β-tubulin在肾脏D5R与CCKBR相互调节中的重要作用

Mechanistic study of β-tubulin and its interaction with the dopamine D5 receptor,cholecystokinin B receptor,and water-sodium metabolism

目的探讨β-tubulin在胆囊收缩素B受体(cholecystokinin B receptor,CCKBR)和多巴胺D5受体(dopamine D5 receptor,D5R)相互作用的调节机制。方法选用正常人和高血压患者肾脏近曲小管细胞(renal proximal tubular cells,RPTC),各分为3组:CCKBR激动剂(gastrin)处理组;D5R激动剂(fenoldopam)处理组;gastrin+微管抑制剂(nocodazole)组。免疫荧光法测定β-tubulin、CCKBR和D5R的定位,免疫印迹方法检测CCKBR、D5R和Na+,K+-ATP酶的表达。结果 Gastrin处理正常RPTC后D5R表达显著增加(P<0.05),Na+,K+-ATP酶的表达降低(P<0.05),fenoldopam同样能够刺激CCKBR表达增加(P<0.05),但nocodazole预处理后再加入gastrin并不能够增加D5R的表达,也不能降低Na+,K+-ATP酶的表达。高血压患者RPTC中CCKBR和D5R的相互作用以及Na+,K+-ATP酶的表达变化并不明显。免疫荧光结果显示,正常RPTC中CCKBR和D5R蛋白能够被诱导转移到细胞膜上,微管抑制剂能够显著阻断转移过程,但高血压患者RPTC中β-tubulin杂乱无章,CCKBR和D5R蛋白也没能有效转移到细胞膜上。结论 tubulin通过促进CCKBR和D5R从细胞质向细胞膜转运,从而发挥生物学功能,可能在二者相互作用和钠水代谢中起到重要作用。

Objective To examine the role of β-tubulin on the interaction between the cholecystokinin B receptor（CCKBR）,dopamine D5 receptor（D5 R）,and water-sodium metabolism. Methods Normotensive and hypertensive renal proximal tubular cells（RPTC） were equally randomized into three separate groups： a gastrin group,fenoldopam group,and gastrin ＋ nocodazole group. Immunofluorescence was used to determine localization of β-tubulin,CCKBR,and D5 R. Western blotting was used to detect CCKBR,D5 R,and Na-K-ATP expression. Results Gastrin stimulation innormotensive RPTC increased D5 R expression（P〈0. 05） and decreased Na-K-ATP expression（P〈0. 05）. These changes were blocked by a tubulin inhibitor（P〈0. 05）. However,interaction between CCKBR,D5 R,and Na-K-ATP expression was not significantly affected in hypertensive RPTC. Immunofluorescence showed that CCKBR and D5 R can induce one another,followed by transport to the plasma membrane,which can prevented by a tubulin inhibitor. Further,tubulin is disordered in hypertensive RPTC,which cannot support intracellular CCKBR and D5 R transport. Conclusions tubulin plays a key role in the interaction between CCKBR,D5 R,and water-sodium metabolism by improving protein transfer from the cytoplasm to cell membrane.