ELSEVIER

PII SOO24-3205(99)00345-S

Life Sciences, Vol. 65, No. 11, pp. 1115-1124, 1999 CopyiSM 0 1999 Elsevia Science Inc. Printed in the USA. All rights rewed 0024_32OS/99/&see front matter

ENDOCYTOSIS

OF GENTAMICIN

IN A PROXIMAL TUBULAR RENAL CELL LINE

Giuliana Decorti, Noelia Malush*, Gabriele Furlan*, Luigi Candussio, Fiora Bartoli Klugmann Department of Biomedical Sciences and *Toxicology Laboratory, University of Trieste, Trieste, Italy. (Receivedin final form May 6, 1999)

Summary The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 3301 megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PKr cell line, derived from the pig proximal tubule, to explore I&her the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 OC. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PKi cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PKi cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins. Key Words: gentamicin, renal proximal tubular cells, LLC-PK, cell line, clathrin dependent endocytosis, clathrin independent endocytosis, adsorptive endocytosis, mastoparan Endocytosis is a property of eukariotic cells whereby components of the extracellular medium are taken up in membrane bound vesicles. In the renal proximal tubular cells endocytosis is extremely active, but the endocytotic pathways followed by renal transporters are not clearly defined (1, 2); both fluid phase endocytosis of substances such as Lucifer Yellow and Horseradish peroxidase, adsorptive endocytosis of proteins and receptor mediated endocytosis have been described. Adsorptive mediated endocytosis is characterized by a lower affinity and higher capacity to internalize molecules in comparison with receptor mediated endocytosis (3). In addition it has been Corresponding Author: G.Decorti, M.D., Via L. Giorgieri n7, I-34100 Trieste, Italy; Telephone: 39 40 6767949; Fax 39 40 577435; E-mail decorti@univ.trieste.it

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suggested that endocytosis offluid and surface adsorbed proteins is clathrin independent, whereas receptor mediated endocytosis initiated at coated pits is clathrin mediated (4). Most endocytic sites in proximal tubular cells are clathrin coated pits (5) however there is increasing evidence for clathrin independent pathways, mediated by caveolae or non coated vesicles (6). Gentamicin is an aminoglycoside antibiotic that is transported and accumulated in the renal proximal tubular cells. The transport of gentamicin and other aminoglycosides in the kidney has been extensively studied, but the exact mechanism remains controversial. Several studies have suggested that gentamicin interacts with brush border membrane and is subsequently taken up by an adsorptive endocytosis, via a common pathway for cationic proteins such as lysozime (7, 8). The initial step of adsorptive endocytosis is binding of the molecule to a binding site on the apical plasma membrane, triggered by an electrostatic interaction between the positively charged moiety of the molecule, and the negatively charged plasma membrane surface region On the contrary, other authors (9) have suggested that gentamicin is internalized in the proximal renal tubular cells by receptor mediated endocytosis and have identified glycoprotein 330Imegalin as a multiligand endocytic receptor that mediates endocytosis of several polybasic drugs. On proximal tubular cell apical membranes large quantities of G proteins have been identified; the functions of these proteins are not immediately apparent, and it has been suggested that they may have a regulatory role in endocytosis (10). The various steps of clathrin mediated and clathrin independent endocytosis seem to be dependent on GTP-binding proteins (11) and heterotrimeric G proteins are known to be involved in regulation of vesicle formation and transport in general In this context we decided to study the regulation of gentamicin endocytosis in renal tubular cells, and to determine the role of G proteins in this function We used the LLC-PKI cell line as a model system, as it has been shown that these cells, derived from the pig proximal tubule (12) are extremely useful for elucidating transport mechanisms of solutes at the cellular level Methods Cell cultures LLC-PK, cells obtained from the American Type Culture Collection (Rockville, MD) (ATCC-CRL-1392) were grown in medium 199 containing 3 % fetal bovine serum without antibiotics under an atmosphere of 95 % air and 5 % CO, at 37 C, and subcultured twice weekly using 0 02 % EDTA and 0.05 % trypsin The cells were used in the passages 199-230 For experiments, 100 mm dishes were inoculated with 2 x IO4 cells/ml in 10 ml of complete culture medium. The uptake of gentamicin was measured on confluent cells, on the 5th day after inoculation After removal of the culture medium, each dish was washed twice with D-PBS at 4 C, then cells were preincubated in incubation buffer containing the inhibitors (141 mM NaCl, 4 rnh4 KCI, 2.8 mM CaC12, 1 mM MgS04, IO mM HEPES, 10 mM D-glucose, 0.1 % bovine serum albumin, pH 7 4) at 37 C for predetermined experimental times A solution of gentamicin was then added in the presence or absence of various compounds At the end of the incubation period, the medium was removed by suction and the dish was rinsed 3 times with ice cold D-PBS buffer. To estimate total gentamicin binding, cells were immediately scraped with a rubber policeman into 2 ml of ice cold saline and the dishes were then rinsed again with 4 ml of ice cold saline to improve the recovery of cells As it has been reported (8) that, to evaluate the intracellular uptake (endocytosis) of gentamicin the cells should be incubated for 30 min at 37 C in the absence of gentamicin, atIer uptake procedures, the cells were incubated in drug free saline solution for 30 additional min., and, only after this procedure, the monolayers were scraped and processed as above. The cells obtained from both procedures, were centrifuged at 4 C for 5 min. at 150 xg. The supematants were aspirated and the cell pellet was resuspended gently in 6 ml of ice cold PBS buffer and centrifuged again. The final pellet was resuspended in 0 5 ml of PBS and the cells were homogenized with a

