Microarray comparison of normal and W/Wv mice in the gastric fundus indicates a supersensitive phenotype
Gerard P. Sergeant,
Roddy J. Large,
Elizabeth A. H. Beckett,
Cathrine M. McGeough,
Sean M. Ward and
Burton Horowitz
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046
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ABSTRACT
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Interstitial cells of Cajal (ICC) have been identified in specific areas throughout the smooth musculature of the gastrointestinal (GI) tract. Located within the circular and longitudinal muscle layers of the gastric fundus lies a specific type of ICC, termed "intramuscular" ICC or IC-IM. The principal function of this cell type is to act as "mediators of excitatory and inhibitory enteric neurotransmission." The functional role of these cells has been investigated using W/Wv mutant mice that specifically lack IC-IM, resulting in disrupted enteric neurotransmission. The aim of the present study was to investigate differential gene expression in W/Wv mutant mice, from the tunica muscularis of the gastric fundus using a mouse cDNA microarray containing 1,081 known genes. Verification of the microarray data was attained using real-time "quantitative" PCR (qPCR). Of the 1,081 arrayed genes, 36 demonstrated differential expression by >2-fold in the W/Wv mice. An agreement rate of 50% (7 of 14 tested) was obtained using qPCR. Of the seven confirmed changes in expression, several were indicative of a supersensitive phenotype, observed in denervation models. Expression of several putative neurotransmitter receptors including P2Y, the receptor for the inhibitory neurotransmitter ATP, was upregulated. The functional role of the P2Y receptor was also investigated using electrophysiological recordings. These results offer a new insight into the molecular changes that occur in W/Wv fundic smooth muscle and may also provide novel information with regard to the importance of IC-IM in enteric neurotransmission.
microarray; quantitative polymerase chain reaction; fundic smooth muscle; interstitial cells of Cajal
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INTRODUCTION
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OUR UNDERSTANDING of gastrointestinal (GI) physiology has improved in recent years with the discovery that interstitial cells of Cajal (ICC) provide the pacemaker activity typical of phasic GI smooth muscles (25). This issue is complicated by the fact that there are specific subclasses of ICC, located in discrete anatomical regions and possessing specific physiological functions. For example, ICC located in the myenteric region in the gastric antrum (IC-MY) were found to initiate electrical slow wave activity (7), whereas ICC located in the deep muscular plexus of the small intestine (IC-DMP) are innervated, playing an important role in enteric nerve signaling (19, 27). ICC that are also important for enteric nerve signaling are intramuscular ICC (IC-IM), which are distributed within the circular and longitudinal smooth muscle layers of the stomach and colon and also within sphincteric regions of the GI tract including the lower esophageal and pyloric sphincters (5, 28, 30).
ICC located throughout the GI tract express the proto-oncogene c-kit, which encodes for the receptor tyrosine kinase, Kit. Kit is essential for the development and maintenance of ICC networks and electrical pacemaking activity in the GI tract (20). Mice with mutations in the W locus, which is allelic for kit, lack kit expression and possess abnormal GI activity (20). For example, the compound heterozygote W/Wv mouse has reduced kit expression and exhibits dramatic differences in the distribution of ICC networks throughout the GI tract (14, 29). Although information from W/Wv mice has greatly enhanced ICC and GI motility research, W/Wv mice do not serve as complete ICC "knockouts" in most tissues with the exception of the gastric fundus. The fundus contains only one population of ICC, IC-IM, which is completely absent in W/Wv mice (5). Molecular analysis of this region of the GI tract in W/Wv mice represents a unique opportunity to investigate the function of IC-IM as well as the implications for cells which are dependent upon IC-IM signaling, such as neighboring smooth muscle cells (5, 9, 28).
DNA microarrays are powerful tools, which have provided a means for studying large-scale gene expression in a range of biological systems. Examples of their use include the identification of genes implicated in the development of heart disease and neurological disorders (12, 15) as well those associated with ageing and immune and liver function (1). The ability to pinpoint genes specifically involved with such diverse manifestations may not only provide a basis for possible future therapeutic treatments but also for a more complete understanding of the processes that occur during normal functioning of these tissues. In the present study we utilized cDNA microarrays to compare the expression levels of 1,081 genes in wild-type and W/Wv murine gastric fundus smooth muscles and assayed the functional role of changes in these genes using an electrophysiological approach.
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METHODS
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Probe synthesis and hybridization.
