j pharmacol exp ther 1994 takano 669 74

8/14/2019 J Pharmacol Exp Ther 1994 Takano 669 74

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A B B R EV IA T iO NS : H AP , ho rse rad ish pe rox idase ; H EP E S , 4 - 2 -hyd roxye thy l -1 -p ipe raz inee thanesu lfon ic ac id ; P B S bu ffe r, D ulb ecco s p hospha te -b uf fe re d s ali ne .

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0022-3566 /94 /2682-0669 03 .00 /0T HE JO UR NA L O F P HA RM AC OL OG Y A ND E XP E RIM EN TA L T hE RA PE UT IC SC opy rig h t C 1 994 by Th e Amer ican Society f or P h ar m a co lo g y and E xp erim en tal T he rape utic s

T ra n s p o rt o f G e n ta m ic in a n d F lu id P h a s e E n d o c y to s is M a rk e rs inth e L L C P K 1 K id n e y E p ith e lia l C e ll L in e 1M IK IH IS A T A K A N O , Y O S H IK O O H IS H I, M A S A H IR O O K U D A , M A S A TO Y A S U H A R A and R Y O H E I H O R I2D ep artm en t o f P ha rm ac y K yo to U niv ers ity H os pita l F ac ulty o f M ed ic in e K yo to U niv ers ity S ak yo k u K yo to 6 06 0 1 J ap anA ccep ted fo r pu blic atio n O ctobe r 25 , 1993

A B S T R A C TIn o rde r to cha rac te r ize the t ranspo r t o f am inog lycos ide in thecu ltu red k idney ep ithe lia l ce ll lin e LLC -P K 1 ce llu la r up take o fgen tam ic in w as s tud ied in com pa rison w ith those o f flu id -phaseendocy tos is m arke rs , lu c ife r ye llow and ho rse rad ish pe rox idase .T he up take o f gentam ic in and flu id -phase m a rke rs w e re tim e -dependen t and w e re reduced a t low tem pe ra tu re . T he ra te o fgen tam ic in up take w as, how eve r, fa s te r than those o f lu c ife rye llow and ho rse rad ish perox idase , and w as sa tu rab le . Theaccum u la tion o f gen tam ic in in the ce lls w as in creas ing even a fte r6 days of in cuba tion , w he reas tha t o f lu c ife r ye llow reached a

s teady s ta te by 1 day . R e lease o f in tra ce llu la r gen tam ic in fromLLC -P K 1 ce lls w as m uch s low e r than tha t o f lu c ife r ye llow .G en tam ic in up take , bu t no t lu c ife r ye llow up take , w as in creasedby reduc ing the am b ien t ion ic s treng th . U nde r the low ion ics treng th cond itions , gen tam ic in up take w as inh ib ited by thep resence o f o the r am inog lycos ides , ly sozym e , po lyam ines suchas spe rm ine and ino rgan ic ca tions such as ca lc ium . These resu ltsind ica te tha t gen tam ic in e le c tro s ta tic a lly b inds to the ce ll ap ica lm em b rane and is subsequen tly ta ken up by an adso rp tiv e en -docy tos is in LLC -P K 1 ce lls .

N eph ro tox ic ity is one of the m ajor lim iting facto rs in the u seo f am inog lycos ide an tib io tics fo r the trea tm ent o f G ram -n eg a-tiv e in fec tiou s d iseases (S an de and M and ell, 19 85) . B ecau sethe am inog ly co side an tib io tics are accum ula ted se lec tiv ely inth e rena l co rtex an d rem a in th ere fo r a long tim e , it ha s beensuggested tha t am inog lycos ides- induced neph ro tox ic ity has ac lo se r ela tio ns hip w ith the ir transport and accum ula tion inproximal tu bu la r cells (K a loy an ides and P asto r iza-M unoz ,1980 ; H um es and O Connor, 1988).