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sonicator. Gentamicin concentrations were determined immunoenzymatic technique (EMIT AMD, Bracco, Italy).

in the

whole

homogenate

by

an

Protein concentration of samples was determined by the method of Lowry et al. (13). with bovine serum albumin as the standard. Chemicals: Gentamicin, monensin, phenylarsine oxide, dansylcadaverine, nystatin, cytochalasin B, cytochalasin D, colchicine, nocodazole, mastoparan, forskolin, cholera toxin and 8-Br-CAMP were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were of analytical grade.

0.14 z 0.6 L g 0.5 P 2 0.4 t .S .,o gz 0.2 0.3 0.12 0.10 0.08 0.06

0.04 -

E m 0.1

-0

10

20

30

40

50

60

10

20

30
time

40
(min)

50

60

time (min)

0.6

gentamicin

mM

Fig. 1 EfTect of temperature on total binding (A) and endocytosis (B) of gen?amicin in LLC-PKr cells. Cells were incubated with 1 mg/ml of gentamicin in buffered solution for up to 60 min. at 37 C (0) or 4 C (0). A. At the stated times, dishes were washed with ice-cold D-PBS and total gentamicin uptake was determined. B To evaluate gentamicin endocytosis, dishes were then incubated in drug-free buffered solution at 37 C for 30 additional min. C. Gentamicin uptake for 10 min. at concentrations between 1 and 20 mg/ml was determined at 37 C (0) or 4 C (a). The osmolarity of the solution was kept constant by adding an adequate concentration of mannitol. After incubation, dishes were processed as in B. Each point represents mean + SE of data from three to six wells

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Averages f SE of the means were calculated, statistical evaluation of results was carried out using analysis of variance (ANOVA) followed by Bonferronis adjustment. Values of p < 0.05 were considered significant Results Figure 1 A and B show the effect of low temperature on the total and intracellular uptake. Both total binding and uptake increased with time The total uptake of gentamicin was decreased only slightly by lowering the temperature, and, till 60 min . there was not significant difference between the total uptake measured at 4 and 37 C (fig I A) In contrast, as shown in figure 1 B, intracellular uptake for 60 minutes decrease markedly at 4 C compared with that at 37 C, showing a marked effect of temperature Figure IC shows the relationship between drug concentration and the initial gentamicin uptake (10 min ) Even at higher concentrations, gentamicin endocytosis was not saturated

