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1 University of Florence
2 Univ. of Florence
3 University of florence
4 Bioxell
* To whom correspondence should be addressed. E-mail: m.maggi{at}dfc.unifi.it.
Human bladder contraction mainly depends on Ca2+ influx, via L-type voltage-gated Ca2+ channels, and on RhoA/Rho-kinase contractile signalling, which is up-regulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho-kinase signalling in rat and human bladder. Since in normal bladder from Sprague-Dawley (SD) rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness, and increased sensitivity to the L-type Ca2+ channel antagonist isradipine, we investigated whether elocalcitol up-regulated L-type Ca2+ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca2+], which was abrogated by the L-type Ca2+ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca2+ currents (ICa), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist Bay-K8644. Addition of elocalcitol (10-7 M) increased L-type ICa size and specific conductance, by inducing faster activation and inactivation kinetics than control and Bay-K8644, while determining a significant negative shift of the activation and inactivation curves, comparable to Bay-K8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10-8 M, 48h), which also showed increased mRNA and protein expression of pore forming L-Type
1C subunit. In bladder from SD rats, Bay-K8644 induced a dose-dependent increase in tension, that was significantly enhanced by elocalcitol treatment (30 µg/kg/day, 2 weeks). In conclusion, elocalcitol up-regulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho-kinase signalling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.
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