The tran sport o f g en tam icin , an am in og ly coside an tib io tic ,in the k idney has b een s tu d ied by various techn iques such asin vivo rena l co rtex up tak e (Jo sep ov itz et a t 1982), iso latedpe rfused k idn ey (C o llie r e t aL 1979), m ic ro in jec tio n (P asto riza -Mun o z e t a L 1979) , iso la ted b rush-bord er m em b rane (L ipskyet aL 1 980 ; Sas trasinh et a l 1982 ; S oko l et a L 1989) andau to ra d iog ra ph y S ilve rb la tt an d K uehn , 1979 ; V andew a lle etal 1981 ; W ed een e t a l., 19 83). M o st bu t n o t a ll o f these resu ltsin d ica ted tha t gen ta .m ic in in te rac ts w ith b rush -bord er m em -b rane , and is su bsequ en tly taken u p by an adso rp tiv e end ocy-tos is . How eve r , ev id ences su gg estin g th e invo lvem en t o f th ea dso rp tive e nd oc yto sis in gen tam ic in up take by rena l ep ithe lia lce l ls a re de rived m os tly from au to rad iog raph ic s tud ie s w h ich

Rece ived fo r public ation July 12 1993 .1 T h is w o rk was supported in par t by a G ran t-in -A id fo r Sc ien tific Resear ch

from the M inis try of E du cation , Sc ien ce and Cultu re i n J ap an .2 Presen t a dd re ss : P h ar m ac eu ti ca l Resear ch and T echno log y In stitu te, K ink i

Univers i ty . 3 4 1 K o wa ka e, H ig as hi -o sa k a 5 77 , Ja pa n.

a re r ela ti ve ly qua litative , an d the b iochem ica l charac te ris ticsof th e transpo rt p rocess n eed to b e c lar ified fu rthe r .

R ecen t deve lopm en t o f cel l cu ltu re te chn iques has p rov idedano the r approach fo r th e stud y of v ariou s transp ort p rocesse s(Hand l e r e t a t 1980 ; Kraye r -Paw lowska e t a t 1991). LLC -PK 1 and OK are ce ll line s de riv ed from th e p ig and the opo ssumk idney , re spec tiv e ly H u ll e t a L 1976 ; K oyam a e t a t 1978), an dthey possess a s tr u ct ur e and func tion sim ila r to those o f rena lp rox im a l tub u la r ce lls . These ce ll lin es have been used fo re lu c ida ting transpo rt m echan ism s o f so lu te s such as nu trien tsand organ ic ions a t the ce llu la r leve l (M a lstr { 246 }m e t a t 1987;Takano e t a t. 1 9 9 2 ; Sa i to et at. 1992 ; H on et a t 1993 ). W ehave repo rted recen tly th e tran spo rt o f g en tam icin in L LC -P K 1 ce lls (H on e t a t 1992 ). In th a t study , w e found tha t it isessen t ia l to es tim a te the in tra ce llu la r gen tam ic in sepa ra te lyfrom th e drug lo ca liz ed in o th er com partm en ts such as cellsu rfa ce m em brane and dom es (m an ifesta tion o f transp ort andaccumula t ion o f flu id and so lu te be tw een the ep ithe lia l m ono -laye r an d the cu ltu re d ish ) , an d sugg es ted tha t gen tam ic in istaken u p by LLC -PK 1 ce lls vi a an endocy tos is w h ich dependson m e tabo lic ene rgy and cy toske le ta l func tion .

The presen t s tudy w as u nde rtak en to elu cid ate fu r the r thetranspo rt cha rac te ris tics o f gen tam ic in in LLC-PK1 ce l ls incom pa ris on w ith those o f m a rke rs fo r flu id -phase endocy tos is .T he e ffe c ts of va rious ca tio n ic m olecu le s on gen tam ic in up takew ere a lso stud ied . The re su lts sh ow ed tha t the up take ra te o fgen tamicin w as fas te r and the drug rema ined in the ce lls fo r a


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670 Takanoetal V o l 2 6 8l o n g e r period than fluid-phase endocytosis markers. Electro-s ta tic in te ra c tio n b e tw e en c a tio n ic g e nta m ic in and c e ll m e m-b ra n e s ho u ld be an important determinant for the endocyticuptake of th e a m in o g ly c o s id e in L L C-PK 1 cells.

Ma te ria ls a n d M e t h o d sM aterials. Gentamicin sulfate, type I I H RP, amikacin, kanamycin

su lf at e, t ob ram yci n and neomycin sulfate w ere obtained from SigmaChemical Co. (St. L ouis, M O ). L ucifer yellow-CH was obtained fromAldrich C hemicals (M ilw aukee, W I). L ysozyme chloride, cadaverinech loride, putrescine chl ori de, sp er mi ne and spermidine were obtainedfrom N acalai T esque (K yoto, Japan) . A ll other chemicals used for theexperiments w ere of the highest p ur it y av ai lab le.

