July 1999
Volume 40, Issue 8
Free
Physiology and Pharmacology  |   July 1999
Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-Glycoprotein Transport Activity and Binding to Cyclophilin
Author Affiliations
  • Kouichi Kawazu
    From the Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmic Research Division, Ikoma-chi, Japan.
  • Kazuhito Yamada
    From the Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmic Research Division, Ikoma-chi, Japan.
  • Masatsugu Nakamura
    From the Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmic Research Division, Ikoma-chi, Japan.
  • Atsutoshi Ota
    From the Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmic Research Division, Ikoma-chi, Japan.
Investigative Ophthalmology & Visual Science July 1999, Vol.40, 1738-1744. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Kouichi Kawazu, Kazuhito Yamada, Masatsugu Nakamura, Atsutoshi Ota; Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-Glycoprotein Transport Activity and Binding to Cyclophilin. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1738-1744.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. The purpose of this study was to characterize cyclosporin A (CsA) uptake and transport in cultured rabbit corneal epithelial cells (RCECs) .

methods. CsA uptake was evaluated by measuring time-dependent 3H-CsA accumulation in confluent RCECs. Bidirectional 3H-CsA fluxes were measured across the RCEC layers grown on Transwell-COL culture plate inserts. The anti-P-gp monoclonal antibody C219 was used in western blot analysis to probe for the presence of P-gp in these cells.

results. The accumulation of 3H-CsA was time and temperature dependent. Steady state was reached by 60 minutes. The initial uptake was saturable and was suppressed as a function of increases in preloading with unlabeled CsA. This uptake process was enhanced by metabolic inhibition with either 3-O-methylglucose, MG, or 10 mM NaN3 and 3-O-MG. The largest increase was obtained with 10 mM NaN3 in combination with 3-O-MG. In their presence, uptake increased by 40%. A multidrug-resistance (MDR)–reversing agent (i.e., 500 μM verapamil, 100 μM vincristine, 100 μM progesterone, 100 μM testosterone, 500μ M quinidine, or 100 μM chlorpromazine) significantly increased 3H-CsA accumulation. The largest increase was obtained with 500 μM quinidine (i.e., 36%). Conversely, verapamil and vincristine produced the largest inhibition of 3H-CsA efflux (i.e., 19% and 28%, respectively). However, in the presence of 10 μM unlabeled CsA, 3H-CsA efflux increased. 3H-CsA flux across RCEC layers showed marked directional asymmetry. The stromal (S) to tear (T) side transcellular 3H-CsA permeability coefficient (P trans) was approximately seven times higher than that in the T-to-S direction. The S-to-T P trans was reduced by an MDR-reversing agent by up to 40%. Western blot analysis of lysates revealed a 170-kDa membrane protein band.

conclusions. These results suggest that in RCEC the tear-side–facing membrane has a P-gp–mediated drug efflux pump. In addition, there is suggestive evidence for the presence of the cytosolic protein, cyclophilin. The presence of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.

