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

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 ≫ ATP > ADP ≫ UTP, and removal of external Ca2+ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). 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 P2X7 gene were used to evaluate the role of the P2X7 receptor in nucleotide-evoked fluid secretion. P2X7 receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X7-null mice. Consistent with these observations, the ATP-induced increases in [Ca2+]i were nearly abolished in P2X7–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X7 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 activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca 2ϩ ] i (5) that is unlikely to appreciably contribute to fluid secretion. The purinergic P2 receptor family is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated by extracellular di-and triphosphate nucleotides. Activation of both subfamilies of P2 receptors causes an increase in [Ca 2ϩ ] i . 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).
Our results revealed that ATP acts in isolation to stimulate fluid secretion from the mouse submandibular gland, but secretion is inhibited when ATP is simultaneously presented with a muscarinic receptor agonist. 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 Ϫ/Ϫ 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. Activation of the P2X 7 receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion.

EXPERIMENTAL PROCEDURES
General Methods-Mice were housed in microisolator cages with ad libitum access to laboratory chow and water during 12-hour light/dark cycles. 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. Reagents were obtained from Sigma unless otherwise specified.
Once the gland began to secrete fluid (defined as time 0), stimulation was continued for an additional 10 min. Saliva was collected in pre-calibrated capillary tubes (Sigma-Aldrich), and volumes were recorded every 0.5 or 1 min to calculate the flow rate (l/min). Following saliva collection, the gland was blot dried and weighed. Saliva samples were stored at Ϫ86°C until further analysis. Na ϩ and K ϩ concentrations were analyzed by atomic absorption using a Perkin-Elmer 3030 spectrophotometer. The Cl Ϫ concentration was analyzed with an Expandable Ion Analyzer EA 940 (Orion Research), and the pH and the osmolality were measured with a pH-sensitive electrode (Thermo Scientific, Beverly, MA) and a Wescor 5500 Vapor Pressure Osmometer (Logan, Utah), respectively.
In Vitro [Ca 2ϩ ] i Measurement-SMG ductal and acinar cells were prepared by enzyme digestion as previously reported (14). In brief, mice were euthanized by 100% CO 2 exposure followed by cardiac puncture. Acinar cells were dispersed in Minimum Essential Medium (SMEM, Invitrogen) supplemented with 1% bovine serum albumin (BSA), 0.17 mg/ml Liberase-RI (Roche Applied Science), and 2 mM L-glutamine, whereas duct cells were dispersed in Minimum Essential Medium (SMEM, Invitrogen) supplemented with 1% BSA, 0.012% trypsin, 0.05 mM EDTA, and 2 mM L-glutamine. Trypsin digestion was stopped with 2 mg/ml of soybean trypsin inhibitor. Duct cells were further dispersed by additional digestion in Minimum Essential Medium (SMEM) supplemented with 1% BSA, 0.15 units/ml Liberase-RI, and 2 mM L-glutamine. Following digestion, acinar and duct cells were rinsed in Basal Medium Eagle (BME, Invitrogen) supplemented with 1% BSA.
The fluorescent dye Fura-2 was used for [Ca 2ϩ ] i measurement. Cells were loaded by incubation with 2 M Fura-2 AM (Invitrogen) for 20 min at room temperature. Imaging was performed using an inverted microscope (Nikon Diaphot 200) equipped with an imaging system (Till Photonics, Pleasanton, CA). Images from Fura-2-loaded cells were acquired at a rate of 1 Hz by alternate excitation of light at 340 nm and 380 nm, and emission was captured at 510 nm using a high speed digital camera (Till Photonics). Chamber volume was maintained at ϳ400 l. Cells were superfused at a rate of 4 ml/min with the ex vivo perfusion solution (37°C). The fluorescence ratio of 340 nm over 380 nm was calculated, and all data are presented as the change in ratio units.
