The purpose of the present study was to determine whether cyclic ADP- ribose (cADPR) acts as a second messenger for Ca2+ release through ryanodine receptor (RyR) channels in tracheal smooth muscle (TSM). Freshly dissociated porcine TSM cells were permeabilized with β-escin, and real- time confocal microscopy was used to examine changes in intracellular Ca2+ concentration ([Ca2+](i)). cADPR (10 nM-10 μM) induced a dose-dependent increase in [Ca2+](i), which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 μM) and by the RyR blockers ruthenium red (10 μM) and ryanodine (10 μM), but not by the inositol 1,4,5-trisphosphate receptor blocker heparin (0.5 mg/ml). During steady-state [Ca2+](i) oscillations induced by acetylcholine (ACh), addition of 100 nM and 1 μM cADPR increased oscillation frequency and decreased peak-to-trough amplitude. ACh-induced [Ca2+](i) oscillations were blocked by 8-amino-cADPR; however, 8-amino- cADPR did not block the [Ca2+](i) response to a subsequent exposure to caffeine. These results indicate that cADPR acts as a second messenger for Ca2+ release through RyR channels in TSM cells and may be necessary for initiating ACh-induced [Ca2+](i) oscillations.
- Confocal microscopy
- Intracellular calcium concentration
- Ryanodine receptor
- Sarcoplasmic reticulum
- Second messenger