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Keywords:

  • 24S-hydroxycholesterol;
  • blood–brain barrier;
  • brain efflux index method;
  • efflux transport;
  • organic anion transporting polypeptide 2

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

24S-Hydroxycholesterol (24S-OH-chol), a major cerebral cholesterol metabolite, is an endogenous ligand for the liver X receptor and is a potential stimulant of cholesterol release from glial cells. The elimination mechanism of 24S-OH-chol from the brain is one of the key issues for understanding cerebral cholesterol homeostasis. The purpose of the present study was to clarify the molecular mechanism of the elimination process of 24S-OH-chol across the blood–brain barrier (BBB). After an intracerebral injection in rats, [3H]24S-OH-chol was eliminated from the brain and the radioactivity derived from [3H]24S-OH-chol was detected in the plasma, while [3H]cholesterol was not significantly eliminated from the brain. Co-administration of unlabeled 24S-OH-chol significantly inhibited the [3H]24S-OH-chol elimination, while no inhibitory effect was seen at the same concentration of cholesterol. The [3H]24S-OH-chol elimination was inhibited by co-administration of probenecid, but not by benzylpenicillin. Pre-administration of digoxin completely inhibited the elimination. Xenopus laevis oocytes expressing rat oatp2 exhibited significant transport of [3H]24S-OH-chol, and this was inhibited by unlabeled 24S-OH-chol and digoxin, indicating that rat oatp2 transports 24S-OH-chol. These results are the first direct demonstration that 24S-OH-chol undergoes elimination from the brain to blood across the BBB via a carrier-mediated process, which involves oatp2 expressed at the BBB in rats.

Abbreviations used
24S-OH-chol

24S-hydroxycholesterol

BBB

blood–brain barrier

BEI

brain efflux index

ISF

interstitial fluid

LXR

liver X receptor

mdr1a

multidrug resistance protein 1a

Mrp4

multidrug resistance-associated protein 4

OAT

organic anion transporter

oatp2

organic anion transporting polypeptide 2

Par2

parietal cortex area 2

24S-Hydroxycholesterol (24S-OH-chol) is the main cholesterol metabolite in the brain (Bjorkhem et al. 1997). Following conversion from cholesterol, 24S-OH-chol is eliminated from the brain to the circulating blood (Bjorkhem et al. 1997, 1998). This is considered to be the main elimination route of brain cholesterol. 24S-OH-chol is also an endogenous ligand for liver X receptor (LXR), and an LXR ligand has been reported to increase cholesterol release from primary glial cells associated with induction of ATP-binding cassette transporter A1 and G1 (Whitney et al. 2002). Therefore, 24S-OH-chol would act as a regulator involved in the autoregulatory mechanism for CNS cholesterol homeostasis (Pfrieger 2003).

A previous 18O-inhalation study has estimated the half-life of 24S-OH-chol in rat brain to be about 15 h (Bjorkhem et al. 1997). To undergo elimination from the brain to the circulation, 24S-OH-chol needs to cross the blood–brain barrier (BBB). However, there is no direct evidence of the brain-to-blood efflux transport of 24S-OH-chol across the BBB. In a study using erythrocytes (Meaney et al. 2002), it was hypothesized that 24S-OH-chol traverses the BBB by diffusion, as hydroxylation of the side chain of cholesterol allows oxysterol to be transferred across the lipid bilayer.

As a major part of plasma 24S-OH-chol originates from brain (Bjorkhem et al. 1998), its plasma levels have been investigated as a biomarker reflecting brain cholesterol homeostasis for the diagnosis of a number of neurodegenerative diseases, including Alzheimer’s disease. However, several reports have demonstrated that the 24S-OH-chol level in CSF is a better biomarker for Alzheimer’s disease, and the plasma levels of 24S-OH-chol showed only a weak correlation with the CSF levels (Papassotiropoulos et al. 2002; Schonknecht et al. 2002). Furthermore, in mouse, the 24S-OH-chol levels in the brain increased with aging associated with increased 24-hydroxylase (CYP46) expression in the brain, whereas its plasma levels in adults fell slightly with aging (Lund et al. 1999). These findings pose the question as to whether 24S-OH-chol can cross the BBB by diffusion.

The BBB expresses several kinds of transporters to regulate the exchange between the brain and circulating blood (Ohtsuki 2004). Organic anion transporters, such as organic anion transporter 3 (OAT3) and organic anion transporting polypeptide 2 (oatp2/oatp1a4), mediate the brain-to-blood efflux transport (Asaba et al. 2000; Mori et al. 2004). These OATs transport compounds structurally related to cholesterol, including bile acids (cholate and taurocholate) and steroid conjugates (estrone sulfate and dehydroepiandrosterone sulfate) (Noe et al. 1997; Kusuhara et al. 1999; Ohtsuki et al. 2004). However, there is no published report showing that 24S-OH-chol is a substrate of these transporters.

