Physiol. Genomics Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 31: 492-509, 2007. First published September 25, 2007; doi:10.1152/physiolgenomics.00113.2007
1094-8341/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figure and Tables
Right arrow All Versions of this Article:
31/3/492    most recent
00113.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.
Right arrow Articles by Sanders, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.
Right arrow Articles by Sanders, K. M.
Received 18 May 2007; accepted in final form 15 September 2007.
Physiological Genomics 31:492-509 (2007)
1094-8341/06 $8.00 © 2007 American Physiological Society

Differential gene expression in functional classes of interstitial cells of Cajal in murine small intestine

Hui Chen 1, Tamás Ördög 1, Junwei Chen 1, David L. Young 1, Michael R. Bardsley 1, Doug Redelman 1,2,3, Sean M. Ward 1 and Kenton M. Sanders 1

1 Department of Physiology and Cell Biology
2 Cytometry Center, University of Nevada, Reno
3 Sierra Cytometry, Reno, Nevada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Interstitial cells of Cajal (ICC) have important functions in regulation of motor activity in the gastrointestinal tract. In murine small intestine, ICC are gathered in the regions of the myenteric plexus (ICC-MY) and the deep muscular plexus (ICC-DMP). These two classes of ICC have different physiological functions. ICC-MY are pacemaker cells and generate the slow-wave electrical rhythmicity of gastrointestinal organs. ICC-DMP form synaptic connections with the varicose nerve terminals of enteric motor neurons and are involved in reception and transduction of motor neurotransmission. Gene expression underlying specific functions of ICC classes is incompletely understood. In the present study, we used recently developed highly selective techniques to isolate the two functional ICC classes from enzymatically dispersed intestinal muscles by fluorescence-activated cell sorting. The transcriptomes of ICC-MY and ICC-DMP were investigated using oligonucleotide microarray analysis. Differential expression of functional groups of genes defined by standard gene ontology terms was also studied. There were substantial numbers of genes expressed more abundantly in ICC than in the tunica muscularis, and we also detected marked phenotypic differences between ICC-MY and ICC-DMP. Notably, genes related to cell junction, process guidance, and vesicle trafficking were upregulated in ICC. Consistent with their specific functions, metabolic and Ca2+ transport genes were relatively upregulated in ICC-MY, whereas genes for signaling proteins involved in transduction of neurotransmitter functions were relatively upregulated in ICC-DMP. Our results may lead to the identification of novel biomarkers for ICC and provide directions for further studies designed to understand ICC function in health and disease.

Affymetrix Mouse Genome 430.2 GeneChips; Bioconductor; enteric nervous system; ion channel; signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
MOTOR PATTERNS of the gastrointestinal (GI) tract require the coordinated contractions of the smooth muscle cell component of the organs. One level of coordination comes from the fact that the smooth muscle cells are electrically coupled, and their electrical activity is driven by pacemaker cells known as interstitial cells of Cajal (ICC) that lie in the plane of the myenteric plexus between the circular and longitudinal muscle layers (ICC-MY) (16, 58). ICC-MY are electrically coupled to each other via gap junctions and form a network of cells that extends around the circumference and along the length of the small intestine (1, 39, 58). The ICC-MY network is electrically coupled to smooth muscle cells of both layers, providing a low-resistance pathway for the conduction of pacemaker activity to the musculature (9). ICC-MY possess the ionic conductances necessary to initiate electrical slow waves and to propagate these events actively (cf. Ref. 43).

A second level of control of GI motor function comes from the intrinsic motor neurons arising from enteric ganglia. Both inhibitory and excitatory motor neurons innervate the muscle layers, and their regulation is superimposed on the ongoing electrical rhythmicity of slow-wave activity. Recent studies have suggested that the bulk of motor innervation in the GI tract occurs via a second class of intramuscular ICC (ICC-IM) lying within muscle bundles (5, 49, 59). These cells form synaptic contacts with the varicose terminals of enteric motor neurons (2). In the small intestine of the mouse, ICC-IM are clustered together within the deep muscular plexus near the submucosal surface of the circular muscle layer and are referred to as ICC-DMP.

ICC comprise a minor component of the tunica muscularis (2–6% of all cells; Ref. 36), which has made it difficult to isolate ICC for physiological studies to examine their mechanisms of function. ICC of both classes express Kit (CD117), a receptor tyrosine kinase, and these cells are dependent on Kit signaling for their survival and continued functional activity (27, 51, 58). The expression of Kit has been used widely as a marker for ICC in a variety of species (see Ref. 42), but Kit expression alone does not distinguish between the different classes of ICC. Recently, work in our laboratory (7) has exploited the fact that ICC-DMP are engaged in neurotransmission and express neurokinin 1 (NK1) receptors (17, 24) to isolate ICC-DMP from the greater population of Kit-positive cells in cell dispersions from the small intestine. We found that ICC-DMP could be labeled by exposing muscles to Oregon green 488-conjugated substance P (OG488-SP), which is internalized along with the NK1 receptor. Using fluorescently labeled anti-Kit antibodies for ICC identification and uptake of OG488-SP to identify ICC-DMP specifically, we developed flow cytometry (FCM) procedures to analyze the relative quantities of ICC classes and fluorescence-activated cell sorting (FACS) techniques to isolate specific classes of ICC to a high level of purity (7).

In the present study, we have harvested large numbers of ICC-DMP and ICC-MY cells, using FACS, and have extracted RNA from these cells for microarray analysis of gene expression. We have compared expression patterns in ICC-MY and ICC-DMP to begin to understand the basis for the phenotypic differences in these cells and what makes them unique compared with the whole tunica muscularis tissues. This study represents the first genome-wide analysis of the specific expression patterns in ICC and in specific classes of ICC. For this report, we have extracted data from the databases relevant to the comparative expression of ion channels, mitochondria, receptors, and signaling molecules that may be important for the differential functions of ICC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Mice and tissue preparation.
BALB/c mice aged 6–9-days were obtained from breeder pairs purchased from Charles River Laboratories (Wilmington, MA). The animals were anesthetized with isoflurane (AErrane; Baxter Healthcare, Deerfield, IL) inhalation and killed by decapitation. The jejunum and ileum were excised and opened along the mesentery, and their contents were washed away with ice-cold Krebs-Ringer buffer solution. The mucosa and submucosa were removed by peeling, and only the tunica muscularis of the entire jejunum and ileum was used. Mice were maintained and the experiments performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and the American Physiological Society's "Guiding Principles in the Care and Use of Animals." All protocols were approved by the Institutional Animal Care and Use Committee at the University of Nevada, Reno.

