Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct.

Arginine vasopressin (AVP) regulates the osmotic water permeability (Pf) of renal collecting duct, conferring the precise control of renal excretion of water. AVP increases Pf in collecting ducts by triggering translocation and insertion of aquaporin-2 (AQP2) to the apical membrane of principal cells in the collecting duct (Wade et al. 1981; Nielsen et al. 1995a). These responses depend on the binding of AVP to basolateral V2 vasopressin receptors in collecting duct cells, which leads to the activation of adenylyl cyclase and an increase of cyclic AMP level in the cells (Chabardes et al. 1996). Cyclic AMP-dependent protein kinase A is then activated, which subsequently phosphorylates AQP2 at Ser256 (Nishimoto et al. 1999). However, the steps that lead to the eventual insertion of AQP2 into apical membrane are not known. A number of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are known to regulate exocytosis of synaptic vesicles, have been identified in renal collecting duct (Sollner et al. 1993). These include syaptobrevin-2 (Nielsen et al. 1995b), synaptotagmin VIII (Kishore et al. 1998), syntaxin-4 (Mandon et al. 1996) and SNAP23 (Inoue et al. 1998). It was hypothesized that the trafficking and targeting of AQP2 vesicles might resemble that of synaptic vesicles (Knepper & Inoue, 1997). Studies of a wide variety of vesicular-trafficking processes have pointed to a key role for localized increases in intracellular Ca2+ concentration ([Ca2+]i) in triggering the fusion of vesicles to their target membranes (Burgoyne & Morgan, 1998). This raises the possibility that the exocytotic insertion of AQP2 is also Ca2+ dependent. Studies from other investigators showed that 10 nm AVP was required to induce a detectable increase of [Ca2+]i in perfused inner medullary collecting duct (IMCD) (Star et al. 1988). However, 0.01–0.1 nm AVP is sufficient to increase cyclic AMP production and to induce a submaximal increase of Pf in IMCD (Star et al. 1988). Detection of Ca2+ mobilization from a perfused IMCD based on conventional fluorescence imaging might be compromised by out-of-focus fluorescence and asynchronous [Ca2+]i oscillations in individual cells. Conventional methods for measuring changes in Pf of a perfused IMCD also do not provide sufficient spatial and temporal resolution to capture the dynamics of AVP-induced exocytosis in individual IMCD cells. In this study the confocal imaging technique developed in rabbit cortical collecting duct was adopted to rat IMCD to monitor the spatial and temporal variations of [Ca2+]i induced by AVP in individual cells (Yip & Kurtz, 1995). To investigate the Ca2+ dependence of the AVP signalling cascade and exocytosis in intact IMCD, a novel confocal imaging technique using a styryl dye (FM1-43) was implemented to detect the real-time apical exocytosis in IMCD cells. The results provide direct evidence that 0.1 nm AVP triggers intracellular Ca2+ mobilization and apical exocytosis in intact IMCD, and that the manifestation of apical exocytosis in IMCD is Ca2+ dependent.