Cftr and ENaC ion channels mediate NaCl absorption in the mouse submandibular gland

Abstract

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR-mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma-like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl-depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in F508 mice (Cftr(F/F)) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole-cell recordings demonstrated that Cftr and the epithelial Na(+) (ENaC) channels are co-expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in Cftr(F/F) mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in Cftr(F/F) mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in Cftr(F/F) mice, saliva was collected from ex vivo perfused SMGs. Cftr(F/F) glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the Cftr(F/F) phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

Figure 1

Immunohistochemical localization of Cftr channel protein in mouse SMG A and B, immunoperoxidase labelling for Cftr in submandibular glands from male (A) and female (B) Cftr^+/+ mice. Sections show staining of striated (SD) and granular ducts (GD) with no apparent immunolabelling in acinar cells (AC). C, immunostaining is not present in SMG from ΔF508 (Cftr^ΔF/ΔF) mice. Asterisks show luminal spaces. Bars = 50 μm.

Figure 2

Cftr-mediated Cl⁻ currents in granular duct cells Whole-cell Cl⁻ currents were recorded as described in Methods. A, cAMP-activated Cl⁻ currents elicited in response to square voltage steps in Cftr^+/+ granular duct cells (from −100 to +100 mV, 20 mV steps). CFTR(INH)-172 at 10 μm fully blocked Cftr-mediated currents. Scale: current 200 pA and time 100 ms. B, current–voltage relations from data like those shown in panel A (n= 5). C, c-AMP-activated Cl⁻ currents were not present in granular duct cells isolated from ΔF508 (Cftr^ΔF/ΔF) mice (n= 4). D, submandibular acinar cells isolated from Cftr^+/+ mice also failed to display cAMP-activated Cl⁻ currents (n= 4).

Figure 3

In vivo effects of the CftrΔF508 mutation on fluid secretion and ion composition of SMG saliva Secretion was stimulated by intraperitoneal injection of pilocarpine HCl (10 mg (kg body weight)⁻¹). In vivo fluid secretion data were normalized by body weight (μl (kg body weight)⁻¹). A, saliva flow in response to stimulation was slightly affected in ΔF508 (Cftr^ΔF/ΔF) mice. B, total submandibular saliva secreted. C, Na⁺ and Cl⁻ saliva concentrations were significantly increased in the ΔF508 CF mouse model. Data are given as the mean ±s.e.m.**P < 0.05, t test; n= 16 for each genotype.

Figure 4

Ex vivo effects of the CftrΔF508 mutation on fluid secretion and ion composition of SMG saliva Ex vivo submandibular glands were used to determine the flow rate and ion composition of saliva in Cftr^+/+ and ΔF508 (Cftr^ΔF/ΔF) mice. Carbachol (CCh, 0.3 μm) and isoproterenol (IPR, 5 μm) were used to stimulate secretion. Flow rate is expressed as μl min⁻¹. A, saliva flow in response to stimulation (Cftr^+/+, open squares, n= 13; Cftr^ΔF/ΔF, grey squares, n= 8). B, ion composition of saliva collected in panel A. Data are given as the mean ± the s.e.m.**P < 0.05, t test.

Figure 5

Pharmacological blockade of Cftr and ENaC ion channels mimics the Cftr^ΔF/ΔF phenotype Wild-type ex vivo submandibular glands (Control, Cftr^+/+) were perfused with 3 μm CFTR(INH)-172 or 10 μm amiloride for 30 min prior to and during stimulation. Carbachol (CCh, 0.3 μm) and isoproterenol (IPR, 5 μm) were used to stimulate secretion. A, saliva flow in response to stimulation (Control, open squares, n= 8; CFTR(INH)-172 perfused glands, grey squares, n= 8). B, ion composition of saliva collected in panel A. C, saliva flow in response to stimulation (Control, open squares, n= 15; amiloride-perfused glands, grey squares, n= 12). D, ion composition of saliva collected in panel C. Data are given as the mean ± the s.e.m.**P < 0.05, t test.

Figure 6

Immunohistochemical and functional evidence for α-ENaC expression in mouse SMG Immunoperoxidase labelling of α-ENaC in wild-type submandibular glands (Control, Cftr^+/+). Transverse (A) and longitudinal (B) sections showing the staining of striated (SD) and granular ducts (GD) with no apparent immunolabelling in acinar cells (AC). Bars = 50 μm. C, whole-cell Na⁺ currents were recorded as described in Methods. Na⁺ currents elicited in response square voltage steps in wild-type (Control, Cftr^+/+) granular duct cells (from −120 to +100 mV, 20 mV steps). Amiloride at 10 μm blocked ENaC-mediated currents. Scale: current 250 pA and time 100 ms, respectively. Dashed line indicates zero current level. D, ENaC-mediated current–voltage relation was obtained by subtracting whole-cell currents after amiloride treatment from those recorded before amiloride addition (n= 8). Data are given as the mean ±s.e.m.

Figure 7

α-ENaC currents and protein level are reduced in Cftr^ΔF/ΔF SMG A, whole-cell Na⁺ currents were recorded as described in Methods. ENaC-mediated current densities versus voltage plots in Cftr^+/+ (open squares) and ΔF508 (Cftr^ΔF/ΔF; grey squares) SMG duct cells. Current densities were obtained by dividing the current magnitude by the cell capacitance and expressing as pA pF⁻¹ (n= 8 for Cftr^+/+ and n= 9 for Cftr^ΔF/ΔF). Data are given as the mean ±s.e.m.**P < 0.05, t test. Data for Cftr^+/+ mice are the same as those shown in Fig. 6C. B, α-ENaC immunoreactivity in submandibular plasma membrane-enriched proteins from Cftr^+/+ and Cftr^ΔF/ΔF mice. C, α-, β- and γ-ENaC immunoreactivities in submandibular whole-cell lysates from Cftr^+/+ and Cftr^ΔF/ΔF mice. α-, β- and γ-ENaC immunoreactive bands ran at molecular weights of approximately 90, 73 and 80 kDa, respectively, similar to the expected sizes of these proteins in mice.