Molecular mechanisms of expression and regulation of water channel proteins aquaporins in the exocrine gland cells

本研究的目的是研究水通道蛋白（AQPs）之一AQP5（外分泌型水通道）的转运机制和功能。为了了解水分转运的分子机制及其调控，我们设置了了解AQP5环- d中pka -靶基序的实验。为此，我们利用体外诱变技术制备了缺乏丝氨酸、苏氨酸或两种氨基酸的突变cdna。将绿色荧光蛋白（green fluorescence protein， GFP）序列分别导入到这些cdna的5′端或3′端，制备GFP- aqp5和AQP5-GFP蛋白。这些aqp将被引入MDCK细胞和HSG细胞，以评估信号通路抑制剂或激活剂的作用。水通道蛋白2 （Aquaporin 2, AQP2）在加压素的调控下负责肾收集小管尿液的浓度。在多饮STR/N小鼠中，AQP2 Mrna水平的降低在3周龄时不明显，此时饮水量完全没有增加。然而，在10周龄时，AQP2 Mrna水平降低到对照小鼠的10%，而饮水量增加了36%。当STR/N小鼠44周龄时，摄水量是对照ICR小鼠的5倍，而这些多饮小鼠的AQP2 Mrna水平仅为约。是对照组的5%这些数据表明，烦渴导致的饮水量增加可能影响了肾脏AQP2 Mrna的转录，导致AQP2表达减少，从而有助于减少水分的过度潴持。采用RT-PCR、northern blotting、免疫组化等方法分析水通道蛋白家族成员AQP1-AQP5在发育大鼠颌下腺中的表达和定位，探讨其与颌下腺发育的关系。RT-PCR分析显示了各AQP的独特表达模式。AQP1在产前发育过程中组成性表达，而AQP5在发育过程中表达更加强烈，尽管RT-PCR检测到，但Northern blotting未检测到。在出生后发育过程中，AQP5和AQP1 mrna持续表达，而AQP3和AQP4 mrna未被检测到。AQP2mRNA在该组织的产前和产后发育过程中均未检测到。我们还通过免疫组化实验来探索这些aqp的精确定位。少

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 … More 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