Enrichment of Biotinylated Plasma Membrane Proteins-Isolated SMG acinar cells (14) were biotinylated according to the manufacturer's instructions (Pierce), and the plasma membrane proteins enriched as previously described (15). In brief, biotinylated cells were collected by centrifugation at 1,000 ϫ g for 45 s, and the pellet homogenized twice in ice-cold homogenizing buffer containing: 250 mM sucrose, 10 mM triethanolamine, 1 g/ml leupeptin, and 0.1 mg/ml phenylmethanesulfonyl fluoride. Unbroken cells and nuclei were pelleted at 4,000 ϫ g for 10 min at 4°C and discarded. The supernatants were centrifuged at 22,000 ϫ g for 20 min at 4°C. The resulting pellet was resuspended in homogenization buffer, centrifuged at 46,000 ϫ g (Beckman SW28 rotor) for 30 min at 4°C, and the crude pellet was resuspended in 1 ml of hypotonic buffer (100 mM NH 4 HCO 3 , pH 7.5, 5 mM MgCl 2 ) followed by incubation overnight with 200 l of Dynabeads M-280 streptavidin (Invitrogen Dynal AS; Carlsbad, CA) at 4°C. Beads were collected with a magnetic plate and washed with hypotonic buffer. Streptavidin beads carrying the enriched plasma membrane fractions were suspended in 100 mM dithiothreitol for 2 h, centrifuged at 10,600 ϫ g for 3 min, and the supernatants collected and used for immunoblotting.
Electrophoresis and Immunoblot Analysis-Protein samples (30 g) were boiled for 5 min prior to separation in a 10% SDS-PAGE Tris-glycine mini-gel (Bio-Rad). Protein was transferred overnight at 4°C onto polyvinylidene difluoride membranes (Invitrogen) as described previously (15). Membranes were blocked overnight at 4°C with 5% nonfat dry milk in 25 mM Tris pH 7.5, 150 mM NaCl (TBS) and then incubated at 4°C overnight with primary antibody raised against the 576 -595 C terminus of the rat P2X 7 receptor (Millipore-Chemicon International Inc., Temecula, CA) at a dilution of 1:300 in a 2.5% nonfat dry milk solution. After washing with TBS containing 0.05% Tween-20 (TBS-T), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Pierce) at a dilution of 1:2,500 in TBS-T/2.5% nonfat dry milk for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (GE-Amersham Biosciences, Piscataway, NJ).
Electrophysiological Recordings-Single SMG acinar cells were prepared by enzymatic digestion (13). In brief, SMG acinar cells were digested for 15 min in SMEM containing 0.02% trypsin (Invitrogen), then centrifuged and resuspended in medium containing soybean trypsin inhibitor (Type 1-S, Sigma), followed by 2 sequential digestions for 25 min each in 0.17 mg/ml Liberase RI Enzyme (Roche Applied Science, Indianapolis, IN). The cell suspension was gently centrifuged and the supernatant filtrated through a 53-m nylon mesh. Finally, the suspension was centrifuged, and the cell pellet was resuspended in BME supplemented with 2 mM L-glutamine (Invitrogen). Cells were maintained at 37°C in a 5% CO 2 humidified incubator until use.
Electrophysiological data were acquired at room temperature using a PC-501A amplifier (Warner Instrument, Hamden, CT) or an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Voltage pulses were generated with Clampex 9 software through a Digidata 1320A interface (Molecular Devices), which also served to acquire the currents. Voltage clamp experiments were performed using the standard whole-cell configuration of the patch clamp technique. Glass pipettes (Warner Instrument) were pulled to give a resistance of 2-3 M⍀ in the solutions described below. The external solution contained (in mM): 150 NaCl, 1 CaCl 2 , 1 MgCl 2 , 10 HEPES, 20 sucrose, pH 7.4. The internal pipette solution contained (in mM): 130 Cs glutamate (130 CsOH ϩ 130 glutamic acid), 10 NaCl, 1 MgCl 2 , 1.5 EGTA, 10 HEPES, pH 7.3. Single mouse SMG acinar cells were voltage clamped at E Cl (Ϫ63.8 mV). Liquid junction potential was calculated to be 17.3 mV, and the correction was applied to the voltage.
Statistical Analysis-Results are presented as the mean Ϯ S.E. Statistical significance was determined using Student's t test or ANOVA analysis, followed by a Bonferroni's test for multiple comparisons with Origin 7.0 Software (OriginLab, Northampton, MA). p values of less than 0.05 were considered statistically significant. All experiments were performed using three or more separate preparations.

