Diven, WF

Sanghvi, A

Starzl, T

Warty, VS

English

PubMed

D-Scholarship@Pitt

/IJOO Receptor-mediated Cellular Uptake of Cyclosporlne Ajit Sanghvi, Vijay S. Warty, Warren F. Diven, and Thomas Starzl THE PROCESS of cellular uptake and metabolism of cyclosporine (CsA) is poorly understood. Earlier stud-ies in this laboratory indicated that transport of CsA in blood occurs in association with plasma lipoproteins, with low density lipoprotein (LDL) and high density lipoprotein (HOL) carrying about 32% each of the total CsA in blood. This observation suggests the possibility that the cellular uptake of CsA may occur through the LDL receptor path-way. To further identify this phenomenon, we incubated CsA-LOL complex with freshly isolated peripheral blood lymphocytes (PBL) and also with PBL in which LDL receptors synthesis was enhanced by incubation in lipid free serum. Results of these experiments show that cellular uptake of CsA is increased about 25% in cells incubated in lipid-free serum relative to controls. This increased uptake of CsA could be in part due to the proliferation of LDL recep-tors in the stimulated cells. Since at least 32% of the drug circulates in association with LDL, this pathway provides an important mechanism for the meta bolism of CsA. The aims of this study were to examine the kinetics of binding and uptake of CsA by the human peripheral blood lymphocytes. and the enhancement of CsA uptake by periph-eral blood lymphocytes via LOL receptor pathway. Cyclosporine (CsA) is widely used in transplantation as the immunosuppressive agent of choice. In our earlier stud-ies. we have shown that transport of CsA in blood occurs mainly in association with plasma lipoproteins. 70% of the drug being in LDL and HOL fractions.! This suggests that cellular uptake and metabolism of CsA may. at least in part. follow the lipoprotein metabolism. specifically. the LOL-receptor mediated pathway. MATERIALS AND METHODS Tritiated dihydrocyclosporine A (CsA) (60 mCi/mmol) and CsA standards were obtained from Sandoz. Ltd (Basel. Switzerland) as ethanolic solutions. FK506 was a gift from Fujisawa Pharmaceutical Co. Ltd (Osaka. lapan). Methanolic solution of FK506 was used in the experiments. Culture media and antibiotics were obtained from GIBCO (Grand Island. NY). All other reagents and supplies were from Fisher Scientific Co. Pittsburgh. Human LDL (d - 1.019 to 1.063) was prepared from blood collected in 0.1 % EOT A from healthy subjects who had fasted overnight. Lipoproteins were fractionated by sequential floatation in a Beckman preparative ultracentrifuge (Beckman Instruments. Inc. Fullerton. CAl according to Havel et aLl Protein determinations were performed by the Bradford method. Peripheral blood mononuclear cells were isolated as previously described) from approximately 100 ml of blood obtained from healthy volunteers for each experiment. The monocyte content of these cells was depleted by the method of lerrells et a!. Cell viability was assessed by using the dye exclusion method. LOL (100 I'g in 1 ml RPMI 1640 medium) was added dropwise with stirring to an ethanolic [IHJ CsA solution (50 Ill). The mixture was incubated with constant shaking for one hour at room tempera-ture. The specific activity of the eHJ CsA-LOL complex thus obtained in three separate preparations was 20 ± 0.8 x 10' dpm/Ilg LOL. lymphocytes were incubated in RPMI 1640 medium containing 1 % bovine serum albumin (BSA) in the presence or absence of CsA and/or FK506 for 24 hours. After these preincubations, the cells were washed twice with ice-cold 0.85% NaCI solution and then resuspended in 1% BSA-RPMI medium. The cells were placed in culture wells (10' cells/well) and exposed to increasing molar concentrations of [IHJ CsA (specific activity. 900 to 1, I 00 dpm/ pmol) and equivalent concentrations of cold CsA and FK506. as indicated in the legends to the figures. for one hour at 37°C in the humidified atmosphere of 95% O2:5% CO2, Transport flux was stopped by adding ice-cold saline and cooling the mixture further for ten minutes over ice. The cells were washed three times with cold saline and incubated in 0.2 mL I N KOH for one hour at 70° C. The digested solution was used for the measurement of radioactivity and protein determinations. RESULTS CsA uptake by lymphocytes from two healthy donors was studied by incubating cells with the [lHJ CsA-LDL complex. The kinetics of the uptake of CsA in these experiments are shown in Figure I. panels A and B. In both individuals studied. the CsA uptake appears to reach saturation at an LDL concentration of about 50 Ilg/ml medium. The corre-sponding CsA concentration is about 900 ng/mL. In a previous studyl on the cell surface binding and subsequent metabolism of LOL by T and B lymphocytes. we have observed that the LOL receptor is saturated at 50 Ilg LOL/ml medium. Moreover. [lHJ CsA uptake by lympho-cytes was suppressed in the presence of cold CsA and LOL. Combined. these observations suggest that internalization of lipoprotein-associated CsA may occur via the high-affinity specific receptor for LOL on the cell surface. The experimen-tal conditions used in these studies imply that the CsA