Cyclosporin A (CsA) is a potent immunosuppressive agent with specific T-cell inhibitory activity. It has been suggested that systemically or topically applied CsA could be beneficial in the treatment of various ocular diseases, such as some forms of uveitis, keratoconjunctivitis, immune-mediated keratitis, necrotizing scleritis, Behçet’s syndrome, Sjögren’s syndrome, and corneal transplantation. 1 2 3 Several investigators have reported on the ocular pharmacokinetics of CsA uptake and transport after topical administration. 4 5 6 7 8 9 10 11 12 13 It was suggested that topically applied CsA is more effective in the treatment of external ocular surface diseases than in intraocular disorders. This could be due to the fact that CsA does not penetrate well through the cornea and conjunctiva into the eye. In some other tissues, parameters have been described that affect CsA permeability. Among these are its limited solubility in aqueous solution resulting in low membrane permeability and marked decrease in its permeability subsequent to a decline in temperature. 1 14 15  
Another consideration that could also affect CsA permeability is the presence of a P-glycoprotein (P-gp) pump, which has been described in a number of tissues including the conjunctiva. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 If the P-gp pump is also present in the corneal epithelium, it would decrease CsA penetration. This decrease would occur by the active removal of intracellular CsA to the external environment. Today, there are no published reports on the presence of such a CsA efflux mechanism in the corneal epithelium. 
We report here on our initial characterization of P-gp–mediated drug efflux in rabbit corneal epithelial cells (RCECs). Its presence suggests that these cells can protect themselves from damage by toxic substances which could otherwise accumulate and compromise cell function. In addition, the data suggest that these cells also contain cyclophilin, which could act as a depot for CsA. 
Materials and Methods
Materials
CsA was purchased from Sigma (St. Louis, MO). Primary cultures of rabbit corneal epithelial cells (RCECs) were obtained from Kurabo Industries (Osaka, Japan). 30 Transwell COL cell culture chambers (pore size 0.4 μm, diameter 12 mm, surface area 1 cm2) were from Costar (Bedford, MA). Dulbecco’s modified Eagle’s medium-nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and other tissue culture reagents were from Gibco (Grand Island, NY). Epidermal growth factor, cholera toxin , hydrocortisone, and insulin-transferrin sodium selenite media supplement were from Sigma. Penicillin G and streptomycin were from Wako Pure Industries (Osaka, Japan). Human fibronectin was from Boehringer Mannheim (Mannheim, Germany). [mebmt-β-3H]CsA (specific activity, 366 GBq/millimole), d-[1-14C]-mannitol (specific activity, 2.11 GBq/millimole), and[ 14C]-urea (specific activity, 2.00 GBq/millimole) were purchased from Amersham Life Science (Buckinghamshire, UK). d-[1-3H(N)]-mannitol (specific activity, 728.9 GBq/millimole) was from NEN (Boston, MA). All radio-labeled compounds were stored at −20°C before use. Vincristine sulfate, progesterone, quinidine, and chlorpromazine hydrochloride were obtained from Sigma. Verapamil hydrochloride, testosterone, 3-O-methyl-α-d-glucopyranose (3-O-MG) was from Nacalai tesque (Kyoto, Japan). Sodium azide was purchased from Wako. The anti-P-gp monoclonal antibody C219 was purchased from Centocor (Malvern, PA). All other chemicals were commercial products of reagent grade. 
Cell Culture
RCECs were grown using DMEM/F-12 medium (pH 7.4), which was supplemented with 5% FBS, 10 ng/ml epidermal growth factor, 0.1μ g/ml cholera toxin, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, and antibiotics (100 IU/ml penicillin G and 100 μg/ml streptomycin). 31 For uptake and efflux studies, RCECs were inoculated on culture plates (16 mm diameter) at 8 × 103 cells/cm2. For transport studies, Transwell-COL inserts were precoated with 4.0 μg human fibronectin, before seeding at a density of 4 × 104 cells/cm2. Cells were cultured at 37°C under 95% air and 5% CO2, and the culture medium was replaced daily. 
CsA Uptake, Efflux, and Transcellular Flux
Uptake experiments were performed after the RCECs reached confluence in 8 to 9 days. Uptake of 3H-CsA by cultured RCEC was measured as reported previously by Tsuji et al. 21 32 Briefly, the cultured cell layer was washed three times with 1 ml incubation buffer (Hanks’ balanced salt solution ([HBSS pH 7.4]; osmolarity 315 mOsm/kg; at 37°C) whose composition was (in millimolar): 1.3 CaCl2, 5.0 KCl, 0.3 KH2PO4, 0.8 MgCl2, 138 NaCl, 0.3 Na2HPO4, 5.6 d-glucose, and 10 HEPES. Cultured RCECs were preincubated at 37°C for 30 minutes in this incubation solution. Immediately after the end of the preincubation period, the solution was removed by suction and replaced with 250 μl of the same medium containing 3H-CsA. 14C-Mannitol was used as the extracellular space marker. To terminate uptake, the RCECs were washed with 1 ml ice-cold incubation medium at a designated time. RCECs in each well were solubilized by incubating them overnight in 500μ l 1 N NaOH at 37°C, and samples were taken for quantifying 3H-CsA and 14C-mannitol content. The same procedure was followed to evaluate efflux. Efflux was determined from the remaining amount of 3H-CsA. Protein content was determined by modified Lowry et al. method using bovine serum albumin as the standard. 33 Transport studies were performed after RCECs reached confluence in 8 to 9 days. It was measured as previously described. 31 The cells were grown on inserts and then were washed three times with transport buffer (HBSS; pH 7.4) at 37°C and placed in a 12-well cluster plate, which was maintained at 37°C. They were then preincubated at 37°C for 30 minutes in the transport buffer. Immediately after the end of the preincubation period, the solution on the donor side was removed by suction, and was replaced with fresh incubation medium containing 3H-CsA (0.5 ml tear [T] side, 1.5 ml stromal[ S] side). At appropriate time intervals over 4 hours, samples were withdrawn from the receiver side, and replaced with an equal volume of HBSS. 14C-Mannitol was used as the paracellular permeable marker. Concerning the adsorption of CsA to cell culture plates, dish, and pipet tips, 0.1% polysorbate 80 and 0.1% dimethyl sulfoxide were added in HBSS to inhibit absorption of CSA. The maximum recovery of 3H-CsA was approximately 95%. The radioactivity was measured by using a liquid scintillation counter (Tri-Carb 2100TR; Packard, Meriden, CT). The transepithelial electrical resistance (in ohms per square centimeter) on Transwell COL was measured with an electrical resistance meter (Millicell ERS; Millipore, Bedford MA). The transepithelial electrical resistance was 152.4 ± 4.0 Ω/cm2 (n =2 4, mean ± SEM). 
Apparent Cell Volume Determination
The apparent cell volume was evaluated as the difference between 14C-urea and 3H-mannitol space measured after 10 minutes of uptake. 34 This time was chosen because steady state levels were attained in both cases by 10 minutes. The uptake method was the same as described earlier. The cell volume was 1.51 ± 0.09 μl/mg protein (n = 12, mean ± SEM). 
Western Blot Analysis
Western blot analysis was performed using the procedure of Hosoya et al. 35 with anti-P-gp monoclonal antibody C219. RCECs grown on culture dishes were lysed for 45 minutes in ice-cold phosphate-buffered saline containing 3% sodium dodecyl sulfate (SDS) and protease inhibitors (83 μM antipain, 73 μM pepstatin A, and 0.1 mM leupeptin). The cell lysate was then centrifuged, and the supernatant was used for further analysis. Total cell protein was measured by the method of Lowry et al. 33 Ten micrograms of cell proteins were electrophoresed on SDS-polyacrylamide gel (7.5%, Daiichi Pure Chemicals, Tokyo, Japan) and subsequently electrotransferred to a nitrocellulose membrane (Millipore). Immunoblot procedure using the enhanced chemiluminescence method was performed, according to the manufacturer’s protocol (Amersham). To validate that the presence of P-gp in RCECs was not a culture artifact, P-gp expression was also evaluated in intact epithelial cells. Male Japanese white albino rabbits (2.0 kg) were purchased from Kitayama Laboratories (Kyoto, Japan). In accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, rabbits were individually housed in cages in an air-conditioned room and maintained on a standard laboratory diet (RC-4; Oriental Yeast, Tokyo, Japan) and water provided ad libitum. Rabbits were killed with an overdose of a pentobarbital sodium (Nembutal Sodium Solution, Abbott Labs, North Chicago, IL) administered through a marginal ear vein. The corneal epithelium was scraped from the eyes with a surgical knife and processed as has been described. 
Analytical Method
Uptake, expressed as cell-to-medium concentration ratio (in microliters per milligram protein), was obtained by dividing the apparent uptake amount per milligram protein by the CsA concentration in the incubation medium. These uptakes were corrected for extracellular adsorbed CsA, which was estimated from the apparent uptake of 14C-mannitol. Flux across cultured RCEC layers was expressed as the transcellular permeability coefficient (P trans,cell in centimeters per second). P trans,cell was calculated as described. 31 Briefly the apparent permeability coefficient (P tot in centimeters per second) were calculated using equation 1:  
\[P{=}Vdc/dtACo\]
where dc/dt is the flux across the RCEC layers (in millimolar per second), V the volume in the receiver chamber (in milliliters), A the surface area of the RCEC layers, and Co the initial concentration (in millimolar) in the donor compartment. The flux of CsA across the RCEC layers, indicated as the amount transported versus time, was evaluated from the slope of the regression line obtained from the linear portion of CsA transport versus time curve. The permeability coefficient, P filt,app, was obtained from the drug flux through the fibronectin-coated membrane. The permeability coefficient of drugs through the RCEC layers, P cell,app, was calculated by equation 2. From equation 2, P cell,app was calculated by equation 3.  
\[1/P_{\mathrm{tot}}{=}1/P_{\mathrm{cell,app}}{+}1/P_{\mathrm{filt,app}}\]
 