FACS and FCM analysis.
ICC-DMP and ICC-MY were identified and isolated using approaches described previously (7). ICC in murine tissues were labeled vitally by incubation with R-phycoerythrin (PE)-conjugated rat monoclonal anti-mouse Kit antibody (clone ACK2, 1 µg/ml; eBioscience, San Diego, CA). ICC-DMP were selectively identified by uptake of OG488-SP (1 µM; Molecular Probes, Eugene, OR). Neurons that might take up OG488-SP are negative for Kit labeling. Macrophages and dendritic cells that might take up antibodies and fluorescent labels nonspecifically were identified with PE-cyanine 5 (PC5) tandem conjugates of rat monoclonal (IgG2b) anti-mouse F4/80 (clone CI:A3-1, 0.5 µg; Caltag, Burlingame, CA) and PC5-conjugated rat monoclonal (IgG2b) anti-mouse CD11b (clone M1/70.15, 0.5 µg; Caltag). Mast cells that also express Kit but are strongly CD45 positive were labeled with PC5-conjugated rat monoclonal anti-mouse CD45 (Ly-5 or leukocyte common antigen, clone 30-F11, 0.2 µg; eBioscience). Fibroblasts and endothelial cells that also might contaminate the sorted ICC populations were identified by labeling with biotin-anti-CD34 (clone RAM34, 0.5 µg; BD Pharmingen) plus PC5-streptavidin (0.025 µg; eBioscience). Control cell suspensions were only labeled with either OG488-SP or anti-Kit or with the PC5-conjugated antibodies. FACS was performed on a Beckman Coulter EPICS Elite ESP sorter equipped with a flow cell with a 100-µm orifice and optical filters to measure 525-, 575-, 610-, and 670-nm emissions. Initial enrichment of all Kit+ ICC (ICC-MY + ICC-DMP) was performed using a three-drop recovery sort mode. For final purification of ICC-MY and ICC-DMP, the enriched cells were resorted using a one-drop sort mode. FCM was performed with a Beckman Coulter XL/MCL flow cytometer (Fullerton, CA) equipped with an argon ion laser (excitation wavelength 488 nm), a photodiode, and photomultiplier tubes to detect orthogonal light scatter and fluorescence at 525, 575, 670, and >740 nm. Listmode data files were analyzed with FlowJo software (Tree Star, Ashland, OR).

RNA preparation and microarray analysis.
More than 3 x 105 ICC-DMP and 106 ICC-MY cells were recovered from 15 FACS experiments (3 animals per experiment). ICC-MY and ICC-DMP from these experiments were sorted and pooled into three independent samples per ICC class for microarray experiments. Small intestinal tunica muscularis from age-matched BALB/c mice were used as controls (2 independent samples from 3 mice per sample). Total RNA was isolated and purified using Trizol (Invitrogen, Carlsbad, CA) and the RNeasy Mini kit (Qiagen, Valencia, CA), respectively. RNA was quantified and its quality tested using the RNA 6000 Nano assay (Agilent Technologies, Palo Alto, CA). Affymetrix Mouse Genome 430 2.0 microarrays (Santa Clara, CA), which cover most of the transcribed mouse genome (more than 39,000 transcripts targeted by more than 45,000 probe sets on a single array), were used for expression profiling of ICC-DMP (3 microarrays), ICC-MY (3 microarrays), and control tissues (2 microarrays). Two-cycle complementary RNA synthesis and hybridization were performed (Nevada Genomics Center) following the manufacturer's protocols. Microarrays were scanned using an Affymetrix GeneChip 3000 system.

Analysis of microarray data.
Analysis was performed using Bioconductor, a publicly available group of software packages. The packages used included Simpleaffy, Limma, Annaffy, and GOstats (14, 31, 45, 46, 62). Simpleaffy was used to preprocess individual probe intensities from CEL files into expression values from which fold changes were derived using Limma. Robust multiple-array analysis (RMA), which uses quantile normalization for cross-chip normalization, was used to preprocess the data. The main advantage of quantile normalization is that it controls outliers while not significantly reducing sensitivity (18). Significance of differential expression was determined using an empirical Bayes approach (Limma) for controlling the standard error of intensity of each probe set based on the standard errors of the intensities of all other probe sets in the comparison. After P values were obtained for each gene, they were adjusted using the Benjamini-Hochberg method (3). This method converts P values, which are measures of the false positive rate, into Q values, which are measures of false discovery rate. The Benjamini-Hochberg method allows a more direct control of false results while not reducing sensitivity as much as other methods for P value adjustment (3). Annaffy was used to annotate each gene probe set and to tabulate information such as GenBank accession numbers, chromosome locations, and gene ontology terms.

Gene expression was compared between ICC-DMP and intestinal tissues, ICC-MY and intestinal tissues, and ICC-DMP and ICC-MY in terms of fold changes. To determine the significantly different expression, we set the cutoff point of the Q value at 0.05 and the cutoff of the log2 fold change as either above 1 or below –1, corresponding to a fold change of 2 or 1/2. Differentially expressed genes were assigned to functional groups by gene ontology programs. GOstats contains a function called GOHyperG that can be used to find gene ontology terms that may be enriched in a subset of genes. It uses an algorithm that detects whether a certain gene ontology term occurs more frequently in the set of genes than it would be expected based on the frequency of its occurrence in the entire genome. For comparisons in terms of gene ontology terms, we used P < 0.05 as the cutoff point to determine whether the gene ontology term was significantly enriched. Probe sets that contributed to the significant gene ontology terms were then extracted and listed with their log2 fold change and Q value. For genes with multiple probe sets, only the probe set with the greatest fold change was selected to be listed.

In the present study, the sorted populations of ICC-DMP and ICC-MY cells were expected to have a small degree of contamination from other cell types. FCM analysis of the sorted populations also suggested a somewhat greater degree of contamination of the ICC-DMP than ICC-MY (purity of the harvested ICC-DMP was 94.1 ± 3.45%, and purity of the harvested ICC-MY was 96.4 ± 2.06%). Differences in contamination between the compared populations could be falsely attributed to differential gene expression by ICC-DMP and ICC-MY if the two populations were directly compared with each other. Therefore, only probe sets with significantly higher expression in either one of the sorted populations than in the control tissues were included in the comparative analyses of ICC-DMP and ICC-MY. This approach was based on the assumption that mRNA species that are more abundant in purified cells than in tissues should be from ICC-DMP or ICC-MY cells but not from contaminating cells. The data discussed in this article have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE7809.

Quantitative real-time reverse transcription-polymerase chain reaction.
The performance of the microarrays and statistical analyses used was assessed using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) on selected genes (n = 50) within each GO term category selected for detailed analysis. Gene expression was compared between ICC-DMP and intestinal tissues, ICC-MY and intestinal tissues, and ICC-DMP and ICC-MY, respectively, in terms of fold changes. In the case of cell type marker genes, the qRT-PCR experiments were performed in three independent samples. Technical triplicates were used for the verification of the other genes. Gene expression detected by qRT-PCR was considered significantly different if the mean fold change was either above 2 or below 1/2 and the P value from the statistical comparisons (unpaired t-test) was <0.05. Gene expression differences confirmed in this manner are shown in bold type in Tables 3–17. The mean and standard error of log2 fold change for the qRT-PCR of each gene are shown in Supplemental Fig. 1. (Supplemental data for this article is available online at the Physiological Genomics website.)


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 1. Isolation by fluorescence-activated cell sorting of interstitial cells of Cajal (ICC) in the region of the myenteric plexus (ICC-MY) and the deep muscular plexus (ICC-DMP) of the murine small intestines. See MATERIALS AND METHODS for details on labeling and preparation of small intestinal cell suspensions. Identification of ICC-DMP and ICC-MY cells was based on the receptor-mediated internalization of Oregon green 488-conjugated substance P (OG488-SP) by ICC-DMP but not by ICC-MY (6). Since some neurons also express neurokinin 1 receptor (NK1R), ICC were identified with a second label, R-phycoerythrin-conjugated rat monoclonal anti-mouse Kit antibody (PE-ACK2). The clusters of ICC-DMP and ICC-MY cells could be isolated by 2-step sorting, first enriching Kit+ cells and then sorting Kit+OG488-SP+ (Kit+SP+) and Kit+OG488-SP– cells (Kit+SP–) in high-purity mode. PC5– LS cells, PE-cyanin 5– cells with light scatter properties characteristic of live cells.

