Na+-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex

Na-dependen D-mannose anspo a he apical memb ane o a small in es ine and kidney co ex M.C. De la Ho a, M. Cano, M.J. Pe al, M. Ga c| ¨a-Delgado, J.M. Du a ¨n, M.L. Calonge, A.A. Ilunda ¨in * Depa amen o de Fisiolog| ¨a y Biolog| ¨a Animal, Facul ad de Fa macia, Uni e sidad de Se illa, C) T amon ana s/n, 41012 Se illa, Spain Recei ed 30 Janua y 2001; accep ed 22 Ma ch 2001 Abs ac The p esence o a Na/D-mannose co anspo ac i i y in b ush-bo de memb ane esicles (BBMV), isola ed om ei he a small in es ine o a kidney co ex, is examined. In he p esence o an elec ochemical Nag adien , bu no in i s absence, D-mannose was ansien ly accumula ed by he BBMV. D-Mannose up ake in o he BBMV was ene gized by bo h he elec ical memb ane po en ial and he Nachemical g adien . D-Mannose anspo s. ex e nal D-mannose concen a ion can be desc ibed by an equa ion ha ep esen s a supe posi ion o a sa u able componen and ano he componen ha canno be sa u a ed up o 50 WMD-mannose. D-Mannose up ake was inhibi ed by D-mannoseED- glucose sphlo izin, whe eas o K-me hyl glucopy anoside he o de was D-glucose = phlo izinED-mannose. The ini ial a e o D-mannose up ake inc eased as he ex a esicula Naconcen a ion inc eased, wi h a Hill coe icien o 1, sugges ing ha he Na:D-mannose co anspo s oichiome y is 1:1. I is concluded ha bo h a in es inal and enal apical memb ane ha e a concen a i e, sa u able, elec ogenic and Na-dependen D-mannose anspo mechanism, which is di e en om SGLT1. ß 2001 Else ie Science B.V. All igh s ese ed. Keywo ds: In es ine; Kidney; B ush-bo de memb ane esicle; Sodium/D-mannose 1. In oduc ion Mannose is a C2 epime o D-glucose and i s ans- po ac oss epi helial cells was hough o ake place ia he glucose anspo e s. Recen ly, Halaihel e al. [1] epo ed ha D-mannose is anspo ed ac oss pig in es inal b ush-bo de memb ane by a Na-inde- penden , di¡usi e anspo sys em. Howe e , a Na/D-mannose co anspo e was epo ed o be p esen a he apical memb ane o dog kidney [2,3], Caco-2 cells [4] and, while we we e p epa ing he cu en manusc ip , in he apical memb ane o a kidney [5]. In bo h a and dog kidney, he Na/D- mannose co anspo sys em is sa u able and elec o- genic and has a subs a e speci¢ci y and kine ic p op- e ies di¡e en om he SGLT. The Na-dependen D-mannose anspo sys em o Caco-2 cells was no cha ac e ized. We ha e ecen ly shown ha he ap- ical memb ane o chicken en e ocy es has an ac i e and elec ogenic Na/D-mannose anspo sys em [6]. In he cu en wo k, we demons a e ha he ap- ical memb ane o a kidney p oximal ubule and a small in es ine p esen a Na/D-mannose co anspo 0005-2736 / 01 / $ ^ see on ma e ß 2001 Else ie Science B.V. All igh s ese ed. PII: S0005-2736(01)00322-4 * Co esponding au ho . Fax: +34-5-423-3765; E-mail: [email p o ec ed] BBAMEM 78092 28-5-01 Biochimica e Biophysica Ac a 1512 (2001) 225^230 www.bba-di ec .com sys em, wi h p ope ies simila o hose desc ibed o he Na/D-mannose co anspo e o chicken en e - ocy es. A p elimina y accoun o he da a desc ibed he e was gi en a he 3 h `Cong eso de la Fesbe' (Spain, 2000). 