Recently we have described the presence of T-type calcium current in smooth muscle isolated from the human proximal urethra (Hollywood et al. 2002) and preliminary data suggested that this current is present in rabbit urethral smooth muscle. The aim of the present study was to characterise the T current in isolated rabbit urethral myocytes using the patch clamp technique and examine the effects of blocking this current on spontaneous activity recorded from strips of urethra with sharp microelectrodes.

Rabbits were humanely killed with pentobarbitone (I.V.) and their urethras removed. Cells were isolated as previously described (Sergeant et al. 2000). These were perfused with Hanks’ solution at 37 °C and studied using the amphotericin B perforated patch technique with Cs⁺-rich pipette solutions. When cells were held at -100 mV and depolarised in 10 mV steps for 500 ms, inward currents were evoked at potentials positive to -70 mV. When the current voltage (I-V) relationship was plotted, it consisted of two peaks – one at ~-40 mV and the other at 0 mV. To dissect these two components, currents were evoked from holding potentials of -100 mV and -60 mV. When the I-V curve obtained at a holding potential of -60 mV was plotted, the negatively activating component of inward current was abolished and little inward current was observed at potentials negative to -40 mV. Subtraction of the currents obtained at these two holding potentials revealed a current that activated at potentials positive to -70 mV, peaked at -30 mV and reversed at approximately 30 mV. To examine the voltage dependence of inactivation of both currents, cells were stepped to either -40 or 0 mV after a series of 2 s preconditioning potentials from -100 mV through to 0 mV. Under these conditions the current evoked by a step to -40 mV half-maximally inactivated at -76 ± 2 mV (mean ± S.E.M., n = 5) compared with -41 ± 3 mV (n = 5) for the current evoked by a step to 0 mV. We next examined the effects of Ni²⁺ on both components of inward current. Ni²⁺ (100 mM) reduced currents evoked at -40 mV from -66 ± 26 to -11 ± 9 pA but only reduced the current at 0 mV from -659 ± 102 to -559 ± 100 pA (n = 3).

To examine the contribution of T current to spontaneous electrical activity, we investigated the effects of Ni²⁺ on spike complexes in strips of urethra using sharp microelectrodes. Application of Ni²⁺ at concentrations of 10 mM, 30 mM and 100 mM decreased the frequency of spike complexes from 4.2 ± 0.5 min^-1 to 2.4 ± 0.5, 2.0 ± 0.4 and 0.3 ± 0.3 min^-1, respectively (n = 5 cells, 3 animals). These data suggest that the negatively activating T current may play an important role in modulating action potential frequency in the rabbit urethra.

This work was supported by Action Research, the Wellcome Trust and the Ralph Shackman Trust.