 
RNA was extracted from sorted ICC-DMP and ICC-MY cells and small intestinal tunica muscularis tissues using Trizol reagent. Genomic DNA was removed with the Absolutely RNA Nanoprep kit (Stratagene, La Jolla, CA). Total RNA was reverse transcribed with 200 units of SuperScript II RNase H– reverse transcriptase (Invitrogen) in a reaction containing 500 ng of oligo d(pT)18 primer (Invitrogen), 10 mM each dNTP, 5x First-Strand buffer, and 100 mM dithiothreitol, followed by heat inactivation. RNA fragments and potential inhibitors of the PCR reaction were removed using 0.5 µl of ribonuclease H (Invitrogen) and the Minelute PCR purification kit (Qiagen, Valencia, CA), respectively. The cDNA reverse transcription product was amplified with specific primers by qRT-PCR using SYBR green chemistry on a 7300 real-time PCR system (Applied Biosystems, Foster City, CA) and the following amplification profile: 95°C for 10 min to activate the AmpliTaq polymerase (Applied Biosystems), followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Transcriptional quantification was obtained relative to GAPDH (a noninducible housekeeping gene). Primers were designed using the Vector NTI 7.1 software (Invitrogen), and the specificity of the primers was tested with the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/) and by sequencing the PCR products. Nonspecific amplification was determined by omitting the template from the PCR. The primer sequences are listed in Supplemental Table 1. Statistical analysis was performed using Student's t-tests.


View this table:
[in this window]
[in a new window]

  		Table 1. Comparison of transcriptional profiling of ICC-DMP, ICC-MY, and tunica muscularis tissues

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
FACS and FCM analysis.
ICC-DMP and ICC-MY were sorted and pooled separately for microarray analysis and qRT-PCR. Identification of these cell types was based on their common expression of Kit and their differential expression of functional NK1 receptor protein (Tacr1) as revealed by receptor-mediated internalization of OG488-SP by ICC-DMP but not by ICC-MY (7) (see Fig. 1 and FACS and FCM analysis for details). Altogether, 28 FACS experiments were performed for microarray and qPCR experiments. Each experiment used jejunal and ileal tunica muscularis tissues from three mice. Up to ~20,000 ICC-DMP and ~65,000 ICC-MY cells per small intestine were collected by FACS. FCM analysis showed that the purity of ICC-DMP used for microarray analysis was 94.1 ± 3.45%, and the purity of ICC-MY was 96.4 ± 2.06%.

Gene expression profiling.
We summarized the numbers of genes and gene ontology terms that were differentially expressed in ICC-DMP and ICC-MY relative to their source tissues and in ICC-DMP relative to ICC-MY (Table 1). As described in Analysis of microarray data, only gene probe sets that were expressed at significantly higher levels in either ICC-MY or ICC-DMP than in the control tissues were included in the comparative analyses of ICC-DMP and ICC-MY genes. The file of complete lists of differentially expressed gene probe sets are provided in Supplemental Table 2, which serves as a database of comparative transcriptional profiling of ICC-DMP and ICC-MY. In this report we focused on genes that may be related to the specific functional roles of ICC-DMP and ICC-MY. However, these genes represent only a small subset of the entire database of differentially expressed genes.

Expression of cell type markers in sorted ICC-DMP and ICC-MY cells.
The results of the microarray analysis for cell type markers illustrate how the microarray analysis alone may lead to erroneous conclusions. Microarray analysis (Table 2) and qRT-PCR (Fig. 2) showed that the expression of ICC marker Kit was enriched in both sorted ICC-DMP and ICC-MY cells. Although NK1 receptor binding was used as a means to distinguish between ICC-MY and ICC-DMP, implying greater expression of NK1 receptors in the latter cells, microarray analysis failed to detect differential expression of Tacr1 mRNA in ICC-DMP and ICC-MY. Quantitative RT-PCR analysis, however, indicated that expression of Tacr1 was about 20-fold higher in ICC-DMP than in ICC-MY. Thus the lack of a significant difference in the microarray analysis likely represents an instance of a false negative result arising, for example, from low mRNA abundance in the compared samples. In contrast to Kit, the expression of smooth muscle cell marker myosin (Myh11), neuron marker ubiquitin carboxy-terminal hydrolase L1 (Uchl1), or macrophage marker Cd68 was much lower in sorted ICC-DMP/ICC-MY cells than in tissues. Although the expression of the mast cell marker mast cell protease 6 (Mcpt6) was not significantly lower in ICC-DMP or ICC-MY relative to whole tissues, qRT-PCR indicated a profound depletion of this marker in the sorted cells. The false negative result reported by the microarrays was likely due to the low number of intramuscular mast cells and the consequent low abundance of the Mcpt6 transcript in the tunica muscularis tissues. Thus the results from the qRT-PCR analyses were consistent with the sorted populations being relatively free of contaminating cells. These results also illustrate that the microarray data can be subject to false negatives and emphasize that the expression of critical genes should be verified. In contrast, qRT-PCR verified the differential expression detected by the microarray analysis in every gene tested (n = 50), indicating that adjusting the P values for false discovery rate very effectively controlled false positives.


View this table:
[in this window]
[in a new window]

  		Table 2. Expression of cell type markers in ICC-DMP and ICC-MY by microarray analysis

 

Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 2. Expression of cell type markers in ICC-DMP and ICC-MY by quantitative RT-PCR. The expression of cell type markers Kit (ICC), Myh11 (smooth muscle cell), Uchl1 (neuron), Cd68 (macrophages and dendritic cells), and Mcpt6 (mast cells) in sorted ICC-DMP (open bars) and ICC-MY (filled bars) were compared with the expression of these genes in tunica muscularis tissues. The expression of Kit was enriched in ICC-DMP/ICC-MY cells, whereas the expression of other cell type markers was much lower compared with source tissues (P < 0.05). The expression of Tacr1 was higher in ICC-DMP than in tissues but lower in ICC-MY than in tissues.

 
Expression of genes related to cell junction in ICC-DMP and ICC-MY.
ICC form networks, and there are gap junctions between ICC and between ICC and smooth muscle cells. Synaptic contacts have been demonstrated between ICC and terminals of motor neurons (2). In the present study, genes related to cell junctions were more abundantly expressed in ICC-MY than in whole tissue (Table 3). Gja1, which encodes connexin 43, is a prominent gap junction protein expressed in ICC and smooth muscle cell gap junctions in GI muscles (57). Several genes related to cell-matrix junctions and adherens junctions were also enriched in ICC-DMP (Table 4). Lasp1 encodes Lasp-1, a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and extracellular matrix proteins (25). Flnb encodes filamin, which is an actin-binding protein that takes part in focal adhesion and participates in the MAPK signaling pathway.


