Sialin (SLC17A5) functions as a nitrate transporter inthe plasma membraneLizheng Qina,1, Xibao Liub,1, Qifei Suna, Zhipeng Fana, Dengsheng Xiaa, Gang Dinga, Hwei Ling Ongb, David Adamsc,William A. Gahlc, Changyu Zhengb, Senrong Qia, Luyuan Jina, Chunmei Zhanga, Liankun Gud, Junqi Hee, Dajun Dengd,2,Indu S. Ambudkarb,2, and Songlin Wanga,e,2

aSalivary Gland Disease Center and Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School ofStomatology, Beijing 100050, China; bMolecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD20892; cMedical Genetics Branch, National Human Genome Research Institute, Bethesda, MD 20892; dKey Laboratory of Carcinogenesis and TranslationalResearch, Peking University Cancer Hospital and Institute, Beijing 100142, China; and eDepartment of Biochemistry and Molecular Biology, Capital MedicalUniversity School of Basic Medicine, Beijing 100069, China

Edited by Mark Gladwin, University of Pittsburg Medical Center, Pittsburgh, PA and accepted by the Editorial Board June 5, 2012 (received for review October13, 2011)

In vivo recycling of nitrate (NO3−) and nitrite (NO2

−) is an importantalternative pathway for the generation of nitric oxide (NO) andmaintenance of systemic nitrate–nitrite–NO balance. More than25% of the circulating NO3

− is actively removed and secreted bysalivary glands. Oral commensal bacteria convert salivary NO3

− toNO2

−, which enters circulation and leads to NO generation. Thetransporters for NO3

− in salivary glands have not yet been identi-fied. Herewe report that sialin (SLC17A5), mutations in which causeSalla disease and infantile sialic acid storage disorder (ISSD), func-tions as an electrogenic 2NO3

−/H+ cotransporter in the plasmamembrane of salivary gland acinar cells. We have identified an ex-tracellular pH-dependent anion current that is carried by NO3

− orsialic acid (SA), but not by Br−, and is accompanied by intracellularacidification. Both responses were reduced by knockdown of sialinexpression and increased by the plasma membrane-targeted sialinmutant (L22A-L23A). Fibroblasts from patients with ISSD displayedreduced SA- and NO3

−-induced currents compared with healthycontrols. Furthermore, expression of disease-associated sialin mu-tants in fibroblasts and salivary gland cells suppressed the H+-de-pendent NO3

− conductance. Importantly, adenovirus-dependentexpression of the sialinH183R mutant in vivo in pig salivary glandsdecreased NO3

− secretion in saliva after intake of a NO3−-rich diet.

Taken together, these data demonstrate that sialinmediates nitrateinflux into salivary gland and other cell types. We suggest that the2NO3

−/H+ transport function of sialin in salivary glands can contrib-ute significantly to clearance of serum nitrate, as well as nitraterecycling and physiological nitrite-NO homeostasis.

pH | proton

The anions NO3− and NO2

− were once thought to be inert endproducts of NO metabolism. However, it is now evident that

nitrate and nitrite can be recycled in vivo to form NO, and thusthese anions complement the nitric oxide synthase (NOS)-de-pendent activity (1). The nitrate-nitrite-NO pathway is emergingas a potential therapeutic target in such diseases as myocardialinfarction, stroke, gastric ulcers, and pulmonary hypertension(1, 2). There are two major sources of nitrate and nitrite: theL-arginine–NO synthase pathway and diet. Dietary intake of ni-trate leads to a relatively rapid increase in NO3

− concentrationin serum. Although a large part of the anion is excreted via thekidneys, up to 25% of the circulating nitrate is actively taken upby the salivary glands and concentrated ∼10-fold in the salivasecreted from the glands (3–5). Conditions that compromisesalivary gland function have been linked to decreased NO3

− se-cretion from the salivary glands and increased NO3

− levels in theserum and urine (5, 6). Although nitrate can be reduced to nitriteby the commensal bacteria in the oral cavity, most of the salivarynitrite escapes gastric conversion to NO and enters the systemiccirculation, where it generates NO. Thus, salivary nitrate isrecycled back to NO2

− and is critical for the maintenance ofphysiological levels of NO and NO2

− in the serum (1, 2, 7).

