Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m808597200
ISSN1083-351X
AutoresTetsuji Nakamoto, David A. Brown, Marcelo A. Catalán, Mireya González–Begne, Victor G. Romanenko, James E. Melvin,
Tópico(s)Restless Legs Syndrome Research
ResumoSalivary 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. 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 Ca2+-dependent process (1Cook D.I. Van Lennep E.W. Roberts M.L. Young J.A. Secretion by the Major Salivary Glands. 3rd Ed. Raven Press, New York1994Google Scholar, 2Melvin J.E. Yule D. Shuttleworth T. Begenisich T. Annu. Rev. Physiol. 2005; 67: 445-469Crossref PubMed Scopus (330) Google Scholar) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates α-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase Cβ (PLCβ) during salivary gland stimulation. PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP3) production. Activation of Ca2+-selective InsP3 receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca2+]i). 4The abbreviations used are: [Ca2+]i, intracellular free calcium concentration; BzATP, 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; SMG, submandibular gland(s); CCh, carbachol; GPCR, G protein-coupled receptors; SLG, sublingual; PG, parotid; ANOVA, analysis of variance; BSA, bovine serum albumin.4The abbreviations used are: [Ca2+]i, intracellular free calcium concentration; BzATP, 2′,3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate; SMG, submandibular gland(s); CCh, carbachol; GPCR, G protein-coupled receptors; SLG, sublingual; PG, parotid; ANOVA, analysis of variance; BSA, bovine serum albumin. Depletion of the endoplasmic reticulum Ca2+ pool triggers extracellular Ca2+ influx and a sustained elevation in [Ca2+]i. This increase in [Ca2+]i activates Ca2+-dependent K+ and Cl– channels promoting Cl– secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na+ 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 KHCO3 producing a final saliva that is hypotonic (1Cook D.I. Van Lennep E.W. Roberts M.L. Young J.A. Secretion by the Major Salivary Glands. 3rd Ed. Raven Press, New York1994Google Scholar, 2Melvin J.E. Yule D. Shuttleworth T. Begenisich T. Annu. Rev. Physiol. 2005; 67: 445-469Crossref PubMed Scopus (330) Google Scholar). Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3Ekstrom J. Mansson B. Olgart L. Tobin G. Quart. J. Exp. Physiol. 1988; 73: 163-173Crossref PubMed Scopus (44) Google Scholar). In addition to muscarinic and α-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca2+]i such as purinergic P2 and substance P receptors. Like muscarinic and α-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca2+]i in salivary acinar cells (4Soltoff S.P. McMillian M.K. Cragoe Jr., E.J. Cantley L.C. Talamo B.R. J. Gen. Physiol. 1990; 95: 319-346Crossref PubMed Scopus (61) Google Scholar). In contrast, substance P receptor activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca2+]i (5Merritt J.E. Rink T.J. J. Biol. Chem. 1987; 262: 14912-14916Abstract Full Text PDF PubMed Google Scholar) 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 [Ca2+]i. P2Y receptors increase [Ca2+]i via InsP3-induced Ca2+ mobilization from intracellular stores (similar to α-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca2+ influx (6Burnstock G. Physiol. Rev. 2007; 87: 659-797Crossref PubMed Scopus (1273) Google Scholar). Salivary gland tissues express at least four isoforms of P2X (P2X4 and P2X7) and P2Y (P2Y1 and P2Y2) subtypes; however, their in vivo physiological significance has yet to be characterized (7Lee M.G. Zeng W. Muallem S. J. Biol. Chem. 1997; 272: 32951-32955Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Li Q. Luo X. Zeng W. Muallem S. J. Biol. Chem. 2003; 278: 47554-47561Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Brown D.A. Bruce J.I. Straub S.V. Yule D.I. J. Biol. Chem. 2004; 279: 39485-39494Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 10Turner J.T. Landon L.A. Gibbons S.J. Talamo B.R. Crit. Rev. Oral. Biol. Med. 1999; 10: 210-224Crossref PubMed Scopus (61) Google Scholar, 11Novak I. News Physiol. Sci. 2003; 18: 12-17Crossref PubMed Scopus (155) Google Scholar). 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 P2X7 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 P2X7–/– animals had dramatically impaired ATP-activated Ca2+ 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 P2X7 receptor. Activation of the P2X7 receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion. 