The purpose of the present study was, therefore, to clarify the mechanism governing the elimination of 24S-OH-chol at the BBB by direct measurement of its elimination process using the brain efflux index (BEI) method (Kakee et al. 1996) and to identify the molecules involved in the elimination process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Adult male Wistar rats, weighing 180–250 g, were purchased from Japan SLC (Hamamatsu, Japan). Mature female Xenopus laevis were purchased from Hamamatsu Kyozai (Hamamatsu, Japan) and maintained in a controlled environment. All experiments were approved by the Animal Care Committee, Graduate School of Pharmaceutical Sciences, Tohoku University.

Reagents

24(S)[22,23-3H]Hydroxycholesterol (50 Ci/mmol) and [1,2,6, 7-3H]cholesterol (60 Ci/mmol) were purchased from American Radiolabeled Chemicals (St Louis, MO, USA). [Carboxyl-14C]Inulin ([14C]inulin, 1.92 mCi/g) was purchased from ICN Biochemicals (Costa Mesa, CA, USA). Unlabeled 24S-OH-chol was purchased from BIOMOL (Plymouth Meeting, PA, USA), while cholesterol and digoxin were purchased from Sigma Chemicals (St Louis, MO, USA). Benzylpenicillin potassium and probenecid were purchased from Wako Pure Chemicals (Osaka, Japan).

Brain efflux index method

In vivo brain efflux experiments were performed by the intracerebral microinjection technique (Kakee et al. 1996; Ohtsuki et al. 2002). In brief, a Wistar rat was anesthetized by intramuscular injection of ketamine–xylazine and placed in a stereotaxic frame (SR-6; Narishige, Tokyo, Japan). [3H]24S-OH-chol or [3H]cholesterol was dissolved in ethanol. The applied solution (0.50 μL) containing [3H]24S-OH-chol (50 nCi) or [3H]cholesterol (50 nCi) with 15% ethanol (final cerebral concentration of less than 0.5%) and [14C]inulin (5 nCi) in an extracellular fluid buffer (122 mmol/L NaCl, 25 mmol/L NaHCO3, 3 mmol/L KCl, 1.4 mmol/L CaCl2, 1.2 mmol/L MgSO4, 0.4 mmol/L K2HPO4, 10 mmol/L d-glucose, and 10 mmol/L HEPES, pH 7.4) in the presence or absence of unlabeled compounds, was then administered into the parietal cortex area 2 (Par2) region of the brain. [14C]Inulin is an impermeable marker used to normalize the actual injection volume, as the injection volume is small (0.5 μL). Our previous report demonstrated that elimination from the CSF by interstitial fluid (ISF) bulk flow was negligible when the test solution was injected into the Par2 region (Kakee et al. 1996). The concentration of [3H]24S-OH-chol is 2 μmol/L in the injectate, and is estimated to be 66 nmol/L at the injection site, as calculated by using the reported dilution factor of 30.3 (Kakee et al. 1996). The osmolarity of inhibitors in the injectate was adjusted by changing the concentration of NaCl. In a pre-administration study, 50 μL of the inhibitor solution at the indicated concentration was injected into the Par2 region 30 s prior to administration of the applied solution. Our previous report demonstrated that pre-administration of 50 μL of extracellular fluid buffer did not significantly change the remaining percentage of indoxyl sulfate or the elimination rate of labeled human amyloid β peptide (1–40) from the rat brain (Ohtsuki et al. 2002; Shiiki et al. 2004). The radioactivity remaining in the brain was measured in a liquid scintillation counter equipped with an appropriate crossover correction for 3H and 14C (LS-6500; Beckman Coulter, Fullerton, CA, USA). The BEI was defined by eqn (1) and the percentage of substrate remaining in the ipsilateral cerebrum (100-BEI) was determined from eqn (2).

  • image(1)
  • image(2)

The apparent elimination rate constant was determined by fitting a semilogarithmic plot of (100-BEI), i.e. the percentage remaining in the ipsilateral cerebrum, versus time, using the non-linear least-squares regression analysis program multi (Yamaoka et al., 1981).

Thin-layer chromatographic analysis

To determine [3H]24S-OH-chol in the plasma and brain after microinjection, 2 μCi of [3H]24S-OH-chol was administered to the Par2 region of rat brain. Blood was collected via the ipsilateral jugular vein at 5 min after administration. Rats were then decapitated 10 min after microinjection, the ipsilateral cerebrum was removed, and the region around the injection site was excised. Plasma was obtained by centrifugation at 7800 g for 5 min at 4°C. For deproteinization, 300 μL of plasma was thoroughly mixed with 1.2 mL of ethanol. The mixture was centrifuged at 15 000 g for 20 min at 4°C, the supernatant was evaporated in a centrifugal evaporator, and the residue was dissolved in 10 μL of ethanol. The brain sample was homogenized in 10 volumes of ethanol. The homogenate was centrifuged at 15 000 g for 20 min at 4°C, and the supernatant was obtained and used for TLC. The samples were separated by TLC on an 8 × 5 cm silica plate (Silica gel 60F254; Merck Co., Japan) with 30% ethyl acetate/hexane according to the manufacturer’s instructions. Then, the plate was cut into 5 mm slices and the 3H-radioacitivity in each slice was measured with a liquid scintillation counter (LS-6500; Beckman Coulter).

