I. Introduction

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Disorders of the central nervous system (CNS²) remain difficult to treat pharmacologically due to poor drug permeability across brain barriers such as the blood-brain and blood-cerebrospinal fluid (CSF) barriers. Therapeutic agents can, however, cross these barriers by a variety of different mechanisms other than passive diffusion including transcytosis, receptor-mediated absorptive endocytosis, and/or facilitated/active transport systems. Once across these initial barriers, drug accumulation in the brain can be further restricted by a number of mechanisms including passive efflux in the bulk flow of the cerebrospinal fluid (sink effect), metabolic degradation, and active efflux transport including mechanisms dependent on P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). Although the mechanisms of drug transfer in and out of the CNS have been fairly well characterized at the interfaces themselves, little information exists on the role of the brain parenchyma in the disposition of drugs. Recent studies demonstrating the existence of both influx and efflux transporters within glial cells such as astrocytes and microglia (Hong et al., 2000, 2001; Declèves et al., 2000; Dallas et al., 2001; Lee et al., 2001) highlight the complexity of drug distribution within the CNS.

The primary interfaces between the CNS and the peripheral circulation are the blood-brain barrier (BBB) and the blood-CSF barrier (Fig. 1, A and B) (Mooradian, 1994; Groothius and Levy, 1997). Tight junctions between the cerebral endothelial cells at the blood-brain interface and between the choroid plexus (CP) epithelial cells represent the structural basis of these barriers and permit the brain to function in a highly regulated and stable environment. The CNS contains two regulated fluid compartments, the interstitial fluid that surrounds the neurons and glia and the CSF that fills the ventricles and cushions the external surfaces of the brain. Due to its large surface area (i.e., 1000-fold larger than that of the CP), the BBB serves as the primary interface between the CNS and the peripheral circulation, whereas the blood-CSF barrier plays a less prominent role. Therefore, the subordinate role of the blood-CSF barrier to CNS drug delivery results from substrates present in the CSF having access only to the brain parenchyma directly neighboring the ventricles and CSF space. The BBB, on the other hand, interfaces with the entire interstitial fluid compartment of the CNS (Pardridge, 1997).

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Anatomy of blood-brain and blood-CSF interfaces. A, the BBB is formed by brain capillary (C) endothelial cells that are joined together by tight junctions. The brain endothelium and pericytes (P) are surrounded by a basement membrane and glial foot processes (G). Blood-borne solutes must cross from the lumen to the brain extracellular fluid (ECF). B, CP capillaries are fenestrated and surrounded by a basement membrane, which may also envelope pericytes (P). Solutes leaving the choroid capillary reside in the extracellular space containing fibroblasts (F) and collagen (C) and must cross the CP before entering the CSF. The CP has ciliated apices that are joined by tight junctions. C, ventricular surfaces are lined by ciliated ependymal cells (E), which are joined by tight junctions. A basement membrane is present between the ependymal cells and astrocytic processes. Solutes in the CSF may traverse across or between ependymal cells to enter brain ECF. D, lining of the brain surfaces. The CSF percolates through the subarachnoid space [containing collagen (C) and the pia matter (P)], and comes in contact with the glia limitans. The glia limitans, covered by a basement membrane, consists of flat astrocytic processes (A) with no intercellular junctions. CSF may exchange with brain ECF by traversing across or between glial processes. Reprinted with permission from Groothuis and Levy (1997).

In addition to the physical barriers provided by the tight junctions along the BBB and blood-CSF barrier, the presence of drug-metabolizing enzymes at the two interfaces provides an additional enzymatic barrier. Drug-metabolizing enzymes have been identified in the cerebral microvessels, choroid plexuses, leptomeninges, and in some circumventricular organs (Ghersi-Egea et al., 1995). These include the cytochrome P450 hemoproteins, several cytochrome P450-dependent monooxygenases, NADPH-cytochrome P450 reductases, UDP-glucuronosyltransferases, alkaline phosphatases, glutathione (GSH) peroxidases, and epoxide hydrolases (Ghersi-Egea et al., 1988, 1993, 1994; Meyer et al., 1990; Perrin et al., 1990). Degradation or biotransformation products are likely eliminated from the brain either by specific transport systems within the BBB or by diffusion from the parenchyma into the CSF by bulk flow (Ghersi-Egea et al., 1995).

The objective of this review is to discuss the location and functional expression of membrane drug transporters in brain barriers (i.e., BBB and CP) and in brain parenchyma (i.e., astrocytes and microglia).

