Regulation of AQP expression by serotonin and pathophisiology of IBS

一些5-羟色胺受体拮抗剂已被用于肠易激综合征的治疗。这种作用的机制被认为是通过下调肠道神经元中的5-羟色胺来控制肠道运动。我们报道了血管活性肠肽上调培养的人结肠上皮细胞HT-29中AQP3的表达。尽管Ht-29细胞表达5-HT2A、5-HT3和5-HT4受体，但加入5-羟色胺并不改变AQP3mRNA的表达。为了研究心房利钠肽(ANP)和脑利钠肽(BNP)对水通道蛋白3(AQP3)表达的调节作用，我们用Northern印迹法研究了心房利钠肽(ANP)和脑钠尿肽(BNP)对Ht-29细胞AQP3表达的调节作用，用Western blotting方法研究了AQP3的蛋白表达，用凝胶迁移率改变实验(EMSA)研究了Ht-29细胞的DNA结合活性。我们还使用了几种抑制剂来研究信号转导。除ANP(>100 nM)和BNP(>10 nM)外，AQP3基因表达上调。加入ANP和BNP后1h，AQP3蛋白表达增强。蛋白激酶-A(PK-A)和蛋白激酶-G(PK-G)抑制剂联合应用可完全抑制ANP或BNP诱导的AQP3mRNA表达，使其达到基础水平。HT-29细胞的环磷酸腺苷反应元件(CRE)的EMSA显示单一条带。这些结果表明，ANP和BNP上调AQP3mRNA和蛋白的表达，PK-A和PK-G依赖的通路都介导了这一作用。

Some serotonin receptor antagonists have been used as a therapy for irritable bowel syndrome. The mechanism of this action was thought to control intestinal movement via down regulation of serotonin in the intestinal neuron. We reported that the expression of AQP3 in cultured human colonic epithelial cells HT-29 was upregulated by vasoactive intestinal polypeptide. Although Ht-29 cells express 5-HT2a,5-HT3 and 5-HT4 receptors, the addition of serotonin did not alter the expression of AQP3 mRNA. To characterize the regulation of AQP3 expression by atrial natriuretic peptide(ANP) and brain natriuretic peptide(BNP), we studied mRNA expression by Northern blotting, protein expression by Western blotting and DNA binding activity by electrophoretic mobility shift assay(EMSA) in HT-29. We also used several inhibitors to investigate signal transduction. AQP3 mRNA was up-regulated in addition to ANP (more than 100 nM) and BNP (more than 10 nM). The expression of AQP3 protein was enhanced at 1 hour after the addition of ANP and BNP. The combination of protein kinase-A(PK-A) and protein kinase-G(PK-G) inhibitors completely inhibited the expression of AQP3 mRNA enhanced by ANP or BNP to its basal level. The EMSA of the cyclic-AMP response element(CRE) in HT-29 cells revealed a single band. These results indicate that ANP and BNP up-regulated the expression of AQP3 mRNA and protein, and both PK-A and PK-G dependent pathways mediated this effect.