Lysosomal Trapping Is Present in Retinal Capillary Endothelial Cells: Insight into Its Influence on Cationic Drug Transport at the Inner Blood–Retinal Barrier

Abstract

Lysosomal trapping was investigated in the retinal capillary endothelial cells that are responsible for the inner blood–retinal barrier (BRB) using LysoTracker^® Red (LTR). Using confocal microscopy on TR-iBRB2 cells, an in vitro model of the inner BRB, the presence of lysosomal trapping in retinal capillary endothelial cells was suggested since TR-iBRB2 cells exhibited punctuate intracellular localization of LTR that was attenuated by NH₄Cl treatment. The study confirmed that LTR uptake by retinal capillary endothelial cells took place in a time- and temperature-dependent manner, and exhibited the 1.58-fold greater uptake at pH 8.4 than that at pH 7.4 while there was no change in uptake between pH 6.4 and pH 7.4, suggesting that passive diffusion is not enough to explain LTR uptake. The inhibition study showed the possible influence of lysosomal trapping on cationic drug transport by retinal capillary endothelial cells since LTR uptake was significantly inhibited by cationic amphiphilic drugs. Inhibition profiling and the estimation of IC₅₀ suggested the influence of lysosomal trapping on propranolol and low-affinity pyrilamine transport while lysosomal trapping had only a partial effect on verapamil, clonidine, nicotine and high-affinity pyrilamine transport in retinal capillary endothelial cells.

Recent progress in neurochemistry research has demonstrated that several cationic drugs exert a neuroprotective effect in optic nerve injury and cerebral ischemia, and it has been suggested that cationic neuroprotective drugs can contribute to the effective treatment of neurological dysfunction.^1–7) In particular, their future application is expected in the treatment of macular degeneration and diabetic retinopathy that are known as representative retinal diseases, since their symptoms include blindness and an impaired quality of life.^8,9)

In the retina, the circulating blood and neural retina are separated by the blood–retinal barrier (BRB), and the study of blood-to-retina transport at the BRB is essential to improve drug development for retinal diseases, since it is a major challenge to deliver drugs to the retina efficiently and safely.^8,9) The BRB involves the inner and outer BRB and the key cells are retinal capillary endothelial cells and retinal pigment epithelial cells, respectively, and the blood-to-retina transport of low-molecular weight molecules, including nutrients and drugs, is largely carried out by transcellular transport in these cells since paracellular transport is limited by tight junctions.^8–11)

In particular, the contribution of the inner BRB to blood-to-retina transport is noteworthy, and recent research has shown that membrane transporters, such as glucose transporter (GLUT1/SLC2A1),¹²⁾ Na⁺-dependent multivitamin transporter (SMVT/SLC5A6),¹³⁾ cationic amino acid transporter (CAT1/SLC7A1)¹⁴⁾ and equilibrative nucleoside transporter 2 (ENT2/SLC29A2),¹⁵⁾ are involved in the blood-to-retina transport of nutrients across the inner BRB. In addition, studies of the inner BRB have revealed the novel transport systems involved in the blood-to-retina transport of cationic drugs, such as verapamil, propranolol, pyrilamine, clonidine and nicotine,^16–19) and these transport systems are suggested to be useful for the efficient and safe drug delivery to the retina because of its interaction with neuroprotective cations, such as desipramine, imipramine, memantine and clonidine.^16–18) In addition, their usefulness is supported by our recent report on the blood-to-retina transport of clonidine that exerts a neuroprotective effect against the loss of neurons in the partially-injured optic nerve system in rats.¹⁹⁾

Cumulative evidence suggests that the blood-to-retina transport of cationic drugs at the inner BRB are carrier-mediated and saturable, and unknown organic cation transporter molecules are supposed to be involved.^16–19) In addition, it seems that their substrates are cationic amphiphilic drugs based on their physicochemical properties,²⁰⁾ and it is thought that these drugs are possibly subjected to lysosomal trapping which is the sequestration of drugs in lysosomes, an acidic organelle, through physicochemical processes such as drug ionization in the acidic environment of lysosomes.^21–23)

In the study of lysosomal trapping, LysoTracker^® Red (LTR; pK_a=7.5, log P=2.1), a cell biological lysosomal probe, has been adopted as a probe, and previous studies with LTR suggest that the sequestration of cationic drugs in lysosomes is reversible, and that sequestration in lysosomes is reduced by the treatment with NH₄Cl or H⁺-ionophore (e.g. carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; FCCP) that reduces the pH-gradient between the cytosol and lysosomes.^22–25) The importance of lysosomal trapping in the distribution of cationic drugs has been suggested in lysosome-rich tissues such as liver and lung,^26–29) implying a possibility that lysosomal trapping resides in retinal capillary endothelial cells to exert influence on the blood-to-retina transport of neuroprotective cations.