	II. The Blood-Brain Barrier and the Choroid Plexus

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The BBB is composed of a monolayer of brain capillary endothelial cells that are fused together by tight junctions. Under normal physiological conditions, these tight junctions form a continuous, almost impermeable, cellular barrier that prevents the passive influx of a variety of substances with the exception of the smallest, lipid-soluble molecules (Reese and Karnovsky, 1967). The absence of fenestrations, vesicular traffic, and pinocytosis in brain capillary endothelia further restrict free flow between brain interstitium and blood.

The endothelial cells of the BBB contain numerous membrane transporters involved in the influx/efflux of various essential substrates such as electrolytes, nucleosides, amino acids, and glucose (Fig. 2). Membrane permeation mechanisms can involve passive diffusion, carrier-mediated (facilitative), and/or ATP-dependent (active) processes and are similar to well characterized transport systems in other tissues (i.e., D-glucose, L-amino acid carrier systems, Na⁺/K⁺-ATPase), although the capacity and rate of transport can vary widely. There appears to be an asymmetric distribution of membrane-bound nutrient carriers across the BBB. One example of this asymmetry involves the facilitative glucose transporter, GLUT-1. This transporter is highly expressed by BBB microvessels, with higher levels of expression at the abluminal membranes compared with the luminal side (Pardridge and Boado, 1993). In general, Na⁺/K⁺-ATPase and the A-system amino acid transporters are primarily located on the abluminal side of cerebral endothelial cells (Sanchez del Pino et al., 1995) whereas Ca²⁺-ATPases are expressed on both luminal and abluminal endothelial membranes and in the plasmalemmal vesicles of the endothelium (Vorbrodt, 1988).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Selected transport mechanisms along the BBB. A general depiction of the polarized expression of transporters for drugs and essential nutrients on a BBB endothelial cell. The arrows indicate the direction of transport. For a more descriptive representation of the major drug transport systems in the BBB [organic cation and anion transporters, nucleoside transporters (N2, es, and ei), and efflux systems (P-gp and MRP)], please refer to Figs. 4, 5, 6, and 7. Adapted from Betz et al., 1980; van Asperen et al., 1997.

In addition to carrier-mediated mechanisms, transcytosis of macromolecules in and out of the brain or CSF has also been reported (van Deurs, 1979; Broadwell, 1989). Furthermore, receptor-mediated and adsorptive endocytosis processes at the BBB exist for both hormones and plasma proteins (Abbott and Romero, 1996). Examples of these receptors include the endothelial barrier antigen (function undetermined), OX-47 (an integral plasma membrane glycoprotein that is involved in cell-to-cell recognition), and the endothelial glycocalyx (possible role in vascular permeability and surface charge) (Vorbrodt, 1988; Rippe and Haraldsson, 1994).

In addition to the presence of numerous receptors and transporters, the endothelial cells of the BBB also express metabolic enzymes such as alkaline phosphatase, peptidases, several cytochrome P450 isozymes (IIE1/IIB1/IIB2), UDP-glucuronosyltransferase, and GSH S-transferase. The enzyme alkaline phosphatase, which hydrolyzes phosphorylated metabolites, is present on both luminal and abluminal membranes of the endothelial cell. However, it is more heavily concentrated on the luminal side (Lawrenson et al., 1999). Cytochrome P450 IIE1 is expressed in most cerebral microvessels as well as in the astrocytic foot processes whereas cytochrome P450 IIB1/2 has been detected in both endothelial cells and neighboring pericytes (Volk et al., 1991). The conjugating enzyme UDP-glucuronsyltransferase is localized to rat brain capillaries (Ghersi-Egea et al., 1994) and one -class GSH S-transferase has been detected in both cerebral capillaries and astrocytic foot processes (Johnson et al., 1993).

The blood-CSF barrier plays a vital role in the selectivity and permeability of the CP membrane to various nutrients and xenobiotics. The CP is a leaf-like highly vascular organ that protrudes into the ventricles. It is comprised of fenestrated capillaries that are surrounded by a monolayer of epithelial cells joined together by tight junctions (Fig. 1B) (Groothius and Levy, 1997; Segal, 2000). These tight junctions form the structural basis of the blood-CSF barrier and seal together adjacent polarized epithelial cells (also known as ependymal cells). Thus, once a solute has crossed the capillary wall, it must also penetrate the ependymal cells before entering the CSF (Fig. 1C).

