Neuropharmaceutical Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, North Campus, University of Delhi, Delhi 110007, India.
Neuropharmaceutical Chemistry Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, North Campus, University of Delhi, Delhi 110007, India.
Biochim Biophys Acta Gen Subj. 2022 Dec;1866(12):130242. doi: 10.1016/j.bbagen.2022.130242. Epub 2022 Sep 6.
Stimulation of A receptors (A R) coupled to G/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A R in the modulation of free cytosolic Ca concentration ([Ca]) involving IP, cAMP and PKA was investigated in HEK 293-A R. The levels of IP and cAMP were observed by enzyme immunoassay detection method and [Ca] using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A R agonist NECA showed increased levels of cAMP, PKA, IP and [Ca] levels. However, the reverse effect was observed with A R antagonists (ZM241385 and caffeine). Blocking the G/PLC/DAG/IP pathway with neomycin, a PLC inhibitor did not affect the modulation of IP and [Ca] levels in HEK 293-A R cells. To investigate the G/AC/cAMP/PKA, HEK 293-A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A R agonist (NECA) showed no effect on cAMP levels. Further, G/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP mediated [Ca] modulation. In the HEK 293-A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP and [Ca] levels. The study distinctly demonstrated that A R modulates IP levels to release the [Ca] via cAMP-dependent PKA. The role of A R mediated G pathway inducing IP mediated [Ca] release may open new avenues in the therapy of neurodegenerative disorder.
A 受体（A R）与 G/olf 蛋白偶联的刺激会激活腺苷酸环化酶（AC），导致 cAMP 的释放，从而激活 cAMP 依赖性蛋白激酶 A（PKA）的磷酸化。在 HEK 293-A R 中，研究了 A R 对涉及 IP、cAMP 和 PKA 的游离细胞浆 Ca 浓度 ([Ca]) 调节的可能作用。通过酶免疫检测法观察 IP 和 cAMP 的水平，通过 Fluo-4 AM 观察 [Ca]。此外，使用 PKA 比色活性试剂盒测定 cAMP 依赖性 PKA。我们观察到，用 A R 激动剂 NECA 预处理的细胞显示 cAMP、PKA、IP 和 [Ca]水平升高。然而，用 A R 拮抗剂（ZM241385 和咖啡因）观察到相反的效果。用 PLC 抑制剂新霉素阻断 G/PLC/DAG/IP 途径，不会影响 HEK 293-A R 细胞中 IP 和 [Ca]水平的调节。为了研究 G/AC/cAMP/PKA，用百日咳毒素预处理 HEK 293-A R 细胞，然后在 A R 激动剂（NECA）存在下用 forskolin处理，对 cAMP 水平没有影响。此外，研究了 G/AC/cAMP/PKA 途径，以阐明 cAMP 依赖性 PKA 在 IP 介导的 [Ca]调节中的作用。在先用 PKA 抑制剂 KT5720 预处理然后用 NECA 处理的 HEK 293-A R 细胞中，IP 和 [Ca]水平受到抑制。该研究明确表明，A R 通过 cAMP 依赖性 PKA 调节 IP 水平来释放 [Ca]。A R 介导的 G 途径诱导 IP 介导的 [Ca]释放的作用可能为神经退行性疾病的治疗开辟新途径。