Uptake study using a Xenopus laevis oocyte system

Rat oatp2 cDNA inserted in pGEM-HEN was constructed as reported previously (Abe et al. 1998). Capped cRNAs were transcribed from NotI-linearized plasmid with T7 RNA polymerase as described (Mori et al. 2004). Defolliculated oocytes were injected with 50 nL of water or the capped cRNAs (25 ng) and incubated in freshly prepared Barth’s solution (88 mmol/L NaCl, 1 mmol/L KCl, 0.33 mmol/L Ca(NO3)2, 0.4 mmol/L CaCl2, 0.8 mmol/L MgSO4, 2.4 mmol/L NaHCO3, and 10 mmol/L HEPES, pH 7.4) containing 100 U/mL benzylpenicillin and 100 μg/mL streptomycin at 18°C. After incubation for 3 days, the oocytes were pre-incubated with 500 μL of uptake buffer (100 mmol/L NaCl, 2 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 10 mmol/L HEPES, pH 7.4) for 20 min at 20°C. The uptake experiment was initiated by replacing the uptake buffer with 200 μL of the same solution containing 0.25 μCi/mL [3H]24S-OH-chol in the presence or absence of inhibitors and terminated by addition of ice-cold uptake buffer after incubation for a designated time at 20°C. Oocytes were solubilized in 5% sodium dodecyl sulfate solution, and the accumulated radioactivity was determined in a liquid scintillation counter (LS-6500; Beckman Coulter). The amount of [3H]24S-OH-chol taken up into oocytes is expressed as the oocyte/medium ratio per oocyte (μL/oocyte), which is determined as the amount of [3H]24S-OH-chol in oocytes (dpm/oocyte) divided by the concentration of [3H]24S-OH-chol in the medium (dpm/μL). The oocyte/medium ratio thus represents the uptake amount in terms of the volume of medium that contains an amount of [3H]24S-OH-chol equal to that taken up into the oocyte.

Statistical analysis

All data, except the apparent elimination rate constant in Fig. 1, represent the mean ± SEM values. An unpaired, two-tailed Student’s t-test was used to determine the significance of differences between the means of two groups. The statistical significance of differences among means of more than two groups was determined by one-way anovafollowed by the Bonferroni multiple comparisons test.

image

Figure 1.  Time-course of [3H]24S-OH-chol remaining in the ipsilateral cerebrum following intracerebral microinjection. [3H]24S-OH-chol (50 nCi) dissolved in 0.50 μL of extracellular fluid buffer was injected into the Par2 region of the brain. The solid line was obtained with the non-linear least-squares regression analysis program, multi. Each point represents the mean ± SEM (n = 4–13). *p < 0.05 and **p < 0.01, significantly different from the percentage remaining at 2 min after intracerebral administration.

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Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Elimination of [3H]24S-OH-chol and [3H]cholesterol from rat brain across the BBB

Figure 1 shows the time profile of the percentage of [3H]24S-OH-chol remaining in the ipsilateral cerebrum after microinjection into the Par2 region of rat brain. The remaining percentage at 20, 60, and 90 min is significantly lower than that at 2 min, indicating that [3H]24S-OH-chol was eliminated in a time-dependent manner across the BBB in rat brain. The apparent elimination rate constant, determined from the slope, was 6.83 × 10−3 ± 1.27 × 10−3/min (mean ± SD) and the half-life was 101 min. No radioactivity associated with this efflux transport process was detected in the contralateral cerebrum or cerebellum (data not shown).

To demonstrate that 24S-OH-chol crosses the BBB, [3H]24S-OH-chol in the plasma was analyzed by TLC after its microinjection into rat brain (Fig. 2). The highest 3H-radioactivity was detected in slice 3 in the plasma at 5 min after injection and in the brain at 10 min after injection (arrows in Fig. 2a and b). In plasma, a smaller peak was also detected in slice 8. When [3H]24S-OH-chol solution was analyzed, the highest 3H-radioactivity was detected in slice 3 (Fig. 2c). This result suggests that [3H]24S-OH-chol crossed the BBB, at least in part, in the intact form.

image

Figure 2.  Identification of [3H]24S-OH-chol in ipsilateral jugular venous plasma (a) and ipsilateral cerebrum (b) using TLC. An aliquot of [3H]24S-OH-chol (2 μCi) was injected into the Par2 region of the brain. Venous blood was collected from the ipsilateral jugular vein at 5 min. Ipsilateral cerebrum was removed at 10 min. (c) Thin-layer chromatogram of [3H]24S-OH-chol solution. Each point represents the 3H-radioactivity in the indicated slice. The arrows show slice 3, which exhibited the highest level of radioactivity in each case.

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In contrast, the percentage of [3H]cholesterol remaining in the brain at each time point up to 90 min after intracerebral administration is not significantly different from that at 2 min (Fig. 3). This result indicates that [3H]cholesterol was not significantly eliminated from rat brain up to 90 min.

image

Figure 3.  Percentage remaining of [3H]cholesterol in the ipsilateral cerebrum following intracerebral microinjection. [3H]Cholesterol (50 nCi) dissolved in 0.50 μL of extracellular fluid buffer was injected into the Par2 region of the brain. Each value is the mean ± SEM (n = 3–4). NS, not significantly different (p > 0.05) from the percentage remaining at 2 min after intracerebral administration.

