Presence and passage dependent loss of biochemical M3 muscarinic receptor function in human detrusor cultured smooth muscle cells.

Abstract

We explored morphological and biochemical aspects of human detrusor cells in culture as a tool for investigating the physiological mechanisms underlying bladder function and disturbances. Primary cultures of smooth muscle cells were derived from human bladder specimens of patients with an average age of 70 years undergoing cystectomy. Cultured cells were investigated by morphological, immunocytochemical and Western blot analysis. The alpha-actin content as well as the presence of muscarinic M2 and M3 receptors was determined in cell lysates and in fresh tissue homogenate for comparison. The functional response to muscarinic stimulation was assessed by measuring IP3 production induced by 1 mM. carbachol. Cultured smooth muscle cells showed a characteristic spindle-shaped morphology at early passages. Similarly immunocytochemical and Western blot analysis revealed an alpha-actin cell content that was unmodified up to passage 3. Conversely this marker protein sharply decreased during further passages. M3 muscarinic receptor was present in cultured cells and fresh tissue homogenates, whereas the M2 subtype was evident only in homogenates. Carbachol produced a time dependent increase in IP3 cell content, reaching maximal production after 20 minutes of exposure. This response was passage sensitive. Cultured human detrusor smooth muscle cells maintain their morphological and biochemical characteristics up to passage 3. With this caveat such cells can be an appropriate tool for investigating the molecular pathways underlying cholinergic activation in normal physiological and pathological bladders, and accordingly for detecting putative targets for pharmacological intervention.