Na+-H+ exchange activity in brush-border membrane vesicles isolated from chick small intestine

ELSEVIER Biochimica e Biophysica Ac a 1235 (1995) 313-316 BB Biochi~ic~a e BiophysicaAc a Na +-H + exchange ac i i y in b ush-bo de memb ane esicles isola ed om chick small in es ine M.I. Fe e o, M. Cano, A. Ilund iin * Depa amen o de Fisiologla y Biologla Animal, Facul ad de Fa maeia, Uni e sidad de Se illa, c) T amon ana s / n, 41012 Se illa, Spain Recei ed 4 Oc obe 1994; accep ed 16 Decembe 1994 Abs ac This s udy was unde aken o in es iga e he p esence o a Na +-H + an ipo e in b ush-bo de memb ane esicles (BBMV) isola ed om chick small in es ine. An ou wa dly di ec ed p o on g adien (pH 5.5 inside, 7.5 ou side) s imula ed Na ÷ up ake in o BBMV and esul ed in a ansien accumula ion. No accumula ion was obse ed in he absence o a p o on g adien . Vol age clamping he memb ane wi h K ÷ and alinomycin dec eased he Na ÷ o e shoo . Amilo ide inhibi ed pH g adien -d i en Na ÷ up ake in a dose-dependen manne wi h an IC5o o 44 /xM. The ela ionship be ween pH g adien -d i en Na + up ake and ex e nal Na + concen a ion ollowed simple, sa u a ing Michaelis-Men en kine ics. Eadie-Ho s ee analysis o he pH g adien -d i en Na + up ake indica ed a single anspo sys em wi h a lima x o 33 nmol/mg p o ein pe 15 s and a K m o Na ÷ o 12 mM. The ini ial a e o pH-d i en Na + up ake inc eased as he in a esicula pH dec eased, wi h a Hill coe icien close o 1. These indings indica e ha BBMV isola ed om chicken small in es ine posses a Na+-H + exchange . This exchange does no appea o be he one in ol ed in cell pH egula ion. Keywo ds: BBMV; Sodium ion-p o on exchange; Kine ics 1. In oduc ion The Na+-H + exchange is an ubiqui ous memb ane anspo p o ein ha has been implica ed in di e se physiological unc ions including he con ol o cell ol- ume and pH [1-4]. We ha e epo ed [5] ha chicken en e ocy es p esen a Na+-H ÷ exchange ha con ols es ing pH i and helps o eco e pH i om an acid load. Isola ed en e ocy es lose hei pola i y and Na÷-H + exchange is p esen in he apical and basola e al memb ane domains o in es inal epi helial cells (see [3] o a ecen e iew). Se e al lines o e idence sugges ha he apical and basola e al Na ÷-H ÷ exchange a e wo dis inc o ms wi h di e en celh la unc ions. The basola e al exchange may ep esen a 'house-keeping' an ipo e used o cell pH egula ion and he apical may be in ol ed in he ansepi helial anspo o sal and luid (see [3] o a ecen e iew). In his s udy we ha e in es iga ed he p esence o he Na÷-H ÷ exchange in b ush-bo de memb ane esicles (BBMV) isola ed om chicken small in es ine. * Co esponding au ho . Fax: +34 5 4233765. 0005-2736/95/$09.50 © 1995 Else ie Science B.V. All igh s ese ed SSDI 0005-2736(94)00322-X 2. Ma e ials and me hods 2.1. B ush-bo de memb ane esicles p epa a ion BBMV we e isola ed om he small in es ine o 4- o 6-week-old Hubba d chickens, by double Mg 2+ p ecipi a- ion me hod as desc ibed in Re . [6]. Pu i ica ion o he BBMV p epa a ion was assessed as p e iously desc ibed [6]. Unless o he wise s a ed he BBMV we e loaded wi h a pH 5.5 bu e consis ing in 300 mM manni ol, 50 mM po assium glucona e and 20 mM Mes-T is. 2.2. 