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Inhibitory effects of various compounds on the elimination of [3H]24S-OH-chol from the brain

To characterize the elimination of 24S-OH-chol across the BBB in vivo, the inhibitory effects of various co- or pre-administered compounds on the elimination of [3H]24S-OH-chol were investigated (Table 1). Co-administration of unlabeled 24S-OH-chol (10 μmol/L) significantly increased the percentage of [3H]24S-OH-chol remaining in the brain to 131% at 90 min after administration (Table 1), while no inhibitory effect on the percentage remaining was detected when cholesterol was co-administered at the same concentration (Table 1). This indicates that excess unlabeled 24S-OH-chol, but not cholesterol, inhibited the elimination of [3H]24S-OH-chol from the brain.

Table 1.   Effect of test compounds on the elimination of [3H]24S-OH-chol from rat brain
 Injectate concentration of test compounds (mmol/L)No. StudiedPercentage remaining in the brain (100-BEI) (%)
  1. Percentage remaining in the brain (100-BEI) was determined at 90 min after intracerebral injection of [3H]24S-OH-chol (50 nCi). In the co-administration experiment, the solution containing [3H]24S-OH-chol and a test compound at the indicated concentration was injected. For the control, only [3H]24S-OH-chol was injected. a,bCo-administration experiments were conducted separately, and the inhibitory effects of compounds were evaluated compared with the corresponding control. In the pre-administration experiment, digoxin or ECF buffer (control) was injected 30 s prior to administration of [3H]24S-OH-chol. Each value represents the mean ± SEM. *p < 0.05 and **p < 0.01, significantly different from the control. BEI, brain efflux index; ECF, extracellular fluid; 24S-OH-chol, 24S-hydroxycholesterol.

Co-administrationa
 Control (tracer only) 578.1 ± 7.1
 24S-OH-chol0.014131 ± 3**
Co-administrationb
 Control (tracer only) 969.0 ± 12.5
 Cholesterol0.01361.3 ± 11.3
 Probenecid1004105 ± 10.7*
 Benzylpenicillin100470.7 ± 8.91
Pre-administration
 Control (ECF buffer) 477.1 ± 8.33
 Digoxin0.24122 ± 11.3**

Probenecid, benzylpenicillin, and digoxin are inhibitors of OAT. However, because of its low solubility, digoxin was tested at lower concentrations by pre-administration, when the dilution effect at the injection site is minimal compared with co-administration. The remaining percentage in the control was not significantly different among co-administrations and pre-administration. As shown in Table 1, co-administration of probenecid (100 mmol/L) increased the percentage of [3H]24S-OH-chol remaining at 90 min to 105%, while benzylpenicillin (100 mmol/L) had no significant inhibitory effect. In the case of pre-administration of digoxin (200 μmol/L), the percentage remaining at 90 min was increased to 122%.

Transport of 24S-OH-chol by rat oatp2

The inhibitory effects shown in Table 1 suggest the involvement of oatp2 in the elimination of 24S-OH-chol at the BBB, as digoxin has been reported to selectively inhibit oatp2 among OATs at the BBB (Sugiyama et al. 2001). Transport of rat oatp2 was examined using a Xenopus laevis oocyte expression system. As shown in Fig. 4, [3H]24S-OH-chol was significantly transported into rat oatp2 cRNA-injected oocytes in a time-dependent manner, compared with water-injected oocytes.

image

Figure 4.  Time-course of [3H]24S-OH-chol uptake by rat oatp2-expressing oocytes. The uptake of [3H]24S-OH-chol (5 nmol/L) by rat oatp2 cDNA-injected oocytes (closed circle) and water-injected oocytes (open circles) was measured at the indicated incubation time. Each point represents the mean ± SEM (n = 7–10). *p < 0.05 and **p < 0.01, significantly different from the uptake by water-injected oocytes.

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The transport of [3H]24S-OH-chol into oatp2 cRNA-injected oocytes was significantly inhibited by unlabeled 24S-OH-chol in a concentration-dependent manner (Fig. 5). The oocyte/medium ratio in water-injected oocytes was not significantly reduced by 1.3 μmol/L unlabeled 24S-OH-chol, while 10 μmol/L unlabeled 24S-OH-chol significantly inhibited the uptake by water-injected oocytes, suggesting the existence of an endogenous transport system for 24S-OH-chol in oocytes (Fig. 5). The oatp2-dependent transport of [3H]24S-OH-chol was determined from the difference between the oocyte/medium ratio in oatp2 cRNA- and water-injected oocytes. Unlabeled 24S-OH-chol inhibited the oatp2-dependent transport of [3H]24S-OH-chol by 83.2% at 1.3 μmol/L, and inhibited it completely at 10 μmol/L. As shown in Fig. 6, digoxin at 10 μmol/L also inhibited the oatp2-dependent transport of [3H]24S-OH-chol by 98.1%.

image

Figure 5.  Effect of unlabeled 24S-OH-chol on the uptake of [3H]24S-OH-chol by oatp2-expressing oocytes. [3H]24S-OH-chol uptake by rat oatp2 cRNA-injected oocytes (closed column) and water injected (open column) was measured in the absence or presence of 1.3 and 10 μmol/L unlabeled 24S-OH-chol. The uptake of [3H]24S-OH-chol (5 nmol/L) was measured at 30 min. Each bar represents the mean ± SEM. (n = 8–10). *p < 0.05 and **p < 0.01, significant difference. NS, no significant difference (p > 0.05).