2. Ma e ial and me hods 2.1. B ush-bo de memb ane esicle (BBMV) p epa a ion BBMV we e isola ed om he kidney co ex o male Wis a a s ollowing he me hod o Bibe e al. [7]. B ie£y, kidney co ex slices om one a we e homogenized in 15 ml isola ion bu¡e (in mM, 300 manni ol, 5 EGTA, 12 T is-HCl, pH 7.4) wi h he Ys al Poly on on se ing 5 o 2 min. 21 ml o cold bi-dis illed wa e and MgCl2, up o a ¢nal concen- a ion o 10 mM, we e added o he homogena e. The suspension was gen ly s i ed o 20 min and hen cen i uged a 1900Ug o 15 min. The esul an supe na an was cen i uged a 30 000Ug o 30 min and he esul an pelle was esuspended in 30 ml o 150 mM manni ol, 6 mM T is-HCl (pH 7.4), 2.5 mM EGTA and homogenized wi h a glass-Te£on po e . MgCl2, a a ¢nal concen a ion o 10 mM, was added o he homogena e. The suspension was gen ly s i ed o 20 min and hen cen i uged a 1900Ug o 10 min. The esul an supe na an was cen i- uged a 30 000Ug o 30 min and he esul an pelle was esuspended in 0.5 ml o he app op ia ed load- ing bu¡e . The suspension was made homogeneous by passing i h ough a 20-gauge needle se e al imes and dilu ed up o 30 ml o he loading bu¡e . The suspension was cen i uged a 30 000Ug o 30 min. The isola ed apical memb anes we e made homoge- neous by passing hem h ough a 25- and a 28-gauge needle se e al imes, and s o ed in liquid ni ogen un il use. All he s eps we e ca ied ou a 4³C. Small in es inal BBMV we e ob ained ollowing he me hod desc ibed by S iege and Mu « e [8]. The jejunum o wo a s we e insed wi h ice-cold 0.9% NaCl solu ion and e e ed. The mucosa was sc aped o¡ and pu in 60 ml o 300 mM manni ol, 5 mM EGTA, 12 mM T is-HCl pH 7.1 and he mix u e dilu ed six imes wi h ice-cold wa e and hen homogenized in a Ys al Poly on on se ing 4 o 2 min. MgCl2was added o a ¢nal concen a ion o 10 mM and a e 20 min he homogena e was cen i uged o 15 min a 2300Ug. The supe na an was cen i uged o 30 min a 21 000Ug. The esul - ing pelle was esuspended in 35 ml o 60 mM man- ni ol, 5 mM EGTA-T is pH 7.1 and homogenized wi h a glass-Te£on po e . A e he addi ion o MgCl2 o a ¢nal concen a ion o 10 mM, he solu- ion was allowed o s and o 20 min and hen he wo cen i uga ions abo e we e epea ed. The esicles we e esuspended in 35 ml o he bu¡e needed o he expe imen , homogenized wi h a glass-Te£on po e and cen i uged o 40 min a 21 000Ug. Fi- nally, he esicles we e esuspended in a small ol- ume o he loading bu¡e and made homogeneous by passing hem h ough a 25- and a 28-gauge needle se e al imes, and s o ed in liquid ni ogen un il use. All he s eps we e ca ied ou a 4³C. Unless o he wise s a ed he BBMV we e loaded wi h a pH 7.5 bu¡e consis ing in 140 mM manni ol, 50 mM K-glucona e and 50 mM HEPES-T is. P o ein was measu ed by he me hod o B ad o d [9], using Q-globulin as he s anda d. 2.2. Suga up ake s udies Suga up ake was measu ed a 25³C by a apid ¢l a ion echnique as desc ibed [10]. Excep whe e indica ed o he wise, he up ake bu¡e consis ed o 140 mM manni ol, 50 mM Na-glucona e, 50 mM HEPES-T is (pH 7.5) and ei he 0.1 WMD- [3H]mannose o he desi ed K-[14C]me hyl glucopy - anoside (K-MG) concen a ion. The amoun o p o- ein in he assay ube anged om 100 o 150 Wg/100 Wl o up ake bu¡e . 2.3. Ma e ials D-[3H]Mannose and K-[14C]me hyl glucopy ano- side (ca ie ee) we e pu chased om Ame sham. The o he compounds and sal s used we e ob ained om Sigma (Mad id, Spain). 2.4. S a is ical analysis Indi idual expe imen s we e ca ied ou in ipli- ca e. Da a a e p esen ed as mean þ S.E.M. In he BBAMEM 78092 28-5-01 M.C. De la Ho a e al. / Biochimica e Biophysica Ac a 1512 (2001) 225^230226 ¢gu es e ical ba s, ha ep esen he S.E.M., a e absen when hey a e less han symbol heigh . Com- pa ison be ween di¡e en expe imen al g oups was e alua ed by wo- ailed S uden 's - es . 