Cell culture. L L C-PK 1 cells obtained from the A merican T ypeC ul ture C ollecti on (A TCC CRL -1392) w er e gr ow n on p last i c d ish es(Corning Glass W orks, Corning, N Y ) in m e d i u m 199 ( IC N B iom edi cals,Costa M esa, CA ), supplemented w ith 10% fetal bovine serum (W hit-taker, W alkersville, M D) without antibiotics, in an atmosphere of 5%C02-95% air at 37 C, and w ere subcultured every 4 to 5 days using0.02% ED T A and 0.05% trypsin (H ori e t a t., 19 92; T ak ano e t a t., 1992).I n a ll experiments, 60-mm d ish es w er e inocu l ated with 4 x 10 cells in5 ml of culture medium. T he cells w ere given fresh medium on th e 4thday after inoculation. I n t he p r esen t stu dy the L L C -PK 1 cel l s w er eused betw een the 218th and 2 4 7t h passages.Transport s t u d i e s . T he transport of gentamicin w as m easur ed inL L C-PK 1 cells attached to the culture dishes (60-mm) as describedpreviously with some modifications (H on e t a t., 1992). U sually, con-fluent L L C-PK 1 cells w ere used on the 5th day after inoculation andthe uptake w ere performed in exper imenta l m e d i u m (bicarbonate-freemedium 199 supplemented with 10 mM H EPES, pH 7 .4 , in PB S buffer(millimolar): N aCl, 137; K C1, 3; N a2H PO4, 8; K H 2PO4, 1.5; CaCl2, 1;and M gC 12, 0.5, pH 7.4, or in mannitol buffer (300 mM mannitol and1 0 mM H EPES, pH 7.4). A f ter removal of the culture medium, eachdish was washed with PBS buffer or mannitol buffer. U ptake m e d i u mcontaining gentamicin w as then added to each dish and the cells w ereincubated at 37 {176}Cor a specified period. A t the end of the incubation,the medium was aspirated and the dishes were rinsed rapidly 3 timeswith 5 ml of ice-cold PBS buffer. T o estimate the intracellular uptakeof gentamicin, the cells w ere scraped w ith rubber policeman into 2 mlof ice-cold PBS buffer. T he dishes were then rinsed again with 2 ml ofice-cold PBS buffer to improve the recovery of cells. T he cells w erecentrifuged at 4 C for 5 mm at 1000 rpm. T he supernatant wasaspirated and the cell pellet was resuspended gently in 2 ml of ice-coldPBS buffer and centrifuged again. For the measurement of gentamicin,the final cell pellet w as homogenized in PBS buffer w ith a bath- typesonicator (model G112SP1, L aboratory Supplies, H icksville, N Y ), andthe homogenate was used for gentamicin and protein assays.

T he uptake of H RP and lucifer yellow in L L C-PK 1 cells was per-fo rm e d w ith the s a m e m e th o d a s gentamicin except that the uptakeme d iu m fo r H RP contai ned 0.1% bovine serum albumin. For themeasurement of H R P, the final cell pellet w as homogenized in 0.05%T riton X -100 with the sonicator. For the measurement oflucifer yellow,the cells w ere extracted w ith 10% perchloric acid, and the perchloricacid-insoluble residue w as solubilized with 0.1 N N aO H to determinethe protein content.W e have r e p or te d prev ious ly that intracellular uptake of gentamicinshould be esti mated separatel y from the drug in other com partm entssuch as cell surface membrane and domes, and therefore the assaysamples w ere prepared from the cells incubated in the absence ofgentamicin for 30 mm at 37 C after uptake procedure in that study(Hori et a t., 1992). I n the present study how ever, the cells after uptakeprocedure w ere scraped and washed in PB S buffer as described above,in order to estimate the uptake of three substrates by the sameexperimental condition and to minimize possible degradation of H RPin the cells. Preliminary experiments showed that the uptake values ofgentamicin as w el l as th ose of f lu i d -p hase en docy t osis m ar k er s w er eessential ly the same in both methods (data not show n) .

