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Desipramine Inhibits Na⁺/H⁺ Exchanger in Human Submandibular Cells

¹ Department of Physiology and
³ Department of Oral and Maxillofacial Surgery, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, Korea; and
² Department of Physiology, Cheju National University College of Medicine, Jeju 690-756, Korea

^* corresponding author, kppark{at}snu.ac.kr

	ABSTRACT

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A common and significant side-effect of the antidepressant desipramine is xerostomia (dry mouth). We investigated the effect of desipramine on Na⁺/H⁺ exchanger, which is an important modulator of salivary secretion. In dissociated human submandibular acinar cells, desipramine inhibited intracellular pH recovery in a concentration-dependent manner. Likewise, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺ exchanger inhibitor, had the same effect as desipramine, whereas the effect of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), a Na⁺/HCO₃^– co-transporter inhibitor, was not dramatic. Although desipramine is known to inhibit catecholamine re-uptake, desipramine also inhibited pH recovery in the human submandibular gland cell line, HSG cells, which lack nerve inputs. Our results suggest that desipramine directly inhibits Na⁺/H⁺ exchange in human submandibular glands without the involvement of catecholamine re-uptake, revealing the cellular mechanism of desipramine-evoked xerostomia.

KEY WORDS: desipramine • xerostomia • intracellular pH • Na⁺/H⁺ exchange • secretion

	INTRODUCTION

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Xerostomia is a subjective complaint of oral dryness due to a decrease in the secretion of saliva (Guggenheimer and Moore, 2003; Scully, 2003). Because secretion in the salivary gland can be affected by many intrinsic and/or extrinsic factors, xerostomia is a common worldwide oral problem. It is generally thought that xerostomia is frequently caused by drug treatment that induces hyposalivation. Series of widely prescribed drugs have been reported to decrease salivary secretion as a common side-effect (Bergdahl and Bergdahl, 2000; Scully, 2003).

Desipramine is a tricyclic antidepressant delivered orally to treat patients with major depression disorders. However, desipramine is reported to induce xerostomia (Scarpace et al., 1993; Koller et al., 2000; Galanter et al., 2002). Xerostomia is the most prominent side-effect among all desipramine-induced symptoms, which also include decreased appetite, nausea, and diarrhea (Nelson et al., 1984). Although a depressed mental state can reduce salivary secretion, a major factor involving hyposalivation is a side-effect of antidepressant drugs (Hunter and Wilson, 1995). Desipramine acts as a potent inhibitor of catecholamine re-uptake (Nelson et al., 1984; Potter et al., 1991), which is known to accumulate extracellular catecholamine and potentiate adrenergic signaling.

The increase in cytosolic Ca²⁺ plays a critical role in stimulated secretion of saliva (Ambudkar, 2000), and several medications (such as anticholinergic drugs) exert their xerogenic effects by modulation of receptor-mediated Ca²⁺ signaling in salivary gland cells. However, mechanisms by which most tricyclic antidepressants, including desipramine, exert xerogenic effects are still unclear, because, to date, there has been no reported modulatory effect on receptor-mediated salivary Ca²⁺ signaling. Not only intracellular Ca²⁺, but also Na⁺-dependent ion exchangers (such as Na⁺/H⁺ exchanger and Na⁺/K⁺/2Cl^–co-transporter), are known to be significant modulators of salivary secretion.

Here, we studied the effect of desipramine and tested the hypothesis that desipramine inhibits Na⁺-dependent ion exchanger and causes a decrease in salivary secretion. There are two possible cellular mechanisms whereby desipramine can inhibit salivary secretion. One is the catecholamine re-uptake pathway, and the other is by direct inhibition of one of the key factors without catecholamine re-uptake. In this report, we tried to elucidate the cellular mechanism of desipramine action as well as its modulatory target.

