Chern Y, Lai H L, Fong J C, Liang Y
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Mol Pharmacol. 1993 Nov;44(5):950-8.
To understand the regulation of A2a adenosine receptor (A2a-R) response, we examined the molecular mechanisms underlying the desensitization of A2a response in rat pheochromocytoma PC12 cells, which possess an A2a-R identical with the A2a receptor we recently cloned from rat brain. Prolonged exposure of PC12 cells to adenosine agonists significantly inhibited the response of the cells to subsequent stimulation with an A2a-selective adenosine agonist (CGS21680). No significant change in the number of binding sites and affinity for CGS21680 was observed in desensitized cells, nor did we find any significant change in the transcript level of A2a-R in cells pretreated with adenosine agonists. However, the basal adenylyl cyclase activity and the cyclase activities stimulated by adenosine agonists, by GTP gamma S, and by forskolin were reduced in desensitized cells. Prolonged exposure of PC12 cells to dibutyryl-cAMP did not significantly change either the basal or the adenosine agonist-evoked adenylyl cyclase activity. Therefore, elevation of cellular cAMP content is by itself not sufficient to produce the observed reductions of adenylyl cyclase activity with A2a desensitization. Inhibition of adenylyl cyclase activity in desensitized cells occurred after short-term (30 min) incubation with CGS21680 and could be blocked by the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine. Gs alpha protein levels did not significantly change after a 30-min exposure to CGS21680. In contrast, long-term exposure (12-20 hr) of PC12 cells to adenosine agonists resulted in a slight further reduction of adenylyl cyclase activity and a consistent decline in the Gs alpha protein level. In addition, long-term incubation with adenosine agonists or with forskolin-enhanced phosphodiesterase (PDE) activity in the cytosolic and membrane fractions by 57 +/- 9% and 53 +/- 18%, respectively. Hydrolysis of cAMP was significantly faster in agonist-desensitized cells than in control cells. PDE might therefore play an important role in desensitization of the A2a response in PC12 cells. Polymerase chain reaction-based analysis of the mRNA for A2a-R and A2b-R indicated that both A2a-R and A2b-R were present in PC12 cells; the A2b response was also diminished in A2a-desensitized cells. Our data suggest that inhibition of adenylyl cyclase after short-term agonist treatment, down-regulation of Gs alpha protein level after long-term agonist treatment, and activation of PDE after long-term agonist treatment account for desensitization of the A2a-mediated response in PC12 cells.
为了解A2a腺苷受体（A2a-R）反应的调节机制，我们研究了大鼠嗜铬细胞瘤PC12细胞中A2a反应脱敏的分子机制，该细胞所具有的A2a-R与我们最近从大鼠脑中克隆的A2a受体相同。将PC12细胞长时间暴露于腺苷激动剂中，可显著抑制细胞对随后用A2a选择性腺苷激动剂（CGS21680）刺激的反应。在脱敏细胞中，未观察到CGS21680结合位点数量和亲和力的显著变化，在用腺苷激动剂预处理的细胞中，我们也未发现A2a-R转录水平有任何显著变化。然而，脱敏细胞中的基础腺苷酸环化酶活性以及由腺苷激动剂、GTPγS和福斯可林刺激的环化酶活性均降低。将PC12细胞长时间暴露于二丁酰环磷腺苷（dibutyryl-cAMP）中，基础或腺苷激动剂诱发的腺苷酸环化酶活性均未显著改变。因此，细胞内cAMP含量的升高本身不足以导致观察到的A2a脱敏时腺苷酸环化酶活性的降低。脱敏细胞中腺苷酸环化酶活性的抑制在与CGS21680短期（30分钟）孵育后发生，并且可被腺苷拮抗剂8-环戊基-1,3-二丙基黄嘌呤阻断。暴露于CGS21680 30分钟后，Gsα蛋白水平未显著变化。相反，将PC12细胞长时间（12 - 20小时）暴露于腺苷激动剂中，导致腺苷酸环化酶活性进一步轻微降低，且Gsα蛋白水平持续下降。此外，长时间用腺苷激动剂或福斯可林孵育，可使胞质和膜部分中的磷酸二酯酶（PDE）活性分别提高57±9%和53±18%。激动剂脱敏细胞中cAMP的水解明显快于对照细胞。因此，PDE可能在PC12细胞中A2a反应的脱敏中起重要作用。基于聚合酶链反应对A2a-R和A2b-R mRNA的分析表明，PC12细胞中同时存在A2a-R和A2b-R；在A2a脱敏细胞中，A2b反应也减弱。我们的数据表明，短期激动剂处理后腺苷酸环化酶的抑制、长期激动剂处理后Gsα蛋白水平的下调以及长期激动剂处理后PDE的激活，共同导致了PC12细胞中A2a介导反应的脱敏。