A nalytical methods. G entamicin was determined by f luorescencepolarization immunoassay by using the T D X system (D ainabot, T okyo,Japan) (H on et a t., 1992). H RP w as determined w ith a very sensitivecolorimetric enzyme assay by using hydrogen peroxide and o-dianisi-dine (Steinman e t a L , 1974). Lucifer yellow w as determined by fluores-cence measurement (excitation, 430 nm and emission, 535 nm) w ith aShimadzu sp ec t ro f l u o ro p h o t o m et er RF-5000 (K yoto, Japan). Proteinwas determined by the method of Bradford (1976), using the Bio-R adProtein A ssay K it w ith bovine r-globulin as the standard.

R e s u l t sFirst, the time course and temperature dependence of intra-

cel lu lar uptake of gentamicin, H RP and lucifer yellow w ereexamined to c o m p a re th e g e n e ra l tra n s p o rt c h a ra c te ris tic s o fthese substrates in L L C-PK 1 cells. A s show n in figure 1, theuptake of gentamicin, H R P and lucifer yellow increased withtim e , a n d in all substrates the uptake w as reduced markedly atlow temperature (4 { 176} C)ompared with that at 37 { 176} C.y using3 0 m in data , the temperature-sensitive uptake of these sub-s t ra t e s w as compared. T he uptake rates of gentamicin, H RPa n d lu c ife r y e llo w w e re 190.0 15.0, 19.0 1.4 and 66.0 0.5n l /m g o f p ro te in per 3 0 m m (m e a n S.E. o f th re e s e p a ra teexperiments),respectively. T hus, the uptake rate of gentamicinwa s 10- and 3-fold faster th a n th o s e o f H R P and lucifer yellow,re sp e ctive ly , in L L C - P K 1 c e l l s . T he same experiment w as per-fo rm e d in a n o th e r c u ltu re d re n a l c e ll lin e , O K. As o b s e rve d inL L C - P K 1 cells, the uptake ofthese substrates w as temperature-d e p e n d e n t, a n d th e ra te o f te m pe ra tu re -s e n s itiv e u p ta ke o fgentamicin w as faster than those of H RP and lucifer yellow inO K cells (gentamicin, 235.8 10.6; H R P, 47.7 5.1; and luciferye llo w , 9 6 .0 86 nl/mg of protein per 30 mm, mean S.E. ofs ix d e te rm in a tio n s fro m tw o s e pa ra te e xp e rim e nts ).

Figure 2 show s the concentration-dependence of gentamicinuptake in L L C-PK 1 cells. T he uptake for 10 mm w as measureda t va rio us c on c e ntra tio ns of gentamicin. T he relationship be-tw e e n th e c o n c e n tra tio n a n d th e ra te o f g e n ta m ic in u p ta ke wasnonlinear, providing evidence for saturability. To a n a lyz e th e

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0 15 30 45 60 0 15 30 45 60TIME m m

0 15 30 45 60

F ig . 1 . Tim e c o u rs e a n d te mp e ra tu re d e pe nd e nc e o f ge n tamic in ( A), H RP(B ) a nd lu cife r y e llo w (C ) up ta ke in LLC -P K1 c e lls . LLC -P K1 c e lls w e rein c u b a te d in 2 m l o f e xp e rim e nta l m e diu m c o n ta in in g 2 m M gentarnicin,1 m g /m I o f HA P o r 0 .4 4 m M (0 .2 m g /m I) lu c ife r ye llo w e ith e r a t 3 7 {1 7 6 }C0 )o r 4 {1 7 6}C {149} ) .h e u p ta ke w a s normalized b y th e c o n c e ntra tio n o f e ac hsubstrate in th e e xpe rim e nta l m e dium , a nd the d a ta a re e xp re s s e d a sm u cro lite rs p e r m illig ra m o f p ro te in fo r c o m p a ris o n . E ac h p o in t rep resen t sth e m ea n S .E . o f th re e s epa ra te e xp e rim en ts p e rfo rm ed in du p lic a tedeterminat ions .


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1994 G e n t a m l c i n Tra ns po rt In LLC -P 1 C1 6 7 1

2

1

4

2

2 4 6

1 0 2 0 3 0

0zz

w

100

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0 12 6 (hr) 1TIME

2 3 (d a y )h e r e V is the initial rate of uptake, [G M ] is the initialc on c en tra tio n o f g en ta mic in , V is the m a x i m u m uptake rateby saturable process, K m is the M ichaelis constant and K d isthe coefficient of nonsaturable process. T he apparent K ,,,, Vand K were estimated w ith the aid of a computer to be 2.8mM , 0.053 nmol/mg of protein per mm and 0.0073 nmol/mg ofprotein per mm/mM , respectively.

