Nebigil C, Malik K U
Department of Pharmacology, College of Medicine, University of Tennessee, Memphis.
J Pharmacol Exp Ther. 1993 Aug;266(2):1113-24.
Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.
此前，我们已经表明，α-2C和α-1A肾上腺素能受体（AR）通过血管平滑肌细胞（VSMC）中一种对百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白（G蛋白）刺激前列环素（PGI2）的合成。本研究的目的是评估Ca++在α-AR激活引发的PGI2产生中的作用，并研究与这些α-AR偶联的G蛋白对VSMC中Ca++通道的调节作用。通过放射免疫测定法将PGI2测定为免疫反应性6-酮-PGF1α，并使用fura-2通过荧光分光光度法测定胞质钙（[Ca++]i）。去甲肾上腺素、甲氧明和UK-14304可增强6-酮-PGF1α的产生和[Ca++]i，而细胞外Ca++耗尽和Ca++通道拮抗剂（维拉帕米、硝苯地平和PN 200-110）可抑制这种增强作用。此外，Ca++通道激活剂Bay K 8644以硝苯地平敏感的方式增加6-酮-PGF1α的产生，表明二氢吡啶敏感的Ca++通道参与了VSMC的活动。百日咳毒素可抑制AR激动剂诱导的6-酮-PGF1α产生和[Ca++]i的增加。在存在鸟苷5'-O-（2-硫代二磷酸）（GTP-γ-S）的情况下，α-AR激动剂可增加Ca++内流，而在存在鸟苷5'-O-（2-硫代二磷酸）（GDP-β-S）和针对Giα1-2蛋白的抗血清时，这种作用在经β-七叶皂苷可逆通透化的细胞中被阻断。兔主动脉的VSMC含有一种G蛋白，在1:200稀释时可被Giα1-2识别，但不能被Giα3或G0抗体识别。钙调蛋白抑制剂W-7可阻断AR激动剂和Bay K 8644刺激的6-酮-PGF1α产生。磷脂酶A2抑制剂7,7-二甲基二十碳二烯酸和油酰氧基乙基磷酸胆碱可降低VSMC中6-酮-PGF1α的产生，但磷脂酶C抑制剂U-73122则不能。这些数据表明，与α-AR偶联的一种对百日咳毒素敏感的G蛋白，可能是Giα1-2，调节Ca++内流，进而通过与钙调蛋白相互作用，增加磷脂酶A2的活性，以释放花生四烯酸用于兔主动脉VSMC中PGI2的合成。