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 age-matched (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 P2X7–/– 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. Ex Vivo Submandibular Gland (SMG) Perfusion—Ex vivo SMG perfusion was performed as previously reported (12Nakamoto T. Romanenko V.G. Takahashi A. Begenisich T. Melvin J.E. Am. J. Physiol. Cell Physiol. 2008; 294: C810-C819Crossref PubMed Scopus (41) Google Scholar, 13Romanenko V.G. Nakamoto T. Srivastava A. Begenisich T. Melvin J.E. J. Physiol. 2007; 581: 801-817Crossref PubMed Scopus (57) Google Scholar). In brief, mice were anesthetized with an intraperitoneal injection of chloral hydrate (400 mg/kg body weight). Following ligation of all branches of the common carotid artery except the SMG artery, the SMG was removed, cannulated, and perfused. The ex vivo perfusion solution contained (in mm): 4.3 KCl, 120 NaCl, 25 NaHCO3, 5 glucose, 10 HEPES, 1 CaCl2, 1 MgCl2, pH 7.4. Extracellular Ca2+-free solutions were made by removing CaCl2. Solutions maintained at 37 °C were gassed with 95% O2, 5% CO2, and perfused at 0.8 ml/min using a peristaltic pump. When stimulating with only purinergic receptor agonists, muscarinic and β-adrenergic receptor antagonists were included (0.5 μm atropine and 20 μm propranolol, respectively). 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 (μl/min). Following saliva collection, the gland was blot dried and weighed. Saliva samples were stored at –86 °C until further analysis. Na+ and K+ concentrations were analyzed by atomic absorption using a Perkin-Elmer 3030 spectrophotometer. The Cl– 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 [Ca2+]i Measurement—SMG ductal and acinar cells were prepared by enzyme digestion as previously reported (14Romanenko V. Nakamoto T. Srivastava A. Melvin J.E. Begenisich T. J. Biol. Chem. 2006; 281: 27964-27972Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In brief, mice were euthanized by 100% CO2 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 [Ca2+]i measurement. Cells were loaded by incubation with 2 μm 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 ∼400 μl. 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 (14Romanenko V. Nakamoto T. Srivastava A. Melvin J.E. Begenisich T. J. Biol. Chem. 2006; 281: 27964-27972Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) were biotinylated according to the manufacturer's instructions (Pierce), and the plasma membrane proteins enriched as previously described (15Gonzalez-Begne M. Nakamoto T. Nguyen H.V. Stewart A.K. Alper S.L. Melvin J.E. J. Biol. Chem. 2007; 282: 35125-35132Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In brief, biotinylated cells were collected by centrifugation at 1,000 × g for 45 s, and the pellet homogenized twice in ice-cold homogenizing buffer containing: 250 mm sucrose, 10 mm triethanolamine, 1 μg/ml leupeptin, and 0.1 mg/ml phenylmethanesulfonyl fluoride. Unbroken cells and nuclei were pelleted at 4,000 × g for 10 min at 4 °C and discarded. The supernatants were centrifuged at 22,000 × g for 20 min at 4 °C. The resulting pellet was resuspended in homogenization buffer, centrifuged at 46,000 × g (Beckman SW28 rotor) for 30 min at 4 °C, and the crude pellet was resuspended in 1 ml of hypotonic buffer (100 mm NH4HCO3, pH 7.5, 5 mm MgCl2) followed by incubation overnight with 200 μl 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 × g for 3 min, and the supernatants collected and used for immunoblotting. Electrophoresis and Immunoblot Analysis—Protein samples (30 μg) 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 (15Gonzalez-Begne M. Nakamoto T. Nguyen H.V. Stewart A.K. Alper S.L. Melvin J.E. J. Biol. Chem. 2007; 282: 35125-35132Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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 P2X7 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 (13Romanenko V.G. Nakamoto T. Srivastava A. Begenisich T. Melvin J.E. J. Physiol. 2007; 581: 801-817Crossref PubMed Scopus (57) Google Scholar). 