View this table:
[in this window]
[in a new window]

  		Table 3. Genes related to cell junction that were expressed more abundantly in ICC-MY than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 4. Genes related to cell junction that were expressed more abundantly in ICC-DMP than in tissues

 
Expression of signal transduction genes in ICC-DMP.
ICC-DMP are in close association with the terminals of enteric motor neurons (12, 50, 54, 56, 63, 67), and these cells play an important role in mediating enteric motor neurotransmission and signal transduction (17, 55, 61). Previous studies showed that ICC-DMP expressed neurotransmitter receptors and signaling molecules (7, 15, 40). Most of the receptors that mediate excitatory and inhibitory postjunctional effects in GI muscles are G protein-coupled receptors (41). In the present study, several genes related to G protein-coupled receptor signaling, such as guanine nucleotide binding protein, and regulators of G protein signaling were found more highly expressed in ICC-DMP than in ICC-MY or in control tissues (Tables 5 and 6). Enteric inhibitory neurotransmitters, via their receptor proteins, couple to effector proteins through enhanced cAMP and cGMP synthesis and activation of protein kinases. Thus phosphodiesterases are needed to restore basal levels of cAMP and cGMP, and protein phosphatases are needed to restore prestimulus levels of protein phosphorylation after neural activity. The abundant expression of a variety of protein kinases, phosphatases, nucleotide cyclases, and phosphodiesterases in ICC-DMP (Tables 5 and 6) compared with controls is consistent with the role of these cells in mediating postjunctional responses to neurotransmission.


View this table:
[in this window]
[in a new window]

  		Table 5. Genes related to signal transduction that were expressed more abundantly in ICC-DMP than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 6. Genes related to signal transduction that were expressed more abundantly in ICC-DMP than in ICC-MY

 
Expression of genes encoding kinase signaling pathways.
Genes encoding members of kinase signaling pathways, phosphatases, and phosphodiesterases were more abundantly expressed in both ICC-DMP and ICC-MY compared with tissues (Tables 5 and 7). Development and differentiation of ICC are strongly dependent on stem cell factor (SCF) signaling via Kit, and a plethora of evidence indicates that this signaling mechanism is both necessary and sufficient for the survival of these cells. Therefore, it was surprising that both ICC classes significantly overexpressed several other receptor tyrosine kinases and receptor serine/threonine kinases, including Bmpr1b (bone morphogenetic protein receptor type 1B, upregulated in ICC-DMP), Eng (endoglin, upregulated in ICC-MY), and Epha4 and Pdgfrb (ephrin receptor A4 and platelet-derived growth factor receptor ß polypeptide, upregulated in both). The presence of Pdgfrb in both ICC-DMP and ICC-MY is particularly interesting, because Kit and platelet-derived growth factor receptor belong to the same class III receptor tyrosine kinase family and share similar binding sites for downstream signaling molecules. The findings raise the possibility that peptide growth/survival/differentiation factors other than SCF may also play a role in the development, differentiation, and maintenance of ICC, perhaps by modulating the effects of the SCF/Kit signaling pathways.


View this table:
[in this window]
[in a new window]

  		Table 7. Genes related to signal transduction that were expressed more abundantly in ICC-MY than in tissues

 
Neurotransmission receptors expressed by ICC-DMP.
Previous studies showed that ICC-DMP express excitatory neurotransmitter receptors, such as Tacr1, Chrm2, and Chrm3 (tachykinin receptor 1 and muscarinic cholinergic receptor 2 and 3), and inhibitory neurotransmitter receptors, such as Vipr2, P2ry1, and P2ry4 (vasoactive intestinal peptide receptor 2; purinergic receptor P2Y, G protein-coupled 2; and pyrimidinergic receptor P2Y, G protein-coupled 4) (7). Expression of these receptors is fundamental to the role of ICC-DMP in neuromuscular neurotransmission. It is also well known that smooth muscle cells also express a wide variety of receptors for enteric neurotransmitters, and many of the same receptors are expressed by enteric neurons. Our microarray data show that expression of receptor genes by ICC-DMP was not elevated above control tissues. Thus specialization of ICC-DMP does not appear to include overexpression of neurotransmitter receptor transcripts relative to other cell types of the small intestinal tunica muscularis. The specificity of ICC-DMP in neurotransmission may more likely result from the very close synaptic contacts between motor nerve terminals and ICC (2). Receptors may be clustered to these synaptic structures, as in other synapses, and thus receptor density may be enhanced in postjunctional membranes opposite sites of transmitter release.

Ion channels more abundantly expressed by ICC-DMP.
Genes related to Ca2+ transport were enriched in ICC-DMP compared with tissues (Table 8). The genes related to ion transport and more abundantly expressed in ICC-DMP compared with ICC-MY include voltage-dependent K+ channel Kcnq5 and Na+/Ca2+ exchangers Slc8a1 and Slc8a3 (Table 9). Kcnq5 is a novel member of the KCNQ family, which controls neuronal excitability by producing sustained voltage-dependent current (M current) in the subthreshold range of action potential initiation in central nervous system (66). Members of the KCNQ family have been reported to be expressed in GI smooth muscle tissues (35); however, the role of KCNQ channels, and specifically Kcnq5, in ICC-DMP is not yet known.


View this table:
[in this window]
[in a new window]

  		Table 8. Genes related to calcium ion transport that were expressed more abundantly in ICC-DMP than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 9. Genes related to ion channel and ion transport that were expressed more abundantly in ICC-DMP than in ICC-MY

 
Slc8a1 and Slc8a3 encode isoforms of Na+/Ca2+ exchangers. Slc8a1–/– is embryonic lethal, because the gene product is an integral component of the excitation-contraction coupling mechanism in cardiac muscles (38). Slc8a3–/– results in multiple defects in neuromuscular transmission at the skeletal neuromuscular junction, and the suggestion was that Ca2+ entering nerve terminals was cleared more slowly in the absence of Slc8a3 (47). Expression of Slc8a3 has previously been reported to be restricted to brain and skeletal muscles (33). A recent report (4) suggested that the Na+/Ca2+ exchange may be involved in pacemaker activity in interstitial cells of the urogenital tract, where it functions in the "reverse mode," pumping Ca2+ into cells and driving Ca2+-activated inward currents. This is unlikely to be the role of Slc8a1 and Slc8a3 in ICC-DMP, because these cells are not pacemaker cells in the mouse.

Differential expression of transient receptor potential (TRP) genes showed that several of these genes were upregulated in ICC-DMP (Table 8). Our group (52) has previously reported that TRPC4 has properties that are similar to the pacemaker conductance observed in ICC-MY, and others (20) have suggested that TRPM7 might be responsible for pacemaker currents. These genes were upregulated in ICC-DMP, but neither were elevated in ICC-MY. These observations suggest pacemaker activity is probably not a result of specialized expression of either TRPC4 or TRPM7.