8060-

Fig. 2 Effect of drugs that affect clathrin-dependent endocytosis, monensin 2.5 uM (rising left lines), phenylarsine oxide 250 uM (horizontal lines), dansylcadaverine 500 uM (vertical lines) and caveolae-mediated endocytosis, nystatin 5 &ml (diagonal cross hatch) on gentamicin (empty bar) endocytosis in LLC-PK, cells. The cells were preincubated in buffer containing the inhibitors for 30 min., then in buffer containing gentamicin 1 ms/rni and the inhibitors for 60 additional min. Dishes were then washed and incubated in drug-free buffer at 37 C for 30 additional min Control value 0 126 -fr 0 013 ug/mg protein. Each bar represents mean I? SE of data from three to six wells In the experiments with endocytosis inhibitors, the endocytosis of gentamicin was not affected by substances that suppress receptor mediated, clathrin dependent endocytosis such as monensin, phenylarsine oxide, or dansylcadaverine or inhibit caveolae mediated endocytosis such as nystatin (fig 2). To determine the possible role of actin cytoskeleton and of microtubules, we used cytochalasin B and D, nocodazole and colchicine As shown in figure 3, cytochalasin D significantly reduced the uptake of gentamicin, whereas the effect of cytochalasin B was much less evident and not significant Colchicine and nocodazole, on the contrary, were without effect.

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Fig. 3 Effect of cytochalasin B 10 ug/ml (rising left lines), cytochalasin D 5 ug/ml (horizontal lines), nocodazole 6 @ml (vertical lines), and colchicine 20 up/ml (diagonal cross hatch) on gentamicin (empty bar) endocytosis in LLC-PKr cells. The cells were preincubated in buffer containing the inhibitors for 30 min., then in buffer containing gentamicin 1 mg/ml and the inhibitors for 60 additional min. Dishes were washed and incubated in drug-free buffer at 37 C for 30 additional min. Control value: 0.108 f 0.005 ug/mg protein. Each bar represents mean f SE of data from three to six wells. **: p< 0.01.

0.071

0.6-

5 acetic

10 acid

15 (mM)

20

0.0

I
0.5 sucrose 1.0 (M)

Fig. 4 Effect of cytosolic acidification and of medium hypertonicity on gentamicin endocytosis at 37 C (0) or 4 C (0). A. Cells were preincubated at pH 5.5 with acetic acid for 15 mm. B. Cells were preincubated in buffer containing sucrose for 30 min. A and B. Gentamicin 1 mg/ml was then added and the incubation continued for 60 additional min. Dishes were washed and incubated in drug-free buffer at 37 C for 30 additional min. Each point represents mean f SE of data from three experiments *- p < 0.05, **: p < 0.01.

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Acidification of cytosol or medium hypertonicity prevent the formation of functional clathrin cages and inhibit clathrin dependent endocytosis but have less effect on clathrin independent endocytosis. In figure 4A the effect of acidification of the cytosol is presented; although there was a reduced uptake, gentamicin was still endocytosed in LLC-PKr cells when clathrin mediated endocytosis was blocked. It should be noted that gentamicin endocytosis in a medium with pH 5 5, devoid of acetic acid, is significantly lower than that observed at pH 7.4 The low value obtained with 20 mM acetic acid should hence be compared to the value obtained at 4 C, in the same experimental conditions. When the osmolarity of the incubation medium was increased from 300 to 1400 mOsmo1 under the same conditions, the uptake of gentamicin did not, surprisingly decrease, but increased significantly (fig. 4B)

1 0,

0,
ii

0.0
0

25 mastoparan Fig. 5 (IAM)