RESULTS
Purinergic Receptor Agonists Evoke Fluid 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). However, purinergic receptor activation has not been previously performed in the intact gland. To test this hypothesis we employed an ex vivo, perfused mouse SMG organ system. This ex vivo technique allows for precise control of the content of the vascular perfusate and ameliorates the rapid degradation of purinergic receptor agonists observed in vivo.
Most G protein-coupled P2Y receptors are sensitive to UTP whereas the ionotropic P2X receptors are not, while both classes of P2 receptors are generally activated by ATP and ADP (6). To determine if purinergic receptor activation produced saliva secretion in the ex vivo SMG preparation we initially utilized the ubiquitous P2 receptor activator ATP. Fig. 1A shows that the time course of the secretion generated by stimulation of the ex vivo SMG by ATP (1 mM) was best described as having an initial peak during the first 2 min followed over the next 8 min by a gradual decline to a relatively sustained fluid secretion rate (Fig. 1A, black squares). A relatively rapid loss of secretion was observed after the removal of ATP from the perfusate. To further characterize which P2 receptor was likely involved we also tested the ability of ADP and UTP to stimulate secretion. ADP (1 mM) produced a similar pattern as observed during ATP stimulation (Fig. 1A, gray squares), but the total amount of saliva produced during a 10-min stimulation was significantly less ( Extracellular Ca 2ϩ Depletion Suppresses ATP-induced Salivation by the ex Vivo SMG-UTP does not activate P2X receptors (6). Accordingly, because UTP caused minimal fluid secretion ( Fig. 1), it appeared that P2Y receptors do not play a major role in the purinergic-induced production of saliva. Therefore, we next evaluated the role of P2X receptors in ATP-mediated, Ca 2ϩ -dependent fluid secretion. Although both couple to an increase in [Ca 2ϩ ] i , one fundamental difference between P2Y and P2X receptors is that P2X receptors mediate extracellular Ca 2ϩ entry, whereas P2Y receptors initially trigger intracellular Ca 2ϩ release. Consequently, acute removal of extracellular Ca 2ϩ effectively eliminates Ca 2ϩ influx via P2X channels, but the intracellular Ca 2ϩ release mediated by P2Y receptors is resistant to this maneuver.
Using the muscarinic agonist carbachol (CCh), which activates an identical Ca 2ϩ release GPCR pathway as P2Y receptors, we show that initial secretion by the ex vivo SMG was not significantly affected by acute removal of extracellular Ca 2ϩ . CCh-evoked (0.3 M) SMG fluid secretion in the absence of extracellular Ca 2ϩ (black squares) was similar to secretion in Ca 2ϩ -containing experiments (gray squares) during the first minute of stimulation ( Fig. 2A). However, without extracellular Ca 2ϩ influx to replenish the rapidly depleted intracellular Ca 2ϩ stores, the flow rate was eventually reduced to near zero ( Fig.  2A, black trace at 4-min stimulation). Replacing extracellular Ca 2ϩ after 5 min of CCh stimulation restored the flow rate to control values ( Fig. 2A, black trace at 6 -10 min stimulation). Secretion rapidly returned to basal levels upon removal of CCh from the perfusate. Using the same paradigm, the ATP-evoked (1 mM) fluid secretion was nearly abolished except for a very small volume secreted during the first minute of stimulation (Fig. 2B, black trace; ϳ85% decrease). Re-addition of extracellular Ca 2ϩ resulted in a modest recovery of the fluid secretion flow rate (Fig. 2B, black versus gray trace at 6 -10-min stimulation). Because UTP stimulated little secretion and because the initial ATP-mediated saliva secretion was dependent upon extracellular Ca 2ϩ , our results suggested that ATP activated a member of the P2X family of channels. To determine which P2X isoform was involved, both pharmacological and genetic approaches were utilized in the following experiments.