Figure 3 show s the longer time course (up to 6 days) ofgentamicin and lucifer yellow uptake in L L C-PK 1 cells. T hea ccu m u l a t i o n of lucifer yellow reached a steady state by 1 dayw h e r e a s tha t of gentamicin w as increasing even after 6 days ofincubation. A t day 6, intracellular accumulation of g e n t a m i c i nwas a b o u t 10-fold higher than that o flu c ife r y e llo w . Th e re le as eo f g en ta micin a n d lu c ife r ye llo w fro m LLC -P K1 cells w as alsos t u d i e d . T he cells were preloaded with either gentamicin orlucifer yellow for 2 days and, after w a s h i n g , the cells w erei n cu b a t ed in the absence of the substrate for a specified period,th e n th e in tra c e llu la r re m a in in g o f th e s u bs tra te w a s d e te r-mined. A s show n in figure 4, intracellular lucifer yellow de-

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F ig . 2 . C on ce ntra tio n d ep en de nc e o f g e nta mic in u pta ke in LLC-PK1 c e l l s .G enta rn lc in up ta ke fo r 1 0 m m a t c onc en tra tio ns be tw e e n 1 a n d 3 0 m Mw as d ete rm ine d in P BS b u ffe r a t 3 7 {1 7 6 }C .h e o s mo la lity o f th e u p ta kem e d i u m w a s ke pt c o n s ta n t b y a d d in g a n a d e qu a te concentrat ions o fmannito l . 0 a n d - - -, th e to ta l u p ta ke a n d th e c a lc u la te d v a lu e o fn o n s a tu ra b le u pta ke , re s pe ctiv e ly . (S ), the s a tura b le up ta ke , w hic h w a sca lcu la ted by s ubtra c tin g no n s atu ra b le u pta ke fro m to ta l u p ta ke a t e a c hs ubs tra te c on c e ntra tio n. E a c h p o in t re pre s en ts th e m e a n S .E . o f th re ed e te rm in a tio ns fro m a typ ic al e xp e rim e n t.s a tu ra tio n o f g e n ta m ic in u p ta ke , th e kin e tic p a ra m e te rs w e rec a lc u la te d b y u s in g n o n lin e ar le as t-s qu a re s regression analysis( Y a m a o k a et a L 1981) as described previously (T akayama eta t 1991). T he equation used was:

V = Vm s z [ G M ] / K m + [G M ]) + K [G M

C0)00.0)Ew

0

0TIME (d a y)

F ig . 3 . Tim e c o u r s e o f g en ta mic in a nd luc ife r y e llo w up ta ke in LLC -P K1c e lls . LLC -P K1 c e lls w e re in c ub a te d w fth c u lture m e d iu m c o n ta ln ing 2m M g e n t a m i c i n (0 ) o r 0 . 4 4 m M lu c ife r y e llo w (Lx ) u p to 6 d a y s a t 3 7 {1 7 6 }CE a c h po int re pre s e n ts the m e a n S .E . o f fo ur d e te rm in a tio ns fro m tw os e p ara te e x pe rim e nts .

F ig . 4 . R e le a se o f in tra ce llu la r g e n t a m n a nd lu c e r ye llo w fro m tiC -P K1 c e lls . LLC-PK1 ce l ls w ere in c u ba te d w fth c u ftu re m ed iu m conta ln ing2 m M g e n t a m i c i n (0 ) o r 0 .4 4 m M lu c ife r ye llo w (E s) fo r 2 d a ys . At tim e 0 ,th e c e lls w e re w a s he d a n d in c u b a te d w ith s u bs tra te -fre e e x pe rim e nta lm e d i u m (0 .5 -6 h r) o r in s ub stra te -fre e c ulture m e d iu m (1 -3 d a y s ) a t3 7 { 1 7 6 } C .t th e s ta te d tim e s , th e s ub s tra te remain ing In th e c e lls w a sm e as ure d. Th e d a ta a re e xp re s s e d a s p e rc e nta g e re m a in ing (1 0 0 % a ttim e 0 ). E a c h p o in t re pre s e n ts th e m e a n S E . o f fo ur d e te rm in atio nsfro m tw o s e pa ra te e x pe rim e nts .c r e a s e d rapidly and w as less than 50% of initial value at 6 hr.In c o ntra s t, th e re le a s e o f gentamicin from L L C - P K 1 cells w asmuch slow er than lucifer yellow , w ith th e h a lf-life b e in g a b o u t3.0 d a y s .