The primary role of the CP is to produce and maintain the homeostatic composition of the CSF. The CP continuously secretes CSF, which is reabsorbed back into the circulation primarily by the arachnoid villi located in the superior sagittal sinus. The total volume of CSF (140 ml) is replaced 4 to 5 times daily (Enting et al., 1998). This continuous flow of CSF through the ventricular system into the subarachnoid space (Fig. 1D) and exiting into the venous system provides a "sink" that reduces the steady-state concentration of a molecule penetrating into the brain and CSF (Saunders et al., 1999). The sink effect is greater for large molecular weight and lipid-insoluble molecules. The CSF also contains approximately 0.3% plasma proteins, totaling 15 to 40 mg/ml, depending on sampling site (Felgenhauer, 1974). This is in contrast to the extracellular space of the normal adult brain, which contains no detectable plasma proteins (Azzi et al., 1990). An increase in CSF protein concentration has been observed under pathological circumstances, in some cases due to an increased permeability of the BBB (McAuthur et al., 1992).

As is the case with the BBB, the CP exhibits a polarized expression of receptors, enzymes, ion channels, and transport systems that regulate the CSF composition via processes of secretion and reabsorption (Spector and Johanson, 1989). The apical side expresses the Na⁺/K⁺-ATPase pump, ion channels for Cl, K⁺, and Na⁺/HCO cotransport carriers (Fig. 3). Studies have also revealed the expression of facilitated and sodium-dependent carriers for the transport of nonelectrolytes (Davson and Segal, 1970; Johanson et al., 1990; Garner and Brown, 1992). The basolateral side is lined with Na⁺/H⁺ antiporters, Cl/HCO antiporters, facilitated carriers for nonelectrolytes, and a carbonic anhydrase (Davson and Segal, 1970; Deng and Johanson, 1989; Johanson et al., 1990). In addition to various receptors and transporters, the CP expresses high levels of metabolic enzymes including UDP-glucuronosyltransferase and epoxide hydrolase, as well as cytochrome P450 IIB1/2, - and µ-class GSH S-transferases, and GSH peroxidase (Tayarani et al., 1989; Volk et al., 1991; Johnson et al., 1993; Ghersi-Egea et al., 1994).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Selected transport mechanisms along the blood-CSF barrier. A general description of the polarized expression of transporters for drugs and essential nutrients along a CP epithelial cell. The arrows indicate the direction of transport. For a more descriptive schema of the major drug transport systems in the CP [organic cation and anion transporters, nucleoside transporters (es, ei and N3), and efflux systems (P-g and MRP)], please refer to Figs. 4, 5, 6, and 7. Adapted from Johanson, 1988; Spector and Johanson, 1989.

The endothelial cells of the BBB and the epithelial cells of the CP thus provide more than a physical barrier between the brain and the peripheral circulation. The blood-brain and the blood-CSF barriers actively regulate the passage of solutes, regulatory proteins, metabolic fuels, neurotransmitter precursors, essential nutrients, and xenobiotics between the CNS and the blood. The presence of drug-metabolizing enzymes within the two brain compartments suggests an important role in the detoxification of potentially harmful xenobiotics and pharmacological agents.

	III. Brain Parenchyma

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The brain parenchyma is made up of neurons and the surrounding neuroglia cells. Neuroglia were originally thought to be passive cells that provided only structural support to the surrounding neurons (Compston et al., 1997; Araque et al., 1999). These cells were classified as neuroglia or "nerve glue" due to their spindle-shape and their "soft, medullary, fragile nature". This purely structural role for neuroglia has been abandoned since these cells are now known to have multiple functions in regulating an optimal interstitial environment.

There are two primary types of neuroglial cells that comprise the brain parenchyma, the macroglia, and microglia. The macroglia consist of astrocytes and oligodendrocytes, which like neurons possess an ectodermal origin and proliferate throughout life, particularly in response to injury (Peters et al., 1991). Microglia are smaller than macroglia and are considered to be the resident immune cells of the brain. Microglia are also capable of proliferating in response to injury. However, their origin, whether mesodermal or neuroectodermal, remains under debate (Schelper and Adrian, 1986; Boya et al., 1991).