Each DNA microarray experiment utilized 15 µg of total RNA, collected from eight aged matched sibling wild-type (+/+; black coats) and eight W/Wv (pure white coats) mice. The tunica muscularis was prepared per Epperson et al. (9) and total RNA was synthesized using the Trizol Reagent (Invitrogen, San Diego, CA) per the manufacturers instructions and subsequently purified using the RNeasy mini kit supplied by Qiagen (Valencia, CA). Synthesis of labeled probes for analysis was achieved per the manufacturers instructions (Clontech Laboratories, Palo Alto, CA). Briefly, purified first-strand cDNA was synthesized from RNA collected from both groups of mice, and then coupled to the relevant fluorescent dye (Cy3 or Cy5). Hybridization of the labeled probe to the glass microarray (Atlas Glass Mouse 1.0; Clontech Laboratories, Palo Alto, CA) was again performed per the manufacturers instructions but consisted of incubating the array with the labeled probes overnight at 50°C and subsequently washed several times.
Data acquisition and analysis.
The fluorescent images of hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Scan Array 4000, Packard Biosciences) and images were acquired and analyzed using Scan Array (version 3.1) and Quant Array (version 3.0) software, respectively (PE Biosystems, Hayward, CA). These data were then further analyzed using Microsoft Excel. Background fluorescence intensities for each spot were subtracted to leave actual fluorescence intensities for each spot on the array. Gene expression was considered positive if the fluorescence intensity was higher than that produced by the respective negative control within each block on the array. This allowed a list of expressed genes on each array to be compiled for analysis. A complete list of the genes spotted on this array can be accessed at the web site http://atlasinfo.clontech.com.
Normalization.
It was necessary to normalize the absolute fluorescence intensity for the relevant channel (Cy3 or Cy5) by multiplication with a normalization coefficient to control for differences in dye coupling and cDNA synthesis. In each case the fluorescence intensity for one channel was rescaled by a normalization coefficient to control for these differences. Gene expression ratios were calculated by dividing normalized fluorescence intensities from W/Wv mice by that from wild type, so that values of 1 indicated that there was no significant difference in expression, and values greater than 1 indicated that any particular gene was now more highly expressed or "upregulated" in W/Wv mice. Differences in expression were considered significant by a
2-fold change, therefore expression ratios of less than 0.5 or greater than 2 were deemed significant. A list of genes that were significantly changed in the W/Wv mice compared with the wild type in both arrays was compiled.
Quantitative RT-PCR.
First-strand cDNA was prepared from the RNA preparations using the Superscript II Reverse Transcriptase kit (GIBCO-BRL, Gaithersburg, MD), 500 µg/µl of oligo dT primers were used to reverse transcribe the RNA sample. Real time "quantitative" PCR (qPCR) was performed on a number of "upregulated" genes identified from the array to verify these data. This was achieved using Syber Green (PE Biosystems, Hayward, CA) chemistry on a sequence detector (model ABI 5700; PE Biosystems, Hayward, CA). Details of the specific primers designed for qPCR to determine relative levels of expression are given in Table 1. ß-Actin (V01217) nt 22042224 and 23842402; amplicon = 198 bp was used as the endogenous standard. PCR products generated from each pair of primers were extracted and sequenced to confirm the specificity of the primers. Regression analysis of the mean values of eight multiplex RT-PCRs for the log10 diluted cDNA was used to generate standard curves. Unknown quantities relative to the standard curve for a particular set of primers were calculated yielding transcriptional quantification of the gene products relative to the endogenous standard (ß-actin). These values were subsequently normalized so that comparisons between genes of differing expression levels could be made. The reproducibility of the assay was tested by analysis of variance (ANOVA) comparing repeat runs of samples, and mean values generated at individual time points were compared by a one-tailed paired t-test.
Functional studies.
The entire stomach, including portions of the esophagus and duodenum was removed and placed in Krebs-Ringer buffer (KRB). For electrophysiological measurements, stomachs were opened along the lesser curvature from the most proximal regions of the fundus through to the corpus. Gastric contents were washed with KRB, and the mucosa was removed by sharp dissection, revealing the underlying circular muscle layer of the gastric fundus.