Nitrate uptake into salivary glands represents the key initial stepin NO3

− clearance from the serum; however, the mechanism me-diating transport of NO3

− in salivary gland epithelial cells has notyet been established. In this study, we examined NO3

− influx insalivary gland cells. Here we report that the sialic acid (SA)/H+

cotransporter, sialin (SLC17A5), mutations in which result in Salladisease and ISSD, is involved in nitrate uptake into salivary glands.Our data suggest a similar function for sialin in several other celltypes as well, including fibroblasts. We show that sialin is endoge-nously localized in the lysosomes as well as in the plasmamembraneof salivary gland cells, where it functions as an electrogenic 2NO3

−/H+ cotransporter mediating influx of nitrate into the cell. We alsoprovide evidence that plasma membrane sialin is a multifunctionalanion transporter that can mediate electrogenic A−/H+ cotransportof anions such as SA, glutamate, and aspartate. Importantly, wehave assessed the function of sialin in vivo in pig salivary glands andprovide evidence for the physiological relevance of sialin in medi-ating nitrate uptake NO3

− influx into pig salivary glands. In ag-gregate, our findings suggest that sialin is a versatile aniontransporter, and that functional defects in the protein may havea deleterious impact on several critical physiological functions.

ResultsCoupled NO3

− Currents and Intracellular Acidification in HumanSubmandibular Gland Cells. Human submandibular gland cell line(HSG) cells did not display constitutive currents in standard ex-tracellular solution. Replacement of Cl− with 150 mM NO3

−

produced a relatively slow, but significant, spontaneous increase inthe outward current that was dramatically enhanced by decreasingthe external pH from 7.4 to 4.0 (Fig. 1A); the current density in the150 mM NO3

− solution was 42.4 ± 5.4 pA/pF (n = 21). Both theconstitutive and low pH-induced currents had similar outwardlyrectifying characteristics with a reversal potential of −15 ± 2 mV(n = 19) (Fig. 1B). Low pH also increased the outward current innormal standard extracellular solution (Cl−-containing), althoughthe amplitude of the current was smaller than that seen with NO3

−

(23.1 ± 4.3 pA/pF; n = 18) (Fig. 1C), and the current reversed at

Author contributions: L.Q., X.L., D.D., I.S.A., and S.W. designed research; L.Q., X.L., Q.S., Z.F., D.X., G.D., H.L.O., D.A., W.A.G., C. Zheng, S.Q., L.J., L.G., and S.W. performed research;L.Q., X.L., C. Zheng, and L.J. contributed new reagents/analytic tools; L.Q., X.L., C. Zheng,L.J., C. Zhang, J.H., D.D., I.S.A., and S.W. analyzed data; and L.Q., X.L., D.D., I.S.A., and S.W.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.G. is a guest editor invited by the EditorialBoard.

See Commentary on page 13144.1L.Q. and X.L. contributed equally to this work.2To whom correspondence may be addressed. E-mail: slwang@ccmu.edu.cn, iambudkar@dir.nidcr.nih.gov, or dengdajun@bjmu.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1116633109/-/DCSupplemental.

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0 mV (Fig. 1D). Decreasing the external pH in Br−- or gluconate-containing medium did not generate any detectable current (Fig. 1E and F). Similar findings were observed in primary human sub-mandibular gland (huSMG) cells (Fig. 1G), human parotid glandductal cells, and freshly dispersed acini cells prepared from mousesalivary glands. Cl− channels in salivary gland cells are quite per-meable to NO3

− and Br−, unlike the activity described here (8–11).Nonetheless, NO3

− conductance was inhibited by several anionchannel blockers that block various Cl− channels (Fig. S1). Fur-thermore, unlike known Cl− channels in salivary gland cells, NO3

−

conductance was not regulated by cAMP, intracellular or extra-cellular Ca2+, or muscarinic or purinergic receptor agonists (Fig.S2). Replacing Na+ in the medium with Cs+ also had no affect onthe current (Fig. S3 A and B). Relatively lower (and likely morephysiological) NO3

− concentrations (0.05–0.5 mM) also inducedcurrents in HSG cells, with channel densities of 2.3 ± 0.3 pA/pF(n = 5) and 3.4 ± 0.5 pA/pF (n = 6), respectively (Fig. S3C). Takentogether, these findings demonstrate the unique properties of theNO3