Astrocytes possess a star-shaped morphology and contain numerous cytoplasmic fibrils, of which the glial acidic fibrillary protein is the main constituent (Walz, 2000). There are two main types of astrocytes, fibrous (type-2) and protoplasmic (type-1), and they seem to differ in their location, cytoplasmic filament content, and antibody staining. Fibrous astrocytes are found mainly in the white matter of the brain, possess numerous filaments, and stain positive with the A2B5 antibody. Protoplasmic astrocytes are located primarily in the gray matter, contain less cytoplasmic filaments and stain negatively to the A2B5 antibody (Black et al., 1993).

Astrocytes are not only cytoskeletal support cells for neurons but possess numerous functions that aid in maintaining the normal homeostatic environment of the CNS. Kuffler et al. (1966) first demonstrated that astrocytes were nonexcitable cells with a large membrane potential that was sensitive to changes in extracellular K⁺ concentrations. These results suggested that astrocytes were active participants in the homeostatic maintenance of the CNS by locally removing excess K⁺ that had been released from active neurons (termed K⁺ spatial buffering). Astrocytes are also involved in the initiation and regulation of immune and inflammatory events during injury and infection (Aschner, 1998). Secretion of cytokines such as interleukin-1 and -6, tumor necrosis factor-, interferon-, and granulocyte colony-stimulating factor in response to infection and injury (Malipiero et al., 1990; Benveniste, 1993) may play an important role in the initiation and maintenance of neurotoxic immune responses within the injured CNS and further propagate CNS damage. For example, cytokine secretion by astrocytes and microglia is likely involved in the pathogenesis of human immunodeficiency virus-1 (HIV-1) dementia, a neurologic disorder characterized by destruction and dysfunction of neurons, that is observed in end-stage AIDS patients (Epstein and Gendelman, 1993; Rausch et al., 1999).

In addition to structural and immunological functions, astrocytes also maintain physiological extracellular neurotransmitter concentrations through their removal from the extracellular fluid (Fonnum, 1984; Anderson and Swanson, 2000). The importance of this excess removal is demonstrated by the removal of the excitatory neurotransmitter glutamate (Fonnum, 1984). Elevated brain levels of glutamate have been implicated in the pathogenesis of a variety of CNS disorders including amyotrophic lateral sclerosis, epilepsy, and cerebral infarctions (Anderson and Swanson, 2000).

Studies in both cultured and isolated astrocytes have shown that these cells express a wide variety of neurotransmitter receptors including glutamate, glycine, taurine, -aminobutyric acid, as well as several monoamines (Pearce et al., 1986; Usowicz et al., 1989; Shain and Martin, 1990; Kanner, 1993). The presence and perisynaptic location of these receptors on astrocyte foot processes suggest a signaling mechanism between neurons and astrocytes (Lieberman et al., 1989; Dani et al., 1992; Bruckner et al., 1993). Furthermore, astrocytes also appear to be intimately associated with neighboring glial and endothelial cells. Studies in brain endothelial-astrocyte and microglial-astrocyte cocultures suggest that astrocytes provide a variety of endogenous signals and diffusible factors that may serve to induce the formation of tight junctions, the expression of various proteins, maintain overall BBB integrity and promote differentiation and maturation of microglia (Debault and Cancilla, 1980; Tao-Cheng et al., 1987; Laterra and Goldstein, 1991; Minakawa et al., 1991; Tanaka and Maeda, 1996). In addition, astrocyte expression of various adhesion molecules (i.e., neural cell adhesion molecule, astrotactin, and L1) may guide immature nerves cells from their site of cell division to their final destination during brain maturation (Rakic, 1990). Recent evidence suggests that astrocytes possess a number of nutrient and drug transport proteins including several nucleoside transporters (Hosli and Hosli, 1988; Gu et al., 1996; Sinclair et al., 2000) as well as the ATP-dependent, membrane-bound, drug efflux transporters P-gp and MRP (Pardridge, 1997; Declèves et al., 2000).