50

Effect of mastoparan on gentamicin endocytosis at 37 C and binding at 4 C in LLC-PKr cells Cells were preincubated in buffer containing mastoparan for 30 min. at 37 C, then gentamicin 1 mgiml was added and the incubation continued at 37 or 4 C (insert; gentamicin alone. empty bar, gentamicin + mastoparan SO PM. rising left lines) for 60 additional min Dishes were washed and incubated in drug-free buffer at 37 C for 30 additional min Each point represents mean + SE of data from three to six we!ls **. p< 001 Mastoparan, a peptide found in wasp venom, is a well known stimulator of heterotrimeric G proteins We therefore tested whether this compound had any effect on endocytosis of gentamicin. As shown in figure 5, mastoparan strongly stimulated endocytosis of gentamicin, but preincubation of cells with mastoparan at 37 C had no effect on the binding of gentamicin at 4 C (insert). Another stimulator of heterotrimeric G proteins is aluminum fluoride, and we show that also this substance and fluoride alone increased significantly the uptake of gentamicin (fig 6). To test whether the effect of mastoparan and aluminum fluoride on gentamicin endocytosis was mediated by an activation of adenylyl cyclase we measured endocytosis of gentamicin after addition of forskolin and cholera toxin, but these compounds had no effect on gentamicin endocytosis (fig. 6) Also the direct addition of a membrane permeant CAMP analogue, %Br-CAMP, was without effect (fig 6)

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200 180 160 140 120 100 80 60 40 20 0

Fig. 6 Effect of NaF 10 mM + AlClj 50 pM (rising left lines), NaF 10 mM (horizontal lines), cholera toxin 1 pg/ml (vertical lines), forskolin 100 PM (diagonal cross hatch), and %BrCAMP 1 mM (rising right lines) on gentamicin (empty bar) endocytosis in LLC-PK, cells. The cells were preincubated in buffer containing the inhibitors for 30 min., (except cholera toxin that was preincubated for 4 hr), then in buffer containing gentamicin 1 mg/ml and the inhibitors for 60 additional min. Dishes were washed and incubated in drug-free buffer at 37 C for 30 additional min Control value: 0 114 + 0 008 pg/mg protein. Each bar represents mean f SE of data from three experiments. * p< 0.05 Discussion Aminoglycoside antibiotics are widely used in the treatment of Gram negative infection diseases but their use is often complicated by their nephrotoxicity These agents are mainly eliminated by glomendar filtration and are taken up by proximal renal tubular cells (14). Due to their amino side chains, the aminoglycosides are polybasic at physiological pH, do not cross biological membranes by simple diffusion and hence do not enter in most cells It has been suggested that gentamicin and other aminoglycosides enter renal tubular cells by means of endocytosis, but the exact biochemical mechanisms remain controversial. It has been suggested that gentamicin interacts with the brush border membrane and is subsequently taken up by an adsorptive endocytosis (7, 8), but other authors have identified glycoprotein 330/megalin and have suggested that this is a multiligand endocytic receptor that mediates endocytosis of gentamicin and other polybasic drugs (9) In the present study, we tried to characterize the properties and characteristics of endocytosis of gentamicin in the LLC-PK, cells; several authors have indeed shown that this cell line serves as a suitable model for studying endocytosis in the renal proximal tubule (12). Low temperature (4 C) is the most effective and non invasive method to inhibit various transport processes such as endocytosis and carrier mediated transport The intracellular uptake of gentamicin by LLC-PK1 cells was inhibited markedly by low temperature, whereas total uptake of gentamicin at 4 C for 60 minutes was similar to that at 37 C; it is hence essential to estimate the intracellular uptake of gentamicin separately from the surface binding. Gentamicin uptake in LLCPKI cells was not saturable, suggesting therefore a fluid phase endocytotic process. The effects of various substances that interfere with the endocytic processes have been therefore tested on intracellular uptake of gentamicin. In particular we have used substances that inhibit the clathrin dependent, receptor mediated endocytosis initiated at coated pits, but have less effect on the

