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J. Tiss. Cuh. Me~.13:19 1-194,1 991 1991Tissue Culture Association0271-8057/91 01.50+0,00

Q U A N T I T A T I O N O F F L U I D P H A S E E N D O C Y T O S IS B YP R O X I M A L T U B U L A R C E L L S I N C U L T U R E

M o h a n I . A b r a h a m a n d S t e p h e n A . K e m p s o n I

Department of Physiology and Biophysics India na University Schoo l of MedicineIndianapolis Indiana 46202

SUMMARY: Two proced ures a re describe d for quantitating fluid-phase endocytosis in m onolay er cultures o f opos-sum kidney epithelial cells. Both pr oced ures use the sam e principle which is to determ ine uptake of a specific, easilydetectable com poun d in the extracellular fluid. One m ethod uses luc ifer yellow, the other uses horseradish per-oxidase.Key words: ucif er yellow; hor serad ish peroxidase; k idney; fluid-phase endocytosis.

I . I N T R O D U C T I O NThere is good evidence tha t endocytosis i s an importantactivity of proxim al tubule cells in the mamm alian kidney.Studies on rena l handling of proteins have shown that theproximal tubule is the major site for reabsorption of fil-tered p roteins and that the initial step is endocytosis at theplasma m em brane facing the tubule lumen (2,12 ). Certainpeptide horm ones, such a s insulin, also are internalized b yendocytosis wi thout s ignif icant ext race l lu la r hydroly-

sis (5).The opossum kidney (OK) ce l l l ine (11) i s an estab-lished line o f cells which shares man y characteristics ofthe rena l proximal tubule (13,14 ,16) . OK ce l ls have beenused to stud y mechanisms of hormon al regulation o f trans-port processes (1 ,6 ,17,2 2) and in t race l lu la r processing ofbiologically active peptides, including insulin (18) andparathyroid hormone (21). Our recent studies indicatethat m onolaye r cultures of OK cells display endocytic ac-tivity (6,10), suggesting that OK cells may be a usefulmo del for detailed studies o f endocytosis, an d perhap smembrane recycling, in proximal tubule cells.A reliable and straightforward proc edur e for quantitat-

ing fluid-phase endocy tosis in OK cells is described. T hisprocedure has been appl ied successful ly to o ther rena lepithelial cell types (9 ,10) and may b e genera lly applica-ble to any ce l l type grown in monolayer cul ture (4) . Th eprinciple is to allow the cells to internahze a soluble com-pound w h ic h a c t s a s a m a rk e r fo r t he e x t ra c e l lu la rfluid. The amo unt of fluid-phase mar ker that is internal-ized is directly proportional to the rate of fluid-phase en-docytosis. Two different fluid-phase markers are com-pared. The se are lucifer yellow (LY), detected by fluores-

1 To whom correspondenceshould be addressed at Medical Sciences374, 635 Barnhill Drive, Indianapolis, IN 46 202.

II.A.

B.

1 9 1

cence , and horseradish peroxidase (HRP), d e tec ted by asensitive en zym e assay.MATERIALSEquipmentFluorescence spec t rophotometer , LS-5, Perkin-Elmer1Spectrophotometer , DU-40, Beckman zHigh-speed, bench top microcentrifuge, E ppen dorf mod el541 5, Brinkman 3Digital dispen ser (Volac, 0.5 to 5.0 ml), with 40 0 mlreservoir, no. P-0875-B, Den~lte4Rocker p la t form, IKA-VIBRAX-VXR model , Janke &Kunkel5Chemicals, solutions, and supphes1. Luc ifer yellow metho d

Lucife r ye llow solut ion , 1 .0 mg /ml , prepared from:LY CH, lithium salt , no. L-4 53 , Molecular Probes6Serum -free culture medium containing 0. 1% bovineserum albumin (B SA), fatty-acid free, no. A- 600 3,Sigma 7Filter sterilize and store frozen in aliquots at - 2 0 C

Isotonic washing solution, pH 7.4, prepared from:145 mM NaC1, no . S-671 , F ishers10 mM Trizma base , no . T-15037Double distilled waterAdjust pH b y addi tion of HEPE S, no. H-3 3757Perch loric acid, 10.0 % (vol/vol), prep ared from:Perchlor ic ac id , 60 % , no. A-2288Double distilled water2 . Horseradish peroxidase methodHorseradish peroxidase solu t ion , 10 .0 mg/m l , pre-pared from:HRP, type II , no . P-82507