The P2X 7 Receptor-selective Activator BzATP Stimulates Fluid Secretion-Salivary glands have been shown to express both P2X 4 and P2X 7 receptors (10). To differentiate between potential activation of P2X 4 or P2X 7 receptors in our ex vivo SMG fluid secretion experiments we utilized the ATP-derivative BzATP. P2X 7 receptors are activated preferentially by BzATP over ATP (16); thus, BzATP has served as a classical tool to evaluate P2X 7 receptor function. Concentrations of ATP and BzATP were selected to avoid maximum flow rates. Fluid secretion was often detectable at agonist concentrations as low as 0.1 mM for ATP (data not shown); however, fluid secretion was always observed at agonist concentrations of 0.25 mM and higher. Fig. 3 shows that either BzATP (black squares) or ATP (gray squares) can induce SMG fluid secretion; however at the submaximal concentration tested, BzATP was much more effective (Ͼ6-fold at 0.25 mM). In addition, the ATP response was not typically sustained at this concentration of agonist; i.e. the majority of the glands quit secreting after 5 min of stimulation, whereas secretion was sustained with BzATP (Fig. 3A). Fig. 3B shows that the volume of BzATP-evoked SMG fluid secretion was significantly greater than the volume secreted by an equal concentration of ATP (0.25 mM BzATP; 54.2 Ϯ 6.6 l/10 min versus 0.25 mM ATP; 8.4 Ϯ 1.0 l/10 min, p Ͻ 0.001). Higher concentrations of ATP (1 mM) resembled the results using a lower BzATP concentration (compare Figs. 1A and 3A), suggesting that ATP-stimulated fluid secretion is mediated by the BzATP-sensitive, P2X 7 purinergic receptor.
Loss of the BzATP-induced Cation Current in P2X 7 Receptornull Mice-Collectively, the observations that extracellular Ca 2ϩ was required for fluid secretion (Fig. 2) and BzATP was the most potent agonist (Fig. 3) implied that P2X 7 receptor activation was primarily responsible for the sustained ATP-mediated fluid secretion. One caveat of pharmacological inhibitors of purinergic P2 receptors is that they are not selective and may affect other channels and transporters required for fluid secretion (16). Thus, to confirm the critical role of P2X 7 receptor activation in stimulating fluid secretion we utilized P2X genedisrupted mice.
Western blot analysis using a P2X 7 receptor-specific antibody demonstrated that plasma membrane proteins isolated from P2X 7 Ϫ/Ϫ mice lacked P2X 7 receptor protein expression in SMG, parotid (PG), and sublingual (SLG) salivary glands (Fig.  4A, see arrow). As an additional positive control and to confirm protein size, the cell lysate from HEK-293 cells transiently overexpressing the P2X 7 receptor was included in the analysis (Fig.  4A, lane 7). Fig. 4 also shows that the large BzATP-induced inward cation current present in SMG acinar cells from wildtype mice was absent in P2X 7 Ϫ/Ϫ SMG acinar cells (Fig. 4   the P2X 7 gene results in loss of both P2X 7 receptor protein expression and nucleotide-gated channel activity.

TABLE 1 Carbachol and ATP stimulated fluid secretion and ion composition in the ex vivo SMG of wild type and P2X 7
؊/؊ mice ANOVA analyses, followed by a Bonferroni test, were performed for multiple comparisons (see "Experimental Procedures"). The numbers in parentheses equal the number of glands tested for each condition.  FEBRUARY 20, 2009 • VOLUME 284 • NUMBER 8
As previously described (7,17), we also found that ATP stimulated an increase in [Ca 2ϩ ] i in SMG granular duct cells (Fig.  6C). This [Ca 2ϩ ] i increase peaked in less than 30 s in duct cells and remained elevated (Fig. 6C, black trace). The kinetics of the ATP-induced [Ca 2ϩ ] i increase in granular duct cells were comparable to that observed in acinar cells (i.e. there was a rapid [Ca 2ϩ ] i increase after exposure to ATP), although the magnitude of the [Ca 2ϩ ] i peak and plateau values were about an order of magnitude less in duct cells. ATP stimulation in P2X 7 Ϫ/Ϫ SMG duct cells resulted in a very small, transient elevation of [Ca 2ϩ ] i , (Fig. 6C, gray trace). This suggested that P2X 7 receptor activation plays a role in ATP regulation of duct cell [Ca 2ϩ ] i signaling and possibly ion transport function.