Microglia, first described by the Spanish neuroanatomist del Rio-Hortega (1932), represent 5 to 20% of the total glial population within the CNS (Lawson et al., 1990; Raivich et al., 1999). Although microglia appear to be ubiquitously distributed within the CNS, actual numbers vary according to region. For example, the basal ganglia and cerebellum have considerably greater amounts than the cerebral cortex (Dickson et al., 1991). The origin of microglia has been a long-standing and often controversial issue historically (Ling and Wong, 1993; Theele and Streit, 1993; Cuadros and Navascues, 1998) due in part to the lack of unique cell markers. Several studies support an ectodermal origin for microglia (Hao et al., 1991; Richardson et al., 1993; Fedoroff et al., 1997). However, with the discovery of various histological markers for microglia in the 1980s, most evidence supports a mesodermal origin, possibly through circulating monocytes that colonize the parenchyma following vascularization or via bone derived precursor cells that migrate during gestation (Jordan and Thomas, 1988; Perry and Gordon, 1991; Thomas, 1992; Theele and Streit, 1993). Several different modes of entry of microglial precursors into the developing CNS have been suggested including traversion of the pial surface of the meninges, crossing the endothelial cell wall of blood vessels of the CNS, and traversion of the epithelial cells lining the ventricles (Cuadros and Navascues, 1998; Navascues et al., 2000). Regardless of the specific area of invasion, following CNS entry, microglia precursors distribute throughout the CNS and differentiate into their mature (ramified) form.

Several morphologically distinct microglia have been identified including ramified (or resting), spheroid (or activated), and phagocytic types (Dickson et al., 1991). In normal adult brain, microglia are mostly found in a ramified or resting state and appear as small highly branched cells. Ultrastructural features of microglia, as determined by electron microscopy, include an irregular nucleus, clumped chromatin, and a sparsely occupied cytoplasm (Kitamura et al., 1977; Dickson et al., 1991). Following injury or infection, microglia become activated, which results in retraction of processes, proliferation, and up-regulation of several cell surface factors. The level of microglia activation appears to be graded according to the type and severity of brain injury involved (Raivich et al., 1999). Streit et al. (1988) have demonstrated this phenomenon using rat-derived facial nerves. Following reversible axotomy (crushing of the nerve), microglia proliferate and surround the nerves while emitting several soluble trophic factors such as basic fibroblast growth factor and nerve growth factor (Heumann et al., 1987; Gomez-Pinilla et al., 1990; Araujo and Cotman, 1992). Increased expression of various integrins and major histocompatibility complex class I markers also occurs. Thus, the microglia appear to play a neuroprotective effect in the spheroid or activated stage, and aid in the recovery of reversibly damaged neurons. Conversely, ricin-induced degeneration of neurons (a irreversible and lethal event) results in microglia becoming fully activated phagocytes. This stage of activation is characterized by a significant increase in the expression of markers observed in the phagocytic stage including several integrins (₅₁, ₆₁ and _M2), and major histocompatibility complex class I and II antigens. Thus, microglia show remarkable "functional plasticity" depending on the severity of injury (Streit et al., 1988). A large body of evidence now exists which implicates excessive microglia activation and proliferation in the development of neuronal death in various pathological disease states. Examples include the Wernicke-Korsakoff syndrome, Parkinson's disease, Alzheimer's disease, ischemia, and several HIV-1 related pathologies in the CNS (Todd and Butterworth, 1999; McGeer and McGeer, 1998; Walton et al., 1999; Akiyama et al., 2000; Xiong et al., 2000). A mechanistic commonality observed in these various diseases is microglial production of a variety of neurotoxins in excess, including nitric oxide, tumor necrosis factor- and reactive oxygen species such as peroxide. Excessive production of these factors leads to a cascade of effects including activation of astrocytes, further activation of microglia, and finally neuronal death.

Microglia express a wide variety of ion channels including multiple potassium, calcium, and sodium channels (Eder, 1998). The expression patterns of ion channels in microglia depend on the functional state of the cells and are involved in a variety of physiological functions including proliferation, ramification, maintenance of membrane potential, intracellular pH regulation, and cell volume regulation (Frelin et al., 1988; Faff et al., 1996; Klee et al., 1999). In addition, both ionotropic and metabotropic types of glutamate receptors also appear to be expressed in microglia (Ong et al., 1996; Gottlieb and Matute, 1997; Biber et al., 1999; Lopez-Redondo et al., 2000; Noda et al., 2000). Kainate (GluR5-7), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GluR1-4), and N-methyl-D-aspartate (NR2A/B) ionotropic receptor subtypes have been identified, as well two metabotropic glutamate subtypes (mGlu5a and mGlu5b). Roles for these receptors in microglia are not fully known. However, one possible theory suggests increased production of cytotoxic cytokines, such as tumor necrosis factor-, following ischemia and traumatic brain injuries (Noda et al., 2000). Less is known concerning the expression of the well characterized drug transporters within microglia. We have recently identified two nucleoside drug transporters within a continuous rat microglia cell culture system (Hong et al., 2000, 2001). As well, we have positively identified the existence of a functional form of P-gp within these rat-derived microglia (Lee et al., 2001).