Serum-free cul ture medium contain ing 0 .1% BSAFilter steril ize and store frozen in aliquots at- 2 0 C

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192 ABRAHAM AND KEMPSON

Potass ium phosphate buf fe r , pH 5 .0 , preparedf rom:

Potass ium phosp hate monob as ic , no. P -28 4 sDouble dist i l led waterAdjus t pH by addi tion of sodium hydroxide , no .S - 3 1 8 so-Dianisidine solut ion, 1 .0% (wt/vol) , pre par ed

f rom:o-Dianisidine, no. D-91 437 (p ossible carcinogen )Absolu te methanol , no . A-4 52-4 sMake a fresh solut ion dai ly and protect from l ightHy drog en perox ide solution, 0.3 % (vol /vol) , pre-

p a r e d f r o m :H R P , 3 0 % , n o . H - 1 0 0 9 7Double dist i l led waterHRP assay reagent , prepared da i ly f rom:o-Dianisidine solution, 0.5 mlHyd rogen peroxide so lut ion , 0 .6 mlPotass ium phosphate buf fe r , 60 .0 mlTr i ton so lu t ion , 0 .0 5% , p repare d from:Tr i ton , no . X-1007Double dist i l led waterIsotonic washing solut ion, a s abov eDisposable c lear polys tyrene cuvet t es , 1 cm pa th

length, 1 .6 ml vol , no. 67 .74 2, Sarstedt 9

I I I P R O C E D U R EThe fo llowing procedures a re des igned for use wi th OK

c e l l m o n o l a y e r s g r o wn a s s u b s t r a t e - d e p e n d e n t m o n o -layers in p las ti c 35- ram cul ture d i shes .

A. Luc ifer yel low metho d:1 . At leas t 2 h before the exper iment , rep lace the normalcul ture medium wi th se rum-f ree medium conta in ing0.1 % BSA. Adjust the medium volume to 95 0 1 a tthe s t a r t of the exper iment .2 . Begin the assay by adding 50 g l of s tock LY solut ion toeach d i sh , an d p lace o n a rock er p la t form for 2 ra in toensure comp le te mixing. T he f ina l LY concent ra t ion i s0 .0 5 mg /ml . Return the ce l l s to the CO2 incubator a t37 C for 30 rain.

3 . Af te r comple t ion of the incubat ion wi th LY, removethe med ium by aspirat ion, and rapidly but careful lywash the monolayer 6 t imes wi th 2 .0-ml a l iquot s ofi ce-cold washing so lu t ion . The d ig i t a l d i spenser i sh ighly recomm ended . In t race l lu la r LY i s ex t rac ted byaddi t ion of 1 .0 ml of 10% perchlor i c ac id . Leave a troom temperature for 15 rain without agi tat ion.4 . Blanks should be inc luded to cor rec t for t r apping andsurface binding of LY to the cel ls . In these dishes, theincubat ion of cel ls with LY is terminated immediately(within 5 s) af ter addi t ion of LY solut ion. There isnegl igible endocytic uptake of LY at this very shorttime interval.5 . Rem ove the perchlor i c ac id ext rac t f rom each d ish andtransfer to a 1.5-ml microcentr i fuge tube. Centr i fugea t 13 000 Xg for 5 ra in to sediment any ce l l debr i sthat is prese nt . Centr i fugat ion is usual ly unne cessa rybec aus e the acid-insoluble mate rial tends to st ick tothe plast ic cul ture surface.

.

.

.

B

Measure f luorescence of the ac id ext rac t s as soon aspossible, using wavelengths of 430 nm for exci tat ionand 540 nm for emiss ion . Subt rac t t i l e b lank va lues(usual ly very low) from al l sample readings.Prepare a s t andard curve by measur ing the f luores -cenc e of LY solut ions with concentrat ions in the range20 to 200 ng LY/ml . The s t andard so lu t ions shouldconta in 10% perchlor i c ac id and a re made by appro-priate di lut ions of the stock LY solut ion. At this con-cent ra t ion perchlor i c ac id does not a l t e r the f luores -cence proper t i es of LY (Kem pson and Mont rose , u n-published observat ions) .In separa te repl i ca te cul tures , de te rmine the pro te incontent us ing the modi f i ed Lowry procedu re descr ibedprevious ly (13 ,14) .9 . Use the LY s tandard curve to conver t samp le fluores -cence to LY concen t ra tion . E xpress ce l l uptake o f LYin micrograms per mil l igram protein per 30 rain.