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image

Figure 6.  Effect of digoxin on the uptake of [3H]24S-OH-chol by oatp2-expressing oocytes. [3H]24S-OH-chol uptake by rat oatp2 cRNA-injected oocytes (closed column) and water injected (open column) was measured in the absence or presence of 10 μmol/L digoxin. The uptake of [3H]24S-OH-chol (5 nmol/L) was measured at 30 min. Each bar represents the mean ± SEM. (n = 10). *p < 0.05 and **p < 0.01, significant difference.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Bjorkhem et al. (1997) used an in vivo18O-inhalation technique to show that the rate of conversion of cholesterol into 24S-OH-chol was about two-thirds that of cholesterol synthesis. This balance of synthesis and conversion is important for maintaining a large pool of brain cholesterol. After conversion from cholesterol, 24S-OH-chol is eliminated from the brain to maintain a steady state brain level. The present study is the first to directly demonstrate the elimination of 24S-OH-chol from the brain across the BBB (Figs 1 and 2). As shown in Fig. 2, [3H]24S-OH-chol appears to cross the BBB at least partly in the intact form. The small peak at slice 8 suggests that a part of the [3H]24S-OH-chol was metabolized in the brain and/or peripheral organs.

The half-life of 24S-OH-chol in the brain pool is about 15 h, which includes processes of release from cells into ISF and elimination from ISF to blood. (Bjorkhem et al. 1997). The elimination rate from the brain across the BBB was evaluated by the intracerebral administration of [3H]24S-OH-chol as shown in Fig. 1, and the elimination half-life was determined to be 101 min. This shorter half-life suggests that release of 24S-OH-chol from the cells into ISF is slow and is the rate-limiting step of its elimination from the brain, and that after release into ISF, 24S-OH-chol is rapidly eliminated from ISF across the BBB. 24S-OH-chol functions as an LXR ligand and also exhibits a neurotoxic effect at high concentrations (Kolsch et al. 2001). Therefore, rapid elimination from the brain ISF would play important role in preventing impairment of CNS function resulting from the accumulation of 24S-OH-chol in the brain ISF.

To date, the passage of 24S-OH-chol across the BBB has been explained in terms of diffusion, as 24S-OH-chol is transferred into erythrocytes more rapidly than cholesterol (Meaney et al. 2002). The present study shows that the elimination process is significantly inhibited by unlabeled 24S-OH-chol and several inhibitors, resulting in an increase in the percentage remaining, as shown in Table 1. These forms of inhibition indicate the involvement of a saturable process, such as carrier-mediated transport, in the brain-to-blood efflux transport of 24S-OH-chol at the BBB. In contrast, cholesterol was not significantly eliminated from the brain up to 90 min after intracerebral administration (Fig. 3), and did not inhibit the elimination of 24S-OH-chol (Table 1). These results suggest that the transport system for 24S-OH-chol at the BBB does not accept cholesterol as a substrate. This substrate specificity seems to contribute to the difference in the BBB permeability of 24S-OH-chol and cholesterol.

The effects of OAT inhibitors were examined to identify the transporters involved (Table 1). Probenecid has a broad inhibition spectrum as far as OATs are concerned; Ki = 20 μmol/L for OAT3 and 70 μmol/L for oatp2 (Sugiyama et al. 2001). Benzylpenicillin and digoxin are selective substrates/inhibitors of OAT3 (Km = 40 μmol/L) and oatp2 (Km = 0.24 μmol/L), respectively (Kusuhara et al. 1999; Sugiyama et al. 2001; Ohtsuki et al. 2004). As the injectate was diluted 30-fold in the co-administration experiment and dilution of injectate was minimal in the pre-administration experiment (Kakee et al. 1996), the concentration of inhibitors tested in Table 1 is high enough to produce an inhibitory effect. It has been reported that the elimination of homovanillic acid and 6-mercaptopurine from the brain, which is mediated mainly by OAT3 at the BBB, was inhibited by co-administration of 100 mmol/L benzylpenicillin (Mori et al. 2003, 2004). Similar treatment did not affect the elimination of [3H]24S-OH-chol (Table 1). It has also been reported that pre-administration of 100 μmol/L digoxin did not inhibit the elimination of indoxyl sulfate from the brain, which was mediated mainly by OAT3 at the BBB (Ohtsuki et al. 2002). In the present study, pre-administration of 200 μmol/L digoxin increased the percentage remaining to 122% (Table 1), which is close to the extrapolated value at 0 min in Fig. 1, suggesting that digoxin almost completely inhibited the elimination. Therefore, the brain-to-blood efflux transport of 24S-OH-chol was mainly mediated by a digoxin-sensitive transporter, i.e. oatp2.