− conductance detected in salivary gland cells.To examine the modulation of NO3

− conductance by pH, wemonitored intracellular pH using 2′-7′-bis(carboxyethyl)-5(6)-car-boxyfluorescein (BCECF). Decreasing the external pH in gluco-nate-containing medium (ambient [Cl−] in gluconate-containingmedium was <10 mM) did not change intracellular pH, whereasincluding 5mMNO3

− induced rapid acidification that was reversedby replacing NO3

− with gluconate in the medium (Fig. 1H). Whenexternal pH was varied, keeping [NO3

−] constant at 5 mM, theoutward current was changed as a function of external pH (Fig. 1I).Importantly, measurement of intracellular pH under the same

conditions demonstrated pH- and NO3−-dependent intracellular

acidification (Fig. 1J). The data were fitted (R2 = 0.999) using theHill equation, yielding a Hill coefficient of 2.0 ± 0.1 (Fig. 1K),consistent with the electrophysiological data. These data stronglysuggest that an electrogenic 2NO3

−/H+ cotransporter is involved inmediating NO3

− uptake in salivary gland epithelial cells.

Involvement of Sialin in 2NO3−/H+ Cotransport. To examine nitrate

transport via salivary gland, [NO3−] was measured in the serum

and saliva of miniature pigs fed with regular or nitrate-richfodder. In both cases, the [NO3

−] level in saliva exceeded that inserum within 1 h after feeding (Fig. 2A), suggesting that salivaryglands take up and secrete nitrate. The nitrate transportersidentified to date belong to the highly conserved major facilitatorsuperfamily (MFS) (11–15). To determine the molecular com-ponent mediating 2NO3

−/H+ cotransport in salivary glands, weanalyzed the expression of 127 MFS genes (11, 16, 17) in humanparotid and submandibular glands (n = 3) using AffymetrixU133Plus 2.0 microarrays. SLC17A5 (which encodes sialin) wasexpressed at high levels in both types of salivary glands (Table S1).Sialin functions as a lysosomal SA/H+ transporter involved in SAefflux, although it is detected in the plasma membrane of neurons(18, 19) where it mediates aspartate and glutamate transport. Theprotein is widely expressed in such tissues as brain, heart, lung,liver, kidney, and mouse submandibular glands (20–22). Impor-tantly, mutations in sialin are causative factors in the neurode-generative disorders Salla disease and ISSD (18, 23).We validatedthe expression of sialin in various human tissues by qPCR (Fig.2B) and by Western blot analysis using samples of various tissues

Fig. 1. Coupled NO3− currents and intracellular acidification in HSG cells. NO3

− currents in HSG cells (A–F) and primary huSMG cells (G) measured by thewhole-cell patch-clamp technique. NaCl was replaced with NaNO3, NaBr, or Na-gluconate as indicated. Changes in extracellular pH are shown in the traces(bar). I-V curves are shown in B and D. (H) External pH and NO3

− (5 mM)-dependent acidification of HSG cells measured using BCECF fluorescence. (I) pHdependence of NO3

− currents in HSG cells. (J) Intracellular pH changes under the same experimental conditions as shown in I. (K) Data from I and J were usedto determined the relationship of NO3

− currents and intracellular acidification (Hill coefficient: 2.0 ± 0.1).

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from miniature pigs (Fig. S4). Sialin was highly expressed in sali-vary glands, and liver, with lower levels of expression in brain,spleen and kidney, and even lower in muscle and pancreas. Sialinwas localized in the basolateral regions of human parotid gland

biopsies and in lysosomes as detected by its colocalization with therespective markers Na+/K+ ATPase and LAMP-1 (Fig. 2C).Based on the expression and function of known solute carrier

