Desensitization of human renal D1 dopamine receptors by G protein-coupled receptor kinase 4
Desensitization of human renal D1 dopamine receptors by G protein-coupled receptor kinase 4
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DOI：
10.1046/j.1523-1755.2002.00525.x
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发表时间：
2002-09-01
影响因子：
19.6
通讯作者：
Felder, RA
中科院分区：
文献类型：
--
作者：
Watanabe, H;Xu, J;Felder, RA
背景多巴胺D-1受体表达于多个肾单位节段，参与水和电解质转运的调节。由于肾D1受体在遗传性高血压中是脱敏的，我们试图确定肾近端小管内源性表达的D1受体脱敏的机制。通过测定G蛋白偶联受体激酶（GRK）活性和表达的刺激或抑制对cAMP产生的反应，研究了人肾近端小管细胞D-1受体同源脱敏的机制。通过免疫印迹法评估蛋白表达。非人肾近端小管细胞，D1激动剂，非诺多泮，增加cAMP积累（73 +/- 2%）。非诺多泮预处理降低了对后续非诺多泮刺激的反应性（t（1/2）约为20 min），在30 min时完全脱敏。逐渐恢复（t（1/2）约为20 min），60 min时完全恢复。毛喉素预处理影响最小的非诺多泮效应，表明蛋白激酶A在同源脱敏过程中的轻微参与。由于GRKs参与同源脱敏过程，我们确定了抑制GRK表达和活性的后果。GRK活性抑制剂肝素降低GRK 2和GRK 4的表达，并减弱D1受体的脱敏作用（85 +/- 1%）。反义寡核苷酸（GRK 4> GRK 2）可减弱D-1受体的脱敏作用。然而，前20分钟的同源脱敏不受肝素或GRK反义寡核苷酸的影响。这些研究记录了GRK 4相对于GRK 2在肾近端小管细胞中D-1受体的同源脱敏中的关键作用。然而，早期阶段的同源脱敏是由非GRK介导的途径。
Background. The D-1 dopamine receptor, expressed in several nephron segments, participates in the regulation of water and electrolyte transport. Because the renal D, receptor is desensitized in genetic hypertension, we sought to determine the mechanism(s) of the desensitization of D, receptors endogenously expressed in renal proximal tubules.Methods. The mechanisms involved in the homologous desensitization of the D-1 receptor in human renal proximal tubule cells were studied by measuring the production of cAMP in response to stimulation or inhibition of G protein-coupled receptor kinase (GRK) activity and expression. Protein expression was assessed by immunoblotting.Results. Inhuman renal proximal tubule cells, the D, agonist, fenoldopam, increased cAMP accumulation (73 +/- 2%). Fenoldopam pre-treatment decreased the responsiveness to subsequent fenoldopam stimulation (t(1/2) approximate to 20 min) with complete desensitization at 30 minutes. Recovery occurred gradually (t(1/2) approximate to 20 min) with full recovery at 60 minutes. Forskolin pretreatment minimally affected the fenoldopam effect, indicating a minor involvement of protein kinase A in the homologous desensitization process. Because GRKs are involved in the homologous desensitization process, we determined the consequences of inhibition of GRK expression and activity. Heparin, an inhibitor of GRK activity, decreased the expression of GRK2 and GRK4 and attenuated the desensitization of the D, receptor (85 +/- 1%). Antisense oligonucleotides (GRK4 > GRK2) blunted the D-1 receptor desensitization. However, the first 20 minutes of homologous desensitization were not affected by either heparin or GRK antisense oligonucleotides.Conclusion. These studies document the critical role of GRK4, relative to GRK2, in the homologous desensitization of D-1 receptors in renal proximal tubule cells. However, the early phase of homologous desensitization is regulated by a non-GRK-mediated pathway.