Role of Ca2+ influx
Johnston et al.,  showed that the frequency of Ca2+ waves in urethra ICC was critically dependent on the external Ca2+ concentration, [Ca2+]o. For example, when [Ca2+]o was reduced from 1.8 to 0.9 mM the frequency of Ca2+ waves fell by ~40%. Conversely when [Ca2+]o was increased to 3.6 mM the frequency of the Ca2+ waves increased significantly. Finally, when Ca2+ was removed from the bath the oscillations stopped. Atypical example of these effects is shown in Fig. 5. Although it was originally thought that these effects may have reflected changes in the Ca2+ content of the intracellular stores, Johnston et al.,  demonstrated that 10 mM caffeine was able to evoke Ca2+ transients of similar amplitude in the presence of 1.8 and 0 mM [Ca2+]o. This suggested that the Ca2+ stores remained intact during this brief removal of [Ca2+]o and pointed to the involvement of a direct contribution of Ca2+ influx to the pacemaker mechanism.
The involvement of several Ca2+ influx pathways to the pacemaker mechanism in urethral ICC has been evaluated. Since nifedipine (10 μM) failed to block either STICs at –60 mV or Ca2+ oscillations in unclamped cells [30, 32] it appears that L-type Ca2+ channels are not essential for pacemaking. Capacitative Ca2+ entry (CCE) also appears not be directly involved as blockers of it did not abolish STICs, suggesting that an alternative pathway must be involved . Arecent study by Bradley et al., reported that Ca2+ influx via reverse Na+/Ca2+ exchange (NCX) is of critical importance to the pacemaker mechanism in urethral ICC . The idea that reverse NCX could be important stemmed from an observation by Putney et al.,  who noted that cells which do not display CCE, utilize reverse NCX to replenish their Ca2+ stores. Bradley et al.,  found that the selective reverse NCX inhibitors KB-R7943 and SEA 0400, significantly reduced the frequency of Ca2+ oscillations and STICs at –60 mV and decreased basal Ca2+. However, it appears that these effects were not due to an effect on Ca2+ store replenishment as neither NCX inhibitor blocked Ca2+ release evoked by caffeine or noradrenaline. Instead, it appears that Ca2+ influx via this pathway acts to raise basal Ca2+ levels in urethra ICC sufficiently to activate RyR. Such a mechanism is reminiscent of that reported by Leblanc & Hume  who found that ryanodine sensitive sarcoplasmic reticulum (SR) Ca2+ release in cardiac myocytes was triggered by Ca2+ entry via reverse NCX.