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clathrin-independent endocytosis of fluid and surface-adsorbed substances Monensin, which prevents the reaching of a sufficiently low pH in the endosomes, thus blocking the split of ligandreceptor coupling (1 S), dansylcadaverine which blocks the formation of coated pits by inhibiting the transglutaminase activity in cell membranes (16) and phenylarsine oxide that inhibits receptor mediated endocytosis of growth factor ( 17) and insulin (I 8) were all without effect. On the other side, nystatin, a sterol binding drug that inhibits the caveolae pathway (19), under our experimental conditions was also without effect Literature data (15-19) show that all these substances are effective in inhibiting clathrin- or caveolae-dependent endocytosis in several ceil types, and the dosages employed in this study were highest dosages used by other authors The actin cytoskeleton is extremely important for maintaining cell polarity and shape, and for transporting vesicles, and disassembly of cytoskeleton prevents the formation of coated pits and coated vesicles In proximal tubular cells, actin is finthermore an important constituent of the brush border membrane We have therefore tested the effect of cytochalasin B which disassembles actin microfilaments (20) and prevents the apical formation of vesicles in epithelial cells (2 I) At the dose of IO pg/ml cytochalasin B had no efTect on gentamicin endocytosis. Our data are in contrast with those of Hori et al (8) who found that cytochalasin B (50 pg/ml) significantly reduced the endocytosis of gentamicin This discrepancy is probably due to lower dose of cytochalasin we used, indeed in our experimental model, SO lg/ml was a highly cytotoxic dose and hence could not be used We investigated therefore the effect of cytochalasin D, and this substance significantly limited gentamicin endocytosis Blok et al (22) have indeed shown that cytochalasin D inhibits fluid phase endocytosis as well as clathrin and caveolae dependent endocytic routes, whereas cytochalasin B has hardly any effect on fluid phase endocytosis Colchicine, which disrupts microtubules along which vesicles move in the cytoplasm (2 1) and nocodazoie, which inhibits microtubule polymerization (23) were also without effect Interestingly, in lymphoid cells, nocodazole reduces internalization of transferrin. a marker of receptor mediated endocytosis, but not the clathrin independent endocytosis of interleukin 2 (24) It is possible to perturb the uptake from clathrin coated pits without affecting clathrin independent endocytosis The first method is acidification of cytosol (25), that in many cell types, including MDCK cells, blocks the formation of coated vesicles from coated pits and hence completely inhibits clathrin dependent endocytosis In our study, acidification of cytosol caused a reduced uptake, but gentamicin was still endocytosed from LLC-PKI cells suggesting that, similarly to what could be seen in MDCK cells (26) there is also a clathrin independent endocytosis. Indeed, it is interesting to note that acidification of cytosol completely blocks the endocytosis of transferrin, which is clathrin dependent Another way to interfere with clathrin mediated endocytosis is incubation in hypertonic medium (27) Hypertonic medium interferes with the formation of clathrin triskelion lattice, which is required for the endocytosis of ligands via the clathrin coated pit pathway, and also this maneuver blocks receptor mediated but not fluid phase endocytosis (28) Gentamicin endocytosis was not reduced, but, surprisingly, was significantly increased after incubation in hypertonic medium. It is however known that carbohydrates like sucrose and mannitol induce ultrastructural alterations such as an increase in the frequency and size of apical endocytic vacuoles in the proximal tubular cells (29) In proximal tubular cells large quantities of G proteins are found in the apical membranes and it has been suggested that these proteins may have a regulatory role in endocytosis (10). Therefore we have studied the effect of mastoparan, a peptide found in wasp venom, which is a well known stimulator of heterotrimeric G proteins (30) on the endocytosis of gentamicin in LLC-PKI cells. This substance stimulates ricin endgytosis, but strongly inhibits the clathrin dependent endocytosis of transferrin in the MCDK renal cell line and Authors have therefore suggested that the increased uptake of ricin be mediated by a clathrin independent mechanism (26)

