The relative contributions of active response and passive stiffness on the pharmacological response of human normal and diseased coronary arteries to ET-1
Janet J. Maguire, Chen Yen Ooi, Michael P F Sutcliffe, Anthony P. Davenport · Life Sciences · 2013
Background: Augmentation of Ca2+ entry via transient receptor potential canonical channel 6 (TRPC6) in response to stimulation of endothelin type A receptor (ETAR) with endothelin-1 is involved in the development of pulmonary arterial hypertension (PAH).For the drug therapy against PAH, Gs protein-coupled prostaglandin I2 receptor agonists to activate adenylate cyclase (AC)-cAMP-protein kinase A (PKA) signaling pathway are administered.This study examined the role of AC-cAMP-PKA pathway in regulation of receptor-operated Ca2+ entry (ROCE) via ETAR-activated TRPC6.Methods: In HEK293 cells coexpressing ETAR and TRPC6, intracellular free Ca2+ concentration was monitored by using a fluorescent Ca2+ indicator, fura-2/AM.To identify the target site(s) of TRPC6 for phosphorylation by PKA, serine residues at positions 14, 28, and 321, and threonine at 69 were replaced with alanine.Results: Stimulation of ETAR induced ROCE in TRPC6-expressing HEK293 cells where store-operated Ca2+ channels had been maximally activated by thapsigargin-induced Ca2+-depletion/Ca2+restoration.The ROCE was inhibited by forskolin and papaverine to activate cAMP-PKA pathway, while it was potentiated by Rp-8-Br-cAMP, a PKA inhibitor.Inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 but not Thr69 of TRPC6 with alanine.Immunoblotting with Phos-tag biotin to detect phosphorylated proteins demonstrated that wild-type and mutant TRPC6 proteins were phosphorylated under basal condition.In vitro kinase assay with TRPC6 proteins dephosphorylated with a phosphatase pretreatment revealed that PKA can phosphorylate TRPC6 on Ser28 and Thr69.Conclusions: ROCE via ETAR-activated TRPC6 is negatively regulated by PKA-mediated phosphorylation of TRPC6 at Ser28 but not at Thr69 in vivo, although PKA can phosphorylate TRPC6 on Ser28 and Thr69 in vitro.