Ion channels more abundantly expressed by ICC-MY.
Previous studies have suggested that a dihydropyridine-sensitive, voltage-dependent Ca2+ conductance may be responsible for the upstroke depolarization of slow waves and for active slow-wave propagation in ICC-MY networks (e.g., Refs. 21–23, 37). Pacemaker activity in ICC-MY is associated with robust Ca2+ entry events that are resistant to block by dihydropyridines (37). The molecular identity of the channels responsible for Ca2+ entry and active propagation of slow waves has not been determined. In the present study, we found that several voltage-dependent Ca2+ channels (e.g., Cacna1h, Cacna1d, and Cacna1c) and Ca2+ channel ß- and {gamma}-subunits were more abundantly expressed by ICC-MY than in ICC-DMP or tissues (Tables 10 and 11). Cacna1h and Cacna1d encode Ca2+ channels that are relatively insensitive to dihydropyridines. Thus these conductances may be functionally important for the propagation of pacemaker activity in ICC-MY.


View this table:
[in this window]
[in a new window]

  		Table 10. Genes related to ion channel and ion transport that were expressed more abundantly in ICC-MY than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 11. Genes related to ion channel and ion transport that were expressed more abundantly in ICC-MY than in ICC-DMP

 
Na+/K+ ATPase and Ca2+ ATPase genes were also expressed more abundantly in ICC-MY compared with ICC-DMP or control tissues (Tables 10 and 11), and Slc8a1 was overexpressed in ICC-MY compared with tissues. These pumps and transporters are involved in restoration of ionic gradients and Ca2+ homeostasis, and all of these functions would be extremely important for pacemaker activity where ionic currents and Ca2+ transients are ongoing, essential events. Abundance of Slc8a1 in ICC-MY is interesting in light of the proposed role for Na+/Ca2+ exchanger in pacemaking (4); however, the role of this exchanger in GI pacemaker function has not been investigated. It is possible that the main function of Na+/Ca2+ exchangers in ICC-MY is cycle-by-cycle maintenance of Ca2+, because these cells undergo prominent transient elevations in intracellular Ca2+ during the course of each slow-wave cycle (37).

The amplitude of slow waves recorded from smooth muscle cells is highly dependent on the resting membrane potentials of the smooth muscle cells (23). Studies have recently shown that a pH- and lidocaine-sensitive conductance (TASK), which is a member of the two-pore K+ channel family, contributes significantly to maintenance of resting membrane potential and regulation of excitability in GI muscles (8). In the present study, we found that the expression of Kcnk3 (TASK-1) in ICC-MY was more abundant than that in tissues or ICC-DMP (Tables 10 and 11). In the small intestines of W/Wv mice, ICC-MY are absent, and membrane potential is depolarized by ~9 mV (58). It is possible that Kcnk3 expression by ICC-MY serves to increase the membrane potential and regulate the excitability of smooth muscle cells in GI muscles.

Mitochondrion genes were more abundantly expressed by ICC-MY.
The ion-transporting ATPases and Ca2+ homeostatic transporters in ICC-MY would tend to increase the energy needs for these cells. ICC-MY are often recognized ultrastructurally for their obvious abundance of mitochondria. Mitochondria also are an important organelle in cellular "pacemaker units" that participate in the clocking of pacemaker events (43, 60). Thus it is important to note that several genes and GO terms related to mitochondria or mitochondrial components and oxidative metabolism were enriched in ICC-MY compared with ICC-DMP. Most genes under these GO terms are related to the tricarboxylic acid cycle and contribute to electron transfer and ATP production (Table 12).


View this table:
[in this window]
[in a new window]

  		Table 12. Genes related to mitochondrion that were expressed more abundantly in ICC-MY than in ICC-DMP

 
Ca2+ release from intracellular stores via inositol 1,4,5-trisphosphate (IP3) receptors is a well-documented event within the pacemaker cycle in ICC-MY (60). In contrast to mitochondrial genes, however, IP3 receptor genes (Itpr1, Itpr2, and Itpr3) were not more abundantly expressed in ICC-MY than in ICC-DMP. This may be due to the fact that IP3 receptors also play important roles in signal transduction linked to activation of a variety of G protein-coupled receptors. Genes encoding other Ca2+ release channels (e.g., ryanodine receptors Ryr1, Ryr2, and Ryr3) also were not more abundantly expressed in ICC-MY or ICC-DMP or in ICC compared with whole tissues. This is not surprising, however, since smooth muscle cells express abundant ryanodine receptors, and there does not appear to be a significant role for ryanodine receptors in the functions attributed to ICC (26).

Antiapoptotic mechanisms in ICC.
It is well established that SCF-Kit interactions are required for the continued survival of functional ICC, although the exact mechanisms have not been determined for ICC. Data from other cell types dependent on SCF-Kit interactions and the current microarray results suggest that control of intrinsic apoptosis may be one important mechanism. For example, mast cells (30), melanocytes (19), and a subset of natural killer cells (6) express Kit, and its engagement with SCF is associated with increased expression of Bcl-2, an anti-intrinsic apoptosis protein. In the current microarray results, the expression of Bcl2 is elevated in both classes of ICC (Tables 13 and 14). Increased Bcl-2 expression can also protect cells by altering the intracellular redox potential and preventing oxidant-induced damage (13). This is likely critical for the survival of ICC, which are characterized by an abundance of mitochondria and depend on oxidative metabolism for generating electrical slow waves. In addition to Kit-related effects on intrinsic apoptosis, studies of hematopoietic cells have shown that Kit engagement can also block extrinsic apoptosis such as that resulting from Fas-FasL interaction (11, 34). However, murine genes assigned to the gene ontology term "GO:0008625: induction of apoptosis via death domain receptors" including Fas or Fasl were not differentially expressed in our data sets, so the link to extrinsic apoptosis is less evident. One important protein regulating extrinsic apoptosis is c-FLIP, and the microarray results indicated that the expression of the c-FLIP gene (Cflar) was not significantly upregulated in either class of ICC. Nevertheless, many other apoptosis-related transcripts were found to be overexpressed both in ICC-DMP and ICC-MY (Tables 13 and 14). Clearly, the significance of antiapoptotic mechanisms in ICC and their relationship to Kit signaling requires further investigation.


View this table:
[in this window]
[in a new window]

  		Table 13. Genes related to apoptosis expressed more abundantly in ICC-DMP than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 14. Genes related to apoptosis expressed more abundantly in ICC-MY than in tissues

 
Expression of genes related to process guidance and vesicle trafficking in ICC-DMP and ICC-MY.
Both ICC-DMP and ICC-MY cells have long, thin processes, which are one of the major morphological characteristics of these cells. Microarray analysis showed that several genes related to axon guidance or axon cargo transport are upregulated in these cells. This may indicate that process guidance and vesicle trafficking occur in the long processes of ICC, and these genes also may be involved in the establishment of contacts between cell processes to form ICC networks. In addition, genes associated with coated pits also are upregulated in ICC (Tables 15 and 16), suggesting that ICC might have endocytotic functions. Previous studies showed that Kit can be internalized (64), and these genes may encode proteins that take part in internalization/recycling of Kit and other receptors.


