Characterization and Localization of P2 Receptors in Rat Submandibular Gland Acinar and Duct Cells

Abstract

[Ca 2؉] i and the Cl ؊ current were measured in isolated submandibular gland acinar and duct cells to characterize and localize the purinergic receptors expressed in these cells. In both cell types 2-3-benzoylbenzoyl (Bz)-ATP and ATP increased [Ca 2؉ ] i mainly by activation of Ca 2؉ influx. UTP had only minimal effect on [Ca 2؉ ] i at concentrations between 0.1 and 1 mM. However, a whole cell current recording showed that all nucleotides effectively activated Cl ؊ currents. Inhibition of signal transduction through G proteins by guanyl-5-␤-thiophosphate revealed that the effect of ATP on Cl ؊ current was mediated in part by activation of a G protein-coupled and in part by a G protein-independent receptor. BzATP activated exclusively the G protein-independent portion, whereas UTP activated only the G protein- Ϫ . Acinar cells secrete the primary, NaCl-rich fluid into the duct, which reabsorbs most of the NaCl in exchange for K ϩ -HCO 3 Ϫ (1). As in any other CFTR-expressing tissues (2-4) and Na ϩ -transporting epithelia (5), Cl Ϫ channels play a central role in transcellular ion transport in the two cell types of SMG (1). However, unlike that of other epithelia, little is known of the regulation of Cl Ϫ channels in salivary gland cells, in particular regulation by purinergic (P 2 ) receptors.In the intact salivary gland secretion is regulated by several agonists, which include cholinergic, ␣-adrenergic, and ␤-adrenergic agonists (6 -8). The agonists act on both acinar and duct cells but modulate different activities in the two cell types. In acinar cells the agonists stimulate secretion primarily by stimulation of the basolateral NaKCl 2 cotransporter (9). In the duct the same agonists reduce the transepithelial potential and Na ϩ reabsorption (6 -8). Work on the cellular level showed that cholinergic and ␣-adrenergic agonists act on salivary acinar and duct cells to increase [Ca 2ϩ ] i (10 -15), and ␤-adrenergic agonists increase cAMP (11,16).Salivary acinar and duct cells also respond to purinergic stimulation by a change in [Ca 2ϩ ] i (15,(17)(18)(19)(20)(21)(22). However, the identity of the receptors and their membrane localization is not clear. Response of duct cells to BzATP and several other nucleotides suggested that duct cells express P 2 z and P 2 Y 1 receptors (37). Response of acinar cells to BzATP was interpreted to suggest that acinar cells express P 2 z receptors (20, 22). More recently it was shown that acinar and duct cells in long term culture up-regulate the P 2 Y 2 receptors (23). However, whether native and freshly isolated cells express P 2 Y 2 receptors and how these and other purinergic receptors regulate cellular activity is not known. P 2 receptors are believed to play a central role in regulating Cl Ϫ transport in CFTR-expressing tissues (3, 4, 24 -27). Because of our recent findings of CFTR expression in SMG duct and acinar cells (28) and the expression of P 2 receptors in SMG cells (15,(17)(18)(19)(20)(21)(22), in the present work we studied the regulation of Cl Ϫ channel...

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

“…Ablation of the P2X 7 gene had no effect on the salivary flow rate evoked by muscarinic receptor activation, but markedly reduced ATP-mediated fluid secretion and rescued the inhibitory effects of ATP on muscarinic receptor activation. Submandibular gland acinar cells from P2X 7 animals had dramatically impaired ATP-activated Ca 2ϩ signaling, consistent with this being the mechanism responsible for the reduction in ATP-mediated fluid secretion in these mice. Together, these results demonstrated that ATP regulates salivation, acting mainly through the P2X 7 receptor.…”

“…An equal number of gender-and agematched (2-6-month-old) animals were utilized. Black Swiss/ 129 SvJ hybrid (Rochester colony) and C57BL/6 strain mice were obtained from Jackson Laboratories (Bar Harbor, ME), while P2X 7 Ϫ/Ϫ C57BL/6 mice were obtained from Pfizer (Benton, CT) and used as indicated. All experimental protocols were approved by the University of Rochester Animal Resources Committee.…”

“…Secretion from the ex Vivo SMG-Salivary glands express several types of purinergic receptors which belong to both the P2X (P2X 4 and P2X 7 ) and P2Y (P2Y 1 and P2Y 2 ) receptor subfamilies (10,11). Given that activation of P2X and P2Y receptors evokes an increase in [Ca 2ϩ ] i in salivary gland cells, purinergic stimulation of either subfamily might be expected to result in fluid secretion (2).…”

“…P2Y receptors increase [Ca 2ϩ ] i via InsP 3 -induced Ca 2ϩ mobilization from intracellular stores (similar to ␣-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca 2ϩ influx (6). Salivary gland tissues express at least four isoforms of P2X (P2X 4 and P2X 7 ) and P2Y (P2Y 1 and P2Y 2 ) subtypes; however, their in vivo physiological significance has yet to be characterized (7)(8)(9)(10)(11).…”

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

“…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].…”