Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct

摘要

Arginine vasopressin (AVP) causes increase in intracellular Ca²⁺ concentration with an oscillatory pattern. Ca²⁺ mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca²⁺ oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca²⁺ oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca²⁺ oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca²⁺ oscillations and endogenous cADPR plays a major role in the AVP-induced Ca²⁺ oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP3) induced Ca²⁺ release but did not maintain sustained Ca²⁺ oscillations. Removal of extracellular Ca²⁺ halted ongoing AVP-mediated Ca²⁺ oscillation, suggesting that it requires extracellular Ca²⁺ entry. AVP-induced Ca²⁺ oscillation was unaffected by nifedipine. Intracellular Ca²⁺ store depletion induced by 20 μM thapsigargin in Ca²⁺-free medium triggered store-operated Ca²⁺ entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl3 and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca²⁺-free medium, application of extracellular Ca²⁺ also triggered Ca²⁺ influx, which was sensitive to GdCl3 and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca²⁺ oscillations in IMCD are mediated by the interplay of Ca²⁺ release from RyRs and a Ca²⁺ influx mechanism involving nonselective cation channels that resembles SOCE.