(SLC) transporters (Table S1) and the channel activity described in

Fig. 2. Involvement of sialin in 2NO3−/H+ cotransport. (A) Saliva and serum [NO3

−] in miniature pigs fed on regular fodder or supplemented with 100 mg/kgof nitrate. (B) Validation of gene expression of SLC17A5 based on the MFS gene expression pattern (Table S1). (C) Detection of sialin (i and iv) in humansalivary gland. Na/K-ATPase (ii) or LAMP-1 (v) are markers for basolateral membrane and lysosomes, respectively. Colocalization of the two proteins is shown(iii and vi, yellow, indicated by arrows). (D) Nitrate uptake in HSG cells transfected with sh-sialin or scram-sh. The values indicated by * or ** are significantlydifferent from the unmarked values. (P < 0.05 or P < 0.01; n ≥ 3). (E and F) Knockdown of sialin by sh-sialin and overexpression of WT-sialin. (G and H) Effect ofsialin knockdown on constitutive and low-pH–induced NO3

− current. Levels of current and intracellular pH in control HSG cells transfected with scram-sh (G) orsh-sialin (H) were as described in Fig. 1. (I) Average data obtained from the experiments shown in G and H. The number of cells tested is indicated. Statisticallysignificant differences are indicated by ** (P < 0.01).

Fig. 3. Sialin mediates NO3−/H+ as well as SA/H+ cotransporter in HSG cells. (A) Current measurement in cells perfused with medium at pH 4.0 containing

5 mM SA, NO3−, or both. (B) I-V curves of the current (Erev= 0 mV for 5 mM SA or NO3

−) obtained under the different conditions shown in A. (C) IntracellularpH in medium of pH 4.0 containing either SA or NO3

−. (D) Currents generated by substituting NO3− with glutamate (Glu) or aspartate (Asp), with other anions

transported by sialin. (E and F) SA and NO3− currents induced at pH 4.0 in control HSG cells (E) and cells treated with sh-sialin (F). The y-axis scale is the same in

E and F. (G) Average of data from the experiments shown in E and F. (H) Effect of expression of plasma membrane-targeted mutant of sialin L22A-L23A onNO3

− and SA currents. (I) Average data and statistical evaluation for H. (J) Surface expression of sialin in WT cells and in cells transfected with scram-sh, sh-sialin, or L22A-L23A mutant (AA). (K) NO3

− and SA uptake measured at 10 min after loading in HSG cells transfected with sh-sialin or scram-sh. *Valuessignificantly different from the unmarked values (P < 0.05; n = 6).

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Fig. 1, we assessed the involvement of sialin in 2NO3−/H+ co-

transport. HSG cells accumulated NO3−, and this function was

significantly decreased in cells expressing sh-sialin compared tocells expressing scrambled shRNA (scram-sh) (Fig. 2D; proteinexpression and mRNA levels are shown in Fig. 2 E and F). In-tracellular levels of K+, Na+, and Cl− were not changed by sh-sialinexpression. Importantly, NO3

− current in HSG cells maintained atnormal or low pH (Fig. 2 G–I) was significantly reduced in sh-sialin–treated cells.In addition, incubation of HSG and other cell types, including

human colon carcinoma (RKO), human gastric mucosal epi-thelial cells (GES-1), and human umbilical vein endothelial cells(HE-CV-304), in medium containing NO3

− resulted in increasedlevels of intracellular NO as detected by diaminofluorescein(DAF) (Fig. S5 A and B); nitrite was not detected in the nitrateloading solution. Athough HSG cells displayed nitrate conduc-tance when exposed to physiological levels of nitrate (50–1,000μM), indicating that these cells have an influx pathway that cantransport nitrate at relatively low [NO3

−], generation of NO andcGMP was detected only at relatively high, nonphysiological levelsof the anions (≥15 mM nitrate and 3 mM NO2

−, respectively)(Fig. S5 C and D). Note that other tissues, such as liver, caneffectively metabolize both anions at lower concentrations (Fig.S5E). This indicates that direct nitrate uptake via sialin is not aphysiological pathway for NO generation in these cell types;rather, once secreted, salivary nitrate is metabolized to nitrite by

the action of commensal bacteria in the oral cavity. This is not acompletely unexpected finding, given that the physiologicalfunction of salivary gland cells is to mediate transepithelial NO3

−

transport and concentrate the anion in saliva. A similar conclu-sion was reached in an earlier study, which showed that salivaryglands deliver NO3

− from the serum into the oral cavity withminimal metabolism (2).