measurements shown reflect both the processes of cell sur-face binding and internalization. When the cell content of CsA was compared in an experiment in which two sets of lymphocytes obtained from the same donor were incubated simultaneously with various concentrations of CsA alone or with CsA-LDL complex it was observed that cells incubated with CsA-LOL contained greater amounts of CsA at each of the concentrations studied. Quite importantly. internalization of CsA from CsA-LOL complex during the initial phase was 45% higher From the Departments of Pathology and Surgery. University ot Pittsburgh School ot Medicine. Pittsburgh. Pennsylvania 15261. Address reprint requests to Ajit Sanghvi. PhD. Director of Clinical Chemistry-CLSI. CHP Main Tower. Room 5845.3705 Fifth Avenue at DeSoto Street. Pittsburgh. PA 15213-2583. CI) 1989 by Appleton & Lange. Inc. 0041-1345/89/$3.001 +0 858 Transplantation Proceedings. Vol 21. No 1 (February). 1989: pp 858-859 RE Fi C rE d· rE p I f RECEPTOR·MEDIATED CELLULAR UPTAKE OF CYCLOSPORINE 20C 9 0---- ~oj I "~~ k- ~:: ~ 10 25 50 T~ '00 5 10 25 50 75 100 ~ yKKl~ PROTEIN, il9fml ~TO1K4 16 12 108,44 CYCLOSPORINE nmol" ml ~~K~~D_a ___ mK_l·_b_D~"D~lm_D ____ __ 0'121-44 36 72 108 144 CYC:...CSPORtNE '1."'01" rnl Fig. 1. Cellular uptake of CsA In PBl Incubated with the [IHJ CaA-LDl complex for one hour. The open and closed symbols r.present the total and nonspecific (LDl receptor·lndepen-dent) uptake of [IH] CsA, respectively. Panels A and Bar. r.,ult, In two subjects. relative to control. When the receptor and internalization processes were saturated this difference stabilized at 18% as shown in Figure 2. Cellular CsA uptake in the absence of LOL was studied in lymphocytes from two healthy volunteers (other than for Figs 1 and 2). The results of the experiments shown in Fig 3. panels A and B. indicate that, as in the presence of LOL, (lH]CsA uptake in this instance may also involve a saturable process. DISCUSSION The cellular uptake of CsA appears to involve both specific was well as nonspecific processes. the former taking place when the drug is associated with lipoprotein. In this instance. the CsA-LOL complex may be internalized via the high-affinity. specific cell surface receptor for LOL. RMr---------------------------~ c: 40 Qi 0 ... Q. 30 Qi () QI 20 E C fI () QI 10 c: 0 0 2 4 6 8 1 0 CsA CONCENTRATION x Hf m Fig. 2. lymphocytes were prelncubated In 1°,," BSA In RPMI 1640 for 24 hours. Cells were then exposed with CsA (A-A) and CsA· lDl complex (A-A) and cellular uptake of [3HJ CsA was measured. 05 '0 20 CVClOSPORINE: (I.IO·'M) 40 05'0 20 CYC;.CSPORrNE (I.ltO·1M) 859 40 FIg. 3. Cellular uptake of CsA In PBl Incubated with [3H] CsA (0-0) and unlabeled CsA (A-A). Panels A and B are results In two subjects. Support for this concept also derives partly from the calculations of dissociation constants for various experiments reported here. The double reciprocal plots of data from these experiments yield two quite distinct apparent dissociation constants (KD)' For the cellular uptake of CsA complexed with LOL. KD ~ 2.3 and 0.6 x 10- 9 mol/l (Fig I, panels A and B, respectively). For the data in all of the rest of the experiments, KD = 2 x 1O-7mol/l. The Ko of 10- 9 mol/l for CsA when in association with LOL certainly suggests high-affinity binding. The rate-limiting step in the LOL pathway is the binding of LOL to a specific high-affinity receptor on the cell surface.' It may be expected. therefore. that this would be the initial step in the metabolism of CsA bound to LOL. The significantly reduced cellular content of [lH] CsA in the presence of cold LOL appears to strengthen this view. Whether such a binding of CsA-LOL to the LOL receptor followed by further metabolism along the LOL pathway occurs cannot be inferred from the experiments reported here. If true. however. it could represent a significant path-way for CsA metabolism because some 32'7c of the drug in blood is bound to plasma LOL. 1 The concentrations of CsA required to saturate the cellular mechanisms(s) responsible for CsA uptake are also different in the presence and absence of LOL. 900 ng/ml with LOL and about 300 ng/ml without LOL. This information further segregates the two processes. CONCLUSIONS Cyclosporinc uptake by peripheral blO<..1d lymphocytes may involve specific as well as nonspecific processes. In either case. the kinetics of uptake indicate a saturable phenomenon implying the involvement of a "carrier" or "transport" protein. Cyclosporine uptake by cells when associated with LOL was greater relative to the uptake of cyclosporine alone. The dissociation constant for CsA-LOL complex of 10- 9 M suggests high affinity binding. perhaps to the LOL receptor. REFERENCES I. Gurecki J. Warty V. Sanghvi A: Transplant Proc 17: 1997. 1985. 2. Havel RJ. Eder HA. Bragdon JH: J Clin Invest 63:1345. 1955. 3. Sanghvi A. Warty V: Biochem J 227:397. 1985. 4. Goldstein JL. Brown MS: J Bioi Chern 249:5153. 1974. 

Receptor-mediated cellular uptake of cyclosporine

Starzl, TE

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Receptor-mediated cellular uptake of cyclosporine

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