Therefore, in the present study, lysosomal trapping in retinal capillary endothelial cells was investigated by confocal microscopy and in vitro uptake studies with TR-iBRB2 cells, an in vitro model cell line of inner BRB.^30,31)

MATERIALS AND METHODS

Reagents

Commercially available chemicals of reagent-grade were used in the present study. LysoTracker^® Red DND-99 (LysoTracker^® Red; LTR) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.) and Wako Pure Chemical Industries (Osaka, Japan), respectively. Fetal bovine serum (FBS) was obtained from SAFC bioscience (Lenexa, KS, U.S.A.) to be used for the culture of TR-iBRB2 cells. The detailed composition of buffers contents used in the present study is shown in Supplementary materials.

Confocal Microscopy with LTR

In the investigation of LTR uptake by retinal capillary endothelial cells, TR-iBRB2 cells, a conditionally immortalized rat capillary endothelial cell line, were used.^30,31) TR-iBRB2 cells were cultured on Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals, Tokyo, Japan) with 10% FBS, 20 mM NaHCO₃, and antibiotics in accordance with our previous reports.^13,16) Confocal microscopy was performed with TR-iBRB2 cells cultured on BioCoat™ Collagen I Cellware 8-well culture slides (BD Biosciences, Franklin Lakes, NJ, U.S.A.). TR-iBRB2 cells were seeded at 5×10³ cells/slide onto culture slides, and were cultured at 33°C for 48 h. After washing the cells on the slides with extracellular fluid (ECF)-buffer (pH 7.4, Supplementary materials), the cells were incubated in ECF-buffer containing 300 nM LTR at 37°C, with or without 10 mM NH₄Cl. The assay was stopped by the removing the assay buffer and washing cells with ice-cold ECF-buffer. In a dark place, the cells were incubated in phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 20 min, and a cover slip was placed on top of each slide to protect the cells. The fluorescence signal of LTR taken up by TR-iBRB2 cells was examined using a confocal microscope (TCS-SP5, Leica Microsystems, Wetzlar, Germany).

Cellular Uptake Study of LTR

TR-iBRB2 cells were cultured on BioCoat™ Collagen I Cellware 24-well dishes (BD Biosciences), and the cellular uptake study of LTR was carried out as described previously.^13,16) In brief, after washing the cells with ECF-buffer, the cells were incubated in 200 µL ECF-buffer containing 300 nM LTR at 37°C. The Na⁺- and membrane potential-dependence of LTR uptake was examined with Na⁺-free ECF-buffers, such as choline-, Li⁺- or K⁺-replacement buffer (Supplementary materials). The study of extracellular pH-dependence was performed as described in our previous studies,^19,32) and FCCP was used at a concentration of 50 µM. In the study of intracellular pH-dependence, NH₄Cl was used at a concentration of 30 mM, and the LTR uptake was examined using acute- and pre-treatment with NH₄Cl, as described previously.^19,32) After assay, the air-dried cells were suspended in 200 µL ECF-buffer, and subjected to sonic disruption. The fluorescence intensity of LTR (λ_ex: 565 nm, λ_em: 590 nm) in cell homogenates was determined using a multi-mode microplate reader (SpectraMax i3, Bio-Rad, Hercules, CA, U.S.A.). The protein content was determined using a microplate reader (Model 550, Bio-Rad) and a DC protein assay kit (Bio-Rad). Data analysis was carried out in accordance with previous reports,^13,16) and the LTR uptake by TR-iBRB2 cells was expressed as the cell-to-medium ratio (cell/medium), that was calculated using Eq. 1.   

(1)

To determine the IC₅₀, the nonlinear least-square regression analysis program, MULTI, was used to fit the data obtained to Eq. 2, and the parameters are described in detail in the Supplementary materials.^33,34)   

(2)

Statistical Analysis

To determine which differences are significant, a one-way ANOVA followed by Dunnett’s test was taken for several groups, and an unpaired two-tailed Student’s t-test was taken for two groups. The parameters determined by the least-squares regression analysis were expressed as the mean values±standard deviation (S.D.). Unless otherwise indicated, data represent the mean values±standard error of the mean (S.E.M.).

