Theroux T L, Esbenshade T A, Peavy R D, Minneman K P
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Mol Pharmacol. 1996 Nov;50(5):1376-87.
We compared the efficiencies with which human alpha 1-adrenergic receptor (AR) subtypes activate inositol phosphate (InsP) formation and increase intracellular Ca2+ in transfected cell lines. Expression of human alpha 1a-, alpha 1b-, and alpha 1d-AR cDNAs under the repressible control of anhydrotetracycline in human embryonic kidney (HEK) 293 cells, which normally express no alpha 1-ARs, was used to compare responses to norepinephrine (NE) at different receptor densities. Maximal NE-stimulated InsP formation was found to increase with increasing density of each subtype, whereas basal levels and responses to sodium fluoride did not change. A comparison of multiple subclones over equivalent ranges of receptor expression showed that activation of each subtype resulted in different maximal responses (alpha 1a > alpha 1b > alpha 1d) in HEK 293 cells. Analogous studies were carried out in human SK-N-MC cells, which normally express low levels of all three alpha 1-AR subtypes, using an isopropyl-beta-D-thiogalactoside-inducible expression system. Induction with isopropyl-beta-D-thiogalactoside increased the density of individual alpha 1-AR subtypes by 4-6-fold over the level of endogenous expression. Increased expression of each of these subtypes in SK-N-MC cells did not alter the EC50 value for NE in stimulating InsP formation or releasing [Ca2+]i but did increase maximal responses to NE. Similar to our findings in HEK 293 cells, a comparison of responses at similar expression levels in SK-N-MC cells showed different maximal responses stimulated by each subtype, for both InsP (alpha 1a > alpha 1b > or = alpha 1d) and [Ca2+]i (alpha 1a > alpha 1b > alpha 1d) responses. These studies show that agonist-occupied human alpha 1-AR subtypes have different efficiencies in activating phospholipase C in human cell lines. In both HEK 293 and SK-N-MC cells, alpha 1a-ARs couple most efficiently, whereas alpha 1d-ARs couple very poorly.
我们比较了人类α1 - 肾上腺素能受体（AR）亚型在转染细胞系中激活肌醇磷酸（InsP）形成和增加细胞内Ca2+的效率。在通常不表达α1 - ARs的人胚肾（HEK）293细胞中，利用脱水四环素的可抑制控制来表达人类α1a -、α1b - 和α1d - AR的cDNA，以此比较在不同受体密度下对去甲肾上腺素（NE）的反应。发现最大NE刺激的InsP形成随着每种亚型密度的增加而增加，而基础水平和对氟化钠的反应没有变化。对受体表达等效范围内的多个亚克隆进行比较表明，在HEK 293细胞中，每种亚型的激活导致不同的最大反应（α1a > α1b > α1d）。在通常表达所有三种α1 - AR亚型低水平的人SK - N - MC细胞中，使用异丙基 - β - D - 硫代半乳糖苷诱导表达系统进行了类似研究。用异丙基 - β - D - 硫代半乳糖苷诱导使单个α1 - AR亚型的密度比内源性表达水平增加4 - 6倍。这些亚型在SK - N - MC细胞中表达增加并没有改变NE刺激InsP形成或释放[Ca2+]i的EC50值，但确实增加了对NE的最大反应。与我们在HEK 293细胞中的发现相似，对SK - N - MC细胞中相似表达水平的反应进行比较表明，对于InsP（α1a > α1b > 或 = α1d）和[Ca2+]i（α1a > α1b > α1d）反应，每种亚型刺激的最大反应不同。这些研究表明，激动剂占据的人类α1 - AR亚型在人类细胞系中激活磷脂酶C的效率不同。在HEK 293和SK - N - MC细胞中，α1a - ARs的偶联最有效，而α1d - ARs的偶联非常差。