\[P_{\mathrm{cell,app}}{=}P_{\mathrm{tot}}{\times}P_{\mathrm{filt,app}}/(P_{\mathrm{filt,app}}-P_{\mathrm{tot}})\]
P trans,cell of CsA was obtained by correcting for the paracellular permeability coefficient of CsA. This correction was performed by subtracting the apparent permeability of 14C-mannitol from P cell,app. All data are expressed as mean ± SEM. Statistical analysis was performed using Student’s two-tailed t-test. A difference between means was considered significant if P < 0.05. 
Results
CsA Uptake
Figure 1 shows that the uptake of 0.5μM CsA was time dependent, because it reached steady state after 60 minutes. This result indicates that CsA uptake was saturable, suggesting that it may be an energy-requiring process. The results in Figure 2 show a clear temperature dependence. The uptake at 4°C was almost negligible, whereas at 25°C the uptake was significantly lower than that measured at 37°C. The y intercepts shown in Figures 1 and 2 are nearly equal to zero. These results indicate that there was little nonspecific surface adsorption of 3H-CsA. An Arrhenius plot of the data shown in Figure 2 for 3H-CsA uptake indicates that the calculated activation energy (E act) based on measurements of uptake for 10 minutes, a midpoint in the linear range of uptake, was as high as 36.1 kcal/mole. 
At 10 minutes, CsA concentration-dependent uptake was evaluated from 0.5 to 30.5 μM. Figure 3 shows an Eadie–Hofstee representation of the results and indicates that it was nonlinear, providing further evidence of saturability. To evalaute the kinetic parameters that describe this process, we fitted the data with the nonlinear least-squares regression analysis program (MULTI) 36 The equation used was  
\[V{=}V_{\mathrm{max}}{\times}C/(K_{\mathrm{m}}{+}C){+}k_{\mathrm{d}}{\times}C\]
where V is the initial rate of uptake, C is the initial concentration of 3H-CsA, V max is the maximum uptake rate of a saturable process, K m is the Michaelis constant, and k d is the coefficient of nonsaturable uptake. The values of apparent K m, V max, and k d were estimated to be 8.5 μM, 22.5 picomoles/min/mg protein, and 3.2 μl/min/mg protein, respectively. 
The effect was determined on the kinetics of 3H-CsA uptake at 60 minutes after preloading the cells for 30 minutes with concentrations of unlabeled CsA between 5 and 30 μM, to clarify whether an intracellular CsA binding protein is present in these cells. Our reasoning was that if such a protein was present, the CsA cell-to-medium accumulation ratio would decrease as a function of increases in the preloading CsA concentration. This could occur because the availability of free binding sites would decrease as the preloading CsA concentration increased. As shown in Figure 4 , the cell-to-medium accumulation ratio of labeled CsA maximally decreased by approximately 60%. This result is consistent with the presence of a cytosolic binding protein for cyclophilin. Its occurrence has been documented in a variety of nonocular tissues and cells. 37 38 39 40 41 42 43 44  
We sought evidence for the existence of a P-gp, a polarized drug efflux pump 17 18 19 by evaluating the effects of various multidrug-resistance–reversing agents, which have been described as reducing drug resistance in vitro and which bind to P-gp. 21 32 To test for the involvement of such an efflux mechanism, we measured the individual effects of 500 μM verapamil, 100 μM vincristine, 100 μM progesterone, 100 μM testosterone, 500μ M quinidine, and 100 μM chloropromazine on 3H-CsA uptake. Their effects, shown in Table 1 , indicate that quinidine increased the relative uptake of CsA more than any of the other inhibitors. Therefore, in RCECs, P-gp–mediated efflux appears to be involved in regulating the level of intracellular CsA accumulation through its activity as a drug efflux pump. To validate that CsA efflux is dependent on metabolic support, the effect of adenosine triphosphate depletion on 3H-CsA uptake was studied by evaluating the effect of the metabolic inhibitor, sodium azide in the presence of 3-O-MG on 3H-CsA uptake. The results shown in Table 2 indicate that in all cases inhibition of metabolic support increased CsA uptake. These increases are indicative of energy-dependent P-gp activity, which mediates drug efflux. 
CsA Efflux
Additional evidence was sought for P-gp drug efflux pump activity through measurements of 3H-CsA efflux by loading the cells with 3H-CsA for 1 hour, and then determining the effect of either 500 μM verapamil or 100 μM vincristine on the efflux of 3H-CsA. As shown in Figure 5 , the loss of 3H-CsA after 30 minutes was markedly suppressed in the presence of either one of these inhibitors. To validate further the notion of a drug efflux mechanism, the cells were loaded with unlabeled 10 μM CsA, to determine whether there is cis-side stimulation of drug efflux. Such a stimulation could occur through the replacement of 3H-CsA bound to cyclophilin with unlabeled CsA. As shown in Figure 5 , after 30 minutes of efflux, the remaining amount of CsA in the presence of 10μ M CsA was 40% less than that in control. This enhancement in CsA efflux adds support to the notion of a P-gp drug efflux mechanism. 
Transcellular CsA Flux
