|Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
The purpose of this study focussed on the study of the trafficking mechanism and function of one of aquaporins (AQPs), AQP5, an exocrine type water channel.
1. In order to understand the molecular mechanism of water transport and its regulation, we set the experiment to understand the PKA-target motif in loop-D of AQP5. For this purpose we prepared mutant cDNAs, lacking either seine, threonine or both amino acids by using in vitro mutagenesis techniques. The sequence for green fluorescence protein (GFP) was also introduced into either 5'-or 3'- end of these cDNAs to prepare GFP-AQP5 and AQP5-GFP proteins. These AQPs will be introduced to MDCK cells and HSG cells to assess the effects of inhibitors or activators of signaling pathway.
2. Aquaporin 2 (AQP2) is responsible for the concentration of urine in the kidney collecting tubule under the regulation of vasopressin. In polydipsic STR/N mice, the reduction in the AQP2 Mrna level was not evident at 3 weeks of age, at which time the water
intake was not increased at all. At 10 weeks of age, however, the AQP2 Mrna level was reduced to 10% of that of the control mice, whereas the water intake was increased by 36%. When STR/N mice were 44 weeks old, the water intake became 5 times of that of the control ICR mice, and the AQP2 Mrna level in these polydipsic mice was only approx. 5% of the control one. These data imply that elevated water intake due to polydipsia may have affected tha AQP2 Mrna transcription in the kidney, resultiong in reduced AQP2 expression, which would contribute to a reduction in over-retention of water.
3. The expression and localozation of aquaporins (AQP1-AQP5), members of the water channel family in the developing rat submandibular gland were analyzed by RT-PCR, Northen blotting, and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed uniqui expression patterns of each AQP. AQP1 was constitutively expressed during prenatal development, whereas the expression of AQP5 became more intense in the course of development were not detected by Northern blotting, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were continuously expressed, but no message for AQP3 or AQP4 was detected. AQP2mRNA was not detected during either prenatal or postnatal development in this tissue. An immunohistichemical experiment was also done to explore the precise localozation of these AQPs. Less