	MATERIALS & METHODS

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Materials
Desipramine, EIPA, DIDS, propranolol, and prazosin were purchased from Sigma (St. Louis, MO, USA). The collagenase P was purchased from Roche Molecular Biochemicals. 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) was obtained from Molecular Probes (Eugene, OR, USA). Modified Eagle’s Medium, bovine calf serum, and penicillin/streptomycin were purchased from GIBCO (Grand Island, NY, USA). [³H]cAMP was purchased from NEN (Boston, MA, USA).

Cell Preparation
The dissociation of human submandibular salivary gland cells was performed as previously described (Park et al., 2002). Pieces of human submandibular glands were surgically removed from 12 oral cancer patients, who had provided informed consent. The patients included both males and females, with ages ranging from 38 to 69 yrs. The glands did not contain atypical cells when assessed histologically. The tissues were quickly removed and finely minced in a physiological salt solution (containing [in mM]: 135 NaCl, 5.4 KCl, 1.2 CaCl₂, 0.8 MgSO₄, 0.33 NaH₂PO₄, 0.4 KH₂PO₄, 10 glucose, 2 glutamine, and 20 HEPES adjusted to pH 7.4 with NaOH), supplemented with 10 mM sodium pyruvate, a 0.02% trypsin inhibitor, and 0.1% bovine serum albumin. The cells were then digested in the same solution containing collagenase P (0.3 mg/7.5 ml) at 37°C for 75 min with continuous agitation. The prepared acinar cells were re-suspended in the physiological salt solution containing 0.1% bovine serum albumin. This study was performed according to the guidelines for experimental procedures found in the Declaration of Helsinki, the World Medical Association, and the Human Research Guidelines of Seoul National University.

The human submandibular gland cell line (HSG cells) was grown in Modified Eagle’s Medium supplemented with 10% (v/v) heat-inactivated bovine calf serum, and 1% (v/v) penicillin (5000 U/mL) + streptomycin (5000 µg/mL) solution. The cells were cultured in a humidified atmosphere of 95% air and 5% CO₂. The culture medium was changed every two days, and the cells were subcultured weekly.

Measurement of Intracellular pH Level
The intracellular pH level of cells was monitored with pH-sensitive fluorescent dye, BCECF-AM, as previously described (Park et al., 2002). The cells were incubated with 2 µM BCECF-AM for 20 min at room temperature, washed once with the physiological salt solution containing 0.1% bovine serum albumin, and kept on ice. The cells were placed onto poly-l-lysine-coated coverslips for 10 min in a perfusion chamber. We measured fluorescence by photon counting using a Photon Technology International system (Birmingham, NJ, USA). BCECF fluorescence was recorded at excitation wavelengths of 440 and 490 nm and an emission wavelength of 530 nm. The 490/440 fluorescence ratios were calibrated according to the high potassium nigericin procedure.

Data Analysis
All quantitative data are expressed as means ± SEM. We calculated the half-maximal inhibitory concentration (IC₅₀) with the Wavemetrics IGOR Pro program (Portland, OR, USA). Differences were determined by one-way ANOVA and considered to be significant only for P < 0.05.

	RESULTS

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Desipramine Inhibited Intracellular pH Recovery
To monitor directly the activity of ion exchanger affecting intracellular pH, we treated the cells with 10 mM NH₄Cl for acid loading after their stabilization with normal HCO₃^–- containing solution ([in mM] 115 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 HEPES, 10 glucose, and 25 NaHCO₃, adjusted to pH 7.4 with NaOH). Because most pH modulators are Na⁺-dependent ion exchangers, acid loading can be maximized if the cells are incubated in Na⁺-free solution prepared by the replacement of Na⁺ with N-methyl-D-glucamine⁺. We then measured the slope of intracellular pH recovery, which is closely correlated with the activity of Na⁺-dependent ion exchanger (Geibel et al., 1990). Desipramine itself did not show any effect on pH level (data not shown). However, desipramine decreased the intracellular pH recovery rate, whereas, in the absence of desipramine, the intracellular pH rapidly recovered toward the initial intracellular level (Fig 1A). The effect was reversible, because the recovery rate could be restored after intensive washing. The inhibitory effect was concentration-dependent, with an IC₅₀ of 31.9 ± 2.6 pM (Fig. 1B).

