The role of Ca(2+) influx in spontaneous Ca(2+) wave propagation in interstitial cells of Cajal from the rabbit urethra

Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC). ICC pacemaker activity results from their ability to generate propagating CA(2+) waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised CA(2+) events for the first time in urethral ICC; these were due to CA(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating CA(2+) waves. We show that propagation of CA(2+) waves in urethral ICC is critically dependent upon CA(2+) influx via reverse mode NCX. Our data provide a clearer understanding of the intracellular mechanisms involved in the generation of ICC pacemaker activity. Interstitial cells of Cajal (ICC) are putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous propagating CA(2+) waves that are modulated by [CA(2+)]o and whose propagation is inhibited by inositol tri-phosphate receptor (IP3 R) blockers. The purpose of this study was to further examine the role of CA(2+) influx and CA(2+) release in the propagation of CA(2+) waves. Intracellular CA(2+) was measured in Fluo-4-loaded ICC using a Nipkow spinning disc confocal microscope at fast acquisition rates (50 fps). We identified previously undetected localised CA(2+) events originating from ryanodine receptors (RyRs). Inhibiting CA(2+) influx by removing [CA(2+)]o or blocking reverse mode sodium-calcium exchange (NCX) with KB-R 7943 or SEA-0400 abolished CA(2+) waves, while localised CA(2+) events persisted. Stimulating RyRs with 1 mm caffeine restored propagation. Propagation was also inhibited when CA(2+) release sites were uncoupled by buffering intracellular CA(2+) with EGTA-AM. This was reversed when CA(2+) influx via NCX was increased by reducing [Na(+)]o to 13 mm. Low [Na(+)]o also increased the frequency of CA(2+) waves and this effect was blocked by tetracaine and ryanodine but not 2-aminoethoxydiphenyl borate (2-APB). RT-PCR revealed that isolated ICC expressed both RyR2 and RyR3 subtypes. We conclude: (i) RyRs are required for the initiation of CA(2+) waves, but wave propagation normally depends on activation of IP3 Rs; (ii) under resting conditions, propagation by IP3 Rs requires sensitisation by influx of CA(2+) via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to CA(2+).