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-μm 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% CO2 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 MΩ in the solutions described below. The external solution contained (in mm): 150 NaCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 20 sucrose, pH 7.4. The internal pipette solution contained (in mm): 130 Cs glutamate (130 CsOH + 130 glutamic acid), 10 NaCl, 1 MgCl2, 1.5 EGTA, 10 HEPES, pH 7.3. Single mouse SMG acinar cells were voltage clamped at ECl (–63.8 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 ± 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. 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 (P2X4 and P2X7) and P2Y (P2Y1 and P2Y2) receptor subfamilies (10Turner J.T. Landon L.A. Gibbons S.J. Talamo B.R. Crit. Rev. Oral. Biol. Med. 1999; 10: 210-224Crossref PubMed Scopus (61) Google Scholar, 11Novak I. News Physiol. Sci. 2003; 18: 12-17Crossref PubMed Scopus (155) Google Scholar). Given that activation of P2X and P2Y receptors evokes an increase in [Ca2+]i in salivary gland cells, purinergic stimulation of either subfamily might be expected to result in fluid secretion (2Melvin J.E. Yule D. Shuttleworth T. Begenisich T. Annu. Rev. Physiol. 2005; 67: 445-469Crossref PubMed Scopus (330) Google Scholar). 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 (6Burnstock G. Physiol. Rev. 2007; 87: 659-797Crossref PubMed Scopus (1273) Google Scholar). 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 (Fig. 1B, ADP; 15.2 ± 2.8 μl/10 min versus ATP; 43.7 ± 3.5 μl/10 min, p < 0.001). Stimulation with UTP (1 mm) produced a transient flow (Fig. 1A, white squares) which generated markedly less fluid, ∼6% of the saliva secreted with an identical concentration of ATP (Fig. 1B, UTP; 2.7 ± 0.7 μl/10 min versus ATP; 43.7 ± 3.5 μl/10 min, p < 0.001). Extracellular Ca2+ Depletion Suppresses ATP-induced Salivation by the ex Vivo SMG—UTP does not activate P2X receptors (6Burnstock G. Physiol. Rev. 2007; 87: 659-797Crossref PubMed Scopus (1273) Google Scholar). 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, Ca2+-dependent fluid secretion. Although both couple to an increase in [Ca2+]i, one fundamental difference between P2Y and P2X receptors is that P2X receptors mediate extracellular Ca2+ entry, whereas P2Y receptors initially trigger intracellular Ca2+ release. Consequently, acute removal of extracellular Ca2+ effectively eliminates Ca2+ influx via P2X channels, but the intracellular Ca2+ release mediated by P2Y receptors is resistant to this maneuver. Using the muscarinic agonist carbachol (CCh), which activates an identical Ca2+ 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 Ca2+. CCh-evoked (0.3 μm) SMG fluid secretion in the absence of extracellular Ca2+ (black squares) was similar to secretion in Ca2+-containing experiments (gray squares) during the first minute of stimulation (Fig. 2A). However, without extracellular Ca2+ influx to replenish the rapidly depleted intracellular Ca2+ stores, the flow rate was eventually reduced to near zero (Fig. 2A, black trace at 4-min stimulation). Replacing extracellular Ca2+ 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; ∼85% decrease). Re-addition of extracellular Ca2+ 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 Ca2+, 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 P2X7 Receptor-selective Activator BzATP Stimulates Fluid Secretion—Salivary glands have been shown to express both P2X4 and P2X7 receptors (10Turner J.T. Landon L.A. Gibbons S.J. Talamo B.R. Crit. Rev. Oral. Biol. Med. 1999; 10: 210-224Crossref PubMed Scopus (61) Google Scholar). To differentiate between potential activation of P2X4 or P2X7 receptors in our ex vivo SMG fluid secretion experiments we utilized the ATP-derivative BzATP. P2X7 receptors are activated preferentially by BzATP over ATP (16North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2468) Google Scholar); thus, BzATP has served as a classical tool to evaluate P2X7 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 (>6-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 ± 6.6 μl/10 min versus 0.25 mm ATP; 8.4 ± 1.0 μl/10 min, p < 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, P2X7 purinergic receptor. Loss of the BzATP-induced Cation Current in P2X7 Receptor-null Mice—Collectively, the observations that extracellular Ca2+ was required for fluid secretion (Fig. 2) and BzATP was the most potent agonist (Fig. 3) implied that P2X7 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 (16North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2468) Google Scholar). Thus, to