22Na + up ake s udies 22 Na + up ake was measu ed a 25°C by a apid il a- ion echnique as desc ibed by Cano e al. [6] o CI- up ake measu emen s. Excep whe e indica ed o he wise, he up ake bu e consis ed in 300 mM manni ol, 50 mM ~2o assium glucona e, 0.1 mM sodium glucona e, ace s o Na + and ei he 20 Mes-T is (pH 5.5) o 20 mM Hepes- T is (pH 7.5). The amoun o p o ein in he assay ube anged om 100 o 150 /xg/100 /xl o up ake bu e . 314 M.1. Fe e o e al. /Biochimica e Biophysica Ac a 1235 (1995) 313-316 2.3. Ma e ials 22Na+ (ca ie ee) was pu chased om Ame sham. Valinomycin, amilo ide and all he sal s used in he cu en s udy we e ob ained om Sigma, S . Louis, MO. 2.4. S a is ical analysis Indi idual expe imen s we e ca ied ou in iplica e. Da a a e p esen ed as mean _+ S.E. In he igu es e ical ba s, ha ep esen he S.E., a e absen when hey a e less han symbol heigh . Compa ison be ween di e en expe i- men al g oups was e alua ed by he wo- ailed S uden 's - es . 3. Resul s and discussion 3.1. Binding s. anspo o Na + Nonspeci ic binding o a solu e o he esicle su ace would lead o an o e es ima e o anspo in o he esicle. Na + up ake a equilib ium (1 h) was dec eased wi h inc easing medium osmola i y (Fig. 1), indica ing ha Na + was aken in o a closed in a esicula space. The ela ion- ship be ween he up ake and he ecip ocal o osmola i y was linea and ex apola ion o he line o in ini e osmola - i y (whe e in a esicula olume is ze o) e eals ha bind- ing ep esen s 55% o up ake a 440 mosmol/l. Binding was no a ec ed by he p esence o absence o a ans- memb ane pH g adien 3.2. Na + up ake in o BBMV The ollowing obse a ions a e consis en wi h he p es- ence o a igh ly coupled elec oneu al Na+-H + exchange a he b ush-bo de memb ane o chicken en e ocy es and ule ou an elec ically coupled di usion: 2oo! 150' lOO. 50' O 0 2 4 6 1/osmola i y (MI Fig. 1. E ec o ex e nal osmola i y on sodium up ake in o BBMV isola ed om chicken small in es ine. Medium osmola i y was inc eased by addi ion o ei he so bi ol o manni ol. The composi ion o he in a- and ex a esicula bu e s a e gi en in Ma e ials and me hods. Up ake o 0.1 mM Na + was measu ed du ing 1 h. Line was calcula ed by linea eg ession analysis, y = 64.4+22.86x, = 0.965. Each poin ep esen s he mean alue _+ S.E. o iplica e assays using i e di e en esicle p epa a ions. 0.4' ~ .3 ~ 0,2' + ,-., 0.1' o 0.( 5.51/7.5o I0 20 30 Time (mill) 5.5i/5.5o Fig. 2. Time-cou se o Na + up ake by in es inal BBMV. Na + up ake was measu ed in he p esence (O, A, II) and absence (O) o a p o on g adien . (A), 45 /xM alinomycin o (11), 45 /xM alinomycin and 1 mM amilo ide we e p esen in up ake bu e , pH 7.5. O he de ails as in Fig. 1. Each poin ep esen s he mean alue + S.E. o iplica e assays using six sepa a e memb ane p epa a ions. * P < 0.001 as compa ed wi h he alues ob ained wi h ei he alinomycin o amilo ide. (i) In he p esence o a p o on g adien (pH 5.5 inside/pH 7.5 ou side) Na + up ake o e shoo s he inal s eady-s a e equilib ium alue by a ac o o app ox. 4. No o e shoo was obse ed in he absence o a pH g adien (Fig. 2). (ii) As Na + up ake migh be s imula ed by an accumu- la ion o nega i e cha ges inside he esicles as he pH g adien dissipa es, he measu emen s we e epea ed wi h he ol age ac oss he memb ane b ough o ze o by equal in e nal and ex e nal K + concen a ions in he p esence o alinomycin. These ol age-clamped