Distinct contributions by ionotropic purinoceptor subtypes to ATP‐evoked calcium signals in mouse parotid acinar cells

Abstract

Key points• There are two major ionotropic purinoceptor subtypes (ATP-gated, non-selective, Ca 2+ -conducting ion channels) in the salivary gland.• Relatively little is known about the physiological roles of these purinoceptors regarding compartmentalization and selective activation, their contributions to the spatiotemporal properties of intracellular Ca 2+ signals and their roles in regulating protein exocytosis and ion channel activity.• In this study, we investigated the subtype-specific sub-cellular distribution and functional characterization of purinoceptors in mouse parotid acinar cells.• Selective activation of ionotropic purinergic receptor subtypes was shown to evoke spatially distinct cytosolic Ca 2+ signals as well as protein exocytosis.• This study identifies a subtype of ionotropic purinergic receptors as a potential therapeutic target for treatment of salivary gland hypofunction.Abstract There is emerging consensus that P2X 4 and P2X 7 ionotropic purinoceptors (P2X 4 R and P2X 7 R) are critical players in regulating [Ca 2+ ] i dynamics and fluid secretion in the salivary gland. In contrast, details regarding their compartmentalization and selective activation, contributions to the spatiotemporal properties of intracellular signals and roles in regulating protein exocytosis and ion channel activity have remained largely undefined. To address these concerns, we profiled mouse parotid acinar cells using live-cell imaging to follow the spatial and temporal features of ATP-evoked Ca 2+ dynamics and exocytotic activity. Selective activation of P2X 7 Rs revealed an apical-to-basal [Ca 2+ ] i signal that initiated at the sub-luminal border and propagated with a wave speed estimated at 17.3 ± 4.3 μm s −1 (n = 6). The evoked Ca 2+ spike consisted of Ca 2+ influx and Ca 2+ -induced Ca 2+ release from intracellular Ca 2+ channels. In contrast, selective activation of P2X 4 Rs induced a Ca 2+ signal that initiated basally and propagated toward the lumen with a wave speed of 4.3 ± 0.2 μm s −1 (n = 8) that was largely independent of intracellular Ca 2+ channel blockade. Consistent with these observations, P2X 7 R expression was enriched in the sub-luminal regions of acinar cells while P2X 4 R appeared localized to basal areas. In addition, we showed that P2X 4 R and P2X 7 R activation evokes exocytosis in parotid acinar cells. Our studies also demonstrate that the P2X 4 R-mediated [Ca 2+ ] i rise and subsequent protein exocytosis was enhanced by ivermectin (IVR). Thus, in addition to furthering our understanding of salivary gland physiology, this study identifies P2X 4 R as a potential target for treatment of salivary hypofunction diseases.

TRPV4 activation in mouse submandibular gland modulates Ca2+influx and salivation

“…A similar pattern has been shown for the P2X 4 purinoceptor, a nonselective Ca 2ϩ -permeable cation channel, the expression of which is restricted to the basal region in mouse parotid acinar cells. Selective activation of P2X 4 induces a Ca 2ϩ signal that initiates basally and propagates toward the lumen, independent of the intracellular IP 3 -sensitive Ca 2ϩ store (7). In contrast, if the receptor channel is restricted to the apical region, the Ca 2ϩ wave direction is expected to be from the apical to the basal, like that seen for the apical P2X 7 channel of parotid acinar cells (7).…”

“…Selective activation of P2X 4 induces a Ca 2ϩ signal that initiates basally and propagates toward the lumen, independent of the intracellular IP 3 -sensitive Ca 2ϩ store (7). In contrast, if the receptor channel is restricted to the apical region, the Ca 2ϩ wave direction is expected to be from the apical to the basal, like that seen for the apical P2X 7 channel of parotid acinar cells (7). Thus the plasma membrane location of Ca 2ϩ -conducting cation receptor channels like P2X 4 ] i induced by muscarinic receptor activation would be expected to occur at the apical pole (site of the major IP 3 -sensitive Ca 2ϩ stores) and to propagate toward the basolateral region, as seen in rat parotid acinar cells (35) and mouse submandibular gland acinar cells (Fig.…”

P2X4ou P2X7

“…T Nakamoto et al [20] ont mesuré la sécrétion des glandes sous-maxillaires pendant une stimulation de 10 min avec du carbachol. En accord avec les résultats antérieurs obtenus sur la salive totale [15], ils n'ont pas observé de différence dans le volume de salive sécrétée par les glandes sous-maxillaires chez les deux types de souris en réponse à un agent muscarinique. Il semble donc que ce soit au cours de la phase de sécrétion soutenue (au-delà de 30 min après la stimulation par un agent muscarinique) que l'absence de récepteurs P2X 7 joue un rôle important sur le volume salivaire sécrété [19].…”

“…Les récepteurs P2X 7 sont localisés sur la membrane basale et la membrane apicale. Selon S. Bhattacharya et al [15], dans les glandes parotides de souris, seules les cellules acineuses expriment les récep-P2X 7 -KO. D'autre part, la sécrétion salivaire en réponse au carbachol est inhibée par une stimulation directe des récepteurs purinergiques [20].…”

“…Brown et D. I. Yule, la protéine kinase A (PKA) potentialise les courants électriques et l'augmentation de la [Ca 2+ ] i en réponse à des agonistes de P2X 4 [21], suggérant que la PKA pourrait réguler l'endocytose et le nombre de récepteurs au niveau de la membrane plasmique [21]. Récemment, S. Bhattacharya et al [15] ont confirmé, dans les cellules acineuses, l'augmentation de la [Ca 2+ ] i et la stimulation de l'exocytose en réponse à la stimulation des récepteurs P2X 4 .…”