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Roles of CLCA and CFTR in Electrolyte Re-absorption from Rat Saliva

¹ Departments of Functional Bioscience,
² Physiological Science & Molecular Biology, and
³ Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan

^* corresponding author, junyama{at}college.fdcnet.ac.jp

	ABSTRACT

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A molecular basis for Cl^– re-absorption has not been well-characterized in salivary ductal cells. Previously, we found strong expression of a rat homologue proposed to be Ca²⁺-dependent Cl^– channels (rCLCA) in the intralobular ducts of the rat submandibular gland. To address the question as to whether rCLCA and cystic fibrosis transmembrane conductance regulator (CFTR) are involved in Cl^– re-absorption, we evaluated the electrolyte content of saliva from glands pre-treated with a small interfering RNA (siRNA). Retrograde injection into a given submandibular duct of an siRNA designed to knock down either rCLCA or CFTR reduced the expression of each of the proteins. rCLCA and CFTR siRNAs significantly increased Cl^– concentration in the final saliva during pilocarpine stimulation. These results represent the first in vivo evidence for a physiological significance of rCLCA, along with CFTR, in transepithelial Cl^– transport in the ductal system of the rat submandibular gland.

KEY WORDS: salivary • duct • re-absorption • chloride channel • siRNA

	INTRODUCTION

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Electrolyte secretion from salivary glands is known to be a two-stage process. Apical channels and transporters are assumed to provide an effective mechanism for the secretion of NaCl-rich primary saliva from acinar cells, and this is followed by re-absorption of Na⁺ and Cl^– plus excretion of K⁺ and HCO₃^– while the primary saliva is flowing down through the ductal system. The hypotonicity of the final saliva indicates that ducts are relatively impermeable to water (Melvin et al., 2005). Multiple classes of Cl^– channels have been described in salivary acinar and ductal cells. These include cystic fibrosis transmembrane conductance regulator (CFTR), CLC, and Ca²⁺-activated Cl^– channels (Zeng et al., 1997a).

The physiological relevance of Ca²⁺-activated Cl^– channels in acinar cells has been described, although the channels and transporters present in ducts are less well-understood (Melvin, 1999). It has been reported that CFTRs exist in the apical membrane of the duct and are likely to participate in Cl^– re-absorption, and that a Ca²⁺-activated Cl^– channel is also present in rat salivary ductal cells (Zeng et al., 1997a). Nevertheless, no direct in vivo evidence has been presented to show that these channels play a role in Cl^– re-absorption during activation of muscarinic receptors. We recently cloned the full length of a rat homologue (rCLCA; Yamazaki et al., 2005) of the Ca²⁺-activated Cl^– channel (CLCA) family (Jentsch et al., 2002; Loewen and Forsyth, 2005). In human embryonic kidney 293 (HEK293) cells transfected with the cDNA of this clone, we found expression of Ca²⁺-activated Cl^– conductance, and we also found that the isoform is expressed in the luminal surface of the granular and striated ductal cells (but not acinar cells) of the rat submandibular gland. We therefore hypothesized that the protein is responsible for modulating Ca²⁺-dependent Cl^– transport in the ductal system.

To test this hypothesis, we measured ion concentrations and osmolarity in rat saliva collected during muscarinic receptor stimulation, after retrogradely injecting one salivary duct with a short double-stranded small interfering RNA (siRNA) designed to knock down rCLCA gene function. We also tested the effect of an siRNA made against the mRNA for CFTR. In these studies, we demonstrated efficient RNA interference in the rat submandibular gland epithelium by the use of a transfection tool that takes advantage of the cell-fusion ability of the envelope of the Sendai virus (Hemagglutinating Virus of Japan, HVJ). This methodology allowed us to examine the local function of rCLCA in the epithelium facing the luminal space, and was designed to minimize any influence of the treatment on the systemic functions of an animal given siRNA.

