Vollum Institute, Oregon Health and Science University, Portland, Oregon.
Vollum Institute, Oregon Health and Science University, Portland, Oregon
Mol Pharmacol. 2020 Oct;98(4):497-507. doi: 10.1124/mol.119.119362. Epub 2020 May 3.
Based on studies using mutations of the -opioid receptor (MOR), phosphorylation of multiple sites on the C-terminus has been recognized as a critical step underlying acute desensitization and the development of cellular tolerance. The aim of this study is to explore which kinases mediate desensitization of MOR in brain slices from drug-naïve and morphine-treated animals. Whole-cell recordings from locus coeruleus neurons were made, and the agonist-induced increase in potassium conductance was measured. In slices from naïve animals, pharmacological inhibition of G-protein receptor kinase (GRK2/3) with compound 101 blocked acute desensitization. Following chronic treatment with morphine, compound 101 was less effective at blocking acute desensitization. Compound 101 blocked receptor internalization in tissue from both naïve and morphine-treated animals, suggesting that GRK2/3 remained active. Kinase inhibitors aimed at blocking protein kinase C and c-Jun N-terminal kinase had no effect on desensitization in tissue taken from naïve animals. However, in slices taken from morphine-treated animals, the combination of these blockers along with compound 101 was required to block acute desensitization. Acute desensitization of the potassium conductance induced by the somatostatin receptor was also blocked by compound 101 in slices from naïve but not morphine-treated animals. As was observed with MOR, it was necessary to use the combination of kinase inhibitors to block desensitization of the somatostatin receptor in slices from morphine-treated animals. The results show that chronic treatment with morphine results in a surprising and heterologous adaptation in kinase-dependent desensitization. SIGNIFICANCE STATEMENT: The results show that chronic treatment with morphine induced heterologous adaptations in kinase regulation of G protein coupled receptor (GPCR) desensitization. Although the canonical mechanism for acute desensitization through phosphorylation by G protein-coupled receptor kinase is supported in tissue taken from naïve animals, following chronic treatment with morphine, the acute kinase-dependent desensitization of GPCRs is disrupted such that additional kinases, including protein kinase C and c-Jun N-terminal kinase, contribute to desensitization.
基于对 - 阿片受体（MOR）突变的研究，已经认识到 C 末端多个位点的磷酸化是急性脱敏和细胞耐受发展的关键步骤。本研究旨在探讨哪些激酶介导了来自未用药和吗啡处理动物脑切片中 MOR 的脱敏。从蓝斑神经元进行全细胞膜片钳记录，并测量激动剂诱导的钾电导增加。在来自未用药动物的切片中，用化合物 101 抑制 G 蛋白受体激酶（GRK2/3）可阻断急性脱敏。在慢性吗啡处理后，化合物 101 阻断急性脱敏的效果降低。化合物 101 阻断了来自未用药和吗啡处理动物组织中的受体内化，表明 GRK2/3 仍然活跃。旨在阻断蛋白激酶 C 和 c-Jun N 末端激酶的激酶抑制剂对来自未用药动物组织中的脱敏没有影响。然而，在来自吗啡处理动物的切片中，需要将这些阻滞剂与化合物 101 联合使用才能阻断急性脱敏。在来自未用药动物的切片中，化合物 101 也阻断了生长抑素受体诱导的钾电导的急性脱敏，但在来自吗啡处理动物的切片中则没有。与 MOR 一样，有必要使用激酶抑制剂的组合来阻断来自吗啡处理动物的生长抑素受体脱敏。结果表明，慢性吗啡处理导致激酶依赖性脱敏的惊人的和异源适应。意义陈述：结果表明，慢性吗啡处理诱导了激酶调节 G 蛋白偶联受体（GPCR）脱敏的异源适应。尽管通过 G 蛋白偶联受体激酶磷酸化支持来自未用药动物组织中的急性脱敏的典型机制，但在慢性吗啡处理后，GPCR 的急性激酶依赖性脱敏被破坏，包括蛋白激酶 C 和 c-Jun N 末端激酶在内的其他激酶也有助于脱敏。