To evaluate further polarized P-gp efflux, the characteristics of 3H-CsA fluxes across RCEC layers were investigated by measuring 3H-CsA (0.5 μM) in both flux directions and calculating values for the P trans,cell in each direction. As can be seen in Figure 6 , the CsA flux clearance in the stromal side (S) to tear side (T) direction was significantly more at all times than in the T-to-S direction. As shown in Table 3 , this difference is exemplified by the result that the P trans,cell value for T-to-S flux was seven times lower than the P trans,cell value determined for S-to-T flux. This finding is consistent with the presence of an efflux system on the apical membrane of RCEC layers that transport CsA out of the cells to the external medium. P-gp drug efflux pump activity is further validated by the effects of the P-gp inhibitors, verapamil (500 μM) and vincristine (100 μM), on CsA flux. As shown in Table 3 , both verapamil and vincristine had no significant effect on T-to-S flux (i.e., P trans,cell) whereas S-to-T flux markedly and significantly decreased to 31% and 40%, respectively, of the CsA flux measured in the absence of either of these inhibitors. These reductions for CsA transport in the S-to-T direction further validate the notion that there is P-gp drug efflux activity in these cells. 
Western Blot Analysis
The expression of P-gp in the membrane fraction of cultured RCECs was determined with anti-P-gp monoclonal antibody C219 using the enhanced chemiluminescence method. As shown in Figure 7 , the protein that reacted with C219 had a molecular mass of approximately 170 kDa. Therefore, the present result showed that P-gp was present in the RCEC membrane. We also observed the expression of P-gp in the membrane fraction of intact corneal epithelial cells. 
Discussion
Our characterization of the uptake, efflux, and transcellular transport of CsA coupled with the identification of P-gp protein expression clearly shows that RCECs have P-gp drug efflux pump activity. The evidence is: 1) all the known MDR-reversing agents increased CsA uptake; 2) CsA fluxes were seven times larger in the S-to-T direction than in the T-to-S direction; 3) the MDR reversing agents verapamil and vincristine reduced the transcellular CsA transport in the S-to-T direction; 4) and western blot analysis revealed the expression of the P-gp drug transporter. Our finding that RCECs exhibit P-gp drug transport activity is relevant to a better understanding of the mechanisms that are responsible for drug influx and efflux across the corneal epithelium. These results may help in the design of strategies that could enhance drug delivery into the eye and thereby improve drug efficacy. Such a result could be of value in the treatment of a variety of ocular diseases. 
Our finding of P-gp protein expression in RCECs is in agreement with a preliminary report that showed that this transporter is expressed in these cells. 45 However, in addition we now have extensively evaluated its kinetic properties. The definitive demonstration of P-gp drug efflux activity in RCECs is in agreement with that reported in the endothelial capillaries of retina, iris, and conjunctival epithelial cells. 29 46 47 The demonstration of P-gp drug efflux activity in both the conjunctiva and cornea is pertinent for obtaining higher bioavailability of CsA to the interior of the eye. It is conceivable that this can be achieved through concomitant inhibition of P-gp drug transport efflux activity when administering systemic or topical CsA. 
There is emerging evidence that the CsA-binding protein, cyclophilin, is expressed in a host of tissues. Cyclophilin is a ubiquitous cytosolic protein capable of binding CsA in the microgram-to-milligram range and is believed to play a role in the intracellular accumulation of CsA in many cell types. 37 38 39 40 41 42 43 44 Cyclophilin contains residues that specifically bind CsA, which is needed for CsA to exert its immunosuppressive action. 1 However, there have been no previous reports demonstrating that cyclophilin is expressed in ocular tissue. Our results suggest for the first time that cyclophilin is also expressed in RCECs. The evidence is: 1) the accumulation of CsA was saturable and temperature dependent with high activation energy for CsA uptake (i.e., 36.1 kcal/mole); 2) there appeared to be an inverse relationship between the amount of initial 3H-CsA uptake and the preloading CsA; 3) CsA accumulated to a level that was 147 times higher than that expected for diffusion (i.e., 1.5 μl/mg protein), which is consistent with the presence of a depot for CsA binding (i.e., cyclophilin); 4) cis-side stimulation of CsA efflux by unlabeled CsA is consistent with the presence of cyclophilin. 
Our results are in agreement with the kinetics of CsA uptake in a kidney epithelial cell line LLC-PK1. 39 CsA can diffuse passively across RCEC membrane and be retained at a high-affinity cytoplasmic binding site (K m = 8.5 μM). Competition for cyclophilin binding sites may also be involved in the decreased 3H-CsA uptake (see Fig. 4 ) by RCECs exposed to unlabeled CsA. Although CsA is a substrate of P-gp, we did not find an excess ratio of unlabeled CsA at the P-gp functions (see Fig. 5 ). There may be enough leakage of unlabeled CsA into the cytosol, across the membrane barrier, and its binding to cyclophilin. Thus, irrespective of whether P-gp was totally inhibited or not by unlabeled CsA, the fraction of unlabeled CsA was high enough to compete efficiently for 3H-CsA binding to cyclophilin. Therefore, the presence of cyclophilin in the cornea helps to explain why CsA is an effective immunopressive agent in corneal transplantation. 
In summary, our identification of P-gp protein expression and characterization of its transport activity explains why RCECs mediate vectorial CsA transport from the S to the T side. Our demonstration of this transport activity may be instrumental in the design of more effective strategies of drug delivery to the eye. Furthermore, we have obtained highly suggestive evidence that RCECs also express the CsA binding protein cyclophilin. Its identification helps in our understanding of why CsA is an effective immunosuppressive agent in reducing the likelihood of tissue rejection after corneal transplantation surgery. 
 
