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Life Sciences, Vol. 65, No. 11, pp. 1115-1124, 1999CopyiSM 0 1999 Elsevia Science Inc.Printed n the USA. All rights rewed
ELSEVIER PII SOO24-3205 99)00345-S 0024_32OS/99/ see front matter
ENDOCYTOSIS OF GENTAMICIN IN A PROXIMAL TUBULAR RENAL CELL LINEGiuliana Decorti, Noelia Malush*, Gabriele Furlan*, Luigi Candussio, Fiora Bartoli Klugmann
Department of Biomedical Sciences and *Toxicology Laboratory, University of Trieste, Trieste,Italy.
(Received n final form May 6, 1999)
SummaryThe mechanisms by which aminoglycosides are accumulated in renal proximaltubular cells remain unclear. Adsorptive mediated endocytosis, via a commonpathway for cationic proteins, or receptor endocytosis, mediated by theglycoprotein 3301 megalin, have been proposed to be involved in gentamicintransport in renal cells. We used the LLC-PKr cell line, derived from the pigproximal tubule, to explore I&her the regulation of gentamicin endocytosis in thesecells and to determine the role of clathrin mediated endocytosis and G proteins inthis function. Gentamicin endocytosis was strictly temperature dependent, whereastotal uptake (endocytosis plus binding) did not significantly differ at 4 or 37 OC.Substances that suppress receptor mediated, clathrin dependent endocytosis, suchas monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediatedendocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PKi cells.Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that isactive also on fluid phase endocytosis, significantly reduced the intracellularconcentrations of the aminoglycoside. Other maneuvers that perturb clathrindependent endocytosis without affecting clathrin independent pathway, such asacidification of cytosol or incubation in hypertonic medium, were also withouteffect. Mastoparan, a well known stimulator of heterotrimeric G proteins, stronglyincreased endocytosis of gentamicin, and the same effect was evident with twoother G protein stimulators, aluminum fluoride and fluoride alone; however theeffect seems not to be mediated by an activation of adenylyl cyclase. In conclusion,gentamicin endocytosis in LLC-PKi cells is probably clathrin independent, limitedby cytochalasin D, which interacts with cytoskeleton, and increased by substanceslike mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.
Key ords:entamicin, renal proximal tubular cells, LLC-PK, cell line, clathrin dependent endocytosis, clathrinindependent endocytosis, adsorptive endocytosis, mastoparan
Endocytosis is a property of eukariotic cells whereby components of the extracellular medium aretaken up in membrane bound vesicles. In the renal proximal tubular cells endocytosis is extremelyactive, 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 tointernalize molecules in comparison with receptor mediated endocytosis (3). In addition it has beenCorresponding Author: G.Decorti, M.D., Via L. Giorgieri n7, I-34100 Trieste, Italy; Telephone:39 40 6767949; Fax 39 40 577435; E-mail [email protected]
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1116 Gentamicin Endocytosis in LLC-PK, Cells Vol. 65, No. 11, 1999
suggested that endocytosis of luid and surface adsorbed proteins is clathrin independent, whereasreceptor mediated endocytosis initiated at coated pits is clathrin mediated (4). Most endocytic sitesin proximal tubular cells are clathrin coated pits (5) however there is increasing evidence forclathrin independent pathways, mediated by caveolae or non coated vesicles (6).Gentamicin is an aminoglycoside antibiotic that is transported and accumulated in the renalproximal tubular cells. The transport of gentamicin and other aminoglycosides in the kidney hasbeen extensively studied, but the exact mechanism remains controversial. Several studies havesuggested that gentamicin interacts with brush border membrane and is subsequently taken up by anadsorptive endocytosis, via a common pathway for cationic proteins such as lysozime (7, 8). Theinitial step of adsorptive endocytosis is binding of the molecule to a binding site on the apicalplasma membrane, triggered by an electrostatic interaction between