3. Resul s 3.1. E¡ec o osmola i y on D-mannose up ake The binding o D-mannose o he memb anes was calcula ed as he up ake o D-mannose by he BBMV a in¢ni e osmola i y, ha is, when he e is no in a- esicula space. Up ake o D-mannose in bo h in es- inal and enal BBMV was a linea unc ion o he in e se o he ex a esicula osmola i y (Fig. 1), in- dica ing ha D-mannose is anspo ed in o an os- mo ically ac i e space. The binding ep esen s 32% and 13% o he D-mannose up ake measu ed in he s anda d condi ions in in es inal and enal BBMV, espec i ely. 3.2. Mannose up ake s. ime The ime cou se o D-mannose up ake in o ei he enal o in es inal BBMV (Fig. 2) e ealed ha D- mannose ansien ly accumula es in he esicula space. Mannose up ake o e sho he ¢nal s eady- s a e equilib ium alue by a ac o o app ox. 2 in bo h jejunum and kidney co ex. No o e shoo was obse ed when up ake was measu ed in nominally Na ee condi ions (Nawas eplaced by K). 3.3. E¡ec o suga s and suga anspo inhibi o s on ei he D-mannose o K -me hyl glucopy anoside up ake in o BBMV D-[3H]Mannose and K-[14C]MG up ake in o in es- inal and enal BBMV was measu ed in he absence and p esence o he compounds lis ed in Table 1. The esul s show ha D-[3H]mannose up ake in o enal and in es inal BBMV was inhibi ed by D-man- nose sD-glucose sphlo izin, whe eas K-MG up ake dec eased as ollows: phlo izin = D-glucose sD-man- nose. Fig. 1. Ex e nal osmola i y and D-[3H]mannose up ake in o BBMV. Medium osmola i y was inc eased by he addi ion o manni ol. Up ake o 0.1 WMD-[3H]mannose was measu ed du - ing 30 min in he p esence o ex a esicula Na. The composi- ion o he bu¡e s is gi en in Sec ion 2. Mean alues þ S.E.M., n=3. Fig. 2. Time cou se o D-[3H]mannose up ake in o BBMV. 0.1 WMD-[3H]mannose up ake was measu ed in ei he he absence (Naisosmo ically eplaced by K) o he p esence o ex a e- sicula Naas a unc ion o ime. Mean alues þ S.E.M., n=3. Table 1 E¡ec s o se e al compounds on ei he D-mannose o K-MG up ake Modi¢e s Rela i e up ake (%) D-[3H]mannose D-K-[14C]MG Kidney None 100 100 Mannose (5 mM) 20 þ 2.0* 87 þ 2# Glucose (5 mM) 39 þ 3.0* 13 þ 0.1* Phlo izin (0.1 mM) 71 þ 6.0#7 þ 0.05* In es ine None 100 100 Mannose (5 mM) 46 þ 6.0* 96 þ 2 Glucose (5 mM) 52 þ 6.0* 7 þ 0.01* Phlo izin (0.1 mM) 87 þ 5.0 4 þ 0.01* Up ake o ei he 0.1 WMD-[3H]mannose o 0.1 WMD-K- [14C]MG was measu ed du ing 30 s in he absence and p esence o he indica ed unlabeled modi¢e s. Labeled suga up ake ob- ained in he absence o modi¢e s was se a 100%. Values a e means þ S.E.M. o ou sepa a e memb ane esicle p epa a ions. *P60.001, #P60.05 as compa ed wi h con ol (100% up ake). BBAMEM 78092 28-5-01 M.C. De la Ho a e al. / Biochimica e Biophysica Ac a 1512 (2001) 225^230 227 3.4. E¡ec o Nachemical g adien and elec ical memb ane po en ial on suga up ake in o BBMV D-[3H]Mannose up ake in o BBMV was measu ed in he p esence and absence o an inwa dly di ec ed elec ochemical Nag adien . Elec ical memb ane po en ial was c ea ed by an ou wa dly di ec ed K g adien in he p esence o alinomycin. When e- qui ed, memb ane ol age ac oss he memb anes was b ough o ze o by equal in e nal and ex e nal Kconcen a ions in he p esence o alinomycin. An inside di ec ed Nag adien was c ea ed by add- ing 50 mM Na o he ex a esicula bu¡e , being he in a esicula bu¡e nominally Na ee. Na chemical g adien was abolished by Na emo al om he ex a esicula solu ion. Fig. 3 shows ha in bo h in es inal and enal BBMV memb ane ol age clamped condi ions inhib- i ed D-mannose up ake and his inhibi ion was u - he inc eased by Na ee condi ions. 3.5. Kine ic s udy o he D-mannose up ake D-Mannose up ake in o BBMV was measu ed in he p esence o inc easing concen a ions o unla- beled D-mannose, a ying om 0.1 o 80 WM. The shape o he cu e (Fig. 4) sugges s he exis ence o a sa u able p ocess and he alues ¢ bes ( = 0.997) a anspo model desc ibing a single sa u able ans- po sys em plus a nonsa u able di¡usion compo- nen : VmaxS=KmSKdS Fig. 3. 