North RA. were dispersed as described previously19,18. eGFP-PDGFR cells, eGFP-SMC, and CopGFP-ICC were purified by fluorescence-activated cell sorting (FACS) (Becton Dickinson FACSAriaII) using the blue laser (488 nm) and the GFP emission detector (530/30 nm). Expression of genes in each sorted cell type was compared against expression in the total cell population (TCP) of colonic of corresponding control animals. Regression analysis of the mean values of three multiplex qPCRs for the log10 diluted cDNA was used to generate standard curves. Unknown amounts of messenger RNA (mRNA) were plotted relative to the standard curve for each set of primers and graphically plotted using Microsoft Excel. Primer efficiencies of 90C110% were only accepted for analysis. This gave transcriptional quantification of each gene relative to the endogenous standard after log transformation of the corresponding raw data. In pilot studies was tested on all three cell types used in the present study and represents an appropriate control for qPCR analyses. All data were expressed as means S.E.M. Students < 0. 05 taken to indicate statistically significant differences. RESULTS 1. Cell markers in sorted SMC, PDGFR+ cells and ICC The qRT-PCR analyses exhibited that this FACS-sorted cells were highly enriched with cell specific markers: and were enriched in sorted CopGFP-ICC, eGFP-PDGFR+ cells, and eGFP-SMC, respectively. The or whereas the or or (PDGFR+ cells), (SMC), (ICC) were compared with the expression of these genes in total cell population (TCP) of dispersed and and Sorted ICC (n=3) were minimally positive for and but enriched with transcripts of After sorting, all three populations of purified cells displayed negligible transcripts of Asterisks indicate P<0.05 when compared to the transcript isolated from TCP. 2. Purinergic Receptors 2.1. P1 Receptors Among genes encoding the four types of adenosine receptors (P1 receptors) PDGFR+ cells were enriched with and and transcripts for and were not resolved (Fig. 2). Expression of and was stronger in PDGFR+ cells than in SMC, ICC or TCP. In contrast, was expressed more in SMC than in ICC or in TCP. was modestly expressed in SMC and ICC but less than in TCP, suggesting that this receptor is mainly expressed on neurons or other non-SIP cells. Open in a separate window Fig. 2 Expression of genes for P1 receptors in SMC, ICC, and PDGFR+ cells RMC-4550 of the murine colon by qRT-PCR analysisNote that SMC RMC-4550 and ICC RMC-4550 were enriched with and and particularly with (Fig. 3A, Table 2). Therefore, PDGFR+ cells might be a target for extracellular ATP acting on ionotropic P2X receptors. ICC expressed and more than the TCP. ICC also expressed a low level of that was significantly less than in PDGFR+ cells or the TCP. Among the genes for P2Y receptors SMC were enriched with (Fig. 3B, Table 2). The gene for was also enriched in PDGFR+ cells, but the expression was lower than in SMC. PDGFR cells showed higher expression of than TCP or SMC. ICC were enriched with suggesting that these cells might be targeted by extracellular pyrimidine substances rather than purines. Open in a separate window Fig. 3 Expression of genes for P2X receptors (panel A) and P2Y receptors (panel B) in SMC, ICC, and PDGFR+ cells of the murine colon by qRT-PCR analysisNote that PDGFR+ cells are enriched with and as well as 2purinergic receptor P2Y, G-protein coupled1.080.59291.940.01103.30.0033expression in PDGFR cells appeared higher than in TCP, but this did not reach statistical significance. The expression of in SMC was less than in the TCP. ICC showed negligible expression of (Fig. 5A). Therefore, adenosine generated by extracellular nucleotides that are autocrine and paracrine mediators in the vicinity of the SIP syncytium may have more rapid and possibly greater effects on SMC than on other cells. Open in a separate window Fig. 5 Expression of genes for ecto-nucleotidases (panel A), ecto-nucleotide pyrophosphatases/phosphodiasterases (panel B), and mono-ADP ribosyl transferases (panle C) in SMC, ICC, and PDGFR+ cells of the murine colon by qRT-PCR analysisNote that PDGFR+ cells show relatively higher expression of and and low expression of was resolved in neither cell type. TCP, RMC-4550 total cell population (n=6), Sorted SMC, ICC and PDGFR+ cells (each n=3). Asterisks indicate P<0.05 when compared to the transcript isolated from TCP. 3.2. NAD glycohydrolases CD38 and CD157/Bst1 Colonic express and since both genes were found expressed in TCP (Fig. 5A). None of the cells Rabbit Polyclonal to CYC1 comprising the SIP syncytium expressed levels of Cd157was expressed in all three cell types: interestingly, showed stronger expression in PDGFR+ cells than in the TCP, whereas expression in.