After removing the mucosa, sections of gastric fundus muscle (6 mm x 5 mm) were cut and pinned to the Sylgard elastomer (Dow Corning, Midland, MI) floor of a recording chamber with the submucosal side of the circular muscle facing upward. For electrical field stimulation (EFS) of motor nerves, parallel platinum electrodes were placed on either side of the muscle strips. Circular muscle cells were impaled with glass microelectrodes, and transmembrane potentials were measured with a high-impedance electrometer (WPI Intra 767; World Precision Instruments, Sarasota, FL), and outputs were displayed on a Tektronix 2224 oscilloscope (Wilsonville, OR). Electrical signals were recorded on a PC running Axoscope 8.0 (Axon Instruments, Union City, CA). Neural responses were elicited using square wave pulses of EFS (0.5-ms duration, 150 Hz, supramaximal voltage; model S48 stimulator; Grass Instruments, Quincy, MA).
Solutions and drugs.
For electrophysiological experiments, muscles were maintained in KRB (37.5 ± 0.5°C; pH 7.37.4) containing (in mM) 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg, 134 Cl-, 15.5 HCO3-, 1.2 H2PO4-, and 11.5 dextrose and bubbled with 97% O2-3% CO2. Neural responses to EFS were recorded under control conditions in a solution containing N
-nitro-L-arginine (L-NA, 100 µM), atropine sulfate, and nifedipine (1 µM) to reduce muscle contraction. To determine the contribution of purinergic neurotransmission through P2 receptors, muscles were incubated in pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS, 1030 µM) for 30 min before repeating EFS stimulation. L-NA, atropine sulfate, and PPADS were dissolved in distilled water at 0.10.01 M. Nifedipine was dissolved in ethanol at 0.01 M. All drugs were then diluted in KRB to the stated final concentrations (all obtained from Sigma, St. Louis, MO).
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RESULTS
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Microarray analysis.
An Atlas glass mouse 1.0 array was hybridized with cDNA probes synthesized from RNA collected from gastric fundus smooth muscles from aged-matched sibling wild-type and W/Wv mice. Shown in Fig. 1 are a series of scanned images from a typical microarray experiment in which wild-type cDNA is coupled with Cy5 fluorophore (Fig. 1A) and W/Wv with Cy3 fluorophore (Fig. 1B). Shown in Fig. 1C is an overlay image of Fig. 1, A and B, indicating the relative difference in gene expression between both tissues. In this example, yellow spots indicate a similar level of expression, whereas red or green spots would suggest a higher level of gene expression in either the wild-type or W/Wv tissues, respectively. A feature to note in Fig. 1 is the general low background fluorescence, an essential component of a microarray experiment for reliable information about gene expression levels (31). Also noteworthy is the observation that the spots containing only Cy3-labeled oligonucleotides, which thus serve as Cy3 orientation markers, are only fluorescent in the Cy3 channel. Also those spots where only buffer and no cDNA were printed on the slide, thus serving as negative controls, are blank. Highlighted in Fig. 1 by the red outline is a section of the array which contains sequences for nine housekeeper genes, details of which are documented in Table 2.

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Fig. 1. Typical example of a microarray experiment utilizing cDNA synthesized from gastric fundus smooth muscles of wildtype and W/Wv mice, labeled with the Cy5 (A) and Cy3 (B) fluorophores, respectively. C is an overlay image of A and B. Highlighted in red is a section of the array containing nine housekeeping genes, the expression of which was not different between the two groups. Images are pseudo-colored, and each spot represents the expression of a specific gene.
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Table 2. A list of the housekeeper genes highlighted in Fig. 1 together with their respective "adjusted" fluorescence intensities and expression ratios
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Table 2 comprises a list of the housekeeper genes highlighted in Fig. 1 together with their respective "adjusted" fluorescence intensities and expression ratios. Adjusted fluorescence intensity in this context defines a value calculated by subtracting the background fluorescence for each gene, then multiplying the relevant values (Cy3 or Cy5 channel) by a normalization coefficient, as described in METHODS. The expression ratios were derived by dividing these values for the W/Wv by that for the wild type. Ratios
1.0 indicate no change in expression, whereas values
2 were considered to be significantly changed. All of the housekeeper genes spotted on this array exhibited ratios
1, demonstrating that their expression level does not significantly change in W/Wv mice and therefore also acted as a good internal control since the expression of these housekeeper genes would not have been expected to differ. Expression ratios for all other genes on the array were then compiled using this method and plotted as a frequency histogram with the expression ratios pooled into bins of width 0.25 and ranging from 0 to >4. These data are illustrated in Fig. 2.

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Fig. 2. A frequency histogram of gene expression ratios from the data displayed in Fig. 1, calculated by dividing the fluorescence intensity for theW/Wv model (Cy3) by the wild type (Cy5). Therefore, expression ratios of one or above indicate increased gene expression in W/Wv animals.
