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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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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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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REFERENCES

1.   Epperson, A, Hatton WJ, Callaghan B, Doherty P, Walker RL, Sanders KM, Ward SM, and Horowitz B. Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal. Am J Physiol Cell Physiol 279: C000-C000, 2000.

2.   Faussone-Pellegrini, M-S. Histogenesis, structure, and relationships of interstitial cells of Cajal (ICC): from morphology to functional interpretation. Eur J Morphol 30: 137-148, 1992[ISI][Medline].

3.   Grider, JR, and Murthy KS. Isolated smooth muscle cells. In: Handbook of Methods in Gastrointestinal Pharmacology, edited by Gaginella TS.. Boca Raton, FL: CRC, 1996, chapt. 10, p. 225-246.

4.   Huizinga, JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, and Bernstein A. The W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373: 347-349, 1995[ISI][Medline].

5.   Huizinga, JD, Thuneberg L, Vanderwinden J-M, and Rumessen JJ. Interstitial cells of Cajal as targets for pharmacological intervention in gastrointestinal motor disorders. Trends Pharmacol Sci 18: 393-403, 1997[ISI][Medline].

6.   Kluppel, M, Huizinga JD, Malysz J, and Bernstein A. Developmental origin of the interstitial cells of Cajal in the mammalian small intestine. Dev Dyn 211: 60-71, 1998[ISI][Medline].

7.   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].

8.   Teng, B-Q, Murthy KS, Kuemmerle JF, Grider JR, and Makhlouf GM. Selective expression of vasoactive intestinal peptide (VIP)2/pituitary adenylate cyclase-activating polypeptide (PACAP)3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Regl Pept 77: 127-134, 1998[ISI][Medline].

9.   Thomsen, L, Tobinson TL, Lee JCF, Farraway LA, Hughes MJG, Andrews DW, and Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4: 848-851, 1998[ISI][Medline].

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

11.   Torihashi, S, Ward SM, Nishikawa S-I, Nishi K, Kobayashi S, and Sanders KM. c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280: 97-111, 1995[ISI][Medline].

12.   Torihashi, S, Ward SM, and Sanders KM. Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine. Gastroenterology 112: 144-155, 1997[ISI][Medline].


Am J Physiol Cell Physiol 279(2):C284-C285
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society




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