View larger version (18K): [in this window] [in a new window]   Figure 1. Desipramine inhibits intracellular pH recovery in acidified human submandibular acinar cells. (A) 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded dissociated human submandibular acinar cells were perfused with normal HCO3– buffers and then challenged with 15 mM NH4Cl (hatched bar) for acid loading. Thereafter, the bath solution was changed to a Na+-free HCO3–buffer (blank bar). After stabilization of the pH level, the intracellular pH recovery patterns were monitored in the absence or presence of 300 nM desipramine (filled bar). Typical pH traces from more than 10 separate experiments are presented. (B) Concentration-dependent effects of desipramine on pH recovery. Cells were treated with various concentrations of desipramine after acid-loading, and the rate of pH recovery was monitored. Each point was obtained from more than 3 separate experiments and is the mean ± SEM. The results were reproducible.

View larger version (25K): [in this window] [in a new window]   Figure 2. Desipramine blocked pH recovery by the inhibition of Na+-dependent ion exchanger recovery in human submandibular acinar cells. (A) In HEPES buffer, the BCECF-loaded acinar cells were acid-loaded (hatched bar) and maintained in a Na+-free HEPES buffer (blank bar), and intracellular pH recovery patterns were monitored in the absence or presence of 3 µM (filled bar) desipramine and/or 5 µM 5-(N-ethyl-N-isopropyl)amiloride (EIPA, gray bar), a Na+/H+ exchanger inhibitor. (B) In HCO3– buffer, cells were acid-loaded (hatched bar) and maintained in a Na+-free HCO3– buffer (blank bar), and intracellular pH recovery patterns were monitored in the absence or presence of 3 µM (filled bar) desipramine and/or 500 µM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, gray bar), an Na+/HCO3– co-transporter inhibitor. Typical pH traces from more than 5 separate experiments are presented. (C) The effects of EIPA, DIDS, and desipramine on pH recovery. Cells were treated with 1 or 3 µM EIPA, 500 µM DIDS, and/or 3 µM desipramine (Desi) after the acid-loading, and the rates of pH recovery were monitored. Each point was obtained from more than 3 separate experiments and is the mean ± SEM. The results were reproducible. *P < 0.01 (compared with control).

View larger version (24K): [in this window] [in a new window]   Figure 3. Desipramine blocked Na+-dependent ion-exchanger-mediated pH recovery in the human submandibular gland cell line, HSG cells. (A) In HCO3 – buffer, the BCECF-loaded HSG cells were acid-loaded (hatched bar) and maintained in a Na+-free HCO3– buffer (blank bar), and intracellular pH recovery patterns were monitored in the absence or presence of 3 µM (filled bar) desipramine and/or 5 µM EIPA (gray bar), an Na+/H+ exchanger inhibitor. (B) In HCO3 – buffer, HSG cells were acid-loaded (hatched bar) and maintained in a Na+-free HCO3 – buffer (blank bar), and intracellular pH recovery patterns were monitored in the absence or presence of 3 µM (filled bar) desipramine and/or 500 µM DIDS (gray bar), an Na+/HCO3– co-transporter inhibitor. Typical pH traces from more than 8 separate experiments are presented. (C) The effects of EIPA, DIDS, and desipramine on pH recovery. HSG cells were treated with 3 µM EIPA, 500 µM DIDS, and/or 3 µM desipramine (Desi) after the acid-loading, and the rates of pH recovery were monitored. Each point was obtained from more than 3 separate experiments and is the mean ± SEM. The results were reproducible. *P < 0.01 (compared with control).