Horseradi sh peroxidase method1. Incubate the ce ll monolayers in se rum-f ree medium asdescr ibed above . Adjust the volume of medium to 90 0gl a t the s t a r t of the ex per iment .2 . Begin by adding 100 l of the s tock H RP solut ion toeach d i sh . Place on a rocker p la t form for 2 ra in toensure comp le te mixing . The f ina l HRP concent ra t ioni s 1 .0 m g/m l . Return the ce l l s to the CO 2 incubator a t37 C for 30 min .3 . At the end of the incubation per iod , r emove the me-dium by aspi ra t ion and wash the monolayer 8 t imeswi th 2 .0-ml a l iquot s of i ce-cold washing so lu t ion .Washing s t eps should be car r i ed out over a per iod of10 min . Br ie f incubat ion of the monolayer in eachal iquot o f washing solution wil l increa se rem ova l ofHR P w hich i s bound to the sur faces of the ce l l s and theplastic dish.4 . Add 1 .0 ml of 0 .0 5% Tr i ton so lut ion to each d i sh tosolubihze the washed mon olayer and to re lease in t ra -ce l lu la r HR P. Agi ta te gently on a roc ker p la t form for30 ra in . S epara te sam ples of the ce l l lysa te a re usedfor de te rmina t ion of pro te in content by the modi f i edL o w r y p r o c e d u r e ( 1 3 , 1 4 ) a n d f o r m e a s u r e m e n t o fHRP act ivi ty s e e below).5 . I n c l u d e b l a n k s t o c o r r e c t f o r s u r f a c e b o u n d HRPwhich i s not r emoved by the washing s t eps . A smal lamou nt of HR P a lways remains a t t ached to the p las ti ccul ture sur face . The procedu re for b lanks i s to t e rmi -nate incubat ion of cel ls with HR P imm ediately (within5 s) af ter addit ion of the HR P solut ion. At this veryshort t ime interval there is negl igible endocytic uptak eo f H R P .6 . Horserad i sh peroxidase act iv i ty i s de te rmined by p lac-ing 50 1 of cel l lysate in a disposable cuvet te, fol-l owe d b y 9 5 0 I o f HR P a s s a y r e a g e n t. Ad d th e r e -agent rapidly to ensure eff icient mixing, record thet ime, and p lace the cuve t t e immedia te ly in the spec t ro-photometer . Af te r 1 .0 min has e lapsed , r ecord thea b s o r b a n c e a t 4 6 0 n m . Th e a b s o r b a n c e s h o u l d i n -crea se l inearly with t ime for up to 3 .0 rain. Ch eck thisto ensure that the 1.O-rain t ime point l ies within thel inear range .

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7 . S u b t r a c t t h e b l a n k v a l u e s f r o m a l l s a m p l e r e a d i n g s.V a l u e s f o r th e b l a n k s s h o u l d b e l e ss t h a n 1 0 % o f t h es a m p l e v a l u e s.

8 . D e t e r m i n e H R P a c t i v i ty i n s t a n d a r d s o l u t i o n s w i thc o n c e n t r a ti o n s i n t h e r a n g e 0 . 1 t o 1 . 0 g g H R P / m l .T h e s t a n d a r d s a r e m a d e b y a p p r o p r i a t e d i l u t io n s w i thw a t e r o f t h e H R P s t o c k s o l u ti o n .

9 . U s e th e H R P s t a n d a r d c u r v e t o c o n v e r t a b s o r b a n c e t oc o n c e n t r a ti o n o f H R P . E x p r e s s c e l l u p t a k e o f H R P i nm i c r o g r a m p e r m i l l i g r a m p r o t e i n p e r 3 0 m i n .

I V D I S C U S S I O N

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L u c i f e r y e ll o w a n d H R P a r e u s e d a s f l u i d - p h a sem a r k e r s b e c a u s e t h e y a r e r e a d i l y s o l u b l e , n o t c y t o t o x i c ,e a s i l y d e t e c t a b l e , t h e r e i s n e g l ig i b l e b i n d i n g t o t h e p l a s m am e m b r a n e , a n d e x t r a c e U u l a r d e g r a d a t i o n i s m i n i m a l . E v i-d e n c e t h a t t h e s e m a r k e r s a r e t a k e n u p p r i m a r i l y b y f lu i d -p h a s e e n d o c y t o s i s i n t o O K c e i l s i s p r o v i d e d b y s e v e r a lp r o p e r t i e s o f t h e u p t a k e p r o c e s s .

