1 Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba Canada; and.
Am J Respir Cell Mol Biol. 2014 Jan;50(1):135-43. doi: 10.1165/rcmb.2013-0085OC.
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 prostanoid (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 palmitoylation. We studied the role of Gαq palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition, the effects of hypoxia on palmitoylation, and the effects 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 coexpressed in HEK293T cells stably expressing TPα. We examined membrane localization of Gαq, TP receptor-Gαq association by coimmunoprecipitation, and Ca(2+) responses to U46619 in hypoxic and normoxic cells. Gαq palmitoylation is essential for the Ca(2+) response to TPα stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not in control pulmonary artery. Hypoxia increases palmitoylation of Gαq; the hypoxic. but not the 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 Ca(2+) mobilization. Depalmitoylation of any one Gαq cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.
肺血管收缩是新生儿持续性肺动脉高压（PPHN）的标志。我们报道了在缺氧肺血管中，钙对血栓素的反应增加，并且血栓素前列腺素（TP）与 Gαq 的选择性增加有关。Gαq 的棕榈酰化对于有效的受体-Gαq-磷脂酶 C 相互作用很重要。TPα 受体本身不易发生棕榈酰化。我们使用药理学棕榈酰化抑制、缺氧对棕榈酰化的影响以及 Gαq 特定半胱氨酸取代突变对 Gαq 膜靶向、TPα 关联和对 TP 激动剂的钙剂量反应曲线的影响，研究了 Gαq 棕榈酰化在缺氧肺血管收缩中的作用。PPHN 肺动脉和表达 TPα 的 HEK293T 细胞在接受 TP 激动剂 U46619 之前，用不可逆的棕榈酰化抑制剂 2-溴棕榈酸处理。研究了缺氧和常氧心肌细胞中的棕榈酸摄取。野生型 Gαq 和半胱氨酸到丙氨酸突变体 C9A、C10A 和 C9A/C10A 瞬时共表达在稳定表达 TPα 的 HEK293T 细胞中。我们检查了 Gαq 的膜定位、通过共免疫沉淀的 TP 受体-Gαq 关联以及缺氧和常氧细胞中对 U46619 的 Ca（2+）反应。Gαq 的棕榈酰化对于对 TPα 刺激的 Ca（2+）反应是必不可少的。棕榈酰化抑制减少了 PPHN 中的血栓素收缩力，但对对照肺动脉没有影响。缺氧增加 Gαq 的棕榈酰化；对血栓素的缺氧反应，但不是常氧反应，是棕榈酰化敏感的。一个 N 端半胱氨酸的棕榈酰化对于 Gαq 与 TPα 受体的物理关联是必需的。两个半胱氨酸的棕榈酰化对于 Gαq 的膜定位和 Ca（2+）动员都是必需的。任何一个 Gαq 半胱氨酸的去棕榈酰化都会将缺氧对血栓素挑战的反应降低到与常氧细胞相同的水平。