View this table:
[in this window]
[in a new window]

  		Table 15. Genes related to process guidance and vesicle trafficking that were expressed more abundantly in ICC-DMP than in tissues

 

View this table:
[in this window]
[in a new window]

  		Table 16. Genes related to process guidance and vesicle trafficking that were expressed more abundantly in ICC-MY than in tissues

 
Genes highly expressed in ICC-DMP and/or ICC-MY.
Microarray analysis disclosed several genes that were greatly enriched in ICC-DMP and/or ICC-MY relative to whole tissues (Table 17). Expression of the gene for the suppressor of cytokine signaling 3 (Socs3) was significantly elevated in both classes of ICC. Socs3 is upregulated by IL-6, and in turn, Socs3 modulates the signals from the proinflammatory cytokine IL-6 (32, 44, 53). The results showed that expression of the IL-6 gene (Il6) also was elevated in ICC-MY. IL-6 is not the only way in which Socs3 is upregulated. Sommer et al. (48) recently reported that other receptor tyrosine kinases also can act on Socs3. Thus, in ICC, Socs3 may be involved not only in feedback regulation of IL-6 signaling but also in signaling from the other receptor tyrosine kinases expressed in these cells. Expression of the gene for the neutrophil chemotactic factor KC (Cxcl1) was significantly elevated in both classes of ICC. Under some conditions, IL-6 can increase the expression of Cxcl1 (29), so there may be a direct relationship between the upregulation of these two transcripts. Of all the transcripts overexpressed in one or both classes of ICC, perhaps the most unexpected is the gene for inducible costimulator (Icos), which was significantly upregulated in ICC-MY but not in ICC-DMP. Icos is a cell surface molecule of the CD28 family of costimulatory receptors that occurs on some T lymphocytes. It is considered to be specific for T cells and has not been reported to occur in nonlymphoid tissue (28).


View this table:
[in this window]
[in a new window]

  		Table 17. Genes that are highly expressed in ICC-DMP and/or ICC-MY

 
Synaptotagmins (Syt) also were highly expressed in ICC-MY. Synaptotagmins are a family of transmembrane proteins thought to function as transducers of Ca2+ signaling in membrane fusion events. Three isoforms (Syt I, IV, and VII) appear to mediate general cell biological functions (10), and previous studies have suggested that Syt IV is predominantly expressed in the postsynaptic compartment, possibly to mediate an evolutionarily conserved postsynaptic membrane-trafficking pathway (65). The relatively high expression of Syt4 in ICC-MY may suggest involvement in vesicle-trafficking processes in ICC-MY, e.g., the internalization and recycling of cell-surface receptors.

High levels of expression of several of these genes were verified by qRT-PCR. However, the much lower relative expression of these genes in other cell types in the tunica muscularis does not determine specificity of expression in ICC, and the expression levels of genes are not necessarily related in a linear manner to the expression of proteins. Nevertheless, it is possible that some or many of these highly expressed genes could serve as candidate biomarkers for ICC or for specific classes of ICC. Future studies utilizing immunohistochemical techniques with specific antibodies are needed to explore the specificity of expression of these candidate proteins in ICC. Greatly upregulated expression also may suggest novel specific functions or novel importance of specific pathways in ICC.

In summary, this is the first genome-wide analysis of expression patterns in ICC and in specific classes of ICC found in the murine small intestine. We found quite significant differences in ICC relative to other cells of the tunica muscularis, and there also were many unique features of ICC-MY compared with ICC-DMP that are likely to underlie the different phenotypes and functions of these cells. In all, there were 1,600 genes more abundantly expressed in ICC-DMP and 1,223 genes more abundantly expressed in ICC-MY compared with the tunica muscularis. Some of these transcripts were relatively very highly expressed and may provide new cell-specific proteins to explore as ways to label ICC or that might serve as biomarkers to evaluate the status of ICC in pathological conditions. ICC-MY and ICC-DMP were also surprisingly distinct from each other, and 330 genes were elevated in ICC-MY versus ICC-DMP, whereas 607 genes were elevated in ICC-DMP versus ICC-MY. Again, it may be possible to exploit these differences in the creation of new cell-specific probes for specific classes of ICC, or it may be possible to use cell-specific proteins as biomarkers to evaluate specific damage to one class of cells versus the other in murine models of pathological conditions. Thus the database obtained from these studies (at NCBI) may provide a basis for novel hypotheses in a variety of future studies.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Program Project Grant DK41315. Dr. T. Ördög and Dr. D. Redelman also received support from NIDDK Grant DK58185 and Nevada IDeA Network of Biomedical Research Excellence Grant P20 RR016464. Core laboratories (i.e., immunofluorescence and FACS) were supported by DK41315.


   ACKNOWLEDGMENTS
 
Present addresses: Dr. H. Chen, Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021; Drs. T. Ördög and M. R. Bardsley, Physiology and Biomedical Engineering and Enteric NeuroScience Program, Mayo Clinic College of Medicine, Guggenheim 10, 200 First St. SW, Rochester, MN 55905.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada, Reno, School of Medicine, Anderson Bldg., Mail Stop 352, Reno, NV 89557 (e-mail: kent{at}unr.edu).


