A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland

Abstract

Activation of an apical Ca(2+)-activated Cl(-) channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl(-) current and Cl(-) efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl(-) channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A(-/-) mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr(∆F508/∆F508)) or ClC-2 (Clcn2(-/-)) Cl(-) channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl(-) channel. Indeed, Cl(-) channel blockers abolished fluid secretion, indicating that Cl(-) channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic-induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A(-/-) mice identify Tmem16A as the Cl(-) channel essential for muscarinic, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.

Keywords: Cftr channel; Cl− channel; Tmem16A/Ano1; acinar cell; secretion.

Fig. 1.

Generation of mice lacking Tmem16A Cl⁻ channels in salivary gland acinar cells. (A) SMG sections from mT/mG reporter mice show that mT is expressed in the absence of Cre-recombinase expression. (B) SMG sections from mice obtained by crossing mT/mG reporter mice with ACID mice display mG expression in acinar (white arrows) and intercalated cells (white arrowheads), whereas other duct cells remain red-stained. (C and D) Immunolocalization of Tmem16A on paraffin-embedded SMG sections from littermate controls shows apical brown staining of acinar cells (C), whereas Tmem16A-specific staining is absent in Tmem16A^−/− mice (D). (Scale bars: 10 µm.)

Fig. 2.

Ca²⁺-activated Cl⁻ currents are abolished in adult Tmem16A^−/− acinar cells. (A and B) Whole-cell recordings in isolated acinar cells generated as described in SI Materials and Methods from control (A) and Tmem16A^−/− (B) SMGs showing that the Ca²⁺-activated Cl⁻ conductance is essentially abolished in Tmem16A^−/− mice. (C) Current–voltage relations obtained from experiments like those shown in A and B (n = 7 control and n = 4 Tmem16A^−/− cells, respectively). Currents are normalized by cell capacitance (pA/pF). Data are presented as mean ± SEM.

Fig. 3.

In vivo and ex vivo salivary gland secretion in response to Ca²⁺-mobilizing agonists. (A and B) In vivo SMG (A) and parotid gland (B; Par) secretions from control (gray circles; n = 7 glands) and Tmem16A^−/− (black circles; n = 6 glands) mice in response to i.p. injection of pilocarpine (10 mg/kg body weight). (C and D) Ex vivo SMG secretions collected as described in SI Materials and Methods from control (gray circles) and Tmem16A^−/− (black circles) mice in response to 0.3 μM CCh (C; n = 12 control glands and n = 6 Tmem16A^−/− glands) and 0.25 mM BzATP (D; n = 4 glands from control and Tmem16A^−/− mice). Data are presented as mean ± SEM.

Fig. 4.

Ex vivo SMG secretions in response to β-adrenergic receptor stimulation. (A) Ex vivo SMG secretions collected as described in SI Materials and Methods from control (gray circles; n = 6 glands) and Tmem16A^−/− (black circles; n = 6 glands) mice in response to 5 μM IPR. (B) Total saliva secreted in 10 min from the data shown in A. (C) Ex vivo SMG secretions from WT (gray circles; n = 8 glands) and Cftr^{∆F508/∆F508} (black circles; n = 10 glands) mice in response to 5 μM IPR. (D) Total saliva secreted in 10 min from the data shown in C. Data are presented as mean ± SEM. The P value was obtained by the Student's t test.

Fig. 5.

Involvement of VRAC in β-adrenergic receptor-dependent saliva secretion. (A) Chloride and bicarbonate dependence of β-adrenergic–induced saliva secretion. Shown is the total ex vivo saliva secreted by SMGs in response to 5-μM IPR stimulation for 10 min using control, HCO₃⁻-free, and low-Cl⁻ perfusates (n = 8 glands per condition). Data are presented as mean ± SEM. **P < 0.0003, one-way ANOVA followed by Bonferroni’s post hoc test. (B) Ex vivo SMG secretions from control (black circles; n = 8 glands), Clcn2^−/− (dark-gray circles; n = 8 glands), and control glands treated with 50 µM DCPIB (gray circles; n = 8 glands) or 100 µM NPPB (white circles; n = 6 glands). (C) Acinar cell volume changes in response to 0.3 µM CCh or 5 µM IPR. (D) Nystatin-perforated whole-cell recording showing the increase in macroscopic currents at −60 mV in response to 5 µM IPR and blockade by 50 µM DCPIB. (E) Summary of current magnitude and blockade by DCPIB of the Cl⁻ conductance activated by IPR (n = 4) and hypotonic shock (n = 3) at −60 mV. The increase in current magnitude by IPR and hypotonic shock and the decrease in current magnitude by DCPIB-mediated blockade are shown as absolute values. Values are given as ± SEM. P values were obtained using the Student's t test.