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

The present study examines the cellular distribution of the ryanodine recep tor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R) subt ypes in parotid acini. Using fluorescently labelled 1,4-difluoro-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPY(TM)-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 reducti on in the fluorescence signal. However, inositol 1,4,5-trisphosphate [Ins(1 ,4,5)P-3] did not alter the binding of BODIPY-ryanodine. Using receptor-sub type-specific antisera, InsP(3)Rs (types I, II and III) were located predom inantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific o ligonucleotide probes. Binding studies using a parotid cell-membrane fracti on identified and characterized RyRs and InsP(3)Rs in terms of binding affi nity (K-d) and maximum binding capacity (B-max) and confirmed that cADPR di splaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribos e blocked Ca2+ release in permeabilized acinar cells in response to cADPR a nd cAMP or forskolin, whereas Ins(1,4,5)P-3-induced Ca2+ release was unaffe cted. The localization of the RyRs and InsP(3)Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca2+ channels. The co nsequences of the dual activation of the RyRs and InsP(3)Rs by physiologica lly relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca2+ signalling in the parotid gland.