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 >> ATP > ADP >> UTP, and removal of external Ca(2+) dramatically suppressed the initial ATP-induced fluid secretion ( approximately 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 P2X(7) gene were used to evaluate the role of the P2X(7) receptor in nucleotide-evoked fluid secretion. P2X(7) 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 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.

FIGURE 1.

Purinergic P2 receptor agonists evoked fluid secretion in the ex vivo SMG. Agonists (1 mm) were applied to the ex vivo, perfused SMG for 10 min as indicated by the bar. A, flow rate following stimulation with ATP (black squares), ADP (gray squares), or UTP (white squares). B, summary of the results shown in panel A as the total volume of fluid secreted over the 10-min stimulation period. Data were from an equal number of male and female animals for ATP, ADP, and UTP (n = 12, n = 10, and n = 10 glands, respectively; *, p < 0.001). Black Swiss/129 SvJ mice were used for these experiments.

FIGURE 2.

Extracellular Ca²⁺ removal abolished ATP-induced fluid secretion in the ex vivo SMG. Agonists were applied to the ex vivo, perfused SMG for 10 min as indicated by the gray bar. For Ca²⁺-free experiments (black squares), glands were perfused with extracellular Ca²⁺-free solution for 2 min prior to stimulation and extracellular Ca²⁺ was re-introduced to the perfusate following 5 min of stimulation as indicated by the black bar. A, 0.3 μm CCh-evoked fluid secretion in the presence (gray squares) or absence (black squares) of extracellular Ca²⁺. B, 1 mm ATP-evoked fluid secretion in the presence (gray squares) or absence (black squares) of extracellular Ca²⁺. CCh- and ATP-evoked fluid secretions were from an equal number of male and female animals (n = 8 glands for each condition). Black Swiss/129 SvJ mice were used for these experiments.

FIGURE 3.

Enhanced SMG fluid secretion evoked by the P2X₇-selective agonist BzATP. Agonists were applied to the ex vivo, perfused SMG for 10 min as indicated by the gray bar. A, flow rate from the ex vivo, perfused SMG following stimulation with 0.25 mm of either BzATP (black squares) or ATP (gray squares). B, summary of the total volume of fluid secreted over a 10-min stimulation period. Data were from an equal number of male and female animals (BzATP and ATP, n = 12 and n = 8 glands, respectively; *, p < 0.001). Black Swiss/129 SvJ mice were used for these experiments.

FIGURE 4.

SMG isolated from P2X₇^–/– mice lacked P2X₇ protein expression and BzATP-activated cation currents. A, immunoblot of biotinylated membrane protein samples probed with a P2X₇ receptor specific antibody. P2X₇ receptor protein expression was absent in all three salivary gland tissues from P2X₇^–/– animals. HEK-293 cells were transiently transfected as previously described (9), and cell lysates from mock-transfected and P2X₇ receptor-expressing cells were included as controls and to confirm P2X₇ protein size (see arrow). B, representative traces of BzATP (0.25 mm) induced inward cation currents from SMG acinar cells isolated from P2X₇^+/+ (upper panel) and P2X₇^–/– (lower panel) mice. C, data summary shows the average inward currents following BzATP stimulation (P2X₇^+/+ versus P2X₇^–/–, n = 9 and n = 8, respectively; *, p = 0.003). Data were from experiments from at least three animals. P2X₇^+/+ or P2X₇^–/– C57Bl/6J mice were used for these experiments.

FIGURE 5.