	MATERIALS & METHODS

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RNA Interference
Small interference RNAs (siRNAs) were synthesized by B-Bridge International Inc. (Sunnyvale, CA, USA) in a purified and annealed duplex form. Three siRNA sequences were designed to target rCLCA and rat CFTR genes, the accession numbers of the targeted mRNA sequences being AB119249 (NM_001013202) for rCLCA and XM_342645 for the rat isoform of CFTR. We performed pilot experiments to select the most effective rCLCA siRNA (Appendix Fig.). The sense sequence of the annealed siRNA duplex form was 5'-CAGCGAUGUGACAAGGUUAdTdT-3'. We chose one siRNA from the 3 for CFTR (with the sense sequence 5'-GCUUAAAGGAAGAGGAUAUdTdT-3'). For negative siRNA controls, we scrambled the sequence to design a corresponding negative control with the same GC content and nucleic acid composition. According to a BLAST search (Altschul et al., 1997), these siRNAs possess no significant homology to other mRNAs within existing databases for the rat (GenBank, EMBL, DDBJ, and PDB; mismatching nucleotides 4).

Retrograde Ductal Injection of siRNA
Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.; Nembutal, Abbot Laboratories, Abbot Park, IL, USA), permission for the procedures used having been granted by the Animal Research Committee of Fukuoka Dental College. Two separate experiments were designed to examine the roles of rCLCA and CFTR in saliva formation. For each experiment, the rats were divided into 2 groups: One was injected with siRNA (the siRNA group), and the other with its negative control (the control group). In each rat, 1 submandibular duct (referred to as the injection side) was cannulated intra-orally (via its orifice in the sublingual papilla), by means of a polyethylene tube. A total of 2 nmol of each siRNA was injected retrogradely through the cannula into the submandibular gland on that side. The efficiency of transfection into the intact epithelium was improved when the siRNA was suspended in TE buffer containing 2 arbitrary units (80 µL) of HVJ envelope vector (GenomONE-Neo; Ishihara Sangyo Kaisha Ltd., Osaka, Japan), according to the manufacturer’s directions.

Measurement of Ion Concentrations in Saliva Collected from Submandibular Glands
Forty-eight hrs after the above injection, rats were anesthetized again with sodium pentobarbital and tracheotomized. Both submandibular ducts (injection side and non-injection side) were cannulated intra-orally with 2 polyethylene tubes. After the first drop had been discarded, the saliva from each cannula was collected into separate test tubes for 15 min after the administration of pilocarpine (8 mg/kg, i.p.; Wako, Osaka, Japan), a muscarinic receptor agonist. The collected saliva was used for the measurement of ion concentrations (Radiometer ABL555; Radiometer A/S, Brønshøj, Denmark) and osmolarity (Osmometer Automatic DI-SMO; Knauer, Berlin, Germany).

At the end of the experiment, both submandibular glands were dissected out for immunoblotting and immunohistochemical studies. An anti-rCLCA antibody was generated against a synthetic peptide, based on the region of the N-terminal external domain (SKSEYLMPKRESYDKAD) (Yamazaki et al., 2005). An anti-CFTR antibody (ACL006) was purchased from Alomone Labs Ltd. (Israel). Precise procedures are described in the APPENDIX.

All values are presented as means ± SEM (N, number of observations). Statistical analysis was performed by a one-way ANOVA, followed by a post hoc Bonferroni’s t test (Figs. 2, 3). A paired or grouped t test was used when 2 groups were to be compared (Fig. 1, Table). A P value less than 0.05 was considered to be statistically significant.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of retrograde injection of rCLCA siRNA (N = 11) or its negative control (N = 9) on electrolyte concentrations in final saliva collected from a rat submandibular duct on the injection side (inj) or the non-injection side (no inj). Means ± SEM. *P < 0.05, ***P < 0.001 (ANOVA followed by a post hoc t test).

View larger version (15K): [in this window] [in a new window]   Figure 3. Effects of retrograde injection of CFTR siRNA (N = 11) or its negative control (N = 9) on electrolyte concentrations in final saliva collected from a rat submandibular duct on the injection side (inj) or the non-injection side (no inj). Means ± SEM. ***P < 0.001 (ANOVA followed by a post hoc t test).

