Actions of two main metabolites of propiverine (M-1 and M-2) on voltage-dependent L-type Ca2+ currents and Ca2+ transients in murine urinary bladder myocytes

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca2+ currents (ICa) using patch-clamp techniques and fluorescent Ca2+ imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of ICa in a concentration-dependent manner at a holding potential of –60 mV (propiverine, Ki = 10 μM; M-1, Ki = 118 μM). M-1 shifted the steady-state inactivation curve of ICa to the left at –90 mV by 7 mV. Carbachol (CCh) reversibly inhibited ICa. This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 μM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of ICa was blocked. In fluorescent Ca2+ imaging, M-2 inhibited EFS-induced and ACh-induced Ca2+ transients. These results suggest that M-1 acts, at least in part, as a Ca2+ channel antagonist (as it inhibited ICa), whereas M-2 has more direct antimuscarinic actions.