Several reviews summarize transport of ions, neurotransmitters, and nutrients within neurons and their myelinating cells oligodendrocytes (Vannucci et al., 1997; Seal and Amara, 1999; Verkhratsky and Steinhauser, 2000). However, there are limited studies documenting peripheral drug transporters localized to either the oligodendrocytes or neurons (Busch et al., 1998).

	IV. Methods to Quantitate Drug Transport into/out of the Central Nervous SystemIn Vivo and In Vitro Methods

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In vivo and in vitro techniques utilized to examine drug transport in the brain will only be briefly discussed as a review of these methods is beyond the scope of this paper and can be found elsewhere (Fenstermacher et al., 1981). In vivo BBB models of drug transport can be broadly categorized according to methodological approach. Single passage techniques such as the indicator diffusion/dilution (Crone, 1963, 1965), brain uptake index (Oldendorf, 1970), and external registration (Raichle et al., 1974, 1976) measure the uptake of substances into the CNS following a single passage through the brain upon injection into the blood stream. A major disadvantage of the single passage techniques is that transport estimates of drugs or solutes with extremely slow uptake may be inaccurate due to the short solute exposure times (Enting et al., 1998). Multipassage techniques, then, can be used to allow the test substance longer circulation times. Intravenous administration (Enting et al., 1998) and microdialysis methods are examples of multipassage techniques (Parsons and Justice, 1994; Boschi et al., 1995; de Lange et al., 1995). These techniques are model-dependent, and the method of data analysis (i.e., two-compartment model, three-compartment model, etc.) is normally chosen prior to the experiment. Therefore, once chosen, the results are model specific and may not necessarily be indicative of the actual transport and metabolic processes within the tissue (Fenstermacher et al., 1981). Finally, perfusion techniques, such as the in situ perfusion method, expose the brain tissue to the test substance by perfusion with a physiological buffer (van Bree et al., 1992). This model was developed to provide further control over the experimental conditions (pH, temperature, etc.) and to avoid metabolism of the test substance during transfer across the BBB. Compared with single or multipassage methods, permeability coefficients can be measured accurately over a 10⁴-fold range (Takasato et al., 1984) making this method 100-fold more sensitive. Therefore, measurements of brain uptake of poorly penetrating compounds (P = 10⁸ to 10⁷ cm · s¹) or rapidly penetrating compounds (P = 10⁴ cm · s¹) can be determined allowing for the characterization of carrier-mediated transport at the BBB (Smith et al., 1984). The involvement of complex surgery and the requirement of mathematical models are the main disadvantages of the perfusion models (Takasato et al., 1984; Enting et al., 1998)

Although a number of experimental approaches have been developed to quantify transport of compounds from the blood into the CSF, many of these methodologies have proven to be inaccurate or do not produce useful data. In addition, the experimental procedure is quite complex and requires a certain amount of surgical skill and experience. The more common methodologies include the CNS deconvolution technique (van Bree et al., 1989) and the in situ CP model (Ames et al., 1964). The CNS deconvolution technique is based on serial sampling of the CSF and numerical deconvolution of data to determine a transport profile of the drug in a single living animal. The in situ CP model replaces the endogenous CSF with oil such as ethyl iodophenylundecylate, which allows the CP to be easily visualized. The fluid droplets that are formed on the surface are collected and a steady-state clearance fraction of the drug can be determined (Ames et al., 1964).

In general, in vivo methodologies to study drug transport in the CNS are costly. Furthermore, it is often difficult to maintain control of environmental factors such as pH, temperature, osmotic pressure, oxygen, carbon dioxide, as well as physiological responses (metabolism, tissue distribution, excretion) that occur in the animal under normal and experimental conditions (Freshney, 1994). An alternative to in vivo studies of drug transport is in vitro cell and tissue culture systems. Tissue culture techniques were developed as a method for studying the behavior of a specific population of cells free of systemic variations that may arise in the animal both during normal homeostasis and under stress of an experiment (Harrison, 1907). The development of tissue culture transport systems has revolutionized the drug transport field and has resulted in an explosion of research over the last 50 years. Not only do cell cultures provide a level of control over the environment and various physiological responses, they also provide specific information on the type of transporter(s) involved and relative pharmacokinetic parameters such as carrier affinity and specificity. Nevertheless, these systems are limited in that many of the phenotypic and functional characteristics of the original tissue may be lost (i.e., tight junctions in brain endothelial cells, production of specific factors by cells, expression and activity of various transporters) due to culture conditions and the absence of endogenous factors and signals (Freshney, 1994). For example, gene expression of some drug transporters in the brain (i.e., P-gp and MRP) can be both up- and down-regulated in culture (Regina et al., 1998). This change in gene expression that sometimes occurs in culture may be a consequence of a variety of factors such as culture conditions (presence of serum in media and nature of substratum) and the absence of endogenous factors and signals that are present in vivo. Consequently, caution must be taken when extrapolating in vitro tissue culture data to either in vivo models or clinical practice.

