Kinetics of urothelial ATP release

Abstract

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 ± 6.5 fmol·min−1·cm−2 ( n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol·min−1·cm−2 with a Km of 0.49 μM. The time course of serosal ATP release was modeled as a constant rate of release ( d: mol·min−1·cm−2) and rate constant of hydrolysis ( kh: min−). In control conditions d was 18 fmol·min−1·cm−2 and kh of 0.056 ± 0.01 min− ( n = 18). Steady-state serosal chamber content is 370 ± 90 fmol/cm2, and concentration is 50 ± 1.2 × 10−12 M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 ± 1 pmol/cm2 and serosal chamber ATP by 0.1 ± 0.06 pmol/cm2 ( n = 5).