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Stimulation of Asub2A/subreceptors (Asub2A/subR) coupled to Gsubs/sub/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of Asub2A/subR in the modulation of free cytosolic Casup2+/supconcentration ([Casup2+/sup]subi/sub) involving IPsub3/sub, cAMP and PKA was investigated in HEK 293-Asub2A/subR. The levels of IPsub3/suband cAMP were observed by enzyme immunoassay detection method and [Casup2+/sup]subi/subusing Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with Asub2A/subR agonist NECA showed increased levels of cAMP, PKA, IPsub3/suband [Casup2+/sup]subi/sublevels. However, the reverse effect was observed with Asub2A/subR antagonists (ZM241385 and caffeine). Blocking the Gsubαq/sub/PLC/DAG/IPsub3/subpathway with neomycin, a PLC inhibitor did not affect the modulation of IPsub3/suband [Casup2+/sup]subi/sublevels in HEK 293-Asub2A/subR cells. To investigate the Gsubαi/sub/AC/cAMP/PKA, HEK 293-Asub2A/subR cells pre-treated with pertussis toxin followed by forskolin in the presence of Asub2A/subR agonist (NECA) showed no effect on cAMP levels. Further, Gsubαs/sub/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IPsub3/submediated [Casup2+/sup]subi/submodulation. In the HEK 293-Asub2A/subR cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IPsub3/suband [Casup2+/sup]subi/sublevels. The study distinctly demonstrated that Asub2A/subR modulates IPsub3/sublevels to release the [Casup2+/sup]subi/subvia cAMP-dependent PKA. The role of Asub2A/subR mediated Gsubαs/subpathway inducing IPsub3/submediated [Casup2+/sup]subi/subrelease may open new avenues in the therapy of neurodegenerative disorder. |
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