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.

Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors in Parotid Acinar Cells

“…8B), a result consistent with the lack of effect of forskolin in intact cells and in agreement with similar studies using imaging of calcium green C18-labeled permeabilized parotid acinar cells (20). In contrast, cAMP has been demonstrated to evoke Ca 2ϩ release from static suspensions of permeabilized cells and microsomal vesicles of rat parotid acinar cells (18,(21)(22)(23), a result that was attributed to activation of RyRs. Of interest in these studies is the fact that cAMP elevation evoked a relatively small Ca 2ϩ release (ϳ35 nM) compared with InsP 3 (ϳ150 nM) (18,21) and that this release occurred under conditions where basal [Ca 2ϩ ] was estimated to be as high as 431 nM (22).…”

“…As stated previously, mouse parotid acinar cells express predominantly RyR 3 (62), whereas rat parotid acinar cells (revealed by reverse transcription-PCR) express predominantly RyR 1 (21). Because RyR 1 exhibit a lower threshold for activation by Ca 2ϩ (63), any potentiation of Ca 2ϩ release by cAMP would be amplified under conditions of elevated resting [Ca 2ϩ ] i (18,21,22). Such species differences could also explain the lack of effect of caffeine in mouse acinar cells, as RyR 3 are thought to be caffeineinsensitive (64,65).…”

“…Conceptually, such a region could be established by the relative abundance of Ca 2ϩ release channels and/or expression of the most sensitive channels. Both of these criteria are satisfied in rat parotid acinar cells; immunocytochemical studies have reported that the extreme apical region is highly enriched in all InsP 3 R types (18,19). Furthermore, of the three InsP 3 Rs expressed, the type II InsP 3 R, which has been reported to be the most sensitive to InsP 3 (52,53), is the most abundant InsP 3 R type in parotid acinar cells (18).…”

“…Both of these criteria are satisfied in rat parotid acinar cells; immunocytochemical studies have reported that the extreme apical region is highly enriched in all InsP 3 R types (18,19). Furthermore, of the three InsP 3 Rs expressed, the type II InsP 3 R, which has been reported to be the most sensitive to InsP 3 (52,53), is the most abundant InsP 3 R type in parotid acinar cells (18). Collectively, this supports the idea that cAMP potentiates Ca 2ϩ release from the most sensitive InsP 3 Rs located in the apical region of the cell.…”

“…In contrast, cAMP has been demonstrated to evoke Ca 2ϩ release from static suspensions of permeabilized cells and microsomal vesicles of rat parotid acinar cells (18,(21)(22)(23), a result that was attributed to activation of RyRs. Of interest in these studies is the fact that cAMP elevation evoked a relatively small Ca 2ϩ release (ϳ35 nM) compared with InsP 3 (ϳ150 nM) (18,21) and that this release occurred under conditions where basal [Ca 2ϩ ] was estimated to be as high as 431 nM (22). At these [Ca 2ϩ ], any RyRs would be expected to be in a sensitized state.…”