HK-2 human renal proximal tubule cells as a model for G protein-coupled receptor kinase type 4-mediated dopamine 1 receptor uncoupling.
HK-2 human renal proximal tubule cells as a model for G protein-coupled receptor kinase type 4-mediated dopamine 1 receptor uncoupling.
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DOI：
10.1161/hypertensionaha.110.152256
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发表时间：
2010-09
期刊：
影响因子：
--
通讯作者：
Felder RA
中科院分区：
文献类型：
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作者：
Gildea JJ;Shah I;Weiss R;Casscells ND;McGrath HE;Zhang J;Jones JE;Felder RA
HK-2人肾近端小管细胞（RPTC）通常用于“正常”RPTC的体外研究。我们最近发现，HK-2细胞从多巴胺-1受体（D1 R）腺苷酸环化酶（AC）刺激解偶联。我们假设G蛋白偶联受体激酶4型（GRK 4）单核苷酸多态性（SNP）可能是负责的D1 R/AC解偶联在HK-2。通过对GRK 4 SNP进行基因分型、测量D1样受体激动剂（非诺多泮）刺激的cAMP积累、定量D1 R对钠转运的抑制以及测试GRK 4 siRNA逆转D1 R/AC解偶联的能力来测试该假设。我们比较了HK-2与2个正常偶联的人RPTC细胞系（nRPTC）和2个未偶联的RPTC细胞系（uRPTC）。发现HK-2细胞系具有已知使D1 R与AC解偶联的6种潜在GRK 4 SNP中的4种（即R65 L、A142 V和A486 V）。在两种nRPTC细胞系中，对非诺多泮刺激的AC反应增加（FEN是对照的2.02±0.05倍和2.33±0.19倍，P<0.001，N=4），但在两种未偶联的或HK-2细胞系中没有。GRK 4 siRNA在未偶联的细胞（包括HK-2）中拯救了非诺多泮介导的AC刺激。在HK-2（N=6）和uRPTC（N=6）中不存在预期的非诺多泮介导的钠氢交换器3型抑制，但在表达野生型GRK 4的两种nRPTC中观察到了这种抑制（−25.41±4.7%和−27.36±2.70%（P<0.001，N=6））。尽管HK-2细胞保留了RPTC的许多功能特征，但从多巴胺能功能的角度来看，它们是不正常的。
HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of “normal” RPTCs. We recently discovered that HK-2 cells are uncoupled from dopamine-1 receptor (D1R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms (SNPs) may be responsible for the D1R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 SNPs, measuring D1-like receptor agonist (fenoldopam)stimulated cAMP accumulation, quantifying D1R inhibition of sodium transport, and testing the ability of GRK4 siRNA to reverse the D1R/AC uncoupling. We compared HK-2 to 2 normally coupled human RPTC cell lines (nRPTC) and 2 uncoupled RPTC cell lines (uRPTC). The HK-2 cell line was found to have 4 out of 6 potential GRK4 SNPs known to uncouple the D1R from AC (namely R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the two nRPTC cell lines (FEN 2.02±0.05-fold and 2.33±0.19-fold over control, P<0.001, N=4), but not in the two uncoupled or HK-2 cell lines. GRK4 siRNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam -mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (N=6) and uRPTCs (N=6), but was observed in the two nRPTCs (−25.41±4.7% and −27.36±2.70% (P<0.001, N=6)), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function.