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Functional muscarinic M2 and M3 receptors and beta-adrenoceptor in cultured rat bladder smooth muscle.

机译:培养的大鼠膀胱平滑肌中功能毒蕈碱M2和M3受体以及β-肾上腺素受体。

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摘要

A highly purified rat urinary bladder smooth muscle cell culture was obtained by a modified enzymic isolation method, and the presence of functional muscarinic as well as beta-adrenergic receptors were subsequently determined. At 7-10 days of culture, cells became elongated and spindle-shaped showing a typical "hills and valleys" form. They were stained with anti-alpha-actin and anti-myosin antibodies. Radiolabeled ligand binding using [3H]N-methylscopolamine and [3H]CGP12177 showed that these cells expressed muscarinic and beta-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M3 antagonist. On the other hand, stimulation of cultured cells with isoproterenol enhanced the basal cyclic AMP formation, which was reversed by carbachol. Therefore, the presence of functional muscarinic (both M2 and M3) as well as beta-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.
机译:通过改良的酶分离方法获得了高度纯化的大鼠膀胱平滑肌细胞培养物,随后确定了功能性毒蕈碱受体以及β-肾上腺素能受体的存在。培养7-10天后,细胞拉长且呈纺锤形,呈典型的“丘陵和山谷”形式。它们用抗α-肌动蛋白和抗肌球蛋白抗体染色。使用[3H] N-甲基东pol碱和[3H] CGP12177进行放射性标记的配体结合表明,这些细胞表达毒蕈碱和β-肾上腺素受体。卡巴胆碱对培养细胞的刺激抑制了福司可林刺激的环AMP的形成,导致呋喃2荧光法测量的细胞内Ca2 +浓度升高。后者的反应几乎被选择性的毒蕈碱M3拮抗剂4-DAMP完全阻断。另一方面,用异丙肾上腺素刺激培养的细胞会增强基础环AMP的形成,而其被卡巴胆碱逆转。因此,在通过酶分离法获得的大鼠膀胱平滑肌细胞的纯培养物中,证实了功能性毒蕈碱(M2和M3)以及β-肾上腺素受体的存在。

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