Taguchi K, Yang M, Goepel M, Michel M C
Department of Medicine, Klinikum, University of Essen, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Feb;357(2):100-10. doi: 10.1007/pl00005143.
The coupling of human alpha1-adrenoceptor subtypes to protein kinase C (PKC) and PKC-related signalling events were investigated in rat-1 fibroblasts stably expressing alpha1A-, alpha1B- or alpha1D-adrenoceptors at densities of 1328+/-200, 5030+/-703 and 150+/-14 fmol/mg protein respectively. In functional assays the alpha1-adrenoceptor agonist phenylephrine significantly stimulated PKC (assessed as increased activity in the membrane fraction) in cells expressing alpha1A- or alpha1B- but not alpha1D-adrenoceptors. In immunoblot assays phorbol ester treatment enhanced membrane-associated immunoreactivity of PKCalpha, PKCdelta and PKCepsilon to a similar extent in all three cell lines. Stimulation of alpha1A- and alpha1B-adrenoceptors also increased immunoreactivity of PKCalpha, PKCdelta and PKCepsilon in the membrane fraction, while alpha1D-adrenoceptor stimulation yielded only very small and inconsistent alterations. Immunoreactivity of PKCzeta was not consistently affected by phorbol ester or phenylephrine in any of the cell lines. Stimulation of all three alpha1-adrenoceptors time- and concentration-dependently increased inositol phosphate formation. Maximum activation occurred with the order alpha1A approximately = alpha1B > alpha1D. Phenylephrine also concentration dependently elevated free intracellular [Ca2+] in all three cell lines with the order of efficacy alpha1A > alpha1B > alpha1D. In the presence of ethanol, phenylephrine stimulated phosphatidylethanol formation in alpha1A- and alpha1B-adrenoceptor-expressing cells time and concentration dependently but only weakly and inconsistently in alpha1D-adrenoceptor-expressing cells. The efficacy of phenylephrine (100 microM) relative to that of noradrenaline (100 microM) for stimulation of phosphatidylethanol formation was similar (> or = 75%) for all three subtypes. The alkylating agent phenoxybenzamine concentration dependently reduced alpha1A-adrenoceptor density and phenylephrine-stimulated Ca2+ elevations to levels seen with alpha1D-adrenoceptors but reductions of phenylephrine-stimulated phosphatidylethanol formation were weaker. We conclude that human alpha1A- and alpha1B-adrenoceptors expressed in rat-1 cells couple to activation of PKCalpha, PKCdelta and PKCepsilon but not PKCzeta; this may involve stimulation of phospholipases C and D and intracellular Ca2+ elevations. Activation of these pathways by alpha1D-adrenoceptors appears to be much weaker and was not detected consistently; this was not fully explained by weak partial agonism of phenylephrine at this subtype or by lower receptor densities. Overall the alpha1A-adrenoceptor may have the highest efficiency of stimulus-response coupling among human alpha1-adrenoceptor subtypes.
在稳定表达α1A -、α1B -或α1D -肾上腺素能受体的大鼠-1成纤维细胞中，研究了人α1 -肾上腺素能受体亚型与蛋白激酶C（PKC）及PKC相关信号事件的偶联情况，其受体密度分别为1328±200、5030±703和150±14 fmol/mg蛋白。在功能测定中，α1 -肾上腺素能受体激动剂去氧肾上腺素显著刺激表达α1A -或α1B -但不表达α1D -肾上腺素能受体的细胞中的PKC（以膜组分中活性增加来评估）。在免疫印迹测定中，佛波酯处理在所有三种细胞系中均以相似程度增强了PKCα、PKCδ和PKCε的膜相关免疫反应性。刺激α1A -和α1B -肾上腺素能受体也增加了膜组分中PKCα、PKCδ和PKCε的免疫反应性，而刺激α1D -肾上腺素能受体仅产生非常小且不一致的变化。在任何细胞系中，PKCζ的免疫反应性均未受到佛波酯或去氧肾上腺素的一致影响。刺激所有三种α1 -肾上腺素能受体均以时间和浓度依赖性方式增加肌醇磷酸的形成。最大激活程度按α1A ≈α1B >α1D的顺序出现。去氧肾上腺素在所有三种细胞系中也以浓度依赖性方式升高细胞内游离[Ca2 +]，其效力顺序为α1A >α1B >α1D。在乙醇存在下，去氧肾上腺素在表达α1A -和α1B -肾上腺素能受体的细胞中以时间和浓度依赖性方式刺激磷脂酰乙醇的形成，但在表达α1D -肾上腺素能受体的细胞中仅微弱且不一致地刺激。对于所有三种亚型，去氧肾上腺素（100μM）相对于去甲肾上腺素（100μM）刺激磷脂酰乙醇形成的效力相似（≥75%）。烷基化剂酚苄明浓度依赖性地降低α1A -肾上腺素能受体密度以及去氧肾上腺素刺激的Ca2 +升高至α1D -肾上腺素能受体所见水平，但去氧肾上腺素刺激的磷脂酰乙醇形成的降低较弱。我们得出结论，在大鼠-1细胞中表达的人α1A -和α1B -肾上腺素能受体与PKCα、PKCδ和PKCε的激活偶联，但不与PKCζ偶联；这可能涉及刺激磷脂酶C和D以及细胞内Ca2 +升高。α1D -肾上腺素能受体对这些途径的激活似乎要弱得多且未被一致检测到；这不能完全由去氧肾上腺素在该亚型上的弱部分激动作用或较低的受体密度来解释。总体而言，在人α1 -肾上腺素能受体亚型中，α1A -肾上腺素能受体可能具有最高的刺激-反应偶联效率。