Figure 1.
 
Time course for uptake of CsA by RCECs. The RCECs were incubated with a medium containing 3H-CsA (0.5 μM) at 37°C. Each point represents mean ± SEM of eight determinations.
Figure 1.
 
Time course for uptake of CsA by RCECs. The RCECs were incubated with a medium containing 3H-CsA (0.5 μM) at 37°C. Each point represents mean ± SEM of eight determinations.
Figure 2.
 
Effect of temperature on CsA uptake by RCECs. Time course of 3H-CsA (0.5μM) uptake by RCECs was determined at 4°C (▪), 25°C (▴), and 37°C (•). Each point represents mean ± SEM of four determinations.
Figure 2.
 
Effect of temperature on CsA uptake by RCECs. Time course of 3H-CsA (0.5μM) uptake by RCECs was determined at 4°C (▪), 25°C (▴), and 37°C (•). Each point represents mean ± SEM of four determinations.
Figure 3.
 
Eadie–Hofstee plots of CsA uptake by RCECs. Uptake (V) of 3H-CsA (0.5–30.5μM) was measured at 37°C. Each point represents the mean ± SEM of four determinations. Solid line for V was calculated by nonlinear regression, assuming Michaelis–Menten kinetic fit for the data; V max of 22.5 picomoles/min/mg protein, a K m of 8.5 μM, a k d of 3.2 μl/min/mg protein.
Figure 3.
 
Eadie–Hofstee plots of CsA uptake by RCECs. Uptake (V) of 3H-CsA (0.5–30.5μM) was measured at 37°C. Each point represents the mean ± SEM of four determinations. Solid line for V was calculated by nonlinear regression, assuming Michaelis–Menten kinetic fit for the data; V max of 22.5 picomoles/min/mg protein, a K m of 8.5 μM, a k d of 3.2 μl/min/mg protein.
Figure 4.
 
Effect of preloaded cyclosporin A on 3H-CsA uptake by RCECs. RCECs were exposed for 30 minutes in the medium containing 0 to 30 μM unlabeled CsA. Immediately after preloading, the uptake studies of 3H-CsA (0.5 μM) were performed.
Figure 4.
 
Effect of preloaded cyclosporin A on 3H-CsA uptake by RCECs. RCECs were exposed for 30 minutes in the medium containing 0 to 30 μM unlabeled CsA. Immediately after preloading, the uptake studies of 3H-CsA (0.5 μM) were performed.
Table 1.
 
Effect of MDR-Reversing Agents on 3H-CsA Uptake by RCECs
Table 1.
 
Effect of MDR-Reversing Agents on 3H-CsA Uptake by RCECs
Agents Dose Relative Uptake (% of Control)
Control 100*
Verapamil 500 μM 123.63 ± 4.49
Vincristine 100 μM 132.28 ± 5.33
Progesterone 100 μM 121.90 ± 4.84
Testosterone 100 μM 130.33 ± 3.26
Quinidine 500 μM 136.30 ± 2.55
Chlorpromazine 100 μM 135.59 ± 7.16
Table 2.
 
Effect of Metabolic Inhibitors on 3H-CsA Uptake by RCECs
Table 2.
 
Effect of Metabolic Inhibitors on 3H-CsA Uptake by RCECs
Agents Relative Uptake (% of Control)
Control 100*
3-O-MG (− glucose), † 128.12 ± 5.38
10 mM NaN3+ 3-O-MG 141.34 ± 5.02
Figure 5.
 
Effect of MDR-reversing agents and unlabeled CsA on the efflux of 3H-CsA from RCECs. RCECs were incubated with 3H-CsA (0.5 μM) at 37°C for 60 minutes, and the efflux was measured in the presence or absence of MDR-reversing agent (control• , 500 μM verapamil ○, 100 μM vincristine ▵, 10 μM CsA□ ). Each point represents the mean ± SEM of four determinations (0 time; n = 8; 209.4 ± 5.5 μl/mg protein).* P < 0.05 versus control by Student’s t-test.
Figure 5.
 
Effect of MDR-reversing agents and unlabeled CsA on the efflux of 3H-CsA from RCECs. RCECs were incubated with 3H-CsA (0.5 μM) at 37°C for 60 minutes, and the efflux was measured in the presence or absence of MDR-reversing agent (control• , 500 μM verapamil ○, 100 μM vincristine ▵, 10 μM CsA□ ). Each point represents the mean ± SEM of four determinations (0 time; n = 8; 209.4 ± 5.5 μl/mg protein).* P < 0.05 versus control by Student’s t-test.
Figure 6.
 
Transepithelial flux of 3H-CsA (0.5 μM) across RCEC layer. RCECs were seeded and grown on cell culture plates. Transepithelial flux across the RCEC layer from the tear side (T) to the stromal side (S) (•) or S-to-T (○). In the presence of 500 μM verapamil (A) or 100 μM vincristine (B), transepithelial fluxes were also performed from T to S (▪) or S to T (□). Each point represents the mean ± SEM of three to four determinations. *P < 0.05 versus control by Student’s t-test.
Figure 6.
 
Transepithelial flux of 3H-CsA (0.5 μM) across RCEC layer. RCECs were seeded and grown on cell culture plates. Transepithelial flux across the RCEC layer from the tear side (T) to the stromal side (S) (•) or S-to-T (○). In the presence of 500 μM verapamil (A) or 100 μM vincristine (B), transepithelial fluxes were also performed from T to S (▪) or S to T (□). Each point represents the mean ± SEM of three to four determinations. *P < 0.05 versus control by Student’s t-test.
Table 3.
 
