Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland

The present study examines the cellular distribution of the ryanodine receptor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP₃R) subtypes in parotid acini. Using fluorescently labelled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPY®-ryanodine) and confocal microscopy, RyRs were localized primarily to the perinuclear region (basal pole) of the acinar cell. Ryanodine, Ruthenium Red, cAMP and cADP ribose (cADPR) competed with BODIPY-ryanodine, resulting in a reduction in the fluorescence signal. However, inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] did not alter the binding of BODIPY-ryanodine. Using receptor-subtype-specific antisera, InsP₃Rs (types I, II and III) were located predominantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific oligonucleotide probes. Binding studies using a parotid cell-membrane fraction identified and characterized RyRs and InsP₃Rs in terms of binding affinity (K(d)) and maximum binding capacity (B(max)) and confirmed that cADPR displaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribose blocked Ca²⁺ release in permeabilized acinar cells in response to cADPR and cAMP or forskolin, whereas Ins(1,4,5)P₃-induced Ca²⁺ release was unaffected. The localization of the RyRs and InsP₃Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca²⁺ channels. The consequences of the dual activation of the RyRs and InsP₃Rs by physiologically relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca²⁺ signalling in the parotid gland.