ATP Modulates the Flow Rate of Muscarinic Receptor-stimulated Saliva-It is unlikely that ATP acts in vivo independently of other neurotransmitters like acetylcholine. Therefore, we tested the effects of co-stimulation of the ex vivo gland with ATP and CCh on saliva production and ion composition. We found that ATP inhibited CCh-induced secretion by Ͼ30% in P2X 7 ϩ/ϩ glands (Fig. 7, A and B, compare black squares and bar to dotted trace and bar). In contrast, ATP failed to inhibit CChstimulated secretion in P2X 7 Ϫ/Ϫ glands (Fig. 7, A and B, gray squares and bar, respectively), suggesting that P2X 7 receptor activation likely mediates the ATP-induced inhibition of the muscarinic receptor response.
The primary, plasma-like saliva generated by acinar cells is modified as it passes through the ducts, which reabsorb NaCl Ϫ/Ϫ SMG granular duct cells. P2X 7 ϩ/ϩ or P2X 7 Ϫ/Ϫ C57Bl/6J mice were used for these experiments. and secrete KHCO 3 (1,2). ATP stimulated a very modest increase in [Ca 2ϩ ] i in SMG granular duct cells (Fig. 6C) suggesting that ATP has little influence on duct cell function. Indeed, in P2X 7 ϩ/ϩ mice, ATP did not significantly alter the Na ϩ or Cl Ϫ concentration of the saliva induced by CCh stimulation (Table  1); while the pH and the K ϩ concentration of saliva increased slightly. Consistent with these observations, ATP induced only modest changes in the ion composition of saliva from P2X 7 Ϫ/Ϫ submandibular glands. Taken together, these results suggested that the ATP-stimulated increase in [Ca 2ϩ ] i in SMG duct cells is unlikely to play a major role in the regulation of ductal function.

DISCUSSION
Salivary tissues express multiple types of purinergic P2 receptors. Activation of these receptors increases [Ca 2ϩ ] i suggesting a possible role for P2 receptors in fluid secretion (4,7,8,10,18,19), although this has never been directly tested. Here we show that purinergic stimulation results in a sustained, extracellular Ca 2ϩ -dependent secretion of saliva in an intact mouse submandibular salivary gland preparation. The sensitivity of this response to different purinergic agonists was consistent with P2X 7 receptor activation (BzATP Ͼ Ͼ ATP Ͼ ADP Ͼ Ͼ UTP). Indeed, a Ͼ70% decrease was observed in BzATP-and ATP-evoked fluid secretions from the SMG in P2X 7 -null animals. These results demonstrate that P2X 7 receptors are essential for ATP-mediated saliva production in the mouse submandibular gland.
Our results highlight the significant contribution purinergic receptors may have on the regulation of salivary gland fluid secretion. Earlier reports describe a non-adrenergic, non-cholinergic mediated fluid secretion (3, 20 -24). In sheep, electrical stimulation of the parotid parasympathetic nerve in the presence of atropine produced a significant increase in the flow rate (25). The authors concluded that the flow rate was produced by vasoactive intestinal peptide (VIP) release (25), however this seems unlikely since VIP receptor activation is linked to an increase in cAMP, not [Ca 2ϩ ] i . Indeed, Ekstrom et al. (26) reported that VIP treatment caused little to no fluid secretion in feline parotid glands. Interestingly, the electrically evoked "atropine-resistant" fluid secretion was ϳ30% of the response prior to muscarinic receptor inhibition in the ferret submandibular gland (3). This observation is essentially identical to our results; i.e. the maximal volume of ATP-induced fluid secretion was about 30% of that produced by muscarinic receptor stimulation ( Fig. 1 versus Fig. 5). Thus, our results are consistent with ATP-mediated fluid secretion playing a physiological role in non-adrenergic, non-cholinergic mediated salivation.