	DISCUSSION

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Salivary secretion occurs via two pathways. One is transcellular secretion mediated by "water channels", involving aquaporin, and the other is transepithelial secretion mediated by the osmotic gradient of anions. Na⁺/H⁺ exchanger plays a significant role in the transepithelial pathway (Turner and Sugiya, 2002). The role of Na⁺-dependent ion exchanger in salivary secretion has been extensively studied. Perhaps the most convincing example is a study in which Na⁺/H⁺ exchanger type 1 knock-out mice showed reduced salivary secretion (Park et al., 2001), as well as the Na⁺/K⁺/2Cl^– co-transporter (Evans et al., 2000) and the Clcn₃ Cl^– channel (Arreola et al., 2002). Our finding that desipramine inhibited Na⁺/H⁺ exchanger may be a possible mechanism for desipramine-induced xerostomia.

In addition, we suggest that desipramine’s effect is not mediated by the catecholamine re-uptake process, but by the direct inhibition of Na⁺/H⁺ exchanger. We found that desipramine inhibited pH recovery in the HSG cell line. The HSG is a well-established human submandibular gland cell line, and does not show catecholamine re-uptake. However, desipramine in HSG cells showed inhibitory characteristics similar to those of human submandibular acinar cells. The results imply that the inhibitory effect of desipramine does not require catecholamine re-uptake in the adrenergic nerve terminal in the salivary gland.

We propose that desipramine directly inhibits Na⁺/H⁺ exchanger. Previous studies have reported desipramine’s direct inhibitory ability. Desipramine inhibited voltage-sensitive Na⁺ channels (Pancrazio et al., 1998), voltage-sensitive Ca²⁺ channels (Lavoie et al., 1990), nicotinic acetylcholine receptors (Hennings et al., 1999), the KATP channel (Sakuta, 1994), and small-conductance Ca²⁺-activated K⁺ channels, SKs (Terstappen et al., 2001). Most of these types of channels are either rarely expressed in salivary glands or show negligible effects on salivary secretion (Stummann et al., 2003). Although the details of the mechanisms (such as the binding affinity and the interaction site) are unclear and may be variable, analysis of the data supports the possibility that desipramine directly interacts with Na⁺/H⁺ exchanger in submandibular cells. We are aware of the structural similarity between Na⁺/H⁺ exchanger and catecholamine re-uptake. Human catecholamine re-uptake involves the SLC6A family of Na⁺/Cl^–-dependent transporters. Interestingly, this shows a predicted protein topology of 12 transmembrane domains, with Na⁺-induced changes and a charge distribution similar to that of the Na⁺/H⁺ exchanger (Nelson, 1998). Here, we report, for the first time, the effect of desipramine on Na⁺/H⁺ exchanger.

Another interesting point is the low IC₅₀ for Na⁺/H⁺ exchanger. In clinical applications, the serum level of desipramine reaches 3–10 µM and covers the inhibitory range for catecholamine uptake. However, the IC₅₀ for Na⁺/H⁺ exchanger in human submandibular acinar cells was less than 100 pM from our results. This indicates that the effect of desipramine on salivary Na⁺/H⁺ exchanger is operative in clinical conditions of desipramine administration.

Because the treatment of depression requires long-term medication, we have focused on the changes in salivary secretion mediated by the xerostomic effect of long-term applications of desipramine. It has been reported that chronic treatment with desipramine perturbs the secretory balance of thyroid hormone, which affects exocrine secretion (Campos-Barros and Baumgartner, 1994) and down-regulates salivary gland function directly (Koller et al., 2000). Here, we propose a novel acute action, as well as the chronic effects, of desipramine in submandibular glands.

Taken together, our results suggest that desipramine directly inhibits Na⁺/H⁺ exchanger in human submandibular glands. Because the activity of the Na⁺/H⁺ exchanger is correlated with salivary secretion, our results contribute to an understanding of the cellular mechanism of desipramine-evoked xerostomia.

	ACKNOWLEDGMENTS

 
We thank Dr. J. Ziburkus for comments on the manuscript. This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (RO8-2004-205-102310) and by Seoul National University.

	FOOTNOTES

 
⁴ authors contributing equally to this work

Received June 17, 2004; Last revision March 9, 2006; Accepted May 11, 2006

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