The percentage of [3H]24S-OH-chol remaining at 0 min was extrapolated to be over 100%. A similar result has been reported in the BEI study of GABA (Kakee et al. 2001). In the BEI study, the actual volume of the injected solution was normalized based on [14C]inulin, as some part of the injectate leaked from the brain during injection. When the test compound is bound and/or taken up by neural cells immediately following injection, the compound remains in the brain in a greater amount than that estimated based on [14C]inulin, and the percentage remaining is over 100% at 0 min. Therefore, the result shown in Fig. 1 also suggests that part of the injected 24S-OH-chol was bound and/or taken up by neural cells.

Organic anion transporting polypeptide 2 has been reported to be localized at the abluminal (brain side) and luminal (blood side) membrane of brain capillary endothelial cells (Gao et al. 1999), and to mediate the brain-to-blood efflux transport of dehydroepiandrosterone sulfate and 17β-estradiol-d-17β-glucuronide (Asaba et al. 2000; Sugiyama et al. 2001). The present study indicates that rat oatp2 transports 24S-OH-chol (Fig. 4) and this transport is inhibited by unlabeled 24S-OH-chol and digoxin as well as in vivo (Figs 5 and 6).

It remains possible that OAT3 transports 24S-OH-chol. However, involvement of OAT3 in 24S-OH-chol elimination across the BBB would be minor, even if OAT3 transports 24S-OH-chol, as the elimination of [3H]24S-OH-chol from the brain was inhibited almost completely by 200 μmol/L digoxin (Table 1), and OAT3-mediated transport was not affected by more than 10 mmol/L digoxin (Sugiyama et al. 2001). Taking into account the in vivo and in vitro results, we consider that 24S-OH-chol in the brain ISF is taken up into brain capillary endothelial cells by oatp2 localized at the abluminal membrane.

After 24S-OH-chol is taken up from brain ISF into brain capillary endothelial cells, it must be transported from the cells to the circulating blood to cross the BBB. The present study has not identified the molecule involved in the transport of 24S-OH-chol at the luminal membrane of brain capillary endothelial cells. Oatp2 has been reported to be expressed at the luminal membrane of brain capillary endothelial cells (Gao et al. 1999) and to be a bidirectional exchanger on the basis of a trans-stimulation effect in vitro (Li et al. 2000). Multidrug resistance protein 1a (mdr1a/ABCB1), multidrug resistance-associated protein 4 (ABCC4) and breast cancer resistance protein (ABCG2) have also been reported to be expressed at the luminal membrane of brain capillary endothelial cells and to pump substrates out to the circulating blood (Schinkel et al. 1995; Wakayama et al. 2002; Hori et al. 2004; Leggas et al. 2004). Taurocholate is a low-affinity substrate of Chinese hamster MDR1 (Lam et al. 2005) and inhibits the transport activity of human multidrug resistance-associated protein 4 and breast cancer resistance protein (Imai et al. 2003; Zelcer et al. 2003). It is possible that 24S-OH-chol is a substrate of some of these transporters, as well as oatp2. Furthermore, the distribution of digoxin into the brain was enhanced in mdr1a knockout mice (Schinkel et al. 1995), indicating that digoxin is transported by mdr1a at brain capillary endothelial cells. Therefore, a contribution from an inhibitory effect of digoxin on mdr1a transport at the luminal membrane to the results shown in Table 1 can not be ruled out.

24S-Hydroxycholesterol is an endogenous potent LXR ligand. Two subtypes of LXR, α and β, are expressed in the brain, and LXRα/β double knockout mice have been reported to develop a number of abnormalities in the brain (Wang et al. 2002). 24S-OH-chol induces the expression of ABCA1 in neurons, glial cells and brain capillary endothelial cells (Fukumoto et al. 2002; Panzenboeck et al. 2002; Whitney et al. 2002; Liang et al. 2004). LXR ligand also induces apolipoprotein E secretion from astrocytoma cells (Liang et al. 2004). Therefore, the brain level of 24S-OH-chol influences CNS functions involving cholesterol homeostasis. The present findings indicate that changes in the efflux transport of 24S-OH-chol mediated by oatp2 may affect the brain levels of 24S-OH-chol.

To date, several reports have investigated the possible association between polymorphism of 24-hydroxylase, which converts cholesterol to 24S-OH-chol, and Alzheimer’s disease (Ingelsson et al. 2004; Wang et al. 2004; Golanska et al. 2005), but no final conclusion has been reached. The present study indicates that the molecule(s) responsible for the brain-to-blood efflux transport of 24S-OH-chol could be related to the risk of Alzheimer’s disease. Therefore, it is necessary to clarify the responsible molecule(s) at the BBB in humans, as the subtypes of oatp are not conserved between rodents and humans. The closest human subtype to oatp2 is OATP-A/OATP1A2 in terms of amino acid sequence (Hagenbuch and Meier 2004). OATP-A transports taurocholate and dehydroepiandrosterone sulfate, like oatp2, while OATP-A does not transport digoxin, which is an oatp2 substrate. It has also been reported that OATP-A is localized at brain capillary endothelial cells (Gao et al. 2000). Based on these findings, OATP-A appears to be a candidate transporter for 24S-OH-chol at the human BBB. Nevertheless, as the expression levels of OATP subtypes and their contributions to transport at human BBB are poorly understood, the involvement of other OATP subtypes in 24S-OH-chol elimination at the human BBB can not be ruled out.