O n e o f th e m o s t i m p o r t a n t o b s e r v a t i o n s i s t h a t t h e u p -t a k e p r o c e s s i s n o t s a t u r a b l e a t c o n c e n t r a t i o n s i n t h e m i l li -g r a m p e r m i ll il i te r r a n g e ( 1 9 ) . T h i s i s t ru e f o r b o t h L Y a n dH R P i n O K c e ll s . U p t a k e o f L Y a t 3 7 C is n o t s a t u r a b l ea n d i n c r e a s e s s t e a d il y o v e r th e c o n c e n t r a t io n r a n g e 0 . 0 3t o 0 . 7 0 m g / m l ( F ig . 1 ) . H R P u p t a k e s h o w s a s i m i l ar ,a l m o s t li n e a r i n c r e a se o v e r t h e r a n g e 0 . 1 t o 1 . 0 m g / m l( 6 ) . T h e n o n s a t u r a b l e n a t u r e o f t h e u p t a k e p r o c e s s i n d i-c a t e s t h a t r e c e p t o r - m e d i a t e d e n d o c y t o s i s , o r a s p e c i f i cp l a s m a m e m b r a n e t r a n s p o r t s y s t e m , i s n o t i n v o k , e d .

A n o t h e r i m p o r t a n t o b s e r v a t i o n is th e d e p e n d e n c e o f th eu p t a k e p r o c e s s o n a m e t a b o l i c e n e r g y s u p p l y . T h u s , p r e -

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LY CONCENTRATION mg/mlF I G. 1 . U p t a k e o f L Y b y O K c e l l m o n o l a y e r s a t d if f e r e n t e x t r a c e l l u la rc o n c e n t r a t i o n s o f L Y . A t 3 7 C c e l l u p t a k e o f L Y i s n o t s a t u r a b l e . U p t a k e o f

L Y i s m a r k e d l y d e c r e a s e d i n c e l l s p r e c o o le d a n d m a i n t a i n e d a t 4 C . D a t aa r e m e a n v a l u e s f r o m a t y p i c a l e x p e r i m e n t a n a l y z e d i n t r i p li c a te .

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F iG . 2 . U p t a k e o f H R P b y O K c e l l m o n o l a y c r s i s d e c r e a s e d i n c el l sp r e c o o l e d a n d m a i n t a i n e d a t 4 C . D a t a a r e m e a n v a l u e s f r o m a t y p i c alexper imen t ana lyzed in t r i p l i ca t e .

c o o l in g O K c e ll s to 4 C p r o d u c e s a m a r k e d r e d u c t io n i nc e l l u p t a k e o f b o t h L Y ( F ig . 1 ) a n d H R P ( F ig . 2 ) . N o t e t h a tf o r st u d i e s o n t e m p e r a t u r e d e p e n d e n c e t h e c e il s a re i n c u -b a t e d i n a n i s o t o n i c i n o r g a n i c s a l t s o l u t i o n b u f f e r e d w i t hH E P E S , p H 7 . 4 ( 6 ) to a v o i d t h e n e e d f o r a C O 2 i n c u b a t o r .S i m i l a r l y , m e t a b o l i c i n h i b i t o r s s u c h a s K C N a l s o p r o d u c ei n h ib i t io n o f u p t a k e o f b o t h L Y a n d H R P ( 8 ). D e m o n -s t r a t e d d e p e n d e n c e o n a n e n e r g y s u p p l y r u l e s o u t t h ep o s s i b i l i t y t h a t t h e f l u i d - p h a s e m a r k e r s e n t e r t h e c e l l s b yn o n e n d o c y t i c p a t h w a y s s u c h a s d i f f u s i o n .A m a i o r a d v a n t a g e o f u s in g L Y i s t h e r e la t i v el y s i m p l ep r o c e d u r e f o r q u a n t it a ti n g i n t r a c e il u l a r L Y . F u r t h e r m o r e ,t h e f l u o r e s c e n c e p r o p e r t i e s o f t h e d y e a l lo w a r a p i d v i s u a lc h e c k o f t h e c e ll s u n d e r a f l u o r e s c en c e m i c r o s c o p e . T h ei n t r a c e l l u l a r d i s t r i b u t i o n o f L Y i s t y p i c a l l y n o n - u n i f o r ma n d p u n c t a t e , a s w o u l d b e e x p e c t e d i f t h e d y e i s c o n t a in e dw i t h i n e n d o c y t i c v e s i c l e s ( 7 ) . A p o t e n t i a l p r o b l e m c a na r i s e i f L Y i s u s e d t o m e a s u r e e n d o c y t o s i s a f t e r d r u ga c t i o n o r c h e m i c a l m o d i f i c a t i o n s d e s i g n e d p r i m a r i l y t oi n t e r f e r e w i t h t h e e n d o c y t i c p r o c e s s . T h e m e t a b o l i c in h i b i -t o r i o d o a ce t a te , f o r e x a m p l e , p r o d u c e s a m a r k e d i n c r e a s ei n O K c e ll u p t a k e o f L Y . W e a t t ri b u t e t h is i n c r e a s e t o