Transcellular Permeability Coefficient (P trans,cell) of 3H-CsA across RCEC Layers
Table 3.
 
Transcellular Permeability Coefficient (P trans,cell) of 3H-CsA across RCEC Layers
Control +500 μM Verapamil +100 μM Vincristine
Tear-to-stroma side 0.96 ± 0.07 0.92 ± 0.24 1.26 ± 0.20
Stroma-to-tear side 6.73 ± 0.54* 2.11 ± 0.52, † 2.68 ± 0.60, †
Figure 7.
 
Western blot analysis of P-gp expression in RCECs. The proteins separated on 7.5% SDS-PAGE gels were transferred and stained by the enhanced chemiluminescence method with the use of C219. Lanes 1 and 2 are the plasma membrane from cultured RCECs and intact corneal epithelial cells, respectively. The molecular weight is indicated on the left.
Figure 7.
 
Western blot analysis of P-gp expression in RCECs. The proteins separated on 7.5% SDS-PAGE gels were transferred and stained by the enhanced chemiluminescence method with the use of C219. Lanes 1 and 2 are the plasma membrane from cultured RCECs and intact corneal epithelial cells, respectively. The molecular weight is indicated on the left.
The authors thank Akira Tsuji and Ikumi Tamai, Faculty of Pharmaceutical Sciences, Kanazawa University, for helpful advice and stimulating discussions; and Peter Reinach, SUNY Optometry, State University of New York, for his helpful comments and support in manuscript preparation. 
Faulds D, Goa KL, Benfield P. Cyclosporin. A review of its pharmacodynamic and pharmacokinetics properties, and therapeutic use in immunoregulatory disorders. Drugs.. 1993;45:953–1040.
Smet MD, Nussenblatt RB. Clinical use of cyclosporine in ocular disease. Int Ophthalmol Clin. 1993;33:31–45.
Mochizuki M, Smet M. Use of immunosuppressive agents in ocular diseasea. Prog Retinal Eye Res. 1994;13:479–506. [CrossRef]
Bell TAG, Hunnisett AG. Cyclosporin A: tissue levels following topical and systemic administration to rabbits. Br J Ophthalmol. 1986;70:852–855. [CrossRef] [PubMed]
Wiederholt M, Kössendrup D, Schulz W, et al. Pharmacokinetic of topical cyclosporin A in the rabbit eye. Invest Ophthalmol Vis Sci. 1986;27:519–524. [PubMed]
Kaswan RL. Intraocular penetration of topically applied cyclosporine. Transpl Proc. 1988;20(suppl. 2):650–655.
Llopis MD, Menezo JL. Penetration of 2% cyclosporin eyedrops into human aqueous humor. Br J Ophthalmol. 1989;73:600–603. [CrossRef] [PubMed]
BenEzra D, Maftzir G. Ocular penetration of cyclosporine A in the rat eye. Arch Ophthalmol. 1990;108:584–587. [CrossRef] [PubMed]
BenEzra D, Maftzir G. Ocular penetration of cyclosporin A: the rabbit eye. Invest Ophthalmol Vis Sci. 1990;31:1362–1366. [PubMed]
BenEzra D, Maftzir G, Courten C, et al. Ocular penetration of cyclosporin A III: the human eye. . Br J Ophthalmol. 1990;74:350–352. [CrossRef] [PubMed]
Kanpolat A, Batioglu F, Yilmaz M, et al. Penetration of cyclosporin A into the rabbit cornea and aqueous humor after topical drop and collagen shield administration. CLAO J. 1994;20:119–122. [PubMed]
Oh C, Saville BA, Cheng YL, et al. A compartmental model for the ocular pharmacokinetics of cyclosporine in rabbits. Pharm Res. 1995;12:433–437. [CrossRef] [PubMed]
Bonduelle S, Carrier M, Pimienta C, et al. Tissue concentration of nanoencapsulated radiolabelled cyclosporin following peroral delivery in mice or ophthalmic application in rabbits. Eur J Pharm Biopharm. 1996;42:313–319.
Cheeks L, Kaswan RL, Green K. Influence of vehicle and anterior chamber protein concentration on cyclosporine penetration through the isolated rabbit cornea. Curr Eye Res. 1992;11:641–649. [CrossRef] [PubMed]
Cefalu WT, Pardrige WM. Restrictive transport of a lipid-soluble peptide (cyclosporin) through the blood-brain barrier. J Neurochem. 1985;45:1954–1956. [CrossRef] [PubMed]
Thiebaut F, Tsuruo T, Hamada H, et al. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci. 1987;84:7735–7738. [CrossRef] [PubMed]
Shimabuku AM, Nishimoto T, Ueda K, et al. P-glycoprotein. J Biol Chem. 1992;267:4308–4311. [PubMed]
Borst P, Schinkel AH, Smit JJM, et al. Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals. Pharmacol Ther. 1993;60:289–299. [CrossRef] [PubMed]
Patel NH, Rothenberg ML. Multidrug resistance in cancer chemotherapy. Invest New Drugs. 1994;12:1–13. [CrossRef] [PubMed]
Levéque D, Jehl F. P-glycoprotein and pharmacokinetics. Anticancer Res. 1995;15:331–336. [PubMed]
Tsuji A, Tamai I, Sakata A, et al. Restrict transport of cyclosporin A across the blood-brain barrier by a multidrug transporter, P-glycoprotein. Biochem Pharmacol. 1993;46:1096–1099. [CrossRef] [PubMed]
Augustijns PF, Bradshaw TP, Gan LSL, et al. Evidence for a polarized efflux system in Caco-2 cells capable of modulating cyclosporin A transport. Biochem Biophys Res Commun. 1993;197:360–365. [CrossRef] [PubMed]