RESULTS

Confocal Microscopy of LTR in TR-iBRB2 Cells

To investigate lysosomal trapping in retinal capillary endothelial cells, LTR was used as a fluorescent probe since it has been widely used in the study of lysosomal trapping.^22,23) In confocal microscopy with TR-iBRB2 cells, punctuate intracellular localization was observed for the fluorescent signal of LTR (Fig. 1A), and this localization of the LTR signal was reduced by treatment with NH₄Cl (Fig. 1B), suggesting the sequestration of LTR in the lysosomes of TR-iBRB2 cells.

Fig. 1. Confocal Microscopy of TR-iBRB2 Cells Treated with LTR

TR-iBRB2 cells were incubated with LTR (300 nM) in the absence (A) or presence (B) of NH₄Cl (10 mM) at 37°C for 30 min.

LTR Uptake by TR-iBRB2 Cells

LTR uptake was studied in TR-iBRB2 cells. The results obtained showed that TR-iBRB2 cells exhibited a time-dependent increase of LTR for 10 min at least, with an initial uptake rate of 34.2±4.4 µL/(min·mg protein) (Fig. 2A, Supplementary Fig. 1). Under steady-state uptake (60 min), TR-iBRB2 cells exhibited an LTR uptake of 641±12 µL/(mg protein) (Fig. 2A), that was approximately 200-fold greater than the cell volume of approximately 3 µL/(mg protein).^30,35) In TR-iBRB2 cells, LTR uptake at 4°C for 3 min was significantly reduced by 56%, and time–course uptake at 4°C also showed significant reduction regardless of the treatment with FCCP that dissipates H⁺-gradient in the lysosomes (Fig. 2A), while no alteration was observed in choline-, Li⁺- or K⁺-replacement buffer (Fig. 2B). Confocal microscopy showed punctuate intracellular localization of LTR in TR-iBRB2 cells at 3 min (Supplementary Fig. 2), supporting the sequestration of LTR in the lysosomes.

Fig. 2. Uptake of LTR by TR-iBRB2 Cells

(A) Time-dependent uptake of LTR (300 nM) by TR-iBRB2 cells was investigated at 37°C (closed circles; 1, 3, 10, 30, 60 min) and 4°C (open circles, without FCCP (50 µM); open diamond, with FCCP (50 µM)), respectively. (B) The effects of Na⁺ and membrane potential on LTR uptake were examined at 37°C for 3 min. Each column and point represents the mean±S.E.M. (n=3–4). * p<0.01, significantly different from the control (37°C).

The study using a variety of extracellular pH conditions showed a significant increase (58%) in LTR uptake at pH 8.4 with no change at pH 6.4 (Fig. 3A), and the study with altered intracellular pH showed a significant reduction (69%) produced by acute treatment with NH₄Cl while there was no change following pre-treatment with NH₄Cl (Fig. 3B). Furthermore, LTR uptake was significantly reduced by 36% in the presence of FCCP which reduced the pH-gradient between cytosol and lysosomes, at a concentration of 50 µM (Fig. 3A).

Fig. 3. Effect of pH on the Uptake of LTR (300 nM) by TR-iBRB2 Cells

The effects of extracellular pH (A) and intracellular pH (B) were examined at 37°C for 3 min. Extracellular pH-dependence was also examined with or without FCCP (50 µM). Intracellular pH was increased and decreased by acute treatment and pretreatment (pre) of cells with 30 mM NH₄Cl, respectively. Each column represents the mean±S.E.M. (n=3–9). ** p<0.01, * p<0.05, significantly different from the control (pH 7.4).

Inhibition Study of LTR Uptake by TR-iBRB2 Cells

The inhibitory effect of several compounds on LTR uptake was examined in TR-iBRB2 cells (Table 1), and it was found that cationic drugs such as desipramine, imipramine, propranolol and memantine markedly inhibited LTR uptake by more than 51%. Moderate inhibition (by more than 37%) was shown in the presence of clonidine, verapamil, pyrilamine and quinidine, and nicotine and amantadine also significantly inhibited LTR uptake by more than 28% while no significant effect was observed in the presence of typical cationic and anionic compounds such as cimetidine, 1-methyl-4-phenylpyridinium (MPP⁺), choline, serotonin, tetraethylammonium (TEA) and p-aminohippuric acid (PAH).