condi ions caused a dec ease in pH g adien -dependen Na + up ake a e 1 min incuba ion (Fig. 2). Elec odi usional coupling was hus excluded as he cause o he emaining pH g adien -d i en Na + up ake. The es o he expe imen s in his pape we e ca ied ou unde ol age-clamped condi ions. (iii) pH-d i en, memb ane ol age-independen Na + up- ake was inhibi ed by amilo ide (Fig 2). The log-dose esponse cu e o amilo ide inhibi ion o Na + up ake in he p esence o a pH g adien (5.5 inside, 7.5 ou side) is gi en in Fig. 3. Appa en hal maximal inhibi ion (ICs0) was achie ed a 44 /xM amilo ide unde he condi ions es ed (Fig. 3B). The ICs0 alue o amilo ide inhibi ion is compa able o ha epo ed o he Na+-H + exchange loca ed in he apical memb ane o o he epi helial cell ypes and much highe han he alues epo ed o he basola e al Na+-H + iso o m [3]. In he p esence o 2.5 mM amilo ide Na + up ake was equal o ha obse ed in he absence o p o on g adien . 3.3. Kine ics s udy o he Na +-H + exchange Na+-up ake in o BBMV was measu ed, in he p esence and absence o an ou wa dly di ec ed ansmemb ane pH M.I. Fe e o e al. / Biochimica e Biophysica Ac a 1235 (1995) 313-316 315 ¢J 60 20 o'-// I" -6 -5 -4 -3 -2 Log [ Am lo ide] M 0,06' C.~ 0,03' i,,,,-I 0.00 B 0 20 40 60 80 1 O0 [Amilo ide] laM Fig. 3. E ec o a nilo ide on Na + up ake by in es inal BBMV. (A) Up ake o Na + was measu ed du ing 15 s in he p esence and absence o a pH g adien . Up ake bu e , pH 7.5, con ained 45/xM alinomycin and inc easing concen a ions o amilo ide. (B) Dixon plo o he amilo ide dose- esponse cu e. These da a we e ob ained by sub ac ing pH-inde- penden Na + up ake om Na + up ake measu ed in he p esence o a pH g adien . ICso o amilo ide was calcula ed using linea eg ession analysis (y = 0.0123-(2.8.10-4)x, = 0.90). Each poin ep esen s he mean alue +S.E. o iplica e assays using se en sepa a e memb ane esicle p epa a ions. g adien , a di e en ex e nal Na + concen a ions. In he p esence o a pH g adien Na ÷ up ake plo ed agains i s concen a ion shows an in lexion a low Na concen a ion and i becomes a s aigh line a highe concen a ions, no yielding a sa u a ion cu e (Fig. 4A). In he absence o a pH g adien Na up ake showed a linea ela ionship wi h i s ex a esicula concen a ion. The di e ence be ween o al Na ÷ up ake and ha obse ed in he absence o pH g adien ollows i s -o de kine ics. Kine ic analysis wi h an Eadie-Ho s ee plo (Fig. 4B) yielded a linea ela ion- ship, consis en wi h he exis ence o a single Na÷-H exchange . The appa en anspo cons an s, Km, and maximal a es o anspo o Na , Vmax, we e 12 mM and 33 nmol/mg p o ein pe 15 s, espec i ely. The appa en K m o ex e nal Na is simila o ha epo ed o he apical Na -H an ipo e o a colon [7] and enal [8] epi helial cells, and lowe han ha epo ed o abbi ileum [9]. 3.4. Na +-H + ac i i y and in acesicula pH In a a ie y o cell ypes he Na+-H ÷ an ipo e is i ually silen nea physiological pH i , bu becomes ac i- a ed below a ce ain h eshold pHa and he a e o ex- change inc eases s eeply wi h inc easing pH i (see [3] and [4] o e iews). So a an excep ion is he apical Na ÷-H ÷ an ipo e o a colon which is no modula ed by pHa [7]. The sensi i i y o he apical exchange o chicken small in es ine o pH was in es iga ed by measu ing Na up ake in o BBMV wi h he in a