Okamura N, Hirai M, Tanigawara Y, et al. Digoxin-cyclosporin A interaction: modulation of the multidrug transport P-glycoprotein in the kidney. J Pharmacol Exp Ther. 1993;266:1614–1619. [PubMed]
Sakata A, Tamai I, Kawazu K, et al. In vivo evidence for ATP-dependent and P-glycoprotein-mediated transport of cyclopsporin A at the blood-brain barrier. Biochem Pharmacol. 1994;48:1989–1992. [CrossRef] [PubMed]
Shirai A, Naito M, Tatsuta T, et al. Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein. Biochimi Biophys Acta. 1994;1222:400–404. [CrossRef]
Schinkel AH, Wagenaar E, Deemter L, et al. Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest. 1995;96:1698–1705. [CrossRef] [PubMed]
Fricker G, Drewe J, Huwyler J, et al. Relevance of P-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation. Br J Pharmacol. 1996;118:1841–1847. [CrossRef] [PubMed]
Tanaka K, Hirai M, Tanigawara Y, et al. Effect of cyclosporin analogues and FK506 on transcellular transport of daunrubicin and vincristine via P-glycoprotein. Pharm Res. 1996;13:1073–1077. [CrossRef] [PubMed]
Saha P, Yang JJ, Lee VHL. Existence of a p-glycoprotein drug efflux pump in cultured rabbit conjunctival epithelial cells. Invest Ophthalmol Vis Sci. 1998;39:1221–1226. [PubMed]
Torishima H, Kinoshita S, Nezu E, et al. Serum-free serial culture and frozen storage of rabbit corneal epithelial cells. Atarashii Ganka. 1996;13:613–620.
Kawazu K, Shiono H, Tanioka H, et al. Beta adrenergic antagonist permeation across cultured rabbit corneal epithelial cells grown on permeable supports. Curr Eye Res. 1998;17:125–131. [CrossRef] [PubMed]
Tsuji A, Terasaki T, Takabatake Y, et al. P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells. Life Sci. 1992;151:1427–1437.
Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–275. [PubMed]
O’Donnell ME. Role of Na-K-Cl cotransport in vasucular endothelial cell volume regulation. Am J Physiol. 1993;264:C1316–C1326. [PubMed]
Hosoya K, Kim KJ, Lee VHL. Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers. Pharm Res. 1996;13:885–890. [CrossRef] [PubMed]
Yamaoka K, Tanigawara Y, Nakagawa T, et al. A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharmacobio-Dyn. 1981;4:875–885.
Merker MM, Handschumacher RE. Uptake and nature of the intracellular binding of cyclosporin A in a murine thymoma cell line, BW5147. J Immunol. 1984;132:3064–3070. [PubMed]
Fabre I, Fabre G, Lena N, et al. Kinetics of uptake and intracellular binding of cyclosporine A in RAJI cells, in vitro. Biochem Pharmacol. 1986;35:4261–4266. [CrossRef] [PubMed]
Takayama A, Okazaki Y, Fukuda K, et al. Transport of cyclosporin A in kidney epithelial cell line (LLC-PK1). J Pharmacol Exp Ther. 1991;257:200–204. [PubMed]
Foxwell BMJ, Woerly G, Husi H, et al. Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vitro. Biochim Biophys Acta. 1992;1138:115–121. [CrossRef] [PubMed]
Didier A, Wenger J, Loor F. Decreased uptake of cyclosporin A by P-glycoprotein (P-gp) expressing CEM leukemic cells and restoration of normal retention by P-gp blockers. Anti-Cancer Drugs. 1995;6:669–680. [CrossRef] [PubMed]
Koletsky AJ, Harding MW, Handschumacher RE. Cyclophilin: distribution and variant properties in normal and neoplastic tissues. J Immunol. 1986;137:1054–1059. [PubMed]
Ryffel B, Woerly G, Greiner B, et al. Distribution of the cyclosporine binding protein cyclophilin in human tissues. Immunology. 1991;72:399–404. [PubMed]
Gallo P, Saviano M, Rossi F, et al. Specific interaction between cyclophilin and cyclic peptides. Biopolymers. 1995;36:273–281. [CrossRef] [PubMed]
Chang JE, Basu S, Lee VHL. Characterization of a primary rabbit corneal epithelial cell culture grown on permeable support [ARVO Abstracts]. Invest Ophthalmol Vis Sci. 1998;39(4):S758. Abstract nr 3497.
Greeenwood J. Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro. J Neuroimmunol. 1992;39:123–132. [CrossRef] [PubMed]
Holash JA, Stewart PA. The relationship of astrocyte-like cells to the vessels that contribute to the blood-ocular barriers. Brain Res. 1993;629:218–224. [CrossRef] [PubMed]
Figure 1.
 
Time course for uptake of CsA by RCECs. The RCECs were incubated with a medium containing 3H-CsA (0.5 μM) at 37°C. Each point represents mean ± SEM of eight determinations.
Figure 1.
 
Time course for uptake of CsA by RCECs. The RCECs were incubated with a medium containing 3H-CsA (0.5 μM) at 37°C. Each point represents mean ± SEM of eight determinations.
Figure 2.
 
Effect of temperature on CsA uptake by RCECs. Time course of 3H-CsA (0.5μM) uptake by RCECs was determined at 4°C (▪), 25°C (▴), and 37°C (•). Each point represents mean ± SEM of four determinations.
Figure 2.
 
Effect of temperature on CsA uptake by RCECs. Time course of 3H-CsA (0.5μM) uptake by RCECs was determined at 4°C (▪), 25°C (▴), and 37°C (•). Each point represents mean ± SEM of four determinations.
Figure 3.
 