Table 1. Effect of Several Compounds on LTR Uptake by TR-iBRB2 Cells

Compound	Relative uptake (% of control)	Predicted pKa	Predicted log P
Control	100±2
Desipramine	23.0±2.4**	10.2	4.13
Imipramine	29.3±5.2**	9.2	4.80
Propranolol	34.6±4.8**	9.5	3.10
Memantine	48.5±8.0**	10.7	3.18
Clonidine	56.0±12.0**	7.9	1.54
Verapamil	58.3±7.3**	8.6	3.90
Pyrilamine	61.8±7.1**	4.0(b), 9.1(b)	2.75
Quinidine	62.2±6.8**	9.05(b), 13.9(a)	3.44
Nicotine	70.0±5.9**	3.12(b), 8.02(b)	0.72
Amantadine	71.9±6.6*	10.7	2.22
Timolol	87.1± 9.5	9.76(b), 14.1(a)	0.68
Cimetidine	100±12	6.91(b), 13.4(a)	0.26
MPP+	109±6	—	−1.35
Choline	115±10	−3.2(b), 14.0(a)	−3.70
Serotonin	117±19	4.9(a), 9.8(b)	0.21
TEA	120±6	—	−3.17
PAH	98.8±3.0	2.7(a), 4.24(b)	−0.58

LTR uptake by TR-iBRB2 cells was performed in the absence (control) or presence of compounds (1 mM) at 37°C for 3 min. Each value represents the mean±S.E.M. (n=3–27). ** p<0.01, * p<0.05, significantly different from the control. Predicted log P values were referred from the web site of The Royal Society of Chemistry. Predicted pK_a values were referred from the web site of Drug Bank. MPP⁺, 1-methyl-4-phenylpyridinium; TEA, tetraethylammonium; PAH, p-aminohippuric acid. (a), acid; (b), base.

Furthermore, in a comparison of the effects on LTR uptake and cationic drug uptakes by TR-iBRB2 cells,^16–19) it was found that the inhibition profiles of LTR uptake were not consistent with those of cationic drug uptakes (Supplementary Fig. 3). In particular, the sensitivity of LTR uptake to timolol was different from that of cationic drug uptake where significant inhibition by timolol had been previously reported in TR-iBRB2 cells (Table 1, Supplementary Table 1 and Supplementary Fig. 3).^16–19)

Comparison of the IC₅₀ of Cationic Drugs Estimated for LTR Uptake Inhibition

Our previous reports suggested the involvement of carrier-mediated transport systems in the blood-to-retina transport of cationic drugs, such as verapamil, propranolol, pyrilamine, clonidine and nicotine, across the inner BRB.^16–19) To compare LTR uptake with such cationic transport, concentration-dependent inhibition of LTR uptake was examined in TR-iBRB2 cells, and the concentration for IC₅₀ was determined to be 276±241, 306±224, 350±117, 2740±1350 and >864 µM for verapamil, propranolol, pyrilamine, clonidine and nicotine, respectively (Table 2).

Table 2. Concentration-Dependent Inhibition of LTR Uptake by TR-iBRB2 Cells

Compound	IC50 (µM)	Km (µM)
Verapamil	276±241	61.9
Propranolol	306±224	237
Pyrilamine	350±117	20.2	High-affinity process
		252	Low-affinity process
Clonidine	2740±1350	286
Nicotine	>864	184

Data for analysis were obtained in the uptake of LTR by TR-iBRB2 cells. The uptake was performed in the presence of inhibitors at 37°C for 3 min. Each IC₅₀ value was determined by means of nonlinear least-square regression analysis program, MULTI, and represents the mean±S.D. (n=3). K_m values were used from previous reports.^16–19)

Previously, K_m values were determined for the saturable transport of verapamil, propranolol, pyrilamine, clonidine and nicotine in TR-iBRB2 cells.^16–19) The IC₅₀ values for verapamil, clonidine and nicotine were much greater than the K_m values, such as 61.9, 286 and 184 µM, respectively, that were estimated for their carrier-mediated transports in TR-iBRB2 cells (Table 2). Regarding propranolol, its IC₅₀ value was shown to be similar to its K_m value, 237 µM (Table 2). Pyrilamine uptake by TR-iBRB2 cells was shown to involve high- and low-affinity saturable transport processes with K_m values of 20.2 and 252 µM, respectively, and its IC₅₀ values were similar to the K_m of the low-affinity process and much greater than the K_m of the high-affinity process (Table 2).

