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

Abstract

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 (BODIPYtrade mark-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 (Kd) and maximum binding capacity (Bmax) 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.