Recently we have demonstrated that a specialised population of cells in the urethra (Sergeant et al. 2000) share characteristics typical of interstitial cells described in the gastrointestinal tract. These urethral interstitial cells are non-contractile, possess abundant calcium-activated chloride current and show regular spontaneous transient inward currents when recorded under voltage clamp. The purpose of the present study was to use confocal microscopy to examine the mechanisms underlying this spontaneous activity in freshly dispersed interstitial cells from the rabbit urethra.

Rabbits were humanely killed with pentobarbitone (I.V.) and their urethras removed. Cells were isolated as previously described (Sergeant et al. 2000) and incubated for 15 min at 37 °C with the fluorescent Ca²⁺-sensitive indicator Fluo-4AM (10 µM). Cells were then plated onto glass-bottomed petri dishes for 30 min before being perfused with Hanks’ solution. Single cell imaging was performed at 5 frames s^-1 using a Nipkow spinning disc laser confocal microscope. When cells were maintained at 37 °C either calcium sparks, or more frequently, calcium waves were apparent and usually initiated in the perinuclear region. Wave amplitudes were calculated as a ratio (F/F₀) by dividing the mean intensity of the region of interest (ROI) during the peak of the wave by the mean intensity during quiescent periods. The waves occurred at a mean frequency of 3.1 ± 0.2 min^-1, had peak amplitudes of 1.8 ± 0.21 (F/F₀) and a mean ± S.E.M. duration of 14.3 ± 1.6 s (n = 6 cells). To investigate the contribution of calcium release from intracellular stores we examined the effects of ryanodine, tetracaine, caffeine and 2-aminoethoxydiphenylborane (2APB) on the calcium waves. Application of either ryanodine (30 µM) or tetracaine (100 µM, n = 3) abolished spontaneous activity. In addition, basal fluorescence was reduced to 0.93 ± 0.02 (F/F₀) in the presence of tetracaine. Application of caffeine (10 mM), transiently increased F/F₀ to 1.97 ± 0.2 and reversibly abolished spontaneous waves (n = 5). When release of calcium from IP₃-sensitive stores was inhibited by 2APB (100 µM), basal fluorescence increased and the amplitude of the waves gradually diminished but frequency was not reduced (n = 3). Similar effects were found with 30 µM 2APB (n = 3).

When external calcium was removed from the bathing solution (Mg²⁺ substituted Hanks’, 5 mM EGTA), F/F₀ decreased to 0.87 ± 0.04 and spontaneous activity was immediately abolished (n = 5). To test if the inhibition of spontaneous activity was due to depletion of the intracellular stores, caffeine was reapplied in the absence of external calcium. In four experiments, caffeine application produced a transient increase in F/F₀ to 2.0 ± 0.3 in normal Hanks’ solution. When caffeine was reapplied approximately 1 min after removal of external calcium, spontaneous activity was abolished but F/F₀ increased to 1.8 ± 0.3, suggested that the caffeine-sensitive stores were not significantly depleted.

The abolition of calcium waves in the absence of external calcium suggested that the influx of calcium is an essential step for initiating spontaneous calcium waves in urethral interstitial cells. To test if this influx was through L-type calcium channels we examined the effects of 10 µM nifedipine. In three cells, nifedipine failed to alter the amplitude or frequency of calcium waves.

These data suggest that spontaneous calcium waves in rabbit urethral interstitial cells are dependent on the release of calcium from intracellular stores which is triggered by calcium influx through a nifedipine-insensitive pathway.

This work was supported by the Wellcome Trust and Department of Education and Learning.