The vitamin D receptor agonist elocalcitol up-regulates L-type Ca2+ channel activity in human and rat bladder
Annamaria Morelli1, Roberta Squecco1, Fabio Francini1, Paola Failli1, Sandra Filippi1, Aravinda Chavalmane1, Benedetta Fibbi1, Rosa Mancina1, Linda Vignozzi1, Enrico Silvestrini1, Mauro Gacci1, Enrico Colli2, Luciano Adorini2 & Mario Maggi1
Human bladder contraction mainly depends on Ca2+ influx, via L-type voltage-gated Ca2+ channels, and on RhoA/Rho-kinase contractile signalling, a pathway up-regulated in overactive bladder (OAB) syndrome. Elocalcitol (Elo) is a vitamin D receptor agonist inhibiting RhoA/Rho-kinase signalling in rat and human bladder, shown to ameliorate OAB symptoms in a clinical study. Since in normal bladder from SpragueDawley (SD) rats Elo treatment delayed the carbachol-induced contraction without changing maximal effect, we hypothesized, based on increased sensitivity to the selective L-type Ca2+ channel antagonist isradipine in bladder strips from Elo-treated rats, an up-regulation of L-type Ca2+ channels. Thus, this hypothesis was further investigated. In human bladder smooth muscle cells (hBCs), Elo induced a rapid increase in intracellular [Ca2+], which was abrogated by the specific L-type Ca2+ channel antagonist verapamil and was undetectable in the absence of extracellular Ca2+. Moreover, hBCs exhibited voltage-activated Ca2+ currents (ICa), T-type and L-type (ICa,L). Accordingly, both isradipine and verapamil only blocked the slow ICa,L, which was enhanced by the selective L-type agonist Bay K8644 (Bay). Addition of Elo (10−7 M) increased ICa,L size and specific conductance (Gm/Cm), by inducing faster activation and inactivation kinetics than control and Bay, and determined a significant negative shift of the activation (Va) and inactivation curves (Vh), as Bay. These effects resulted potentiated in long-term treated hBCs with Elo (10−8 M, 48 h), which also showed increased mRNA and protein expression of pore forming L-Type α1C subunit. In bladder strips from SD rats, Bay induced a dose-dependent increase in tension and its maximal contractile effect was significantly enhanced by Elo-treatment (30 μg/kg per day, 2 weeks). In conclusion, Elo upregulated Ca2+ entry through L-type Ca2+ channels in human bladder smooth muscle cells, thus balancing its inhibitory effect on RhoA/Rho-kinase signalling, and providing a mechanistic basis for using this drug in the treatment of OAB.