30 s suga up ake in o BBMV in he p esence and ab- sence o elec ical memb ane po en ial and/o Nachemical g adien . The BBMV we e loaded wi h a bu¡e consis ing o (in mM): 140 manni ol, 50 KG and 50 HEPES-T is (pH 7.5). The up ake bu¡e con ained (in mM): 50 HEPES-T is (pH 7.5), 0.045 alinomycin, 1034D-[3H]mannose, and ei he 140 manni ol, 50 NaG (elec ochemical Nag adien , black ba ); 40 manni ol, 50 NaG, 50 KG (chemical Nag adien , ha ched ba ) o 140 manni ol, 50 KG (absence o elec ochemical Na g adien , s ippled ba ). Mean alues þ S.E.M., n=4. *P60.001, as compa ed wi h elec ochemical Nag adien condi ions. NaG, Na-glucona e; KG, K-glucona e. Kiand Ko abo e he ba s indica e he ela i e [K] (i.e. memb ane po en- ial). Fig. 4. Ini ial a e (15 s) o D-mannose up ake s. inc easing concen a ions o ex e nal D-mannose. The composi ion o he in a- and ex a esicula bu¡e s was as desc ibed in Sec ion 2. The alues o he nonsa u able componen (999) we e de e - mined by ¢ ing he o al anspo da a (b) o a anspo model desc ibing a single sa u able sys em plus a nonsa u able componen . The heo e ically de i ed nonsa u able componen was sub ac ed om o al anspo o calcula e he sa u able componen (a). (Inse s) Eadie-Ho s ee plo s o he di¡e ence da a. Mean alues þ S.E.M., n=4. BBAMEM 78092 28-5-01 M.C. De la Ho a e al. / Biochimica e Biophysica Ac a 1512 (2001) 225^230228 whe e is ini ial a e o up ake, Sis he ex e nal D- mannose concen a ion, Vmax is he maximal ini ial up ake a e, Kmis he Michaelis-Men en cons an , and Kdis he appa en di¡usion cons an . The calcu- la ed appa en Kmand Vmax alues o D-mannose a e gi en in Table 2. The di¡e ence be ween o al D-mannose up ake and he nonsa u able up ake ollows ¢ s -o de ki- ne ics. Analysis wi h an Eadie-Ho s ee plo (Fig. 4) yielded a linea ela ionship, consis en wi h he ex- is ence o a single sa u able D-mannose anspo e in bo h enal and in es inal BBMV. 3.6. Na + :D-mannose s oichiome y Na:D-mannose s oichiome y has been e alua ed by measu ing D-[3H]mannose up ake in o in es inal and enal BBMV as a unc ion o ex a esicula Naconcen a ion, wi h he memb ane ol age clamped o ze o (equal in e nal and ex e nal [K] in he p esence o alinomycin). The Naconcen a- ion anged om 0 o 100 mM, wi h manni ol eplac- ing Na-glucona e o main ain he ex a esicula os- molali y cons an . The esul s (see Fig. 5 and Table 2) e ealed ha he Hill coe¤cien alues we e below 1, indica ing 1:1 Na:D-mannose s oichiome y in bo h in es inal and enal BBMV. 4. Discussion The epi helial anspo o D-mannose has no e- cei ed much a en ion and in he small in es ine D- mannose has been conside ed o be anspo ed by he D-glucose anspo e s, bu a a low a e [11]. Recen epo s, howe e , ha e demons a ed he p esence o a Na/D-mannose co anspo sys em in he apical memb ane o Caco-2 cells [4], dog [2,3], a kidney [5] and chicken small in es ine [6]. The ollowing obse a ions ag ee wi h hose e- po ed o dog kidney [2,3] and a e consis en wi h he p esence in he b ush-bo de memb ane o a small in es ine and kidney co ex o a Na/D-man- nose co anspo e , which is ac i e, sa u able and elec ogenic: (i) D-mannose was aken by an osmo i- cally ac i e space; (ii) D-mannose was