Sialin Mediates NO3−/H+ and SA/H+ Cotransport in HSG Cells. In-

clusion of SA, a major substrate for sialin, in the external me-dium also induced currents when ambient pH was decreased.Although NO3

− and SA individually (5 mM each) generatedoutward currents at low external pH (Fig. 3A), the amplitudewith NO3

− (11.2 ± 2.2 pA/pF; n = 15) was greater than that withSA (5.4 ± 0.7 pA/pF; n = 7). When both anions were presenttogether (10.6 ± 0.7 pA/pF; n = 9), the current amplitude wasslightly more than that seen with SA alone, although it was nota sum of the individual currents. The characteristics of currentswere similar in all of the conditions (Fig. 3B). As seen withNO3

−, decreasing extracellular pH in the presence of 5 mM SAalso led to intracellular acidification (Fig. 3C). In addition,proton-dependent aspartate and glutamate currents, similar toNO3

− current, were detected in these cells (Fig. 3D), consistentwith a previous study indicating that sialin transports both ofthese anions (20). Sialin knockdown decreased low-pH stimu-lated currents in HSG cells perfused with media containing SA +

Fig. 4. Assessment of sialin-mediated NO3− transport in fibroblasts from patients with ISSD, showing the effect of the Salla disease and ISSD sialin mutations

on anion transport. (A–D) NO3− and SA (5 mM each) currents in HSG cells transfected with sialinR39C (B) or sialinH183R (C). (C, Insert) Average data and

statistical evaluation. (D) I-V curve of the NO3− current shown in A and B. (E–H) Sialin-mediated current (150 mM SA or NO3

−) in fibroblasts from healthyvolunteers (HV) (E and F) and patients with ISSD (G and H). (I and J) Effect of sialinH183R expression on anion transport in fibroblasts from patients with ISSD.(K) Average data from the experiments with cells from patients.

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NO3−, SA, or NO3

− (Fig. 3 E–G). Importantly, expression of thesialin mutant L22A-L23A, which reportedly is targeted to theplasma membrane (24, 25), increased the currents by approxi-mately twofold with either NO3

− or SA in the external solution(Fig. 3 H and I). This increase was also detected when the anionswere used at low concentrations (5 mM). Surface biotinylationconfirmed the presence of endogenous sialin in the plasmamembrane, which was decreased by the expression of sh-sialinand increased by expression of sialin L22A-L23A (Fig. 3J). NO3

−

and SA were transported into HSG cells (NO3− uptake > SA

uptake), and uptake of both anions was reduced when sialinexpression was suppressed by sh-sialin (Fig. 3K).Sialin also transports NO2

−, as detected by activation of low-pH currents when either NO3

− or NO2− was included in the bath

solution (Fig. S3D). NO2− current (7.5 ± 1.0 pA/pF; n = 8) was

smaller than NO3− current (11.2 ± 2.2 pA/pF; n = 15), but was

not additive in solutions containing both anions (12.8 ± 2.4 pA/pF; n = 5). Nitrite uptake was confirmed in cells for which in-cluding nitrite or nitrate in the medium led to intracellularincreases in the accumulation of the respective anion, and in cellsfor which including SA with the anions did not significantly alterthe uptake of either anion (Fig. S3E).

Assessment of Sialin-Mediated NO3− Transport in Fibroblasts from

Patients with ISSD and Effect of the Salla Disease and ISSD SialinMutations on Anion Transport. To establish the link between sia-lin and nitrate transport, we examined the effect of two non-functional sialin mutants that have been associated with Salladisease (R39C) and ISSD (H183R) (23–25). Expression of eachof these mutants in HSG cells induced dominant suppression ofboth NO3

− and SA− currents without altering the current-voltage(I-V) characteristics (Fig. 4 A–D). A major finding of the presentstudy was that fibroblasts from patients with ISSD displayeda substantial reduction in pH-dependent NO3

− or SA currentscompared with the current amplitudes in cells from healthyvolunteers (Fig. 4 E and G) with no change in the I-V re-lationship (Fig. 4 F and H). Note that a relatively higher [NO3

