Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl- channel gene

Abstract

Salivary gland acinar cells shrink when Cl(-) currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl(-) channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl(-) channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl(-) currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca(2+)-calmodulin-dependent protein kinase II; however, the magnitude of the Ca(2+)-dependent Cl(-) current was unchanged in the Clcn3(-/-) animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3(-/-) mice. Our results demonstrate that the ClC-3 Cl(-) channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva.

Figure 1. ClC-3 protein and mRNA expression in mouse parotid acinar cells

Protein was isolated from the parotid glands of ClC-3 wild-type and null mutant mice. Upper panel, an antibody to ClC-3 recognized several bands of the appropriate molecular mass for ClC-3 in protein samples from a wild-type mouse parotid gland (lane 1). In a protein sample from parotid glands of Clcn3^−/-mice, a band of the correct molecular mass for ClC-3 protein was not observed (lane 2), although several other bands remained immunoreactive. These most likely represent cross-reactivity of the antibody with non-specific proteins. When probed with an anti-ClC-3 antibody that had been pre-absorbed with a threefold excess of the peptide antigen to which the antibody had been raised (lane 3) or with secondary antibody alone (lane 4), none of the immunoreactive bands in the wild-type protein sample were recognized. Molecular weight markers are shown to the right of the blots. Lower panel, four overlapping RT-PCR primers were designed to amplify the ClC-3 message from isolated parotid acinar cells including two sense primers (U1 and U2) complimentary to codons 34–40 and 384-391, respectively, and two nonsense primers corresponding to codons 209–214 and 590–597 (L1 and L2, respectively). The PCR product sizes using primer pairs U1/L1 and U2/L2 are 542 base pairs (bp) and 638 bp long, respectively. DNA sequencing confirmed 100 % identity of the PCR products as ClC-3. Lane M is a 100 bp DNA ladder size marker.

Figure 2. Immunohistochemical labelling using a ClC-3 antibody in the parotid glands of Clcn3^+/+and Clcn3^−/-mice

The parotid glands were removed from Clcn3^+/+ and Clcn3^−/- mice and fixed in paraformaldehyde overnight prior to imbedding in paraffin and sectioning at 5 μm. The glands were exposed to anti-ClC-3 antibody (1:250) overnight. Reactivity was then detected by the use of an Alexa-594 fluorescent secondary antibody. Images were taken at several magnifications from × 200 to × 600, as indicated below. Upper left panel, section from wild-type parotid gland. Arrows indicate intense staining of blood vessels (magnification, ×200). Upper right panel, section of parotid gland from null mutant mouse. Intense staining of blood vessels is absent, but non-specific staining of duct and acinar cells remains (magnification, ×200). Lower left panel, section from wild-type parotid gland. Arrows indicate intense staining of blood vessels (magnification, ×600). Lower right panel, Nomarski image of the section in the lower left panel (magnification, ×600).

Figure 3. Targeted disruption of the Clcn3 gene fails to inhibit the RVD response in parotid acinar cells

The role of ClC-3 in the regulatory volume decrease (RVD) response was examined in parotid acinar cells loaded with the fluoroprobe calcein, as described in Methods. Upper panel, parotid acini isolated from Clcn3^+/+ (▪) and Clcn3^−/- (▵) mice were superfused in an isosmotic solution and then hyposomotic cell swelling was induced by switching the perfusate to a hypotonic medium (30 % dilution with water). The cell volumes shown are normalized to the maximum volume achieved following exposure to a hypotonic solution. Changes in cell volume are represented as 1/normalized calcein fluorescence (1/calcein Fn). Lower panel, the initial rate of change expressed as 1/calcein Fn × min⁻¹ × 10⁻² (Clcn3^+/+, n = 8; Clcn3^−/-, n = 11; no statistical differences between groups).

Figure 4. Swell-activated Cl⁻ currents in parotid acinar cells from Clcn3^+/+ and Clcn3^−/- mice

Volume-sensitive Cl⁻ currents recorded from acinar cells isolated from Clcn3^+/+ (upper row) and Clcn3^−/- (middle row) mice. Control traces (left column) were recorded after 5 min of dialysis under isotonic conditions; hypotonic-activated currents (centre column) were recorded after 2–3 min in the hypotonic solution; inhibition of Cl⁻ currents (right column) by cell shrinkage was assessed after 5 min exposure to a hypertonic solution. The lower row shows the current-voltage relationship for the data depicted in the upper and middle rows under isotonic (▪), hypotonic (•) and hypertonic (▴) conditions obtained from acinar cells isolated from Clcn3^+/+ and Clcn3^−/- mice. There was no statistical difference between the swell-activated current (current present in the hypotonic solution minus the current after switching to the hypertonic solution) from wild-type and null mutant mice (Clcn3^+/+ = 29.2 ± 8.2 pA pF⁻¹, n = 6; Clcn3^−/- = 32.2 ± 3.9 pA pF⁻¹, n = 7).

Figure 5. Ca²⁺-dependent Cl⁻ currents in parotid acinar cells from Clcn3^+/+and Clcn3^−/-mice

Upper panel, currents from a parotid acinar cell from a Clcn3^+/+ mouse in response to 3 s voltages pulses from −30 to +90 mV in 20 mV increments from a holding potential of −50 mV. Lower panel, currents at the same potentials from a parotid acinar cell from a Clcn3^−/- mouse. There was no statistical difference between the Ca²⁺-activated current from wild-type and null mutant mice (Clcn3^+/+, 140 ± 20 pA pF⁻¹; n = 10; Clcn3^−/-, 135 ± 13 pA pF⁻¹; n = 5).

Figure 6. Targeted disruption of the Clcn3 gene does not inhibit pilocarpine-induced in vivo salivation

The role of ClC-3 in the production of saliva was examined in vivo by stimulating with 10 mg pilocarpine-HCl (kg body weight (BW))⁻¹. Whole saliva was collected from the lower cheek pouch by suctioning at intervals of 5, 10 and 15 min and amounts are expressed as μl 5 min g BW⁻¹. Time zero was designated as the point when saliva was first noted following pilocarpine injection. Clcn3^+/+ (▪), n = 17; Clcn3^−/- (□), n = 12.