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1 9 4 A B R A H A M A N D K E M P S O N

enhanced L Y uptake by nonendocyt ic pa thways 8) . Thisis probably d ue to se cond ary effects of iodoacetate whichalter the permeability p roperties of the plasma m emb ranetoward small mo lecules such as L Y [molecu lar weightMr) 457] .Horseradish peroxidase is a much larger molecule ( M r40 000) and ce l l uptake of HRP is unl ike ly to be in-

f luenced by the k ind o f permeabi l ity changes d iscussedabove. Indeed , iodoacetate prod uces mar ked inhibition ofHR P u ptake by OK ce lls, as expec ted 8) . Another advan-tage of H RP is the availability of a histochemical proce-dure to produce a reac t ion product which can be o bservedby electro n microscopy. Th is allows precise determinationthat intracellular HR P is confined to the interior of apicalendocy tic vesicles 6). Although there is a proce dur e forvisua l iz ing LY in the e lec t ron microsco pe 15) , themethod has not been used widely for s tudies on endocy-tosis.Growth of renal epithelial cells on perm eable supp orts

allows the stu dy o f transcytosis, i .e . the vectorial transfe rof endocy tosed material from the apical to the basal s ideof the cell , or vice-versa. Both LY and HRP have beenused for quantitating transcytosis in primary cultures ofproximal tubule cells 3) and in Madin-Darby canine kid-ney MDC K) cells 20).

V R E F E R E N C E S1. Abraham, M. I.; McAteer, J . A.; Kem pson , S. A. Insulin st imulates phos-phate t ranspor t in opossum kidney ep i thel i a l cel l s . Am. J . Phys io l .2 5 8 : F 1 5 9 2 - F 1 5 9 8 ; 1 9 9 0 .2. Coudrier, E.; Kerjaschki, D.; Louvard, D. Cytoskeletal organization andsubmem branous in teractions in in tes tinal and renal brush borders . K id-n ey In t. 3 4 : 3 0 9 -3 2 0 ; 1 9 8 8 .3. Goligorsky, M. S.; Hruska, K. A. Transcytosis in cultured proximal tubular

cel ls . J . Membr. B io l. 93 :23 7-2 47 ; 1986 .4. Guil lot , F. L.; Audus, K. L.; Raub, T . J . Fluid-ph ase endocytosis by pri-mary cultures o f bovine brain m icrovessel endothelial cell monolayers.Microvasc. Res . 39 :1- 14 ; 1 990.5. Hjelle, J . T.; Oparil, S.; Pete rson , D. R. Subceltular si tes of insulin hydroly-s i s in renal proximal tubules. Am. J . Phys io l . 246 :F40 9-F 416 ; 1984 .6. Ke mps on, S. A.; Helmle, C.; Abraham, M. I., et al. Parathyroid ho rmo ne

action on phosphate transport is inhibited by high osmolali ty. Am. J.P h y si ol . 2 5 8 : F 1 3 3 6 -F 1 3 4 4 ; 1 9 9 0 .7 . Kem pson, S . A.; Helmle, C . ; Murer, H. E ndocytos is and phospha te t rans-p o r t i n O K ep i t h e l ia l c e ll s . R en a l P h y s i o l . B i o ch em . 1 2 : 3 5 9 - 3 6 4 ;1 9 8 9 .8. Kem pson, S. A.; Kunkler, K. J.; Murer, H . Iodoacetate action on i luid-phase endocytosis in OK cells. Physiologist 32:212; 1989 (Abstract).9. Kem pson, S. A.; McAteer, J . A.; AI-Mahrouq, H., et al . Proximal tubulecharacteristics of cultured hum an rena l cortex epithelium. J. Lab. Clln.M ed . 1 1 3 : 2 8 5 -2 9 6 ; 1 9 8 9 .