T he effect of ionic strength on the uptake of gentamicin andlu cife r ye llo w w as examined by using either PBS buffer ( n o rm a lio n ic s tre n g th ) o r m a n n ito l b u ffe r (lo w io n ic s tre n g th ) in L L C -PK 1 c e lls . As s ho w n in figure 5, the uptake of gentamicin (fig.


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5A ) w a s m u c h h ig he r in mannitol buffer th a n in PB S buffer.I n contrast, the uptake of lucifer yellow (fig. 5B ) did notincrease, but rather decreased in mannitol buffer comparedw ith th a t in PB S buff er.

N ext, the effect of various cationic molecules on gentamicinuptake w as examined. T he gentamicin uptake was measured inmannitol buffer in these experiments. Figure 6 show s the effectof various aminoglycosides on gentamicin uptake in L L C -PK 1c e lls . As c a n b e s e e n in fig u re 6 , g e n ta m ic in up ta ke w a s inh ib -ited by all aminoglycosides tested. T he inhibitory potency ofa m i n o g l y c o s i d e s was in the order o f n e om yc in > to br am yci n>amikacin > k a n a m y c i n .

Figure 7 s h o w s the effect of lysozyme and polyamines ongentamicin uptake. T he uptake of gentamicin w as inhibited byly s oz y m e , a c a tio n ic protein. I n addition, all the polycationse xa m in e d (p u tre s c in e , c a d a ve rin e , s p e rm id in e a n d s p e rm in e )inhibited gentamicin uptake markedly.

Figure 8 shows the effect of inorganic cations on gentamicinuptake in L L C-PK 1 cells. T he uptake w as inhibited by increas-in g s od iu m c o n c e ntra tio n in th e b u ffe r, b u t re la tive ly h ig hconcentration w as needed for the inhibition (fig. 8A ). In con-tra s t, d iva le n t c a tio n s w e re m u c h m ore ef fecti ve in in h ib itin ggentamicin u p t a k e , and the inhibitory effect of calcium w ass tro n g e r th a n tha t of m agne si um (fig. SB).

D i s c u s s i o nAm in o g lyc o s id e a n tib io tic s a re e lim in a te d m a in ly b y g lo m e r-

ular filtration in the kidney, and are taken up in part byp ro x im a l tu b u la r c e lls . Th e re n a l h a n d lin g o f aminoglycosideshas been s tu die d e xte n sive ly , but the biochemical mechanismsunderlying the transport of the drugs from lumen into thep ro x im a l tu b u la r c e lls n e e d to be clarif ied further. In thepresent study w e s tu d ie d th e c h a ra c te ris tic s o f g e n ta m ic intransport in comparison w ith those of markers for fluid-phaseendocytosis, by using cultured renal epithelial cells as a models y s t e m . H R P and lucifer yellow are commonly used as fluid-

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F ig . 5 . E ffe ct o f io n ic -s tre ng th o n g e nta m ic in (A) a nd lucifer ye l low (B )u pta ke in LLC -P K1 c e lls . LLC -P K1 c e lls w e re in c u b a te d w ith P BS buffero r m an n ito l b u ffe r c o n ta in in g 2 m M g e nta m ic in o r 0 .4 4 m M lu c ife r ye l lowa t 3 7 {1 7 6}Co r 3 0 m m . E a c h c o lu m n re pre s e n ts th e m e a n S E. o f s ixde te rm in a tio n s fro m tw o s ep a ra te e xp e rim en ts . ** < .0 1 , sign if icantd iffe re nc e fro m th e u p ta ke in P BS b u ffe r b y u s in g a tw o -ta ile d t t e s t .

Fig . 6. E ffect o f v a rio u s a m in o g lyc os id e s o n g e nta m ic in u p ta ke in LLC -P K1 c e lls . LLC -P K1 c e lls w ere in c ub a te d w ith m an n ito l b u ffe r c on ta in in g2 m M ge n tamic in In th e a b s e nc e o r p r e s e n c e o f I 0 m M aminoglycosidea t 3 7 {1 7 6}Co r 3 0 m m . E a c h c o lu m n re p re s e n ts th e m e a n S .E . o f fo u rd e te rm in a tio n s fro m tw o s ep a ra te e xp e rim e nts . < .0 1 , sign if icantd iffe re nc e fro m c on tro l b y u s in g o n e -w a y a n a ly s is o f v aria nc e fo llo we db y D un n e tts t te s t.