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If all genes on the array displayed a similar relationship as the "housekeepers" then a bell-shaped distribution centered on 1.0 might be expected. However, Fig. 2 shows that the distribution curve centers at a value positive of 1.0. Indeed, the mean expression ratio for this array was 1.46, indicating that there is a trend toward higher gene expression levels inW/Wv mice compared with the wild type.
Table 3 is a joint list of genes which consistently showed a twofold or greater change in expression levels in W/Wv mice from two microarray experiments. In the second microarray experiment, the coupling of the Cy3 and Cy5 dyes was reversed to control for any differences in differential dye coupling, so that wild-type cDNA was coupled to Cy3 and W/Wv to Cy5. Thirty-six genes in total, from both arrays, demonstrated a greater than twofold upregulation in the W/Wv whereas none showed any significant downregulation.
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Table 3. A joint list of genes which consistently showed a 2-fold or greater change in expression levels in W/Wv mice from 2 microarray experiments
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Quantitative RT-PCR.
To verify that the cDNA microarray data do reflect changes in gene expression, qPCR experiments were conducted to support these findings. Fourteen genes of interest out of the 36 identified from the array were chosen. In addition, the levels of c-kit were also analyzed to act as a control, since c-kit levels should be significantly decreased in W/Wv mice as the number of Kit-expressing cells is dramatically decreased. These data are plotted in Fig. 3. The data are normalized so that levels of gene expression in the wild type are called 1, and therefore any change in expression levels in W/Wv mice will be represented as a "fold" change. Genes with a twofold or greater change are indicated by an asterisk. These data yielded
50% agreement rate (7/14) between the microarray data and the independent quantitative RT-PCR data, suggesting that in addition to the seven candidate genes which change, there are also a number of false positives in the microarray. This is comparable with other studies, which have noted similar levels of false-positive values (8). Kit expression was decreased 15-fold in W/Wv fundus. Although this was expected, the value does not match the decrease reported in the array experiments in which kit expression was decreased by only 1.1-fold (10%). This discrepancy and the fact that no genes were described as significantly downregulated in the array may indicate a flaw in the array being able to quantitatively detect gene downregulation. In addition, we examined the expression of mast-cell carboxypeptidase in the W/Wv fundus smooth muscles. We found that the levels were lower in the W/Wv fundus than in wild-type fundus, indicating a lower level of mast cells in this phenotype (data not shown). Several purinoceptor genes were found to be upregulated in both the microarray and qPCR that led us to the hypothesis that the W/Wv fundus might be exhibiting a supersensitive phenotype leading to a functional increase in inhibitory neurotransmission. Therefore, we conducted functional studies to test this hypothesis.

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Fig. 3. The relative expression value of genes identified using real-time-quantitative PCR. Data are normalized so that levels of expression levels of all genes in wild-type mice were termed "1" (open bars). Therefore, changes in the W/Wv are represented as "fold" change. Significant differences are indicated by an asterisks.
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Functional studies.
Previous studies have demonstrated that cholinergic excitatory junctional potentials (EJPs) and nitric oxide (NO)-mediated inhibitory junctional potentials (IJPs) are dramatically attenuated in W/Wv fundus muscles (5, 28). In gastric muscles of these mutants, unlike that of wild-type animals, EFS (single pulses, 0.5-ms duration) produced an inhibitory neural response that was insensitive to L-NA. ATP via P2Y receptors has also been proposed as an inhibitory neurotransmitter in the gastric fundus (17). To investigate the functional relevance of P2 receptor upregulation in W/Wv fundus muscles neural responses to EFS (110 Hz, 0.5-ms duration for 1 s, or 30 and 50 Hz delivered for 100 ms) were compared before and in the presence of PPADS, a nonselective antagonist of P2Y and P2X receptors (18, 32). To ensure that responses elicited by neural stimulation were not mediated by release of NO or acetylcholine (ACh), recordings were made in the presence of L-NA (100 µM) and atropine (1 µM). Under these conditions resting membrane potentials of W/Wv fundus smooth muscle cells averaged -48.0 ± 1.5 mV (n = 6 animals) (Fig. 4). Single pulses of EFS (0.5-ms duration, supramaximal voltage) produced tetrodotoxin-sensitive (0.3 µM; data not shown), postjunctional inhibitory responses with average amplitude of 5.4 ± 0.7 mV (n = 6). Multiple pulse stimulation of 3, 5, and 10 pulses delivered in a 1-s time period produced larger amplitude inhibitory events of 8.4 ± 0.8, 10.2 ± 0.8, and 11.6 ± 1.8 mV, respectively (n = 6). A second high-frequency stimulation protocol in which three and five pulses were delivered in a 100-ms time period evoked IJPs averaging 8.9 ± 1.1 and 10.8 ± 1.2 mV. In the continued presence of L-NA and atropine, PPADS (30 µM) attenuated the amplitude of IJPs at all stimulus frequencies. In these conditions inhibitory events evoked by single pulse stimulation had average amplitudes of 2.6 ± 0.4 mV (n = 6; P < 0.05 compared with control conditions). With multiple pulse stimulation of 3, 5, and 10 pulses (delivered in 1 s) IJP amplitude averaged 3.5 ± 0.7, 4.5 ± 1.0, and 4.0 ± 0.9 mV, respectively (P < 0.05 for all stimulation frequencies tested), and the high-frequency pulse delivery of three and five pulses in 100 ms evoked reduced responses of 3.1 ± 0.9 and 3.8 ± 0.9 mV, respectively (P < 0.05 for both frequencies compared with control conditions). The resting membrane potential of fundus smooth muscle cells in the presence of PPADS averaged -50.3 ± 2.6 mV, which was not significantly different than in control conditions (n = 6; P > 0.05).