CCh evoked SMG fluid secretion and [Ca²⁺]_i signals in P2X₇^+/+ and P2X₇^–/– mice. A, ex vivo SMG flow rate (left panel), total saliva volume over a 10-min stimulation period (middle panel), and the osmolality (right panel) in response to CCh (0.3 μm). There was no significant difference between P2X₇^+/+ and P2X₇^–/– animals for the total volume secreted (P2X₇^+/+ versus P2X₇^–/–, n = 20 glands and n = 17 glands, respectively, p = 0.23) or osmolality (P2X₇^+/+ versus P2X₇^–/–, n = 12 and n = 8 respectively, p = 0.74). B, Fura-2 loaded SMG acinar cells were stimulated with CCh (0.3 μm) for 3 min. The representative [Ca²⁺]_i response is shown as a change in ratio units (left panel). The data summary (right panel) shows that there was no significant difference in either the average peak value over baseline [P2X₇^+/+; 0.30 ± 0.02 ratio units versus P2X₇^–/–; 0.33 ± 0.03 ratio units, n = 9 (33 cells) and n = 9 (34 cells), respectively, p = 0.41] or average plateau value over baseline (taken at 1.5 min into the 3-min stimulation) [P2X₇^+/+; 0.17 ± 0.01 ratio units versus P2X₇^–/–; 0.21 ± 0.02 ratio units, n = 9 (33 cells) and n = 9 (34 cells), respectively, p = 0.07]. P2X₇^+/+ and P2X₇^–/– C57Bl/6J mice were used for these experiments.

FIGURE 6.

Purinergic receptor agonist-evoked SMG fluid secretion and [Ca²⁺]_i signals were decreased in P2X₇^–/– animals. A, ex vivo SMG flow rate (left panel) in response to ATP (1 mm). There was a significant decrease in the total volume of SMG fluid secretion (right panel) following ATP stimulation in the P2X₇^–/– animal (P2X₇^+/+ versus P2X₇^–/–, n = 12 glands and n = 15 glands, respectively, *, p < 0.05). B, Fura-2 loaded isolated SMG acinar cells were stimulated with ATP (1 mm) for 3 min. The representative [Ca²⁺]_i response is shown as a change in ratio units (left panel). There was a significant decrease in both the ATP-evoked average Ca²⁺ peak [P2X₇^+/+; 0.50 ± 0.04 ratio units versus P2X₇^–/–; 0.21 ± 0.02 ratio units, n = 8 (25 cells) and n = 9 (29 cells), respectively, *, p < 0.001] and plateau [P2X₇^+/+; 0.38 ± 0.03 ratio units versus P2X₇^–/–; 0.04 ± 0.01 ratio units, n = 8 (25 cells) and n = 9 (29 cells), respectively, *, p < 0.001] in P2X₇^–/– SMG acinar cells. C, similar to panel B, only Fura-2 loaded SMG granular duct cells. There was a significant decrease in both the ATP-evoked average Ca²⁺ peak [P2X₇^+/+; 0.058 ± 0.008 ratio units versus P2X₇^–/–; 0.015 ± 0.004 ratio units, n = 13 (25 cells) and n = 14 (19 cells), respectively, *, p < 0.001] and plateau [P2X₇^+/+; 0.080 ± 0.008 ratio units versus P2X₇^–/–; 0.006 ± 0.002 ratio units, n = 13 (25 cells) and n = 14 (19 cells), respectively, *, p < 0.001] in P2X₇^–/– SMG granular duct cells. P2X₇^+/+ or P2X₇^–/– C57Bl/6J mice were used for these experiments.

FIGURE 7.

Purinergic receptor agonist induced changes in muscarinic receptor-activated SMG fluid secretion. A, flow rate from the ex vivo, perfused SMG from P2X₇^+/+ (black squares) or P2X₇^–/– (gray squares) mice during stimulation with 0.3 μm CCh + 1 mm ATP. For comparison the dotted line represents data from P2X₇^+/+ mice using 0.3 μm CCh alone from 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 P2X₇^+/+ mice stimulated with CCh only (dotted bar) and P2X₇^–/– animals stimulated with CCh + ATP (gray bar) [P2X₇^+/+ with CCh only; 135 ± 5 μl/10 min, versus P2X₇^–/– with CCh + ATP; 130 ± 6 μl/10 min, n = 20 and n = 10 glands, respectively], but both were significantly greater than the total volume of SMG fluid secretion in P2X₇^+/+ mice stimulated with CCh + ATP (black bar) [P2X₇^+/+ with CCh + ATP; 90 ± 7 μl/10 min, n = 21, *, p = 0.01]. P2X₇^+/+ and P2X₇^–/– C57Bl/6J mice were used for these experiments.