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o\Fig. 7. E ffect o f po lyc atio ns o n g e nta mic in u p ta ke in LLC -P KI c e lls . LLC -P K1 c e lls w ere in c ub a te d with m an n ito l b u ffe r c on ta in in g 2 m M g e nta -micin in th e a b s e n c e o r p r e s e n c e o f 1 0 m M p o lyc a tio n a t 3 7 {1 7 6 }Co r 3 0m m . E a c h c o lu m n re pre se n ts th e m e an S .E . o f fo ur d e te rm in atio nsfro m tw o s e pa ra te e xpe rim e nts . < .0 1 , s ig nific an t d iffe re n ce fro mc o n tro l b y u s in g o n e -w a y a n a ly s is o f var iance fo llo we d b y D un ne ttst e s t .


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CO N CEN T RA T IO N (mM )F ig . 8 E ffe c t o f in o rg an ic cat ions on gentamicin u p ta ke in LLC -P K1 c e lls .LLC -P K1 c e lls w e re in c ub a te d with 2 m l o f m an n ito l b uffe r c o nta in in g 2m M g e nta m ic in in th e p re s e nc e o f va rio u s c o n c e ntra tio n s o f N a C l (A, tx ),Mg C I2 (B , 0 ) a n d C a C I2 (B , S ) a t 3 7 { 1 7 6 }Co r 3 0 m m. E a ch p o in t re pre s en tsth e m ea n S E. o f fo u r d e te rm in a tio n s fro m tw o s e p a ra te e x p e r i m e n t s .phase endocytosis markers (Steinman e t a t., 197 4; G oli gorsk ya n d H ru s ka , 1 9 8 6 ; Kempson e t a t., 1991). A s can be expectedfor endocytic processes, th e u p ta ke of gentamicin and fluid-phase markers w as reduced by low ering temperature. H owever,the uptake rate of gentamicin in L L C-PK 1 cells, as w ell as inO K c e l l s , w as faster than those of H RP and lucifer yellow . Inaddition, gentamicin uptake in L L C-PK 1 cells w as saturable.T hus, gentamicin uptake can not be explained simply by afluid-phase endocytosis, but rather seems to occur by an ad-s o rp tive e n do c yto s is .

T he accumulation of gentamicin by L L C-PK 1 cells w as in-creasing even after 6 days of incubation, w hereas th a t o f lucifery e l l o w reached a steady state by 1 day. In addition, the releaserate of gentamicin from the cells w as remarkably slower (half-life , 3 .0 days) th a n th a t o f lucifer yellow . I t is w ell recognizedtha t , w hen gentamicin w as administered in v ivo , the drug wasa cc um ula te d s e le c tive ly in the renal cortex and the eliminationha l f - l i f e fro m th e c o rte x was m u c h lo n g e r th a n th a t from seruma n d other organs (L uft and K leit, 1974; Fabre e t a t, 1976).Th u s , o ur fin d in g s in L L C-PK 1 cells may be closely related tothe renal handling of gentamicin in viv o . T he reason for thedifferent fates of gentamicin and lucifer yellow in L L C-PK 1ce l l s is not known, but it may be due to the polycationic natureof gentamicin .

T he uptake of gentamicin, but not lucifer yellow , w as in-creased by reducing the ambient ionic strength. N akagaki e t a t.(1981) reported that the surface potential of negatively chargedphospholipid membrane was increased by reducing ionicstrength, utilizing the partition of methylene blue. T herefore,t he i ncr eased u p ta ke o f g e n ta m ic in u nd e r the condition of lowionic strength is probably due to the increased interactionbetw een cationic gentamicin and negatively charged L L C-PK 1cell membrane. H ow ever, in our experiments, mannitol w assu b s t i t u t ed for inorganic ions to reduce the ambient ionicstrength. I t is know n that carbohydrates like sucrose andmannitol induce u ltra s tru ctu ra l a lte ra tio ns such a s a n in c re a s ein the frequency and size of the apical endocytic vacuoles andan appearance of large cytoplasmic vacuoles in the proximaltubule cells (M aunsbach and Christensen, 1992). T herefore,