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Fig. 4. The selective P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADs) reduced neural evoked inhibitory junctional potentials (IJPs) in W/Wv mutant fundus muscles. Under control conditions (in the presence of L-NA; 100 µM and atropine; 1 µM), single and multiple pulses of electrical field stimulation (EFS, 110 Hz; delivered at arrow and bars, respectively; 0.5-ms pulse duration at supramaximal voltage) evoked IJPs (A). PPADs (1030 µM) reduced IJP amplitudes at all frequencies tested (B). A summary of these results is shown in C. IJPs evoked by high-frequency EFS (3 and 5 pulses delivered in 100 ms; i.e., 30 and 50 Hz) were also significantly attenuated by PPADs (summarized in D).
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DISCUSSION
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Identification of specific genes involved with particular physiological phenomena has long been a goal of many biological studies. cDNA microarrays have proven to be valuable tools to this end, allowing the identification of genes associated with many disease states and specific physiological phenomena (1). In the present study, use was made of cDNA microarrays to identify genes with differential expression levels in gastric fundus smooth muscles of W/Wv mice compared with age-matched, sibling, wild-type controls. Since this tissue is a model of GI smooth muscle lacking IC-IM, genes that are differentially regulated may be a consequence of this ablation. After subsequent verification with qPCR, seven significantly upregulated genes were identified; the adenosine A1 receptor (AA1 receptor), purinergic receptor P2Y, G-protein coupled 1 (P2Y receptor), phenylethanolamine-N-methyltransferase (PNMT), gap junction membrane channel protein ß3 (connexin 31),
-internexin neuronal intermediate filament protein (AINXN protein), platelet-derived growth factor-
(PDGF-
), and oncostatin M.
A number of functional studies have reported physiological differences between W/Wv and wild-type mice (5, 14, 28, 29). Burns et al. (5) demonstrated that postjunctional inhibitory neural responses elicited by EFS recorded from the circular smooth muscle layers from wild-type controls were largely sensitive to NO synthase (NOS) inhibitors. In W/Wv mice neural responses were markedly attenuated but the remaining responses were insensitive to NOS inhibition, suggesting a key role for IC-IM in NO-dependent inhibitory neurotransmission. Low-frequency EFS in W/Wv mice revealed a more prominent inhibitory neural response or IJP than that of wild-type controls, suggesting that an additional form of inhibitory neurotransmission may be present in mice lacking IC-IM. The identity of the neurotransmitter involved in this IJP was not investigated by Burns et al. (5); however, it was shown that apamin reduced the inhibitory neural responses in W/Wv mice after inhibition of NOS (which had no effect). Apamin is known to block SK potassium channels and it is thought that the effects of ATP, an inhibitory neurotransmitter in the GI tract, are partially blocked by apamin, suggesting that the NOS-independent IJP observed in W/Wv mice may be due to ATP. Studies on rat gastric fundus have also indicated that ATP may be involved in inhibitory neurotransmission, as the P2-purinoceptor antagonist PPADS inhibited a NOS-independent IJP (17), although the exact isoform of the P2 receptor involved with this IJP was not studied. In this respect it is interesting that the data reported in the present study identified the purinergic receptor P2Y, G protein-coupled 1 (P2Y1 receptor) as being dramatically upregulated in W/Wv murine fundus. The exact reason as to why this should be the case is unclear, but it is possible, however, to envisage a scenario where in the absence of normal neural signaling caused by lack of IC-IM, that inhibitory neurotransmitter receptors located on the smooth muscle cells upregulate to fulfill the requirements of the tissue. The mechanism by which the genes identified in this array upregulate has not been investigated in this study, but there are some intriguing possibilities.