View larger version (157K): [in this window] [in a new window]   Figure 1. Western blotting analyses and immunostaining of the rat submandibular gland on the injection side (inj) following injection of rCLCA siRNA (A,C) or CFTR siRNA (B,D), and from the submandibular gland on the non-injection side (no inj). Corresponding antibodies were used (anti-rCLCA for A and C, anti-CFTR for B and D). (A,B) Intensity of the immunoreactive bands (90 kDa for A and 170 kDa for B) is summarized in bar graphs. Means ± SEM. *P < 0.05; **P < 0.01 vs. corresponding "no inj" (paired t test, N = 5 for A and N = 6 for B). Protein loading was 25 µg per lane. (C,D) Bar = 50 µm. ST, striated duct; G, granular convoluted tubule; AC, acini.

	RESULTS

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Immunohistochemical Detection of rCLCA and CFTR Proteins
Immunohistochemical analysis with anti-rCLCA antibody revealed intense staining in the ductal epithelia, but not in the acini, of the rat submandibular gland on the non-injection side (Fig. 1C, no inj). The immunoreactivity exhibited both an intracellular and an apical plasma membranous distribution, much as reported previously (Yamazaki et al., 2005). The immunoreactivity was almost completely absent in the submandibular gland on the rCLCA-siRNA injected side (Fig. 1C, inj).

Immunostaining of the submandibular gland revealed a pattern for CFTR that differed strikingly from that obtained for rCLCA; the signals were widely distributed in the luminal plasma membranes of acinar cells and in the cells of the whole intralobular duct system, and less in the excretory and main ducts (Fig. 1D, no inj). Immunoreactivity was completely absent from the acini and intercalated ducts in the submandibular gland on the CFTR-siRNA injected side (Fig. 1D, inj). The granular convoluted and striated ducts displayed faint immunoreactivity at the luminal membrane.

Effects of Retrograde Injection of siRNA on Electrolyte Concentrations in Rat Saliva
The Cl^– concentration in the saliva was 11.5 mM on the non-injection side (no inj) in the rCLCA-siRNA group, and about the same in the negative control group (Fig. 2), a value approximately one-tenth that reported previously for primary saliva (112 mM; Schneyer et al., 1972). Na⁺ and K⁺ concentrations have been reported to be 136 and 8.4 mM, respectively, in primary saliva (Schneyer et al., 1972). In contrast, Na⁺ concentration was 16–18 mM and K⁺ concentration was 32–34 mM in the final saliva in the present study, suggesting that the concentrations of these cations were altered drastically while the primary saliva flowed down through the ductal system.

In saliva obtained from the rCLCA-siRNA group, Cl^–concentration was significantly greater on the injection side (inj) than on the non-injection side (no inj) (Fig. 2). On the injection side, Cl^– concentration was significantly greater in the rCLCA-siRNA group than in its negative control group. In contrast, neither Na⁺ nor K⁺ concentration differed between the 2 sides in either group. On the injection side, no significant difference was observed between the rCLCA siRNA group and its negative control group, in submandibular gland weight, flow rate, pH, or osmolarity (Table).

In the CFTR-siRNA group, Cl^– concentration was significantly greater in saliva from the injection side (inj) than in that from the non-injection side (no inj) (Fig. 3). On the injection side, Cl^– concentration was significantly greater in the CFTR-siRNA group than in its negative control group. K⁺ concentration was increased significantly on the injection side in the CFTR-siRNA group (vs. its negative control group). In contrast, Na⁺ concentration was not different between the two sides in either group. On the injection side, no significant difference was observed between the CFTR siRNA group and its negative control group in submandibular gland weight, pH, or osmolarity (Table). However, flow rate on the injection side was significantly lower in the CFTR-siRNA group than in its negative control group.

