| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
|
|---|
| Research Abstract |
We applied whole-cell patch clamp to single parietal cells isolated from the guinea-pig then found the ETb-receptor coupled Maxi-ClィイD1-ィエD1 and pH-sensitive Mini-ClィイD1-ィエD1 channels on the parietal cells. In the 10ィイD1thィエD1 year, we detected the expression of ClィイD1-ィエD1 channel mRNA by the method of RT-PCR at the whole gastric epithelial cell level. Then, we made a cDNA library from guinea-pig gastric epithelial cells and determined the cDNA sequence of guinea-pig CLC-2 ClィイD1-ィエD1 channel. We analyzed the ClィイD1-ィエD1 current using whole-cell patch clamp in the CLC-2 gene expression cell system of Xenopus oocytes and CHO cells, but could not detect the channel current previously founded. To analyze the ClィイD1-ィエD1 channel gene expression in parietal level, we amplified the CFTR, CLC-2, 3, 4 mRNA by RT-PCR method using the purified guinea-pig parietal cells. But we could not detect any single band. In 11ィイD1thィエD1 year, we further applied whole-cell patch clamp and single cell mRNA analysis by RT-PCR method to single parietal cell. Single parietal cell did not show any ClィイD1-ィエD1 channel gene expression though G3PDH did the expression in single intestinal cell as positive control. These results did not come from the technical failure. Recent our intestinal work showed that discrepancy between electrophysiological and molecular biological results depended on the difference of cellular maturation. Therefore, we concluded that parietal cells have several grade of maturation and this result induced the discrepancies of these works.
|