ansien ly ac- cumula ed by he BBMV only in he p esence o Na; (iii) D-mannose up ake was dec eased when ei he Nawas emo ed (subs i u ed by manni ol) om he ba hing solu ions o when he elec ical memb ane po en ial was clamped o ze o, and (i ) he ca ie -media ed D-mannose up ake s. D-man- nose concen a ion in he bu¡e solu ion ollows ¢ s o de kine ics. The Km alues a e simila o hose epo ed o D-mannose anspo in dog kidney [2,3] and chicken small in es ine [6], bu an o de o magni ude lowe han ha epo ed o a kidney [5]. The Eadie-Ho s ee plo s o he ca ie -media ed D-mannose up ake (Fig. 4) yielded a linea ela ion- ship, consis en wi h he exis ence o a single D-man- Table 2 Kine ic pa ame e s o he D-mannose up ake in o BBMV iso- la ed om ei he a small in es ine o a kidney co ex Vmax KmHill coe . In es ine 14.3 þ 0.06 14 þ 0.05 1.09 Kidney 14.4 þ 0.01 15 þ 0.02 0.77 The appa en Km(in WM), Vmax (nmol/mg/15 s) and he Hill coe¤cien (coe .) alues we e calcula ed as indica ed in Figs. 4 and 5. Mean alues þ S.E.M., n=4. Fig. 5. S oichiome y o Na/D-mannose co anspo . 0.1 WM D-[3H]mannose up ake was measu ed o 30 s as a unc ion o ex e nal Naconcen a ion. The BBMV we e loaded wi h a bu¡e con aining, in mM: 200 manni ol, 50 KG and 50 HEPES-T is (pH 7.5). The up ake bu¡e con ained (in mM): 50 KG, 0.045 alinomycin, 50 HEPES-T is (pH 7.5), 1034 D-[3H]mannose, and 0^100 NaG wi h manni ol eplacing NaG isosmo ically. The Na-dependen D-mannose up ake s. ex a- esicula Naconcen a ion is shown. Mean alues þ S.E.M., n= 4. (Inse s) Hill (log-log) plo s o he da a. BBAMEM 78092 28-5-01 M.C. De la Ho a e al. / Biochimica e Biophysica Ac a 1512 (2001) 225^230 229 nose anspo e in he wo BBMV p epa a ions. As epo ed o dog kidney [2], he s oichiome y o he in es inal and enal Na/D-mannose co anspo , de- sc ibed in he cu en wo k, is 1:1. Blasco e al. [5] ound a 2:1 s oichiome y o Na-D-mannose in e - ac ion in he BBMV isola ed om a kidney. The cha ac e is ics abo e desc ibed o bo h in es- inal and enal D-mannose anspo could indica e ha D-mannose is anspo ed by he SGLT ans- po e s. Howe e , he ollowing obse a ions clea ly di¡e en ia e he Na/D-mannose anspo e om he SGLTs. Thus, he inhibi ion o D-[3H]mannose up- ake induced by ei he cold D-mannose, D-glucose o phlo izin di¡e s om ha induced by he same com- pounds on K-MG up ake (see Table 1). Also, he e alua ed Vmax o D-mannose (see Table 2) is abou 1000 imes lowe han he Vmax o D-glucose by SGLT1 (nanomola ange). Ano he undamen al di¡e ence is ha SGLT1 has a 2:1 Na:D-glucose s oichiome y [12,13], whe eas we ound a 1:1 Na:D-mannose s oichiome y. The SGLT2 and SAAT1 ha e a 1:1 Na:D-glucose s oichiome y [14^17], bu hey a e low a¤ni y glucose anspo e s (Km o K-MG is a ound 2 mM). I is unlikely, how- e e , ha D-mannose is anspo ed by he low a¤n- i y glucose anspo e s, because phlo izin inhibi s he SGLT1, SGLT2 and SAAT1 wi h equal po ency (Kiclose o 18 WM) [14^17] and he Na/D-mannose anspo e desc ibed he e has low a¤ni y o phlo - izin (see Table 1). In conclusion, he cu en wo k shows ha he apical memb ane o a small in es ine and kidney co ex p esen s a anspo e ha ca alyzes he Na/D-mannose anspo , which is di¡e en om he SGLT1. This anspo e has subs a e speci¢ci y and kine ic p ope ies simila o hose desc ibed o he Na/D-mannose anspo e p esen a he apical memb ane o chicken en e ocy es and dog kidney co ex. Acknowledgemen s This wo k was suppo ed by a g an om he Spanish DGICYT No. PM99-0121. 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