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

  1. Baluk P, Gabella G. Scanning electron microscopy of the muscle coat of the guinea-pig small intestine. Cell Tissue Res 250: 551–561, 1987.[Web of Science][Medline]
  2. Beckett EA, Takeda Y, Yanase H, Sanders KM, Ward SM. Synaptic specializations exist between enteric motor nerves and interstitial cells of Cajal in the murine stomach. J Comp Neurol 493: 193–206, 2005.[CrossRef][Web of Science][Medline]
  3. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B 57: 289–300, 1995.
  4. Bradley E, Hollywood MA, Johnston L, Large RJ, Matsuda T, Baba A, McHale NG, Thornbury KD, Sergeant GP. Contribution of reverse Na+-Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra. J Physiol 574: 651–661, 2006.[Abstract/Free Full Text]
  5. Burns AJ, Lomax AEJ, Torihashi S, Sanders KM, 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]
  6. Carson WE, Haldar S, Baiocchi RA, Croce CM, Caligiuri MA. The Kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2. Proc Natl Acad Sci USA 91: 7553–7557, 1994.[Abstract/Free Full Text]
  7. Chen H, Redelman D, Ro S, Ward SM, Ördög T, Sanders KM. Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine. Am J Physiol Cell Physiol 292: C497–C507, 2007.[Abstract/Free Full Text]
  8. Cho SY, Beckett EA, Baker SA, Han I, Park KJ, Monaghan K, Ward SM, Sanders KM, Koh SD. A pH-sensitive potassium conductance (TASK) and its function in the murine gastrointestinal tract. J Physiol 565: 243–259, 2005.[Abstract/Free Full Text]
  9. Cousins HM, Edwards FR, Hickey H, Hill CE, Hirst GD. Electrical coupling between the myenteric interstitial cells of Cajal and adjacent muscle layers in the guinea-pig gastric antrum. J Physiol 550: 829–844, 2003.[Abstract/Free Full Text]
  10. Craxton M. Synaptotagmin gene content of the sequenced genomes. BMC Genomics 5: 43, 2004.[CrossRef][Medline]
  11. Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/Kit signaling the other. J Exp Med 192: 1707–1718, 2000.[Abstract/Free Full Text]
  12. Duchon G, Henderson R, Daniel EE. Circular muscle layers in the small intestine In: Proceedings of the Fourth International Symposium on Gastrointestinal Motility, edited by Daniel EE, Gilbert JAL, Schofield B, Schnitka TK, Scott G. Vancouver, British Columbia: 1974, p. 635–646.
  13. FitzGerald UF, Gilbey T, Brodie S, Barnett SC. Transcription factor expression and cellular redox in immature oligodendrocyte cell death: effect of Bcl-2. Mol Cell Neurosci 22: 516–529, 2003.[CrossRef][Web of Science][Medline]
  14. Gentlemen R, Falcon S. How to use GOstats: testing gene lists for GO term association. http://bioconductor.org/packages/2.1/bioc/vignettes/GOstats/inst/doc/GOstatsHyperG.pdf.
  15. Huber A, Trudrung P, Storr M, Franck H, Schusdziarra V, Ruth P, Allescher HD. Protein kinase G expression in the small intestine and functional importance for smooth muscle relaxation. Am J Physiol Gastrointest Liver Physiol 275: G629–G637, 1998.[Abstract/Free Full Text]
  16. Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, Bernstein A. W/kit gene required for intestinal pacemaker activity. Nature 373: 347–349, 1995.[CrossRef][Medline]
  17. Iino S, Ward SM, Sanders KM. Interstitial cells of Cajal are functionally innervated by excitatory motor neurons in the murine intestine. J Physiol 556: 521–530, 2004.[Abstract/Free Full Text]
  18. Irizzary RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249–264, 2003.[Abstract]
  19. Ito M, Kawa Y, Ono H, Okura M, Baba T, Kubota Y, Nishikawa SI, Mizoguchi M. Removal of stem cell factor or addition of monoclonal anti-Kit antibody induces apoptosis in murine melanocyte precursors. J Invest Dermatol 112: 796–801, 1999.[CrossRef][Web of Science][Medline]
  20. Kim BJ, Lim HH, Yang DK, Jun JY, Chang IY, Park CS, So I, Stanfield PR, Kim KW. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity. Gastroenterology 129: 1504–1517, 2005.[CrossRef][Web of Science][Medline]
  21. Kim YC, Koh SD, Sanders KM. Voltage-dependent inward currents of interstitial cells of Cajal from murine colon and small intestine. J Physiol 541: 797–810, 2002.[Abstract/Free Full Text]
  22. Kito Y, Suzuki H. Properties of pacemaker potentials recorded from myenteric interstitial cells of Cajal distributed in the mouse small intestine. J Physiol 553: 803–818, 2003.[Abstract/Free Full Text]
  23. Kito Y, Ward SM, Sanders KM. Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine. Am J Physiol Cell Physiol 288: C710–C720, 2005.[Abstract/Free Full Text]
  24. Lavin ST, Southwell BR, Murphy R, Jenkinson KM, Furness JB. Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P. Histochem Cell Biol 110: 263–271, 1998.[CrossRef][Web of Science][Medline]
  25. Lin YH, Park ZY, Lin D, Brahmbhatt AA, Rio MC, Yates JR 3rd, Klemke RL. Regulation of cell migration and survival by focal adhesion targeting of Lasp-1. J Cell Biol 165: 421–432, 2004.[Abstract/Free Full Text]
  26. Liu LW, Thuneberg L, Huizinga JD. Cyclopiazonic acid, inhibiting the endoplasmic reticulum calcium pump, reduces the canine colonic pacemaker frequency. J Pharmacol Exp Ther 275: 1058–1068, 1995.[Abstract/Free Full Text]
  27. Maeda H, Yamagata A, Nishikawa S, Yoshinaga K, Kobayashi S, Nishi K, Nishikawa S. Requirement of Kit for development of intestinal pacemaker system. Development 116: 369–375, 1992.[Web of Science][Medline]
  28. Mages HW, Hutloff A, Heuck C, Buchner K, Himmelbauer H, Oliveri F. Kroczek RA. Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand. Eur J Immunol 30: 1040–1047, 2000.[CrossRef][Web of Science][Medline]
  29. McLoughlin RM, Hurst SM, Nowell MA, Harris DA, Horiuchi S, Morgan LW, Wilkinson TS, Yamamoto N, Topley N, Jones SA. Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms. J Immunol 172: 5676–5683, 2004.[Abstract/Free Full Text]
  30. Mekori YA, Gilfillan AM, Akin C, Hartmann K, Metcalfe DD. Human mast cell apoptosis is regulated through Bcl-2 and Bcl-XL. J Clin Immunol 21: 171–174, 2001.[CrossRef][Web of Science][Medline]
  31. Miller CJ. Description of Simpleaffy: easy analysis routines for affymetrix data. http://bioconductor.org/packages/2.1/bioc/vignettes/simpleaffy/inst/doc/simpleAffy.pdf.
  32. Nicholson SE, Willson TA, Farley A, Starr R, Zhang JG, Baca M, Alexander WS, Metcalf D, Hilton DJ, Nicola NA. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J 18: 375–385, 1999.[CrossRef][Web of Science][Medline]
  33. Nicoll DA, Quednau BD, Qui Z, Xia YR, Lusis AJ, Philipson KD. Cloning of a third mammalian Na+-Ca2+ exchanger, NCX3. J Biol Chem 271: 24914–24921, 1996.[Abstract/Free Full Text]
  34. Nishio M, Oda A, Koizumi K, Satoh I, Sato Y, Endoh T, Tsutsumi A, Fujihara M, Ikebuchi K, Ikeda H, Koike T, Sawada KI. Stem cell factor prevents Fas-mediated apoptosis of human erythroid precursor cells with Src-family kinase dependency. Exp Hematol 29: 19–29, 2001.[CrossRef][Web of Science][Medline]
  35. Ohya S, Asakura K, Muraki K, Watanabe M, Imaizumi Y. Molecular and functional characterization of ERG, KCNQ, and KCNE subtypes in rat stomach smooth muscle. Am J Physiol Gastrointest Liver Physiol 282: G277–G287, 2002.[Abstract/Free Full Text]
  36. Ördög T, Redelman D, Horvath VJ, Miller LJ, Horowitz B, Sanders KM. Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers, and mediators of neurotransmission in the gastrointestinal tract. Cytometry A 62: 139–149, 2004.[CrossRef][Medline]
  37. Park KJ, Hennig GW, Lee HT, Spencer NJ, Ward SM, Smith TK, Sanders KM. Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ. Am J Physiol Cell Physiol 290: C1411–C1427, 2006.[Abstract/Free Full Text]
  38. Reuter H, Henderson SA, Han T, Mottino GA, Frank JS, Ross RS, Goldhaber JI, Philipson KD. Cardiac excitation-contraction coupling in the absence of Na+-Ca2+ exchange. Cell Calcium 34: 19–26, 2003.[CrossRef][Web of Science][Medline]
