Functional muscarinic M 2 and M 3 receptors and β-adrenoceptor in cultured rat bladder smooth muscle

Abstract

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 β-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-α-actin and anti-myosin antibodies. Radiolabeled ligand binding using [ 3H]N-methylscopolamine and [ 3H]CGP12177 showed that these cells expressed muscarinic and β-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca 2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M 3 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 M 2 and M 3) as well as β-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.