the positively charged moietyof 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 cellsby receptor mediated endocytosis and have identified glycoprotein 330Imegalin as a multiligandendocytic receptor that mediates endocytosis of several polybasic drugs.On proximal tubular cell apical membranes large quantities of G proteins have been identified; thefunctions of these proteins are not immediately apparent, and it has been suggested that they mayhave a regulatory role in endocytosis (10). The various steps of clathrin mediated and clathrinindependent endocytosis seem to be dependent on GTP-binding proteins (11) and heterotrimeric Gproteins 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 modelsystem, as it has been shown that these cells, derived from the pig proximal tubule (12) areextremely useful for elucidating transport mechanisms of solutes at the cellular level
MethodsCell 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 withoutantibiotics under an atmosphere of 95 % air and 5 % CO, at 37 C, and subcultured twice weeklyusing 0 02 % EDTA and 0.05 % trypsin The cells were used in the passages 199-230 Forexperiments, 100 mm dishes were inoculated with 2 x IO4 cells/ml in 10 ml of complete culturemedium. The uptake of gentamicin was measured on confluent cells, on the 5th day afterinoculationAfter removal of the culture medium, each dish was washed twice with D-PBS at 4 C, then cellswere preincubated in incubation buffer containing the inhibitors (141 mM NaCl, 4 rnh4 KCI, 2.8mM CaC12, 1 mM MgS04, IO mM HEPES, 10 mM D-glucose, 0.1 bovine serum albumin, pH7 4) at 37 C for predetermined experimental times A solution of gentamicin was then added in thepresence or absence of various compounds At the end of the incubation period, the medium wasremoved by suction and the dish was rinsed 3 times with ice cold D-PBS buffer. To estimate totalgentamicin binding, cells were immediately scraped with a rubber policeman into 2 ml of ice coldsaline and the dishes were then rinsed again with 4 ml of ice cold saline to improve the recovery ofcells As it has been reported (8) that, to evaluate the intracellular uptake (endocytosis) ofgentamicin the cells should be incubated for 30 min at 37 C in the absence of gentamicin, atIeruptake 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 obtainedfrom both procedures, were centrifuged at 4 C for 5 min. at 150 xg. The supematants wereaspirated and the cell pellet was resuspended gently in 6 ml of ice cold PBS buffer and centrifugedagain. The final pellet was resuspended in 0 5 ml of PBS and the cells were homogenized with a
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Vol. 65, No. 11, 1999 Cientamicin Endccytosis in LLC-PK, Cells 1117
sonicator. Gentamicin concentrations were determined in the whole homogenate by animmunoenzymatic technique (EMIT AMD, Bracco, Italy).Protein concentration of samples was determined by the method of Lowry et al. (13). with bovineserum 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 werepurchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were of analytical grade.
z 0.6Lg 0.5P2 0.4
t.S 0.3.,ogz 0.2Em 0.1
-0 10 20 30 40 50 60time min)
0.14 -0.12 -0.10 -0.08 -0.06 -0.04 -
0 10 20 30 40 50 60
0.6time min)
gentamicin mM
Fig 1EfTect of temperature on total binding (A) and endocytosis (B) of gen?amicin in LLC-PKrcells. Cells were incubated with 1 mg/ml of gentamicin in buffered solution for up to 60min. at 37 C (0) or 4 C (0). A. At the stated times, dishes were washed with ice-coldD-PBS and total gentamicin uptake was determined. B To evaluate gentamicinendocytosis, dishes were then incubated in drug-free buffered solution at 37 C for 30additional min. C. Gentamicin uptake for 10 min. at concentrations between 1 and 20mg/ml was determined at 37 C (0) or 4 C (a). The osmolarity of the solution was keptconstant by adding an adequate concentration of mannitol. After incubation, dishes wereprocessed as in B. Each point represents mean + SE of data from three to six wells
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1118 Gentamicin Endocytosis in LLC-PK, Cells Vol. 65, No. 11, 1999