confirm the critical role of P2X7 receptor activation in stimulating fluid secretion we utilized P2X gene-disrupted mice. Western blot analysis using a P2X7 receptor-specific antibody demonstrated that plasma membrane proteins isolated from P2X7–/– mice lacked P2X7 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 P2X7 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 wild-type mice was absent in P2X7–/– SMG acinar cells (Fig. 4, B and C; P2X7+/+; –349.8 ± 88.5 pA versus P2X7–/–; –2.2 ± 2.3 pA, p < 0.01). Together, these data demonstrated that disruption of the P2X7 gene results in loss of both P2X7 receptor protein expression and nucleotide-gated channel activity. P2X7 Receptor Disruption Has No Effect on CCh-evoked Fluid Secretion and [Ca2+]i Signals—We next determined if disruption of P2X7 receptors in the SMG had nonspecific effects on the fluid secretion machinery. To evaluate this we utilized the muscarinic receptor agonist CCh, the response to which should be unaffected in P2X7-null mice. Fig. 5A shows that the ex vivo salivary flow rates in P2X7+/+ (black squares) and P2X7–/– (gray squares) SMG were effectively identical in response to CCh stimulation. There were only subtle differences in the total volumes secreted in 10 min (P2X7+/+; 135 ± 5 μl/10 min versus P2X7–/–; 144 ± 6 μl/10 min, p = 0.25), saliva ion compositions (Table 1), or osmolalities (P2X7+/+; 183.9 ± 4.5 mOsm versus P2X7–/–; 186.0 ± 3.3 mOsm, p = 0.74). The lack of effect of P2X7 receptor ablation on the CCh-stimulated response demonstrated that ion channels and transporters responsible for fluid secretion in SMG are not affected when the P2X7 gene is disrupted.TABLE 1Carbachol and ATP stimulated fluid secretion and ion composition in the ex vivo SMG of wild type and P2X7-/- miceP2X7+/+P2X7-/-CChCCh + ATPCChCCh + ATPNa37.4 ± 2.6 (20)44.1 ± 3.3 (21)39.9 ± 4.6 (17)47.9 ± 5.8 (10)K50.4 ± 3.3 (20)59.6 ± 2.2aValues are different from those labeled b by at least p < 0.03. (21)47.7 ± 2.8b (17)53.8 ± 3.9 (10)Cl89.2 ± 4.6 (20)85.2 ± 1.7 (21)82.9 ± 4.3aValues are different from those labeled b by at least p < 0.03. (17)100.9 ± 3.3b (10)pH7.85 ± 0.07 (8)7.99 ± 0.09aValues are different from those labeled b by at least p < 0.03. (8)7.71 ± 0.03b (9)7.71 ± 0.06b (10)μl135 ± 5aValues are different from those labeled b by at least p < 0.03. (20)90 ± 7b (21)144 ± 6aValues are different from those labeled b by at least p < 0.03. (17)130 ± 6aValues are different from those labeled b by at least p < 0.03. (10)a Values are different from those labeled b by at least p < 0.03. Open table in a new tab Given that salivary gland fluid secretion is dependent on an elevation of [Ca2+]i, we expected that disruption of P2X7 receptors would also have no effect on CCh-evoked Ca2+ signals in SMG acinar cells. To confirm this, SMG acini were isolated and loaded with the Ca2+-sensitive dye Fura-2. In support of the fluid secretion data, Fig. 5B shows that the CCh-induced Ca2+ signals in P2X7+/+ (black trace) and P2X7–/– (gray trace) isolated SMG acinar cells were essentially identical. Analysis of the data showed that the average peak value over baseline of the CCh-induced Ca2+ signal for P2X7+/+ cells was not significantly different from that for P2X7–/– cells (Fig. 5B, P2X7+/+; 0.30 ± 0.02 ratio units versus P2X7–/–; 0.33 ± 0.03 ratio units, p = 0.41). In addition, the average plateau value over baseline (taken at 1.5 min into the 3-min stimulation) of the CCh-induced Ca2+ signal for P2X7+/+ cells was also similar to that for P2X7–/– cells (P2X7+/+; 0.17 ± 0.01 ratio units versus P2X7–/–; 0.21 ± 0.02 ratio units, p = 0.07). Thus, disruption of P2X7 receptors had no significant effect on either CCh-mediated fluid secretion or Ca2+ signaling. Disruption of P2X7 Receptors Decreases ATP-evoked Fluid Secretion and [Ca2+]i Signals—We next determined if loss of P2X7 receptor expression had an effect on purinergic receptor agonist-evoked fluid secretion. Fig. 6 shows that the secretion response to the purinergic receptor agonist ATP was markedly reduced in the P2X7–/– SMG (gray squares). The total volume of saliva collected over a 10-min period from the P2X7–/– SMG was reduced 71% when stimulated with 1 mm ATP (Fig. 6A, P2X7+/+; 28.9 ± 4.3 μl/10 min versus P2X7–/–; 8.4 ± 1.5 μl/10 min, p < 0.001). A similar result was obtained when
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