Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Abstract

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP Ͼ Ͼ ATP > ADP Ͼ Ͼ UTP, and removal of external Ca 2؉ dramatically suppressed the initial ATP-induced fluid secretion (ϳ85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X 7 gene were used to evaluate the role of the P2X 7 receptor in nucleotide-evoked fluid secretion. P2X 7 receptor protein and BzATPactivated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X 7 -null mice. Consistent with these observations, the ATP-induced increases in [Ca 2؉ ] i were nearly abolished in P2X 7 ؊/؊ submandibular acinar and duct cells. ATP appeared to also act through the P2X 7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X 7 receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca 2ϩ -dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates ␣-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase C␤ (PLC␤) during salivary gland stimulation. PLC␤ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP 3 ) production. Activation of Ca 2ϩ -selective InsP 3 receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca 2ϩ ] i ). 4 Depletion of the endoplasmic reticulum Ca 2ϩ pool triggers extracellular Ca 2ϩ influx and a sustained elevation in [Ca 2ϩ ] i . This increase in [Ca 2ϩ ] i activates Ca 2ϩ -dependent K ϩ and Cl Ϫ channels promoting Cl Ϫ secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na ϩ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO 3 producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and ␣-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca 2ϩ ] i such as purinergic P2 and substance P receptors. Like muscarinic and ␣-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca 2ϩ ] i in salivary acinar cells (4). In contrast, substance P receptor ac...

Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities

“…P2X7 receptors would be the main responsible for the ATP response in this tissue. Purinergic receptor agonist-stimulated salivation was suppressed by more than 70 % in submandibular glands from P2X7 null mice [10]. At least one EC 50 is >100 μM ATP for this receptor [8,41], which is consistent with the high ATP Km we measured for microsomal NTPDases.…”

“…In submandibular gland (SMG), metabotropic (P2Y 1 , P2Y 2 ) and ionotropic (P2X4, P2X7) receptor subtypes were found, both in basolateral and luminal membranes in acinar and ductal cells [6][7][8]. It was demonstrated that P2X7 activation regulates ionic currents and saliva volume and also induces reactive oxygen species production and inflammatory responses [9][10][11].…”

P2X4ou P2X7

“…On peut conclure de ces résultats que l'ATP libéré en réponse à une stimulation muscarinique régule les sécrétions exocrines via des récepteurs de type P2X 7 [19]. 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]. En revanche, T. Nakamoto et al [20] ont montré que l'activation directe des récepteurs P2X 7 stimule la sécrétion sous-maxillaire. Cette sécrétion, en réponse à l'ATP et au benzoyl-ATP (Bz-ATP, un agoniste puissant des récepteurs P2X 7 ), nécessite la présence de calcium dans le milieu de perfusion et est fortement réduite (70 %) chez les souris réunis par une grande boucle extra cellulaire contenant cinq ponts disulfures et des sites de N-glycosylation.…”

“…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]. Ce résultat est cohérent avec d'autres travaux qui montraient que, dans les glandes sous-maxillaires, l'ATP bloquait la mobilisation de réservoirs intracellulaires de calcium en réponse au carbachol [14].…”