Dysfunctional D1A receptor-G-protein coupling in proximal tubules of spontaneously hypertensive rats is not due to abnormal G-proteins.
Dysfunctional D1A receptor-G-protein coupling in proximal tubules of spontaneously hypertensive rats is not due to abnormal G-proteins.
复制标题
自发性高血压大鼠近曲小管中功能失调的 D1A 受体 - G 蛋白偶联并不是由异常的 G 蛋白引起的。
DOI：
10.1097/00004872-199715030-00007
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发表时间：
1997
影响因子：
4.9
通讯作者：
Sidhu,A
中科院分区：
文献类型：
--
作者：
Sela,S;White,BH;Uh,M;Kimura,K;Patel,S;Sidhu,A
背景自发性高血压大鼠(SHR)的肾近曲小管(RPT)中存在功能失调的多巴胺神经传递和缺陷的D1A受体-G蛋白偶联。目的确定SHR中的G蛋白是否异常，阻止SHR中激动剂高亲和力位点的形成。方法检测SHR和肾近曲小管中G蛋白α亚基的表达水平，以及与外源添加的正常G蛋白的D1A受体受体偶联。正常血压 Wister-Kyoto (WKY) 大鼠。结果在 110 mmol/l NaCl 存在下，D1 A 多巴胺受体选择性激动剂 SKF R-38393 与从正常血压 Wistar-Kyoto (WKY) 大鼠肾近曲小管中提取的溶解和重建的 D1 A 受体上的高亲和力和低亲和力位点结合。在自发性高血压大鼠 (SHR) 中，SKF R-38393 与重建受体上的单个位点结合，其亲和力值对应于受体的低亲和力状态。蛋白质印迹分析表明，鸟嘌呤核苷酸结合蛋白（G 蛋白）的 α 亚基 G s 以相似的水平表达，而 G o α 在 WKY 大鼠和 SHR 的近曲小管膜中不表达。在 SKF R-38393 存在的情况下，用烷化剂 N-乙基马来酰亚胺预处理近端小管膜会灭活内源性 G 蛋白的 α 亚基，但不会灭活 D1A 受体，导致 WKY 大鼠失去高亲和力结合位点。这些来自 WKY 大鼠的 N-乙基马来酰亚胺处理的 D1A 受体，当用外源性 G 蛋白重构时，能够与这些外源性 G 蛋白偶联，并完全恢复高亲和力位点。此外，这些杂交位点的亲和力值和比例与未处理的受体相似，并且这些亲和力位点受到鸟嘌呤核苷酸类似物的调节。用相同的外源 G 蛋白重构 SHR 的 D1A 受体未能类似地诱导混合重构系统中高亲和力结合位点的形成，并且 SKF R-38393 继续以受体的单一低亲和力状态结合。结论这些结果表明，SHR 中 G 蛋白偶联的缺失是由于受体蛋白内的内在缺陷，而不是内源 G 蛋白本身的任何异常。
BackgroundDysfunctional dopamine neurotransmission and defective D1 A receptor–G protein coupling exist in renal proximal tubules (RPT) of the spontaneously hypertensive rat (SHR).ObjectiveTo determine whether the G proteins in SHR are abnormal, preventing formation of agonist high affinity sites in SHR.MethodsWe examined the expression levels of the α-subunits of G proteins, as well as D1 A receptor receptor coupling to exogenously added normal G proteins, in RPT of SHR and the normotensive Wister–Kyoto (WKY) rat.ResultsIn the presence of 110 mmol/l NaCl, the D1 A dopamine receptor-selective agonist SKF R-38393 binds both to high-and to low-affinity sites on solubilized and reconstituted D1 A receptors extracted from renal proximal tubules of normotensive Wistar-Kyoto (WKY) rats. In the spontaneously hypertensive rat (SHR), SKF R-38393 bound to a single site on the reconstituted receptor with affinity values corresponding to the low-affinity state of the receptor. Western blot analyses indicated that the α-subunit of the guanine nucleotide binding protein (G-protein), G s, was expressed at similar levels, whereas G o α was not expressed in proximal tubule membranes from WKY rats and SHR. Pretreatment of proximal tubule membranes with the alkylating agent N-ethylmaleimidein the presence of SKF R-38393 inactivated α-subunitsof endogenous G-proteins, but not D1 A receptors, resulting in loss of high-affinity binding sites in WKY rats. These N-ethylmaleimide-treated D1 A receptors from WKY rats, when reconstituted with exogenous sources of G-proteins, were able to couple to these exogenous G-proteins, with complete restoration of high-affinity sites. Moreover, the affinity values and the proportion of these hybrid sites were similar to those of untreated receptors, and these affinity sites were regulated by guanine nucleotide analogs. Reconstitution of D1 A receptors from SHR with the same exogenous G-proteins failed to similarly induce formation of the high-affinity binding sites in the hybrid reconstituted systems, and SKF R-38393 continued to bind in a single low-affinity state of the receptor.ConclusionThese results demonstrate that the absence of G-protein coupling in SHR is due to intrinsic defects within the receptor protein, rather than to any abnormalities of the endogenous G-proteins themselves.