NHE1 activity contributes to migration and is necessary for proliferation of human gastric myofibroblasts

Abstract

Myofibroblasts play central roles in wound healing, deposition of the extracellular matrix and epithelial function. Their functions depend on migration and proliferation within the subepithelial matrix, which results in accelerated cellular metabolism. Upregulated metabolic pathways generate protons which need to be excreted to maintain intracellular pH (pH(i)). We isolated human gastric myofibroblasts (HGMs) from surgical specimens of five patients. Then we characterized, for the first time, the expression and functional activities of the Na(+)/H(+) exchanger (NHE) isoforms 1, 2 and 3, and the functional activities of the Na(+)/HCO(3)(-) cotransporter (NBC) and the anion exchanger (AE) in cultured HGMs using microfluorimetry, immunocytochemistry, reverse transcription polymerase chain reaction and immunoblot analysis. We showed that NHE1-3, NBC and AE activities are present in HGMs and that NHE1 is the most active of the NHEs. In scratch wound assays we also demonstrated (using the selective NHE inhibitor HOE-642) that carbachol and insulin like growth factor II (IGF-II) partly stimulate migration of HGMs in a NHE1-dependent manner. EdU incorporation assays revealed that IGF-II induces proliferation of HGMs which is inhibited by HOE-642. The results indicate that NHE1 is necessary for IGF-II-induced proliferation response of HGMs. Overall, we have characterized the pH(i) regulatory mechanisms of HGMs. In addition, we demonstrated that NHE1 activity contributes to both IGF-II- and carbachol-stimulated migration and that it is obligatory for IGF-II-induced proliferation of HGMs.

Na+/Ca2+exchangers regulate the migration and proliferation of human gastric myofibroblasts

“…The details of patients can be found in Table 1. The isolation of myofibroblasts was performed using a previously described method (6). Briefly, the specimens were washed and chopped into ϳ1-to 2-mm 3 pieces and were then bathed in a shaking water bath at 37°C for 15 min with 1 mM dithiothreitol.…”

“…After gentle washing for three times with serum-free media, an inverted light microscope was used to measure and photograph wounds. Reagents were then added to the wells in serum-free media, and HGMs were incubated in a CO 2 incubator at 37°C for 24 h. Migration was evaluated by counting the cells in the same area of the wound after 24 h as reported earlier (6). Proliferation assay.…”

“…Proliferation assay. Proliferation assays were performed as previously described (6). HGMs (routinely 50,000 cells) were seeded on cover glasses.…”

Multimodal Evaluation of Esophageal Epithelial Response in Gastroesophageal Reflux Disease

“…(75,86,87) Using NHE isoform specific inhibitor HOE-642, we determined that NHE-1 and NHE-2 are participating in the restoration of pHi which is rather attributable to NHE-1. This finding was in accordance with Goldman et al who found that 60% of pHi recovery is mediated through NHE-1.…”

“…NBC represents another important pHi regulatory mechanism in several types of epithelial cells (75)(76)(77). In the case of EECs, in standard HCO3 -/CO2-buffered extracellular solution, the pHi of CP-A cells rapidly decreased by the quick diffusion of CO2 into the cytoplasm.…”