Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
Published: May 2017
AI Article Synopsis
Article Abstract
The adrenergic receptor (AR) increases intracellular Ca in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the AR promotes Ca mobilization (pEC = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca following AR stimulation. The increase in cytoplasmic Ca was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the AR activation. Consistent with this mechanism, AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the AR-promoted Ca mobilization. Together, these data support a model whereby the AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP-dependent Ca mobilization from intracellular stores. Given that AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.