Since the W/Wv murine fundus exhibits enhanced inhibitory neural responses, it could be considered as analogous to models of "denervation-induced supersensitivity" where due to impaired physiological stimulus, effector cells exert augmented responsiveness. In denervated rat urinary bladder, for example, which exhibits supersensitivity to muscarinic agonists, the expression of postjunctional M2 receptors are significantly increased (4). It appears that this denervation model unmasks a function of the M2 receptors that is mediated by M3 receptors in normal bladders (4).
Purinoceptors have been divided into two main classes, those for adenosine (P1), including the adenosine A1 and A2 subtypes, and those for ATP (P2), including the P2X and P2Y receptors (6). Therefore, it was interesting that in W/Wv fundus the P2Y and adenosine A1 receptors were upregulated, perhaps suggesting a common underlying mechanism. PDGF and the profibrogenic cytokine, oncostatin M also demonstrated higher expression levels in the W/Wv, and therefore it would be of interest to investigate whether these phenomena are linked. Evidence from a number of recent studies indicates that both these factors can influence either neural growth or expression of neurotransmitter directly. For example, Zhao et al. (33) showed that PDGF colocalized with ACh receptors in mouse neuromuscular junctions (NMJ) leading to the suggestion that PDGF played a role in the interaction between the pre- and postsynaptic components of the NMJ. In addition PDGF-B expression increased after transection of the optic nerve and was implicated in the subsequent cascade of cellular events that followed (23). Oncostatin M has recently been found to be able to regulate vasoactive intestinal peptide expression in neurons innervating the pancreas (3). Furthermore, data reported by Hou et al. (13) demonstrate that cytokines were able to upregulate P2Y receptors located on vascular smooth muscle cells.
The intermediate filament protein (AINIFP) was also positively regulated in W/Wv fundic tissue. This is particularly interesting in the light of recent research which showed that expression of this gene coincides with the onset of neuronal differentiation in the developing rat nervous system (10) and also that AINIFP accumulated more in plastic rather than stable axonal neurites (26).
All of these findings are consistent with the hypothesis that expression of these genes is higher in the W/Wv as a result of abnormal neuronal signaling. Microarray analysis has shed light on this hypothesis that needs to be tested with more direct experimentation. However, microarray analysis should be viewed as a diagnostic tool that allows hypotheses to be formed and tested. Along with these advantages come, of course, a number of pitfalls, some of which were directly encountered in this study. The first and most common type of error encountered were "false positives," i.e., genes that were identified as being changed from the array, but when checked independently were not found to differ. This merely highlights the need to verify microarray data by another means, such as real-time qPCR, as in this study. This appears to be the method of choice for testing array data (11, 24) although other investigators have used RT-PCR, semi-quantitative RT-PCR and Northern blots, respectively (2, 21, 22). The second form of error encountered were false negatives, i.e., genes that expression levels change significantly but are not identified by the array. An example of this in this study is the level of c-kit, which when checked by Q-PCR was shown to be decreased by more than 15-fold in the W/Wv, but curiously did not exhibit significantly decreased fluorescence in the array. In fact, no gene showed a significant drop in expression by the array, suggesting that this may in fact be an inherent problem with this type of array perhaps due to limited sensitivity. Other investigators have also reported similar findings, where the array appears better suited to identify upregulated genes than those that are downregulated (16).
In summary this study highlights the potential use for microarray investigation in research related to GI motility. Seven genes were found to be significantly upregulated in W/Wv fundic smooth muscles compared with wild type. The mechanisms underlying these changes are unclear, but it appears that disruption of normal enteric motor neurotransmission due to loss of IC-IM is an important factor and indicates a supersensitive phenotype in the W/Wv fundic tissue.
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ACKNOWLEDGMENTS
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We thank Lisa Miller, Yulia Bayguinov, and Heather Beck for excellent technical assistance.
National Institutes of Health Grants DK-41315 and DK-57236 supported this work.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, MS352/Anderson Medical Bldg., Reno, NV 89557-0046 (E-mail: burt{at}physio.unr.edu).
10.1152/physiolgenomics.00052.2002.
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