−]level was required to detect NO3

− conductance in fibroblasts.Although we could not induce recovery of function in cellsobtained from patients with ISSD by expressing the WT-sialin(likely due to a dominant negative effect of the endogenouslyexpressed mutant), expression of sialinH183R in cells fromhealthy volunteers strongly suppressed transporter function tolevels seen in the cells from patients with ISSD (Fig. 4 I–K).Although we appreciate that including data demonstrating sali-vary deficiencies in patients would have strengthened our find-ings, several major issues preclude conducting such a study at thepresent time. There are very few patients with Salla disease orISSD worldwide. In Salla disease, found primarily in Finland,newborns develop intellectual impairment gradually. ISSD, al-though not geographically restricted, is more severe, and patientsgenerally die early in childhood or even in utero. Both disorderscause developmental delays. The majority of patients are chil-dren with severe developmental defects and poor overall health.Thus, although assessment of saliva in patients might yield sig-nificant data, this is beyond the scope of the present study. De-spite lack of such data, the findings presented herein establisha strong link between nitrate transport and sialin. We furtherdemonstrate that disease-causing mutants of sialin also decreasesNO3

− transport.

Effect of Adenovirus-Dependent Expression of Sialinh183r Mutant inVivo in Pig Salivary Glands on Salivary Nitrate Secretion. To providefurther evidence that sialin mediates salivary gland nitrate trans-port, we used adenoviral vector containing cytomegalovirus pro-moter (AdCMV)-EGFP-polyethylenimine (PEI) complex asa carrier to deliver sialinH183R orWT-sialin vector into pig salivaryglands. At 3 d after infection, the animals were fed a NO3

−-rich dietfor 30 min and then stimulated with pilocarpine to induce salivasecretion. Compared with control animals or those that receivedWT-sialin, the pigs receiving sialinH183R displayed relatively lower

levels of NO3− in the saliva (152.5 ± 17.3 μM vs. 221.6 ± 8.5 μM at

30min and 204.1± 31.5 vs. 305.3± 27.8 μMafter 60min of feeding;P < 0.05; data obtained from fours pigs and eight parotid glands ineach group) (Fig. 5A). [NO3

−] in the saliva from animals over-expressing WT-sialin was not significantly different than that fromcontrol animals, and although serum and urine [NO3

−] levels werehigher after feeding, there was no difference between the twogroups. Western blot analysis (Fig. 5B) revealed greater sialin ex-pression in parotid glands in pigs receiving the sialinH183R plasmidcompared with controls (receiving AdCMV-EGFP-PEI alone).Furthermore, immunohistochemistry demonstrated a higher sialinsignal in glands receiving sialinH183R compared with those in thecontrol group (Fig. 5 C and D). Taken together with the datashown in Fig. 4, these data strongly suggest that nitrate transportin salivary glands could be potentially altered in patients withISSD or Salla disease.

DiscussionHerein we report a novel function for the lysosomal SA/H+

cotransporter, sialin (SLC17A5), which has been linked to Salladisease and ISSD. We show that sialin can also function as anelectrogenic 2NO3

−/H+ cotransporter in the plasma membrane.Other findings include that (i) sialin mutants associated withSalla disease and ISSD induce dominant suppression of 2NO3

−/H+ cotransporter activity, (ii) cotransporter activity is relativelylow in fibroblasts obtained from patients with ISSD comparedwith healthy volunteers, and (iii) expression of the ISSD-asso-ciated sialin mutant (H183R) suppresses cotransporter functionin cells from healthy volunteers. Taken together, these dataprovide strong evidence that sialin mediates H+-dependentNO3

− uptake into cells. Although it is well established that thelysosomal H+/SA− transport function mediated by sialin con-tributes to lysosomal recycling of SA, further studies are neededto determine the exact physiological function of nitrate transportmediated by sialin in fibroblasts and other cell types. However, ithas been well established that salivary glands serve as a major

Fig. 5. Effect of in vivo suppression of sialin function in salivary glands onsalivary nitrate secretion. (A) Salivary nitrate concentrations at 30 min and 60min after feeding a nitrate-rich diet to miniature pigs after in vivo deliveryof plasmids encoding WT-sialin or sialinH183R (Methods). *Values signifi-cantly different from the control values (P < 0.05). (B) Sialin expression incontrol parotid glands and glands receiving the plasmid-PEI-Ad complex asdetermined by Western blot analysis. (C and D) Detection of sialin in salivaryglands at 3 d after transduction with AdCMV-EGFP-PEI plus sialinH183Rvector (C) or in control parotid gland (D). (Scale bar: 50 μM.)