	DISCUSSION

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In the present study, we used the retrograde siRNA injection technique, taking advantage of the reportedly high transfection efficiency of the HVJ envelope vector. The immunoblotting and immunohistochemical studies showed this methodology to be effective in reducing rCLCA or CFTR protein to a low level, enabling us to detect any subtle changes in electrolyte composition. We obtained in vivo evidence suggesting that rCLCA, like CFTR, is physiologically significant in modulating the Cl^– composition of rat final saliva. These are likely to be molecules playing important roles in transepithelial Cl^– transport, although compensatory actions of other Cl^– channels (other CLCA or CLC channels) and/or exchangers cannot be ruled out. Injection with either of the siRNAs failed to induce any drastic change in the Na⁺ concentration or osmolarity of the saliva. Thus, it is likely that other types of channels or exchangers (e.g., K⁺ or HCO₃^–) compensate for the electrolyte imbalance caused by knocking down rCLCA or CFTR, thus avoiding any secondary change in Na⁺ concentration or osmolarity.

Although mouse CLCA1 mRNA was reported to be present in salivary acini on the basis of an in situ hybridization study (Gruber et al., 1998), no relevant proteins have been shown to be expressed in salivary ductal cells until we showed that rCLCA is expressed in the duct cells of the rat submandibular gland (Yamazaki et al., 2005). The present study revealed that the rCLCA siRNA caused an increase in the Cl^– concentration of the final saliva during stimulation with pilocarpine, a muscarinic receptor agonist. This suggests that muscarinic stimulation most likely increased the apical Ca²⁺-activated Cl^– conductance, leading to a decrease in Cl^– concentration in the luminal fluid, an effect that may have been mediated by rCLCA present specifically in the ductal epithelium.

CFTR is a cAMP-regulated Cl^– channel and is expressed in acinar and duct cells (Trezise and Buchwald, 1991; Zeng et al., 1997b). A previous in vitro study demonstrated the existence of Ca²⁺-insensitive, glibenclamide-sensitive Cl^– channels (possibly CFTR) in rat submandibular gland duct cells (Zeng et al., 1997b). Results from the present study indicate some influence of the cAMP-CFTR Cl^– channel pathway on the Cl^– re-absorption activated by muscarinic stimulation. Recent evidence suggests that Ca²⁺ and cAMP signaling systems are likely to cross-talk (Lundberg et al., 1980, 1982; Bruce et al., 2002; Melvin et al., 2005). Since CFTR is known to modulate other types of Cl^– channels (Schwiebert et al., 1999), expression of CFTR on the apical membrane of duct cells may augment the apical Cl^– channels utilized during muscarinic stimulation. Alternatively, since CFTR may be modulated not only by protein kinase A, but also by protein kinase C (Jia et al., 1997; Yamazaki et al., 1999), it is possible that, in the resting state, the sympathetic system is tonically activating the CFTR Cl^– channel, and that this effect is augmented by the muscarinic receptor-phospholipase C-protein kinase C pathway.

The finding that retrograde injection of CFTR siRNA led to a decrease in the flow rate of saliva is in accordance with the immunohistochemical localization of CFTR in the apical membrane of the acini, which is where water is secreted due to the osmotic driving force caused by Cl^– efflux (Melvin, 1999). There may also be cross-talk between the Ca²⁺- and cAMP-signaling systems in the secretion of water in the acinar system, as discussed for Cl^– re-absorption above. Analysis of the present data revealed an increase in K⁺ concentration in rats injected with CFTR siRNA. Salivary electrolyte concentrations have been reported to differ among cystic fibrosis homozygotes, heterozygotes, and healthy controls, K⁺ concentration being higher in the cystic fibrosis homozygote group than in the other groups (Aps et al., 2002). These results suggest that a defect in the CFTR gene is likely to alter K⁺ secretion across the ductal epithelium, although the precise mechanism remains unknown.

The present technique allowed us to make an in vivo non-invasive evaluation of possible molecular candidates for transepithelial Cl^– transport, since the epithelial cells facing the luminal space can be easily transfected with a specific siRNA. This would be expected to modify the final saliva, and our method permitted it to be collected easily. Consequently, this study has yielded the first in vivo evidence for a physiological significance of rCLCA, along with CFTR, in Cl^– re-absorption in the ductal system of the rat submandibular gland.

	ACKNOWLEDGMENTS

 
This work was supported by grants-in-aid (17591958 and 18059032) from the Ministry of Education, Science, Sports, and Culture of Japan.

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received May 29, 2006; Last revision August 15, 2006; Accepted September 22, 2006

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