DISCUSSION

In the inner BRB, the blood-to-retina transport has been reported for cations, such as verapamil, propranolol, pyrilamine, clonidine and nicotine,^16–19) suggesting the involvement of unknown transporter molecules. Based on their physicochemical properties,^20–23) these substrates are defined as cationic amphiphilic drugs, and they are expected to be subjected to lysosomal trapping. The study in lysosome-rich tissues has suggested the significant influence of lysosomal trapping on drug distribution,^26–29) and this has raised the possibility that lysosomal trapping affects the blood-to-retina transport of cationic drugs. However, little is known about lysosomal trapping in the retinal capillary endothelial cells (inner BRB), and the present study investigated lysosomal trapping in retinal capillary endothelial cells using LTR as a fluorescent probe for lysosomal trapping.^22,23)

The confocal microscopy results suggested the presence of lysosomal trapping in retinal capillary endothelial cells since LTR exhibited punctuate intracellular localization in TR-iBRB2 cells, an in vitro model of retinal capillary endothelial cells, suggesting the sequestration of LTR in lysosomes (Fig. 1A). In addition, lysosomal trapping in retinal capillary endothelial cells is supported by the reduced punctuate intracellular signal following treatment with NH₄Cl since the reduction in LTR sequestration in lysosomes has been reported in the presence of a reduced pH-gradient between cytosol and lysosomes²³⁾ (Fig. 1B).

In the study of LTR uptake by TR-iBRB2 cells, the initial uptake rate was calculated as 34.2 µL/(min·mg protein), and LTR uptake at steady-state (60 min) was much greater than the cell volume of TR-iBRB2 cells to indicates that cellular concentration of LTR is approximately 64 µM that is markedly higher than that of assay buffer (300 nM)^30,35) (Fig. 2A), suggesting the concentrative uptake of LTR by retinal capillary endothelial cells. In addition, the results obtained for the uptake of LTR suggested that the uptake of LTR by retinal capillary endothelial cells takes place in a temperature-dependent and Na⁺- and membrane potential-independent manner (Fig. 2). In the uptake study with a variety of extracellular pH values, TR-iBRB2 cells exhibited the 1.58-fold greater uptake at pH 8.4 than that at pH 7.4 with no change in uptake between pH 6.4 and pH 7.4 (Fig. 3A). Although it was thought that the increase in LTR uptake at pH 8.4 might be caused by the increased unionized form of LTR, these are unexplainable by the pH-partition hypothesis, and the Henderson–Hasselbalch equation estimates that the LTR uptake at pH 8.4 and pH 6.4 is 2-fold greater and 6-fold lower than that at pH 7.4, respectively. This shows that passive diffusion governed by the physicochemical properties is not enough to explain the LTR uptake in TR-iBRB2 cells.

Regarding the effect of intracellular pH, the pH-gradient between lysosomes and cytosol was reduced by acute treatment with NH₄Cl or treatment with FCCP,^23–25) and LTR uptake by TR-iBRB2 cells was also significantly decreased, supporting the reduced sequestration of LTR in lysosomes observed using confocal microscopy (Figs. 1B, 3). Regarding FCCP, it is also known to reduce the pH-gradient at the plasma membrane,¹⁷⁾ and the significant reduction in LTR uptake by FCCP suggests the possible involvement of H⁺-coupled transport in the LTR uptake across the plasma membrane of retinal capillary endothelial cells (Fig. 3A). However, this is supposed to be negligible since the H⁺-coupled transport of LTR was not supported by the results obtained in the study of intracellular and extracellular pH effects (Fig. 3). In addition, LTR uptake was not altered by pretreatment with NH₄Cl (Fig. 3B), and this was inconsistent result with the induction of LTR uptake at pH 8.4 (Fig. 3A), since these conditions have outwardly directed proton gradient. Although the definitive reason for this discrepancy is still unclear, it could be possible story that the acidified intracellular pH caused by pretreatment with NH₄Cl traps ionized LTR in cytosol to interfere proper trapping of LTR by lysosome.

In the study of the inhibitory effect, no alteration in LTR uptake by TR-iBRB2 cells was shown by typical anions and cations, such as PAH, TEA, choline, cimetidine, serotonin and MPP⁺ (Table 1), of which the physicochemical properties differ from those of cationic amphiphilic compounds,²⁰⁾ suggesting that lysosomal trapping is minor for these typical compounds in retinal capillary endothelial cells. On the other hand, it would appear that the lysosomal trapping of cationic amphiphilic drugs was supported in retinal capillary endothelial cells since the LTR uptake by TR-iBRB2 cells was significantly inhibited by cationic drugs, such as desipramine, imipramine, propranolol, memantine, clonidine, verapamil, pyrilamine, quinidine, nicotine and amantadine (Table 1), which are supposed as cationic amphiphilic compounds.²⁰⁾ In particular, the results obtained for cationic neuroprotectants, such as imipramine, clonidine and memantine, suggest the possible impact of lysosomal trapping on their blood-to-retinal transport across the inner BRB.