esicula pH p ese o a ious alues, and a wo di e en ex e nal pH (pH o) 5.5 and 8. A pH o 8 Na ÷ up ake was s imula ed by dec easing in e nal pH (Fig. 5A). The inc ease was comple ely inhib- i ed a pH o 5.5. The Hill plo o he ela ionship be ween IOO ~" 40 + Z A ~ • i • i • i • i 0 10 20 30 40 4 B = 0 1 2 3 INa +1 mM V/[Na *1 Fig. 4. E ec o inc easing concen a ions o ex e nal Na + on he ini ial a e o Na ÷ up ake (15 s). Up ake bu e con ained inc easing concen a ions o sodium glucona e isoosmo ically subs i u ed wi h manni ol. (A) Up ake in he p esence and absence o p o on g adien . Di e ence: o al up ake minus ha in he absence o a pH g adien . (B) Eadie-Ho s ee plo o he di e ence da a. Kine ic pa ame e s (i.e., he appa en K m and Vma x) o he Na+-H ÷ exchange we e calcula ed using linea eg ession analysis (y = 33.5 - 12.17x, = 0.97). Each poin ep esen s he mean alue + S.E. o iplica e assays using se en independen memb ane esicle p epa a ions. 316 M.L Fe e o e al. / Biochimica e Biophysica Ac a 1235 (1995) 313-316 120" O 100- 60 Z N 2O o pHo = 8 O ~ O O O ' O O pHo= 5.5 5 6 7 8 5 6 7 8 pHi pHi Fig. 5. E ec o in a esicula pH on Na + up ake. (A) Vesicles we e loaded wi h 300 mM manni ol, 50 mM po assium glucona e and 20 mM o ei he Hepes o Mes bu e ed wi h T is o he indica ed in a esicula pH. 15 s up ake o 0.1 mM Na ÷ was assayed in he p esence o 300 mM manni ol, 50 mM po assium glucona e, ace s o 22Na+ and ei he 20 Mes-T is (pH o 5.5, C)) o 20 mM Hepes-T is (pH o 8, O). Each poin ep esen s he mean alue + S.E. o iplica e assays using h ee independen memb ane esicle p epa a ions. In lux a pH o 5.5 was sub ac ed om alues a pH o 8 and he Hill plo o da a is gi en in B. Jmax was calcula ed om he Linewea e -Bu k plo o he da a. Line was calcula ed by linea eg ession analysis, y = 7.03 - 1.06x, = 0.93. pH-d i en Na ÷ up ake and in a esicula pH (Fig. 5B) gi es an in e ac ion coe icien (n) o 1 and a [H+]05 o 221 nM. This indica es ha he apical Na÷-H + an ipo e is no allos e ically egula ed by cy osolic p o on. We ha e p e iously epo ed [5] ha he ac i i y o he Na÷-H + an ipo e in ol ed in pH i egula ion in chicken en e o- cy es is allos e ically egula ed by cy osolic p o ons. The p esen esul s sugges ha , as in o he epi helial cells, chicken en e ocy es may ha e a leas wo ypes o Na÷-H + an ipo e s wi h di e en sensi i i y o amilo ide and o egula ion by p o ons, and wi h di e en unc ions. Acknowledgemen s The wo k was suppo ed by a g an om he Spanish DGICYT No. PB89-0616. We hank D . C.M. V izquez o he echnical help. Re e ences [1] Roos, A. and Bo on, W.F. (1981) Physiol. Re . 61,296-434. [2] Ho mann, E.K. and Simonsen, L.O. (1989) Physiol. Re . 69, 315- 382. [3] Cla k, J.D. and Limbi d, L.E. (1991) Am. J. Physiol. 261, C945-C953. [4] Ilund~in, A. (1992) Comp Biochem. Physiol. 101A, 413-424. [5] Calonge, M.L. and Ilund~in, A. (1990) Biochim. Biophys. Ac a 1029, 201-210. [6] Cano, M., Vazquez, C.M. and Ilund~iin, A. (1993) P liige s A ch. Eu . J. Physiol. 425, 395-40. [7] Rajend am, V.M. and Binde , H.J. (1990) J. Biol. Chem. 265, 8408- 8414. [8] Sel aggio, A.M., Schwa z, J.H., Bengele, H.H. and Alexande , E.A. (1986) Am. J. Physiol. 251, C558-C562. [9] Kinckelbein, R.G., A onson, P.S. and Dobbins, J.W. (1990) Am. J. Physiol. 259, G802-G806.