|Budget Amount *help
¥5,500,000 (Direct Cost : ¥5,500,000)
Fiscal Year 1997 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1996 : ¥2,900,000 (Direct Cost : ¥2,900,000)
The purposes of this research was to Identify molecular structure and regulatory mechanisms of vasopressin water channel AQP2 In kidney collecting duct, and to develop functional Inhibitors of AQP2 that will be of use as a water diuretics in clinical medicine.
First, we examined molecular structure of AQP2, which was first cloned by the investigator, using molecular techniques of site-directed mutagenesis and gene expression. We were able to localize the aqueous pore in the AQP2 molecule and proposed a three-dimensional structure model of AQP2. In our model aqueous pore of AQP2 is composed of two of five hydrophilic loops of AQP2, which is a membrane protein with six transmembrane segments connected by five hydrophilic loops.
Second, we found that intracellular trafficking of AQP2 is involved in the vasopressin regulation of osmotic water permeability of kidney collecting duct apical membrane. It was observed that, after vasopressin stimulation, AQP2 stored on endosomal membranes was transferred to the surface membrane with exocytosis of vesicles from cytoplasm to the apical membrane, resulting in the increase of osmotic water permeability of the apical membrane. In these regulatory steps, we proved that phosphorylation of serine 256 by protein kinase A is required for cAMP-dependent regulatory exocytosis of AQP2.
Based on these findings, we looked for AQP2 inhibitors = potential water diuretics by random screening, as agents that bind near the aqueous pore and inhibit water permeability, or as agents that inhibit vasopressin-dependent exocytosis of AQP2. We obtained several agents that inhibited water permeability. To determine the effects in vivo, the agents were given to rats intravenously or intraperitoneally, but because of toxicity of the agents it was not able to determine diuretic effects. Screening will be continued to find AQP2 inhibitor with less toxicity.