The role of Ca2+ influx in spontaneous Ca2+ wave propagation in interstitial cells of Cajal from the rabbit urethra

• Published in: The Journal of physiology Vol. 593; no. 15; pp. 3333 - 3350
• Main Authors: Drumm, Bernard T., Large, Roddy J., Hollywood, Mark A., Thornbury, Keith D., Baker, Salah A., Harvey, Brian J., McHale, Noel G., Sergeant, Gerard P.
• Format: Journal Article
• Language: English
• Published: London Wiley Subscription Services, Inc 01.08.2015; John Wiley & Sons, Ltd
• Subjects: Muscle
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/JP270883

Key points 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 Ca2+ waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised Ca2+ events for the first time in urethral ICC; these were due to Ca2+ release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca2+ waves. We show that propagation of Ca2+ waves in urethral ICC is critically dependent upon Ca2+ 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 Ca2+ waves that are modulated by [Ca2+]o and whose propagation is inhibited by inositol tri‐phosphate receptor (IP3R) blockers. The purpose of this study was to further examine the role of Ca2+ influx and Ca2+ release in the propagation of Ca2+ waves. Intracellular Ca2+ 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 Ca2+ events originating from ryanodine receptors (RyRs). Inhibiting Ca2+ influx by removing [Ca2+]o or blocking reverse mode sodium–calcium exchange (NCX) with KB‐R 7943 or SEA‐0400 abolished Ca2+ waves, while localised Ca2+ events persisted. Stimulating RyRs with 1 mm caffeine restored propagation. Propagation was also inhibited when Ca2+ release sites were uncoupled by buffering intracellular Ca2+ with EGTA‐AM. This was reversed when Ca2+ influx via NCX was increased by reducing [Na+]o to 13 mm. Low [Na+]o also increased the frequency of Ca2+ 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 Ca2+ waves, but wave propagation normally depends on activation of IP3Rs; (ii) under resting conditions, propagation by IP3Rs requires sensitisation by influx of Ca2+ via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to Ca2+.