Averages f SE of the means were calculated, statistical evaluation of results was carried out usinganalysis of variance (ANOVA) followed by Bonferronis adjustment. Values of p < 0.05 wereconsidered significant
ResultsFigure 1 A and B show the effect of low temperature on the total and intracellular uptake. Bothtotal binding and uptake increased with time The total uptake of gentamicin was decreased onlyslightly by lowering the temperature, and, till 60 min . there was not significant difference betweenthe 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 drugconcentration and the initial gentamicin uptake (10 min ) Even at higher concentrations,gentamicin endocytosis was not saturated
80-
60-
Fig. 2Effect of drugs that affect clathrin-dependent endocytosis, monensin 2.5 uM (rising leftlines), phenylarsine oxide 250 uM (horizontal lines), dansylcadaverine 500 uM (verticallines) and caveolae-mediated endocytosis, nystatin 5 &ml (diagonal cross hatch) ongentamicin (empty bar) endocytosis in LLC-PK, cells. The cells were preincubated in buffercontaining the inhibitors for 30 min., then in buffer containing gentamicin 1 ms/rni and theinhibitors for 60 additional min. Dishes were then washed and incubated in drug-free bufferat 37 C for 30 additional min Control value 0 126 -fr 0 013 ug/mg protein. Each barrepresents mean I? SE of data from three to six wells
In the experiments with endocytosis inhibitors, the endocytosis of gentamicin was not affected bysubstances 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 Band D, nocodazole and colchicine As shown in figure 3, cytochalasin D significantly reduced theuptake of gentamicin, whereas the effect of cytochalasin B was much less evident and notsignificant Colchicine and nocodazole, on the contrary, were without effect.
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Vol. 65 No. 11 1999 Gentamicin Endocytosis in LLC-PK Cells 1119
Fig. 3Effect of cytochalasin B 10 ug/ml (rising left lines), cytochalasin D 5 ug/ml (horizontallines), 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 inbuffer containing the inhibitors for 30 min., then in buffer containing gentamicin 1 mg/mland the inhibitors for 60 additional min. Dishes were washed and incubated in drug-freebuffer at 37 C for 30 additional min. Control value: 0.108 f 0.005 ug/mg protein. Eachbar represents mean f SE of data from three to six wells. **: p< 0.01.
0.071
5 10 15 20acetic acid mM)
0.6-
0.0 I0.5 1.0sucrose M)
Fig. 4Effect of cytosolic acidification and of medium hypertonicity on gentamicin endocytosis at37 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. Gentamicin1 mg/ml was then added and the incubation continued for 60 additional min. Dishes werewashed and incubated in drug-free buffer at 37 C for 30 additional min. Each pointrepresents mean f SE of data from three experiments *- p < 0.05, **: p < 0.01.
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112 Gentamicin Endocytosisin LLC-PK, Cells Vol. 65, No. 11, 1999
Acidification of cytosol or medium hypertonicity prevent the formation of functional clathrin cagesand 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 reduceduptake, gentamicin was still endocytosed in LLC-PKr cells when clathrin mediated endocytosis wasblocked. It should be noted that gentamicin endocytosis in a medium with pH 5 5, devoid of aceticacid, is significantly lower than that observed at pH 7.4 The low value obtained with 20 mM aceticacid 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 thesame conditions, the uptake of gentamicin did not, surprisingly decrease, but increased significantly(fig. 4B)
1 0,
ii0, )0.0 1 I
0 25 50mastoparan (IAM)
Fig. 5Effect of mastoparan on gentamicin endocytosis at 37 C and binding at 4 C in LLC-PKrcells Cells were preincubated in buffer containing mastoparan for 30 min. at 37 C, thengentamicin 1 m iml was added and the incubation continued at 37 or 4 C (insert;gentamicin alone. empty bar, gentamicin + mastoparan SO PM. rising left lines) for 60additional min Dishes were washed and incubated in drug-free buffer at 37 C for 30additional min Each point represents mean + SE of data from three to six we ls **. p
