Sikarwar AS, Hinton M, Santhosh KT, Chelikani P, Dakshinamurti S

Pulmonary arterial vasoconstriction is a hallmark of persistent pulmonary hypertension of the newborn (PPHN). We reported increased calcium responses to thromboxane, and selectively increased thromboxane receptor (TP) association with Gαq, in hypoxic pulmonary artery. Palmitoylation of Gαq is important for efficient receptor-Gαq-phospholipase-C interactions. TPα receptor is not itself amenable to ...

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palmitoylation. We studied the role of Gαq palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition; effects of hypoxia on palmitoylation; and of site-specific cysteine substitution mutations of Gαq, on Gαq membrane targeting, TPα association, and calcium dose-response curve to a TP agonist. PPHN pulmonary artery, and HEK293T cells expressing TPα, were exposed to irreversible palmitoylation inhibitor 2-bromopalmitate, before challenge with TP agonist U46619. Palmitate uptake was studied in hypoxic and normoxic myocytes. Wild-type Gαq, and Gαq cysteine-to-alanine mutants C9A, C10A and C9A/C10A were transiently co-expressed in HEK293T cells stably expressing TPα. We examined membrane localization of Gαq, TP receptor-Gαq association by co-immunoprecipitation, and Ca2+ responses to U46619 in hypoxic and normoxic cells. Gαq palmitoylation is essential for Ca2+ response to TPα stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not control pulmonary artery. Hypoxia increases palmitoylation of Gαq; the hypoxic but not normoxic response to thromboxane is palmitoylation-sensitive. Palmitoylation of one N-terminal cysteine is required for physical association of Gαq with the TPα receptor. Palmitoylation of both cysteines is required for Gαq membrane localization and Ca2+ mobilization. Depalmitoylation of any one Gαq cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.

Date: Aug. 20, 2013

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