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route for NO3− clearance from the serum. Approximately 25%

of the circulating NO3− is taken up by the salivary glands, where

it is concentrated and secreted (at millimolar levels) in the saliva.Thus, it is likely that transepithelial flux of NO3

− occurs acrossthe salivary gland cells as the anion is taken up from the serumand secreted into saliva.We propose that sialin can function as the NO3

− uptake systemin salivary gland cells. Consistent with this proposal, we found thatthe protein is localized in the basal and lateral regions. Furtherstudies are needed to identify the NO3

− efflux pathway likely to belocalized in the apical membrane of the cells. The physiologicalrelevance of sialin in NO3

− transport via the salivary gland isconfirmed by our finding that in vivo expression of the dominantnegative sialin mutant (sialinH183R) in pig parotid glands re-duced NO3

− secretion in the saliva in response to a NO3−-rich

diet compared with the function in control animals and thoseexpressing WT-sialin in the glands. Together with our findingsdemonstrating that sialin mediates NO3

− uptake into salivarygland cells, these data provide evidence for a physiological roleof sialin in nitrate uptake into these glands. In addition, sialinappears to be a versatile anion transporter that also has the abilityto mediate H+-dependent transport of SA, NO2

−, aspartate, orglutamate. Given the protein’s relatively wide distribution in dif-ferent tissues, its function in the plasma membrane as well as ly-sosomes can have a significant impact on cell function, includingSA recycling as well as nitrate–NO balance. In neuronal tissues,there might be additional consequences related to sialin’s abilityto transport glutamate and aspartate.In summary, our data provide insight into the function of sialin

and demonstrate its unique function as a nitrate transporter. Inmammals, the diet is a major source of NO3

−; absorption of di-etary NO3

− results in an increase in serum NO3−. Approximately

25% of the circulating NO3− is taken up into salivary gland and

secreted via saliva, where it is reduced to nitrite by the action ofcommensal bacteria in the oral cavity and then converted to NOin the stomach. NO is suggested to have an important role in theprotection of gastric tissues from stress-induced injury; however, a

large amount of ingested nitrite survives hydrolysis in the stomachand enters the systemic circulation, where it is reduced to NO andother bioactive nitrogen oxides (1, 2). Thus, salivary nitratetransport provides an alternative, noncanonical pathway for thegeneration of nitrite and NO. This pathway appears to be par-ticularly significant under conditions of hypoxia and acidosis.Taken together, these findings suggest that NO3

− secretion via thegland can significantly impact the nitrate–nitrite–NO balance inthe serum, which is of major importance in such conditions ashigh blood pressure, platelet aggregation, and vascular damage(26–30). We suggest that disruption of sialin function, as seen inpatients with Salla disease or ISSD, can have significant effectson the nitrate–nitrite–NO balance, in addition to the previouslyrecognized impairment in SA storage and aspartergic neuro-transmission. Furthermore, loss of salivary gland secretory ac-tivity as a result of radiation treatment for head and neck cancersor autoimmune disease (e.g., Sjogren’s syndrome) can have po-tential systemic consequences owing to an imbalance of nitrite–NO homeostasis. Together, the findings presented herein dem-onstrate that sialin, as a nitrate cotransporter in the salivaryglands, can play an important role in the physiological regulationof systemic nitrate–nitrite–NO balance.

MethodsAll reagents and detailed methods are described in SI Methods. These includecell culture, electrophysiology, confocal imaging, and biochemical techni-ques such as surface biotinylation and western blotting. Descriptions ofexperiments with miniature pigs are also provided in SI Methods.

ACKNOWLEDGMENTS. We thank Dr. Jim Turner, Dr. Shmuel Muallem, Dr.Jing Jiang, and Professor Liangbiao Chen for their invaluable help duringthe course of this work. This study was supported by the National NatureScience Foundation of China (Grants 30430690, 30125042, and 81170975),the National Basic Research Program of China (Grants 2007CB947304 and2010CB944801), and the Divisions of Intramural Research of the NationalInstitute of Dental and Craniofacial Research and the National HumanGenome Research Institute.

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