Although both P2X 4 and P2X 7 receptors are expressed in salivary glands, the greater secretion induced by BzATP (Fig. 3) and the severe impairment of secretion in P2X 7 -null mice (Fig.  6) implied that the P2X 7 receptor channel contributes most to salivation. Adding to P2X receptor signaling complexity, functional P2X receptors form heteromeric channels comprised of three P2X subunits (16,27,28). Until recently P2X 7 receptors were the only P2X isoform thought not to form a heteromeric channel. However, recent reports have identified P2X 4 /P2X 7 heteromeric channels (29,30). These latter observations suggest that salivary gland cells might contain both P2X 4 and P2X 7 homotrimers, as well as P2X 4 /P2X 7 heterotrimers. The functional significance of P2X 4 /P2X 7 heterotrimer formation is unclear, but genetic disruption of P2X 7 receptors might also interfere with P2X 4 targeting and/or overall function. Nevertheless, while a P2X 4 /P2X 7 heterotrimer is possible, there is no evidence of P2X 4 /P2X 7 heterotrimer formation in native tissue. Future studies should be directed to determine the relationship, if any, between P2X 4 and P2X 7 homo-and heterotrimers in salivary gland function. P2X 7 receptors have been previously localized to the apical membrane of mouse parotid acinar and duct cells (8). P2X 7 receptor immunostaining was also noted in mouse SMG duct cells, which suggests that this ligand-gated channel may participate in modification of the electrolyte content (31). Li et al. (8) proposed that preassembled P2X 7 receptors in duct cells sense upstream secretion by acinar cells (via ATP release) and respond by augmenting HCO 3 Ϫ secretion. In agreement with this model, we found that ATP produced a modest increase in the pH of CCh-induced secretions, suggesting an increase in the [HCO 3 Ϫ ], and this effect disappeared in P2X 7 -null mice ( Table 1). Given that ATP is not likely to be released independent of other agonists in vivo, ATP might be expected to modulate the response of salivary glands to muscarinic receptor stimulation. Indeed, we also found that the volume of fluid secreted during muscarinic receptor activation was decreased by co-stimulation with ATP and CCh in the ex vivo SMG. These results are consistent with the previous observation that ATP inhibits musca-  Fig. 5A. B, summary of the total volume of fluid secreted over a 10-min stimulation period. There was no difference in the total volume of SMG fluid secretion between P2X 7 ϩ/ϩ mice stimulated with CCh only (dotted bar) and P2X 7 Ϫ/Ϫ animals stimulated with CCh ϩ ATP (gray bar) [P2X 7 ϩ/ϩ with CCh only; 135 Ϯ 5 l/10 min, versus P2X 7 Ϫ/Ϫ with CCh ϩ ATP; 130 Ϯ 6 l/10 min, n ϭ 20 and n ϭ 10 glands, respectively], but both were significantly greater than the total volume of SMG fluid secretion in P2X 7 ϩ/ϩ mice stimulated with CCh ϩ ATP (black bar) [P2X 7 ϩ/ϩ with CCh ϩ ATP; 90 Ϯ 7 l/10 min, n ϭ 21, *, p ϭ 0.01]. P2X 7 ϩ/ϩ and P2X 7 Ϫ/Ϫ C57Bl/6J mice were used for these experiments.
rinic receptor induced Ca 2ϩ mobilization in rat parotid and submandibular gland acinar cells (32)(33)(34). The inhibition of the CCh-induced response by ATP was absent in P2X 7 -null mice (Fig. 7). Additional studies will be required to confirm the mechanism(s) responsible for these observations. Regardless of the mechanism, our results suggest that ATP can stimulate secretion on its own, and when co-released with a muscarinic agonist, ATP likely modulates the muscarinic receptor-stimulated response of salivary glands. Moreover, the in vivo physiologically relevant source of ATP release is unknown. It is well known that ATP is co-released from nerve terminals with acetylcholine and other neurotransmitters (35). Alternatively, ATP is also released from the secretory granules of exocrine acinar cells in response to agonists that stimulate granule fusion (36). The release of ATP from these two distinct sites is likely to have very different functional consequences. In summary, our results demonstrate that ATP regulates fluid secretion in the mouse submandibular gland. This mechanism is extracellular Ca 2ϩ -dependent, sensitive to BzATP, and insensitive to UTP, suggesting activation of a P2X receptor family member. Indeed, ablation of the P2X 7 gene resulted in a dramatic reduction in the amount of fluid secreted during ATP exposure. These new findings contribute to our understanding of the role of purinergic P2 receptors in salivary glands and provide insight into future studies. ATP-mediated fluid secretion is likely an important mechanism for "tuning" the resultant fluid secretion under different physiological conditions.