and
soluble SCF in ICC and SMC cultures suggests reversion to a common
precursor cell with the potential to become either smooth muscle or ICC.
The studies presented in this article in focus demonstrate some of the
molecular and genetic similarities and differences in gut smooth muscle
and ICC. These important findings will likely point the way for future
studies of the functional expression of these gene products and their
relative roles in mediating the electrophysiological and contractile
patterns that regulate movement within the gut.
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FOOTNOTES |
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. §1734 solely to indicate this fact.
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Am J Physiol Cell Physiol 279(2):C284-C285
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