A common method of studying in vitro drug transport of nonpolarized cells involves culture and growth of isolated cells on impermeable polystyrene strata (e.g., 24-well plates) and measurement of the cellular uptake/accumulation or efflux of a radiolabeled substrate or fluorescent probe. Specific transporter characteristics can then be examined utilizing known transporter inhibitors, metabolic inhibitors, etc., which are appropriate for the transporter of interest (Hunter et al., 1991; Hong et al., 2001). Polarized cells, such as epithelial and endothelial cells, can also be grown on porous filter membranes, which provide the option of examining both basal-to-apical and apical-to-basal transport of substrates. Recently, Miller et al. (2000) have developed a novel method to characterize transport properties of substrates in isolated brain capillaries using fluorescent substrates, confocal microscopy, and quantitative image analysis. This method minimizes the possibility of altered transporter expression and activity observed in cell culture systems (Regina et al., 1998; Gaillard et al., 2000) and provides direct evidence of transport in isolated capillaries, which can be masked by other efflux transporters (i.e., MRP) in conventional transport assays. Most importantly, this method provides spatial resolution where the substrate fluorescence can be distinguished from that in the endothelium and associated cells.

By far, the most extensively studied cells of the brain are the endothelial cells of the blood-brain barrier (van Bree et al., 1992). Growth of homogenous cultures of brain microvessel endothelial cells, both primary cultures or immortalized cultures, have been described from various mammalian species including rat, bovine, and human (Tsuji et al., 1992; Begley et al., 1996; Seetharaman et al., 1998). A variety of drug transporters have now been identified and characterized utilizing these methods and will be discussed in detail including organic cation transporters (Wu et al., 1998a), organic anion transporters (Gao et al., 1999), nucleoside transporters (Thomas and Segal, 1996), P-gp (Tatsuta et al., 1992), and MRP (Regina et al., 1998).

The BBB is not an isolated tissue. Therefore, in an attempt to produce a more representative in vitro BBB model, the coculture BBB system was developed (Meyer et al., 1991). Cocultures of endothelial and astrocyte cells allow for greater cell differentiation and may express specific proteins that are not present in monocultures (Regina et al., 1998). For example, primary rat brain capillary endothelial cultures generally do not maintain tight junctions past the fourth cell passage. However, addition of astrocyte-conditioned culture media can re-establish these junctions (Tao-Cheng et al., 1987). Furthermore, compared with single cultures, BBB cocultures have a down-regulated expression of MRP (Regina et al., 1998) and up-regulated P-gp expression (Gaillard et al., 2000), which is reflective of the in vivo expression patterns. Thus, a coculture system provides a more physiologically accurate representation of the BBB and allows for more meaningful studies of drug transport, metabolism, and drug-drug interactions at the cellular level. The coculture system is achieved by growth of the endothelial cells and astrocytes either in the same culture dish or via growth on the opposite sides of a porous filter, which permits cell to cell contact between the astrocyte foot processes and the endothelium (Pardridge, 1999).

Drug transport across the epithelial cells of the CP has also been well characterized (Washington et al., 1996). A variety of mammalian cell culture systems have been described and produce an impermeable cell monolayer that displays many of the characteristics of the CP barrier in vivo (Zheng et al., 1998; Haselbach et al., 2001). Transporters identified to date in the CP include organic anion transporters (Gao and Meier, 2001), P-gp and MRP (Rao et al., 1999), nucleoside transporters (Wu et al., 1994), and organic cation transporters (Suzuki et al., 1986).