In conclusion, the elimination of 24S-OH-chol from the brain is governed by a carrier-mediated process at the BBB in rats, and oatp2 is involved in this brain-to-blood efflux. Identification of the transporter(s) responsible for the elimination of 24S-OH-chol at the human BBB is necessary to evaluate the role of this transporter in CNS cholesterol homeostasis and CNS diseases in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported, in part, by a Grant-in-Aid for Scientific Research on Priority Areas 17081002 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a 21st Century Center of Excellence (COE) Program grant from the Japan Society for the Promotion of Science, and the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization (NEDO) of Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Abe T., Kakyo M., Sakagami H. et al. (1998) Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. J. Biol. Chem. 273, 2239522401.
  • Asaba H., Hosoya K., Takanaga H., Ohtsuki S., Tamura E., Takizawa T. and Terasaki T. (2000) Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2. J. Neurochem. 75, 19071916.
  • Bjorkhem I., Lutjohann D., Breuer O., Sakinis A. and Wennmalm A. (1997) Importance of a novel oxidative mechanism for elimination of brain cholesterol. J. Biol. Chem. 272, 3017830184.
  • Bjorkhem I., Lutjohann D., Diczfalusy U., Stahle L., Ahlborg G. and Wahren J. (1998) Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J. Lipid Res. 39, 15941600.
  • Fukumoto H., Deng A., Irizarry M. C., Fitzgerald M. L. and Rebeck G. W. (2002) Induction of the cholesterol transporter ABCA1 in central nervous system cells by liver X receptor agonists increases secreted Abeta levels. J. Biol. Chem. 277, 4850848513.
  • Gao B., Stieger B., Noe B., Fritschy J. M. and Meier P. J. (1999) Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain. J. Histochem. Cytochem. 47, 12551264.
  • Gao B., Hagenbuch B., Kullak-Ublick G. A., Benke D., Aguzzi A. and Meier P. J. (2000) Organic anion-transporting polypeptides mediate transport of opioid peptides across blood-brain barrier. J. Pharmacol. Exp. Ther. 294, 7379.
  • Golanska E., Hulas-Bigoszewska K., Wojcik I. et al. (2005) CYP46: a risk factor for Alzheimer’s disease or a coincidence? Neurosci. Lett. 383, 105108.
  • Hagenbuch B. and Meier P. J. (2004) Organic anion transporting polypeptides of the OATP/SLC21 family: phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch. 447, 653665.
  • Hori S., Ohtsuki S., Tachikawa M., Kimura N., Kondo T., Watanabe M., Nakashima E. and Terasaki T. (2004) Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s). J. Neurochem. 90, 526536.
  • Imai Y., Asada S., Tsukahara S., Ishikawa E., Tsuruo T. and Sugimoto Y. (2003) Breast cancer resistance protein exports sulfated estrogens but not free estrogens. Mol. Pharmacol. 64, 610618.
  • Ingelsson M., Jesneck J., Irizarry M. C., Hyman B. T. and Rebeck G. W. (2004) Lack of association of the cholesterol 24-hydroxylase (CYP46) intron 2 polymorphism with Alzheimer’s disease. Neurosci. Lett. 367, 228231.
  • Kakee A., Terasaki T. and Sugiyama Y. (1996) Brain efflux index as a novel method of analyzing efflux transport at the blood-brain barrier. J. Pharmacol. Exp. Ther. 277, 15501559.
  • Kakee A., Takanaga H., Terasaki T., Naito M., Tsuruo T. and Sugiyama Y. (2001) Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier. J. Neurochem. 79, 110118.
  • Kolsch H., Ludwig M., Lutjohann D. and Rao M. L. (2001) Neurotoxicity of 24-hydroxycholesterol, an important cholesterol elimination product of the brain, may be prevented by vitamin E and estradiol-17beta. J Neural Transm 108, 475488.
  • Kusuhara H., Sekine T., Utsunomiya-Tate N., Tsuda M., Kojima R., Cha S. H., Sugiyama Y., Kanai Y. and Endou H. (1999) Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J. Biol. Chem. 274, 1367513680.
  • Lam P., Wang R. and Ling V. (2005) Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice. Biochemistry 44, 1259812605.
  • Leggas M., Adachi M., Scheffer G. L. et al. (2004) Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. Mol. Cell. Biol. 24, 76127621.
  • Li L., Meier P. J. and Ballatori N. (2000) Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol. Pharmacol. 58, 335340.
  • Liang Y., Lin S., Beyer T. P. et al. (2004) A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes. J. Neurochem. 88, 623634.
