Mechanisms of Vasopressin-Induced Intracellular CA2+ Oscillations in Rat Inner Medullary Collecting Duct

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caged cyclic ADP-ribose, caged inositol 1, 4, 5-trisphosphate, store-operated calcium entry, laser scanning confocal microscopy

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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.

oscillation of intracellular Ca2+ concentration ([Ca2+]i) serves as the signal transduction mechanism for many physiological stimuli in both excitable and nonexcitable cells, with information being encoded in both the frequency and the amplitude of the Ca2+ signal (2). There is emerging evidence that Ca2+ oscillation in renal epithelium is an integral part of the signaling transduction process for regulation of water reabsorption (8, 45). A physiological dose of AVP has been shown to trigger intracellular Ca2+ mobilization and Ca2+ oscillations in inner medullary collecting duct (IMCD) (32, 45), and the Ca2+ mobilization is necessary for AVP-stimulated apical exocytosis and osmotic water permeability (8, 45). AVP also induced Ca2+ oscillations in mouse thick ascending limb, which was associated with AVP-stimulated secretion of nucleotides (30). Ca2+ oscillations were also reported recently in tubular epithelial cells near the macula densa (23). They are modulated by luminal NaCl concentration and luminal flow, and are possibly related to juxtaglomerular signaling. Furthermore, angiotensin II-triggered Ca2+ oscillations have been recorded in descending vasa recta pericytes (11, 51). It has been suggested that Ca2+ oscillations in pericytes are due to repetitive cycles of ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release and SKF-96365-sensitive refilling of SR Ca2+ stores.

Our previous studies (8, 45) have provided the initial characterization of AVP-induced Ca2+ oscillation in IMCD. With the use of confocal fluorescence microscopy to monitor [Ca2+]i and apical exocytosis in individual cells, AVP was found to trigger a rapid increase in [Ca2+]i followed by sustained repetitive Ca2+ oscillations. These Ca2+ responses were mediated through a cAMP-dependent mechanism and mimicked by an agonist of exchange protein directly activated by cAMP (Epac) (46). Removal of extracellular Ca2+ did not prevent the initial rise of [Ca2+]i induced by AVP but abolished the sustained Ca2+ oscillation (45). In the absence of extracellular Ca2+, ryanodine completely obliterated AVP-induced Ca2+ mobilization and AVP-stimulated increase of osmotic water permeability (8). These results suggested that both extracellular Ca2+ influx and intracellular Ca2+ release contribute to the AVP-induced Ca2+ response.

Ca2+ oscillation can be driven by influx of extracellular Ca2+, Ca2+-induced Ca2+ release (CICR) from intracellular Ca2+ stores, or both (33). Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in endoplasmic reticulum (ER) of IMCD (8, 19, 44). Both receptors are capable of supporting CICR and Ca2+ oscillations (13, 14). Ca2+ release through RyRs or IP3Rs may deplete intracellular Ca2+ stores and trigger store-operated Ca2+ entry (SOCE), which can serve as a mechanism to replenish the depleted Ca2+ stores and sustain Ca2+ oscillations (33, 34). To capture the dynamics of Ca2+ release from RyRs and IP3Rs from the ER of IMCD, flash photolysis of caged cyclic ADP-ribose (cADPR) and caged IP3 was used in the present study to directly activate RyRs and IP3Rs, respectively. The specific contributions of RyRs, IP3Rs, and SOCE in the AVP-induced Ca2+ oscillations were evaluated. Our results indicate that AVP-induced Ca2+ oscillation in IMCD is mediated in part by the endogenous production of cADPR and is maintained through the interplay of Ca2+ release from RyRs and Ca2+ influx through SOCE.

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American Journal of Physiology-Renal Physiology, v. 300, issue 2, p. F540-F548