Ontogeny of Purinergic Receptor-Regulated Ca2+ Signaling in Mouse Cortical Collecting Duct Epithelium

Abstract

Changes in ATP-induced increase in [Ca²⁺]_i during collecting duct ontogeny were studied in primary monolayer cultures of mouse ureteric bud (UB) and cortical collecting duct (CCD) cells by Fura-PE3 fluorescence ratio imaging. In UB (embryonic day E14 and postnatal day P1) the ATP-stimulated increase (EC₅₀ ≈ 1 µM) in fluorescence ratio (ΔR_ATP) was independent of extracellular Ca²⁺ and insensitive to the P2 purinoceptor-antagonist suramin (1 mM). From day P7 onward when CCD morphogenesis had been completed ΔR_ATP increased and became dependent on extracellular Ca²⁺. This ATP-stimulated Ca²⁺ entry into CCD cells was non-capacitative and suramin (1 mM)-insensitive, but sensitive to nifedipine (30 µM) and enhanced by Bay K8644 (15 µM), a blocker and an agonist of L-type Ca²⁺ channels, respectively. Quantitative RT-PCR demonstrated similar mRNA expression of L-type Ca²⁺ channel α1-subunit, P2Y₁, P2Y₂, and P2X_4b purinoceptors in UB and CCD monolayers while the abundance of P2X₄ mRNA increased with CCD morphogenesis. In conclusion, both embryonic and postnatal cells express probably P2Y₂-stimulated Ca²⁺ release from intracellular stores. With development, the CCD epithelium acquires ATP-stimulated Ca²⁺ entry via L-type Ca²⁺ channels. This pathway might by mediated by the increasing expression of P2X₄-receptors resulting in an increasing ATP-dependent membrane depolarization and activation of L-type Ca²⁺ channels.