1 0 . K em p s o n , S . A . ; Y in g , A . L . ; M cA t ee r , J . A . , e t a l . E n d o cy t o s i s an dNa+ /so lu te c o t ran spor t in rena l ep i thel i a l cei l s . J . B iol . Chem.2 6 4 : 1 8 4 5 1 - 1 8 4 5 6 ; 1 9 8 9 .1 t . Koyama, H .; Goedpasture, C.; Miller, M. M., et al. Establishment andcharacterization o f a celt l ine from the American oposs um Didelphysvirginiana). In V i tro 1 4 : 2 3 9 -2 4 6 ; 1 9 7 8 .12. Maack, T.; Park, C . H.; Camargo, M . J. F. Ren al fi l trat ion, transport , an dmetabolism o f proteins. In: Seldin, D. W.; Giebisch, G., eds. Th e kid-n ey : p h y s i o l o g y an d p a t h o p h y s i o l o g y . N ew Y o rk : R av en P re s s ;1 9 8 5 : 1 7 7 3 - 1 8 0 3 ,13. Malmstrom, K.; Murer, H. Parathyroid horm one inhibits phos phate trans-por t in OK cel l s bu t no t in LLC-PK1 and JTC12.P3 cel l s . Am. J .P h ys io l. 2 5 1 : C 2 3 -C 3 1 ; 1 9 8 6 .14. Malmstrom, K .; Stange, G.; Murer, H . Identification of proximal tubulartransport functions in the established kidney cell l ine, OK. Biochim.B i op h y s. A c t a 9 0 2 : 2 6 9 -2 7 7 ; 1 9 8 7 .15. Maranto, A. R. Neuronal mapping: a photooxidation reaction make s luciferyel low usefu l for e lect ron microscopy. Science 21 7:95 3-9 55; 1982.

16. Pollock, A. S.; Warnock, D. G.; Strewler, G. J. Parathyroid h orm one inhibi-t ion of Na+-H+ antiporter activity in a cultured rena l cell l ine. Am. J.P h y si ol . 2 5 0 : F 2 1 7 -F 2 2 5 ; 1 9 8 6 .17 . Quamme, G .; B iber , J . ; Muter , H. Sodium-phosphate c o t ranspor t in OKcel ls : inh ib it ion by PTH a nd 'adaptat ion to low phosphate . Am. J .P h y si ol . 2 5 7 : F 9 6 7 - F 9 7 3 , 1 9 8 9 .18. Rabkiu, R .; Yagil , C.; Frank, B. Basolateral and apical binding, internaliza-t ion, and degradation of insulin by cultured kidney epithelial cells. Am.J . P hy s io l . 2 5 7 : E 8 9 5 -E 9 0 2 ; 1 9 8 9 .

19. Steinman, R . M.; Mellman, I. S.; Muller, W . A., et al . Endocytosis and therecycling of p lasma m embran e. J . Cell B io l . 96 :1-2 7; 1983.

20. Van Bonsdorf, C.-H.; Fuller, S. D.; Simons, K. Apical and basolateralendocytosis in Madin-Darby canin e kidney (MDCK ) cells grown on ni-t rocel lu lose f il t ers . EM BO J . 4 :278 1-2 79 2; t98 5 .21. Yamagu chi, T.; Fukase, M .; Nishikawa, M., et al . Parathyroid horm onedegradation by chymotrypsin-l ike endopep tidase in the opossum kidneycel l. Endocr inology 123:28 12- 281 7; 1988.

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We ar e indebted to Dr. M. H. Montrose Johns Hopkins University, Baltimore) and Prof. H. Murer University ofZurich, Switzerland) for helpful discussions and suggestions during d evelopm ent of the methodology describ ed here.M. I. A. was sup ported b y a P ostdoctoral Fellowship from the American Hea rt Association, Indiana Aifihate Inc.S. A. K. was supported b y a R esearch Career Development Award and grant DK 321 48 from the Nat iona l Ins ti tu tesof H eal th .

1 Perk in-Elmer Corp . , Norwalk , CT2 Beckman Instrum ents Inc., Spinco Division, Palo Alto, CA3 Brinkmau Ins t rum ents Inc. , Wes tbury , NY4 Denvil le Scientific Inc., Denvil le, NJ5 Janke & Kunke l GmbH., S taufen , W . Germany

6 Molecular P robes Inc. , Eugene, OR7 Sigma Chem ical Co., St . Louis, MOs Fisher Scientific, Pit tsburgh, PA9 Sars ted t Inc. , P r inceton , NJ