Our previous publications have reported the blood-to-retina transport of cationic drugs, such as verapamil, propranolol, pyrilamine, clonidine and nicotine,^16–19) suggesting the contribution of novel organic cation transporters. In this study, LTR uptake by TR-iBRB2 cells was significantly reduced by 30, 38, 42, 44 and 65% in the presence of nicotine, pyrilamine, verapamil, clonidine, and propranolol, respectively (Table 1), suggesting their partial sequestration in lysosomes. LTR uptake also exhibited functional properties similar to those for the blood-to-retina transport of these cationic drugs in terms of their temperature-, Na⁺- and membrane potential-dependence^16–19) (Fig. 2), also suggesting the possible effect of lysosomal trapping on cation transport at the inner BRB.

However, apart from these similarities, the illustration of data listed in Table 1 and Supplementary Table 1 suggests that lysosomal trapping is not major influencer on cationic drug transport in retinal capillary endothelial cells since the comparison of the inhibition profiles in TR-iBRB2 cells clearly showed that the inhibition profile for LTR was not consistent with those for verapamil, pyrilamine, propranolol, clonidine and nicotine in terms of the order of inhibitory potency and sensitivity to timolol, that exhibited a significant inhibition of the uptake of these cationic drugs with no effect on the uptake of LTR^16–19) (Supplementary Fig. 3). Timolol, a nonselective beta-blocker often used in treatment of glaucoma, was reported to interact with the cellular uptake of verapamil, and the involvement of transporter was also implied in its transport.^36,37) The possible involvement of carrier-mediated transport in the transport of timolol was also supported by the marked inhibitory effect of timolol on the carrier-mediated transport of cationic drugs (Supplementary Fig. 3). In particular, regarding verapamil, clonidine and nicotine, the comparison of the IC₅₀ and K_m values supported that their transport by the retinal capillary endothelial cells are only partly influenced by lysosomal trapping since their IC₅₀ values for LTR uptake (verapamil, 276 µM; clonidine, 2.74 mM; nicotine, >864 µM) were much greater than their K_m values (verapamil, 61.9 µM; clonidine, 286 µM; nicotine, 184 µM) suggested for their saturable transport processes in retinal capillary endothelial cells^16,18,19) (Table 2). These properties different from LTR supports that the transport of these cationic drugs largely involves carrier-mediated process at the plasma membrane, not lysosomal trapping, and visualization with confocal microscopy will be helpful for further confirmation of this (Supplementary Fig. 4).

On the other hand, the estimated propranolol IC₅₀ for LTR uptake was 306 µM (Table 2), and it is thought that lysosomal trapping affects propranolol transport by retinal capillary endothelial cells since the values were similar to the K_m values suggested for the saturable transport process of propranolol.¹⁷⁾ The influence of lysosomal trapping was also thought for pyrilamine transport, involving high- and low-affinity saturable transport processes with K_m values of 20.2 and 252 µM, respectively.¹⁶⁾ The estimated IC₅₀ of pyrilamine for LTR uptake was 350 µM (Table 2), which was similar to the K_m of the low-affinity process and much greater than the K_m of the high-affinity process, suggesting that the low-affinity process of pyrilamine transport can be affected by lysosomal trapping in retinal capillary endothelial cells (Supplementary Fig. 4).

In conclusion, confocal microscopy of LTR suggests the presence of lysosomal trapping in retinal capillary endothelial cells, and this was also supported by the results of the in vitro LTR uptake study, suggesting that passive diffusion is not enough to explain LTR uptake in TR-iBRB2 cells. The results of the inhibition study, including IC₅₀ estimation, suggest that lysosomal trapping only partly influences the transport of verapamil, clonidine, nicotine, and pyrilamine (high-affinity) while lysosomal trapping significantly affects the transport of propranolol and pyrilamine (low-affinity) in retinal capillary endothelial cells. These findings will increase our understanding of drug transport across the BRB.

Acknowledgments

This study was supported in part by Grant-in-Aids for Scientific Research (B) [KAKENHI: 25293036] and Scientific Research (C) [KAKENHI: 26460193] from the Japan Society for Promotion of Science, and Research Grants from the Tamura Science and Technology Foundation and the Takeda Science Foundation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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