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Another stimulator of heterotrimeric G proteins, aluminum fluoride (3 1) also stimulated endocytosis of gentamicin in the LLC-PKr ceils. This compound was previously shown to stimulate also apical endocytosis of ricin in the MDCK cells (26). In addition, Fe alone, that also stimulates endocytosis of ricin in MDCK and of gentamicin in the LLC-PKi cells, has been previously shown to increase the level of CAMP in cell lysates containing adenylyl cyclase (32) and in MDCK cells (26) In a number of studies were heterotrimeric G proteins are thought to influence membrane traffic, the mediator of the observed effect is not known In the MDCK cells, mastoparan did not increase the level of CAMP (26) but it should be noted that mastoparan is a more potent stimulator of Gi than of G, (10, 33). To test whether the increase in gentamicin endocytosis was mediated by an activation of adenylyl cyclase, we measured endocytosis of gentamicin after addition of forskolin, an activator of adenylyl cyclase, and cholera toxin which ADP rybosilates the a, subunit of heterotrimeric G proteins and thereby activates adenylyl cyclase (34, 35) but none of these compounds stimulates gentamicin endocytosis. Also the addition of S-Br-CAMP, a membrane permeant CAMP analogue, did not stimulate gentamicin endocytosis. It seems therefore that mastoparan and aluminum fluoride affect endocytosis by a different mechanism, not mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PKi cells is probably clathrin independent; indeed, it is not modified by substances that inhibit endocytosis by modifying the functions of clathrin coated vesicles Among substances interacting with the cytoskeleton, only cytochalasin D, which inhibits also fluid phase endocytosis, is active Mastoparan and aluminum fluoride, that activate heterotrimeric G proteins, increase gentamicin endocytosis in these cells, hn-ther suggesting a clathrin independent mechanism.

Acknowledgments This research was supported by grants from the Minister0 Universith e Ricerca Scientifica e Tecnologica 60% (Universities of Trieste and Udine) and 40% (M.U.R.S.T. Targeted Project New Assessment Approaches in Toxicology), and C.N.R, Progetto Finalizzato A C.R.O., contract no 94.01138.PF39 References S.A. KEMPSON, A.L. YTNG, J.A. MCATEER AND H. MURER J. Biol. Chem. 264 1845 l18456 (1989). 2 S A KEMPSON, C HEMLE AND H. MURER, Renal Physiol. Biochem. 12 359-364 (1989). 3. I TAMAI and A TSUJI, Adv. Drug Deliv. Rev. 19 401-424 (1996). 4 P E STRBMHAUG, T.O. BERG, T. GJBEN and P.O. SEGLEN, Eur. J Cell Biol. 73 28-39 (1997). 5 T. MAACK, C.H. PARKS and M.J.F. CAMARGO, 7?re Kidney. Physiology and Pathophysiology, D.W. Seldin and G. Giebisch (Eds), 3005-3038, Raven, New York (1992). 6 C. LAMAZE and S. SCHMID, Curr. Opin. Cell Biol. 7 573-580 (1995). 7. M.TAKANO, Y. OHISHI, M. OKUDA, M. YASUHARA and R. HORI, J Pharmacol. Exp. Ther. 288 669-674 (1994). 8. R. HORI, M. OKUDA Y. OHISHI, M YASUHARA and M. TAKANO, J Pharmacol. Exp. Ther 261 1200-1205 (1992) 9 S.K. MOESTRUP, S. GUI, H. VORUM, C BREGENGARD, S.E. BJBRN and K. NORRIS, J. CIin. Invest. 96 1404-1413 (1995). 10 NJ BRUNSKILL, N. COCKCROFT, S. NAHORSKI and J. WALLS, Am J Physiol. 271 1.

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