  39. Rumessen JJ, Thuneberg L. Interstitial cells of Cajal in human small intestine. Ultrastructural identification and organization between the main smooth muscle layers. Gastroenterology 100: 1417–1431, 1991.[Web of Science][Medline]
  40. Salmhofer H, Neuhuber WL, Ruth P, Huber A, Russwurm M, Allescher HD. Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence. Cell Tissue Res 305: 331–340, 2001.[CrossRef][Web of Science][Medline]
  41. Sanders KM. G protein-coupled receptors in gastrointestinal physiology. IV. Neural regulation of gastrointestinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 275: G1–G7, 1998.[Abstract/Free Full Text]
  42. Sanders KM, Ördög T, Koh SD, Torihashi S, Ward SM. Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil 11: 311–338, 1999.[CrossRef][Web of Science][Medline]
  43. Sanders KM, Koh SD, Ward SM. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 68: 307–343, 2006.[CrossRef][Web of Science][Medline]
  44. Schmitz J, Weissenbach M, Haan S, Heinrich PC, Schaper F. SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J Biol Chem 275: 12848–12856, 2000.[Abstract/Free Full Text]
  45. Smith CA. Textual description of Annaffy. http://bioconductor.org/packages/2.1/bioc/vignettes/annaffy/inst/doc/annaffy.pdf.
  46. Smyth GK. Limma: linear models for microarray data. In: Bioinformatics and Computational Biology Solutions Using R and Bioconductor, edited by Gentlemen R, Carey V, Dudoit S, Irizarry R, Huber W. New York: Springer, 2005, p. 397–420.
  47. Sokolow S, Manto M, Gailly P, Molgo J, Vandebrouck C, Vanderwinden JM, Herchuelz A, Schurmans S. Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3. J Clin Invest 113: 265–273, 2004.[CrossRef][Web of Science][Medline]
  48. Sommer U, Schmid C, Sobota RM, Lehmann U, Stevenson NJ, Johnston JA, Schaper F, Heinrich PC, Haan S. Mechanisms of SOCS3 phosphorylation upon interleukin-6 stimulation. Contributions of Src- and receptor-tyrosine kinases. J Biol Chem 280: 31478–31488, 2005.[Abstract/Free Full Text]
  49. Suzuki H, Ward SM, Bayguinov YR, Edwards F, Hirst GDS. Involvement in intramuscular interstitial cells of Cajal in nitrergic inhibition in the mouse gastric antrum. J Physiol 546: 751–763, 2003.[Abstract/Free Full Text]
  50. Torihashi S, Kobayashi S, Sanders KM. Interstitial cells in deep muscular plexus of canine small intestine may be specialized smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 265: G638–G645, 1993.[Abstract/Free Full Text]
  51. Torihashi S, Ward SM, Nishikawa SI, Nishi K, Kobayashi S, Sanders KM. Kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280: 97–111, 1995.[Web of Science][Medline]
  52. Walker RL, Koh SD, Sergeant GP, Sanders KM, Horowitz B. TRPC4 currents have properties similar to the pacemaker current in interstitial cells of Cajal. Am J Physiol Cell Physiol 283: C1637–C1645, 2002.[Abstract/Free Full Text]
  53. Wang X, Athayde N, Trudinger B. A proinflammatory cytokine response is present in the fetal placental vasculature in placental insufficiency. Am J Obstet Gynecol 189: 1445–1451, 2003.[CrossRef][Web of Science][Medline]
  54. Wang XY, Sanders KM, Ward SM. Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine. Cell Tissue Res 295: 247–256, 1999.[CrossRef][Web of Science][Medline]
  55. Wang XY, Ward SM, Gerthoffer WT, Sanders KM. Protein kinase C{varepsilon} translocation in enteric neurons and interstitial cells of Cajal in response to muscarinic stimulation. Am J Physiol Gastrointest Liver Physiol 285: G593–G601, 2003.[Abstract/Free Full Text]
  56. Wang XY, Paterson C, Huizinga JD. Cholinergic and nitrergic innervation of ICC-DMP and ICC-IM in the human small intestine. Neurogastroenterol Motil 15: 531–543, 2003.[CrossRef][Web of Science][Medline]
  57. Wang YF, Daniel EE. Gap junctions in gastrointestinal muscle contain multiple connexins. Am J Physiol Gastrointest Liver Physiol 281: G533–G543, 2001.[Abstract/Free Full Text]
  58. Ward SM, Burns AJ, Torihashi S, Sanders KM. Mutation of the proto-oncogene Kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol 480: 91–97, 1994.[Abstract/Free Full Text]
  59. Ward SM, Beckett EAH, Wang XY, Baker F, Khoyi M, Sanders KM. Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons. J Neurosci 20: 1393–1403, 2000.[Abstract/Free Full Text]
  60. Ward SM, Ordog T, Koh SD, Baker SA, Jun JY, Amberg G, Monaghan K, Sanders KM. Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol 525: 355–361, 2000.[Abstract/Free Full Text]
  61. Ward SM, McLaren GJ, Sanders KM. Interstitial cells of Cajal in the deep muscular plexus mediate enteric motor neurotransmission in the mouse small intestine. J Physiol 573: 147–159, 2006.[Abstract/Free Full Text]
  62. Yamamoto M. Electron microscopic studies on the innervation of the smooth muscle and the interstitial cell of Cajal in the small intestine of the mouse and bat. Arch Histol Jpn 40: 171–201, 1977.[Medline]
  63. Yee NS, Hsiau CW, Serve H, Vosseller K, Besmer P. Mechanism of down-regulation of Kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C. J Biol Chem 269: 31991–31998, 1994.[Abstract/Free Full Text]
  64. Yoshihara M, Montana ES. The synaptotagmins: calcium sensors for vesicular trafficking. Neuroscientist 10: 566–574, 2004.[Abstract/Free Full Text]
  65. Yus-Najera E, Munoz A, Salvador N, Jensen BS, Rasmussen HB, Defelipe J, Villarroel A. Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation. Neuroscience 120: 353–364, 2003.[CrossRef][Web of Science][Medline]
  66. Zhou DS, Komuro T. The cellular network of interstitial cells associated with the deep muscular plexus of the guinea pig small intestine. Anat Embryol (Berl) 186: 519–527, 1992.[Medline]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
S. J. Hwang, P. J. A. Blair, F. C. Britton, K. E. O'Driscoll, G. Hennig, Y. R. Bayguinov, J. R. Rock, B. D. Harfe, K. M. Sanders, and S. M. Ward
Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles
J. Physiol., October 15, 2009; 587(20): 4887 - 4904.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. H. Zhu, T. W. Kim, S. Ro, W. Yan, S. M. Ward, S. D. Koh, and K. M. Sanders
A Ca2+-activated Cl\#8722; conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity
J. Physiol., October 15, 2009; 587(20): 4905 - 4918.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
T. A. Jepps, I. A. Greenwood, J. D. Moffatt, K. M. Sanders, and S. Ohya
Molecular and functional characterization of Kv7 K+ channel in murine gastrointestinal smooth muscles
Am J Physiol Gastrointest Liver Physiol, July 1, 2009; 297(1): G107 - G115.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
P. J. Gomez-Pinilla, S. J. Gibbons, M. R. Bardsley, A. Lorincz, M. J. Pozo, P. J. Pasricha, M. V. de Rijn, R. B. West, M. G. Sarr, M. L. Kendrick, et al.
Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract
Am J Physiol Gastrointest Liver Physiol, June 1, 2009; 296(6): G1370 - G1381.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. Kim, G. W. Hennig, K. Park, I. S. Han, T. K. Smith, S. D. Koh, and B. A. Perrino
Modulation of murine gastric antrum smooth muscle STOC activity and excitability by phospholamban
J. Physiol., October 15, 2008; 586(20): 4977 - 4991.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
E. J. White, S. J. Park, J. A. Foster, and J. D. Huizinga
Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal
Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G691 - G699.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figure and Tables
Right arrow All Versions of this Article:
31/3/492    most recent
00113.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.
Right arrow Articles by Sanders, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.
Right arrow Articles by Sanders, K. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.