  • Lund E. G., Guileyardo J. M. and Russell D. W. (1999) cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc. Natl Acad. Sci. USA 96, 72387243.
  • Meaney S., Bodin K., Diczfalusy U. and Bjorkhem I. (2002) On the rate of translocation in vitro and kinetics in vivo of the major oxysterols in human circulation: critical importance of the position of the oxygen function. J. Lipid Res. 43, 21302135.
  • Mori S., Takanaga H., Ohtsuki S., Deguchi T., Kang Y. S., Hosoya K. and Terasaki T. (2003) Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells. J. Cereb. Blood Flow Metab. 23, 432440.
  • Mori S., Ohtsuki S., Takanaga H., Kikkawa T., Kang Y. S. and Terasaki T. (2004) Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs. J. Neurochem. 90, 931941.
  • Noe B., Hagenbuch B., Stieger B. and Meier P. J. (1997) Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain. Proc. Natl Acad. Sci. USA 94, 1034610350.
  • Ohtsuki S. (2004) New aspects of the blood-brain barrier transporters; its physiological roles in the central nervous system. Biol. Pharm. Bull. 27, 14891496.
  • Ohtsuki S., Asaba H., Takanaga H., Deguchi T., Hosoya K., Otagiri M. and Terasaki T. (2002) Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain. J. Neurochem. 83, 5766.
  • Ohtsuki S., Kikkawa T., Mori S., Hori S., Takanaga H., Otagiri M. and Terasaki T. (2004) Mouse reduced in osteosclerosis transporter functions as an organic anion transporter 3 and is localized at abluminal membrane of blood-brain barrier. J. Pharmacol. Exp. Ther. 309, 12731281.
  • Panzenboeck U., Balazs Z., Sovic A., Hrzenjak A., Levak-Frank S., Wintersperger A., Malle E. and Sattler W. (2002) ABCA1 and scavenger receptor class B, type I, are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells. J. Biol. Chem. 277, 4278142789.
  • Papassotiropoulos A., Lutjohann D., Bagli M., Locatelli S., Jessen F., Buschfort R., Ptok U., Bjorkhem I., Von Bergmann K. and Heun R. (2002) 24S-hydroxycholesterol in cerebrospinal fluid is elevated in early stages of dementia. J. Psychiatr. Res. 36, 2732.
  • Pfrieger F. W. (2003) Outsourcing in the brain: do neurons depend on cholesterol delivery by astrocytes? Bioessays 25, 7278.
  • Schinkel A. H., Wagenaar E., Van Deemter L., Mol C. A. and Borst P. (1995) Absence of the mdr1a p-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J. Clin. Invest. 96, 16981705.
  • Schonknecht P., Lutjohann D., Pantel J., Bardenheuer H., Hartmann T., Von Bergmann K., Beyreuther K. and Schroder J. (2002) Cerebrospinal fluid 24S-hydroxycholesterol is increased in patients with Alzheimer’s disease compared to healthy controls. Neurosci. Lett. 324, 8385.
  • Shiiki T., Ohtsuki S., Kurihara A., Naganuma H., Nishimura K., Tachikawa M., Hosoya K. and Terasaki T. (2004) Brain insulin impairs amyloid-beta(1-40) clearance from the brain. J. Neurosci. 24, 96329637.
  • Sugiyama D., Kusuhara H., Shitara Y., Abe T., Meier P. J., Sekine T., Endou H., Suzuki H. and Sugiyama Y. (2001) Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from the brain across the blood-brain barrier. J. Pharmacol. Exp. Ther. 298, 316322.
  • Wakayama K., Ohtsuki S., Takanaga H., Hosoya K. and Terasaki T. (2002) Localization of norepinephrine and serotonin transporter in mouse brain capillary endothelial cells. Neurosci. Res. 44, 173180.
  • Wang L., Schuster G. U., Hultenby K., Zhang Q., Andersson S. and Gustafsson J. A. (2002) Liver X receptors in the central nervous system: from lipid homeostasis to neuronal degeneration. Proc. Natl Acad. Sci. USA 99, 1387813883.
  • Wang B., Zhang C., Zheng W., Lu Z., Zheng C., Yang Z., Wang L. and Jin F. (2004) Association between a T/C polymorphism in intron 2 of cholesterol 24S-hydroxylase gene and Alzheimer’s disease in Chinese. Neurosci. Lett. 369, 104107.
  • Whitney K. D., Watson M. A., Collins J. L., Benson W. G., Stone T. M., Numerick M. J., Tippin T. K., Wilson J. G., Winegar D. A. and Kliewer S. A. (2002) Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system. Mol. Endocrinol. 16, 13781385.
  • Yamaoka K., Tanigawara Y., Nakagawa T. and Uno T. (1981) A pharmacokinetics analysis program (MULTI) for microcomputer. J. Pharmacobiodyn. 4, 879885.
  • Zelcer N., Reid G., Wielinga P., Kuil A., Van Der Heijden I., Schuetz J. D. and Borst P. (2003) Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4). Biochem. J. 371, 361367.