a l t h o u g h the influence of these u ltra s tru c tu ra l a lte ra tio n s o nthe cellular function is not w ell understood, the changes insubstrate uptake observed in mannitol buffer may not be dueso l e l y to the reduction in ionic strength. U nder more physio-logical conditions, C hristensen e t a L (1983) studied the effectof molecular charge of protein on renal tubular uptake, andfound tha t 5 times more cationized than anionic albumin and2.7 times more cationic than anionized lysozyme were reab-sorbed b y a n e nd o c yto s is in the proximal tubules. T aken to -gether, cationic charge o f g e n ta m ic in s h o u ld b e a n im p o rta n tdeterminant in its uptake by the adsorptive endocytosis inL L C - P K 1 cells.

T he uptake of gentamicin in L L C-PK 1 cells w as inhibited byaminoglycosides, lysozyme and polyamines. The inhibitory p0-tency of various aminoglycosides was in the order of neomycin> tobramycin > amikacin > kanamycin, which agrees w ith then um be r o f d is s oc ia ta ble amino groups of each aminoglycoside.In a d d itio n , th e in h ib itio n of gentamicin uptake by tri- andte tra a m in e (s pe rm id in e a n d s p e rm in e ) w as st rong er th a n th a tby diamines (putrescine and cadaverine). Josepovitz e t a t.(1982) reported the inhibitory effect of aminoglycosides ando rg an ic p olyc a tio ns on gentamicin uptake in rat renal cortexin viv o . A lthough the order of inhibitory potency of variousaminoglycosides they found is not completely the same w ithth a t o f the present study, th e ir re su lts a re e ss en tia lly c o m p a t-ible w ith ours. T hus, the cationicity of aminoglycosides shouldbe important during the uptake of the drugs from the tubularlumen into renal epithelial cells. G entamicin may be taken upvia a common pathw ay fo r c a tio nic proteins such as lysozyme(Christensen e t a t., 1953).

Ishikawa e t a t. (1985) reported that gentamicin binding torenal brush-border membrane is inhibited by calcium, as wellas by neomycin and spermine, in a dose-dependent manner.K irschbaum (1984) also studied the interaction betw een gen-tamicin and renal brush-border membrane by measuring thea g g reg a t i o n of the membrane induced by the aminoglycoside,a nd fo u n d th a t m o n o - a n d d iva le n t in o rg a n ic c a tio n s inhibitedthe interaction. I n the present study w e found that the uptakeo f gentamicin by L L C -PK 1 cells w as inhibited by inorganicc a tio n s a n d e s p e c ia lly c a lc iu m , a d iva le nt c a tio n , h a d a p o te n tinhibitory effect. O ur results are compatible w ith b in din g stud-ies described above, and therefore it is most likely that thesein o rg a n ic c a tio n s , a s w e ll a s o rg a n ic p o ly c a tio n s , in h ib it gen-tamicin uptake b y re du c ing g e nta m ic in binding to the a p i ca lmembrane of the cells, w hich is the first step during its uptake.I nterestingly, there a re s om e recent reports show ing that g e n -tamicin induces calciuria immediately af ter i ts adm ini strationin v ivo (Elliott and Patchin, 1992; Foster e t a t., 199 2; G arl ande t a t., 1992). T hus, gentamicin and calcium may mutuallyi n h i b i t their binding and subsequent uptake into renal epithe-hal cel ls.

I n conclusion, the characteristics of gentamicin transport inL L C - P K 1 cells w ere evaluated in comparison w ith those off lu id -phase endocytosis markers, and w e demonstrated that therate of gentamicin uptake w as faster and that of eliminationfrom the cells was slow er than those of fluid-phase markers,w h ic h m a y e xp la in the se l ec t i v e and sustained accumulation ofthe drug in the renal cortex in v iv o . In addition, cationicity ofg en t a m i c i n should be an important determinant in its bindingto the apical membrane and subsequent uptake vi a an adsorp-tiv e e n do cy to s is . T he present study should provide useful in-


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Send reprint requeeta to: M ikthisa T akano, Ph.D ., D epartment of Pharmacy,Kyoto U n iver si t y H osp i t al , Sak yo-k u , K yoto 606-01, Japan .

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