Eadie–Hofstee plots of CsA uptake by RCECs. Uptake (V) of 3H-CsA (0.5–30.5μM) was measured at 37°C. Each point represents the mean ± SEM of four determinations. Solid line for V was calculated by nonlinear regression, assuming Michaelis–Menten kinetic fit for the data; V max of 22.5 picomoles/min/mg protein, a K m of 8.5 μM, a k d of 3.2 μl/min/mg protein.
Figure 3.
 
Eadie–Hofstee plots of CsA uptake by RCECs. Uptake (V) of 3H-CsA (0.5–30.5μM) was measured at 37°C. Each point represents the mean ± SEM of four determinations. Solid line for V was calculated by nonlinear regression, assuming Michaelis–Menten kinetic fit for the data; V max of 22.5 picomoles/min/mg protein, a K m of 8.5 μM, a k d of 3.2 μl/min/mg protein.
Figure 4.
 
Effect of preloaded cyclosporin A on 3H-CsA uptake by RCECs. RCECs were exposed for 30 minutes in the medium containing 0 to 30 μM unlabeled CsA. Immediately after preloading, the uptake studies of 3H-CsA (0.5 μM) were performed.
Figure 4.
 
Effect of preloaded cyclosporin A on 3H-CsA uptake by RCECs. RCECs were exposed for 30 minutes in the medium containing 0 to 30 μM unlabeled CsA. Immediately after preloading, the uptake studies of 3H-CsA (0.5 μM) were performed.
Figure 5.
 
Effect of MDR-reversing agents and unlabeled CsA on the efflux of 3H-CsA from RCECs. RCECs were incubated with 3H-CsA (0.5 μM) at 37°C for 60 minutes, and the efflux was measured in the presence or absence of MDR-reversing agent (control• , 500 μM verapamil ○, 100 μM vincristine ▵, 10 μM CsA□ ). Each point represents the mean ± SEM of four determinations (0 time; n = 8; 209.4 ± 5.5 μl/mg protein).* P < 0.05 versus control by Student’s t-test.
Figure 5.
 
Effect of MDR-reversing agents and unlabeled CsA on the efflux of 3H-CsA from RCECs. RCECs were incubated with 3H-CsA (0.5 μM) at 37°C for 60 minutes, and the efflux was measured in the presence or absence of MDR-reversing agent (control• , 500 μM verapamil ○, 100 μM vincristine ▵, 10 μM CsA□ ). Each point represents the mean ± SEM of four determinations (0 time; n = 8; 209.4 ± 5.5 μl/mg protein).* P < 0.05 versus control by Student’s t-test.
Figure 6.
 
Transepithelial flux of 3H-CsA (0.5 μM) across RCEC layer. RCECs were seeded and grown on cell culture plates. Transepithelial flux across the RCEC layer from the tear side (T) to the stromal side (S) (•) or S-to-T (○). In the presence of 500 μM verapamil (A) or 100 μM vincristine (B), transepithelial fluxes were also performed from T to S (▪) or S to T (□). Each point represents the mean ± SEM of three to four determinations. *P < 0.05 versus control by Student’s t-test.
Figure 6.
 
Transepithelial flux of 3H-CsA (0.5 μM) across RCEC layer. RCECs were seeded and grown on cell culture plates. Transepithelial flux across the RCEC layer from the tear side (T) to the stromal side (S) (•) or S-to-T (○). In the presence of 500 μM verapamil (A) or 100 μM vincristine (B), transepithelial fluxes were also performed from T to S (▪) or S to T (□). Each point represents the mean ± SEM of three to four determinations. *P < 0.05 versus control by Student’s t-test.
Figure 7.
 
Western blot analysis of P-gp expression in RCECs. The proteins separated on 7.5% SDS-PAGE gels were transferred and stained by the enhanced chemiluminescence method with the use of C219. Lanes 1 and 2 are the plasma membrane from cultured RCECs and intact corneal epithelial cells, respectively. The molecular weight is indicated on the left.
Figure 7.
 
Western blot analysis of P-gp expression in RCECs. The proteins separated on 7.5% SDS-PAGE gels were transferred and stained by the enhanced chemiluminescence method with the use of C219. Lanes 1 and 2 are the plasma membrane from cultured RCECs and intact corneal epithelial cells, respectively. The molecular weight is indicated on the left.
Table 1.
 
Effect of MDR-Reversing Agents on 3H-CsA Uptake by RCECs
Table 1.
 
Effect of MDR-Reversing Agents on 3H-CsA Uptake by RCECs
Agents Dose Relative Uptake (% of Control)
Control 100*
Verapamil 500 μM 123.63 ± 4.49
Vincristine 100 μM 132.28 ± 5.33
Progesterone 100 μM 121.90 ± 4.84
Testosterone 100 μM 130.33 ± 3.26
Quinidine 500 μM 136.30 ± 2.55
Chlorpromazine 100 μM 135.59 ± 7.16
Table 2.
 
Effect of Metabolic Inhibitors on 3H-CsA Uptake by RCECs
Table 2.
 
Effect of Metabolic Inhibitors on 3H-CsA Uptake by RCECs
Agents Relative Uptake (% of Control)
Control 100*
3-O-MG (− glucose), † 128.12 ± 5.38
10 mM NaN3+ 3-O-MG 141.34 ± 5.02
Table 3.
 
Transcellular Permeability Coefficient (P trans,cell) of 3H-CsA across RCEC Layers
Table 3.
 
Transcellular Permeability Coefficient (P trans,cell) of 3H-CsA across RCEC Layers
Control +500 μM Verapamil +100 μM Vincristine
Tear-to-stroma side 0.96 ± 0.07 0.92 ± 0.24 1.26 ± 0.20
Stroma-to-tear side 6.73 ± 0.54* 2.11 ± 0.52, † 2.68 ± 0.60, †
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×