In contrast to the BBB and CP, primary cultures and continuous cell lines of astrocytes and microglia are not polarized. As a result, only unidirectional cellular accumulation or efflux can be measured (Hertz et al., 1998). Although the functional expression of nucleoside transporters P-gp and MRP has been characterized to some extent in cultures of primary astrocytes (Hosli and Hosli, 1988; Gu et al., 1996; Declèves et al., 2000), drug transport studies in microglia remain extremely limited. Recently, we have characterized a Na⁺-dependent nucleoside transporter (Hong et al., 2000) and a novel electrogenic zidovudine/H⁺-dependent transporter (Hong et al., 2001) in microglia, utilizing a continuous rat brain microglia cell line (MLS-9) developed by Schlichter et al. (1996). These studies provide evidence that microglia express membrane transporters that may be important for drug transport and distribution in brain parenchyma. More recently, we have characterized the functional expression of P-gp (Lee et al., 2001) and MRP (Dallas et al., 2001) within primary and continuous microglia cell cultures.

In general, isolated spheroid microglia cells rarely differentiate into their mature ramified form in the absence of astrocytes (Tanaka and Maeda, 1996). Indeed, the continuous microglia cell line (MLS-9) does not exhibit the morphology of ramified, process-bearing microglia in culture (Hong et al., 2000). At confluence these cells are more characteristic of spheroid microglia precursors or "activated" microglia, with short processes and large egg-shaped cell bodies (Hong et al., 2000). Tanaka and Maeda (1996) have demonstrated that microglia-astrocyte cocultures promote highly differentiated and ramified microglia. As with brain endothelial-astrocyte cocultures, it appears that astrocytes provide a variety of diffusible factors that are present in vivo to promote differentiation of microglia cells in vitro. The study of drug transport in these microglia-astrocyte cocultures certainly warrants future investigation.

	V. Drug Transport Mechanisms in the Brain

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It was originally believed that membrane carriers localized at the brain barriers were solely responsible for the transport of endogenous substances into and out of the brain and that drug transport across the brain barriers was largely dependent on the physicochemical characteristics of the drug such as lipophilicity, molecular weight, and ionic state (Spector, 1990; Tamai and Tsuji, 2000). Generally, small, nonionic, lipid-soluble molecules penetrate easily across the BBB whereas larger, water-soluble, and/or ionic molecules will less likely exhibit passive diffusional processes (Spector, 1977, 1990). For some drugs the rate of entry and distribution in the CNS cannot be explained by passive processes that depend on the physicochemical characteristics listed above (Spector, 1987, 1988; Takasawa et al., 1997b). Many drug transporters that have been well characterized in peripheral tissues and are known to be involved in the influx and efflux of drugs (i.e., the organic cation, organic anion, nucleoside, P-gp, and MRP transporters), have now been identified in the brain. It is now recognized that these drug transporters may influence many pharmacokinetic characteristics of drugs in the processes of absorption, distribution, and elimination.

A diverse group of organic cations, including endogenous bioactive amines (i.e., acetylcholine, choline, dopamine, epinephrine, norepinephrine, guanidine, N¹-methylnicotinamide, thiamine), therapeutic drugs (i.e., cimetidine, amiloride, mepiperphenidol, morphine, quinine, quinidine, tetraethylammonium, verapamil, trimethoprim), and xenobiotics (i.e., paraquat), are actively transported by the OCT system primarily in the liver and kidney (Rennick, 1981; Zhang et al., 1998). At physiological pH, the nitrogen moiety of these compounds (generally primary, secondary, tertiary, or quaternary amines) bears a transient or permanent net positive charge, which is determined by the compound's pK_a value. Two distinct classes of OCT systems have been defined: a potential-sensitive transporter usually involved in the influx of organic cations and an H⁺ gradient-dependent transporter, mediating efflux (Ullrich, 1994). The concerted action of these two OCT subtypes results in the vectorial transfer of cationic compounds from the blood into the luminal fluid across the renal tubular cells (Hsyu and Giacomini, 1987; Dantzler et al., 1989; Bendayan et al., 1990, 1994; Escorbar et al., 1994) or from the blood into the bile across the hepatocyte, intestinal epithelium, and the placental syncytiotrophoblast (Ganapathy et al., 1988; Prasad et al., 1992; Iseki et al., 1993; Zevin et al., 1997; Laforenza et al., 1998). In the brain, the physiological role of the OCT systems includes transport of cationic neurotoxins and neurotransmitters (Murakami et al., 2000).

At present, the OCT family includes three potential-sensitive (i.e., OCT1, OCT2, OCT3) and two H⁺-driven systems (i.e., OCTN1 and OCTN2) (Table 1). In general, OCTs contain 12 transmembrane domains with a large extracellular hydrophobic loop between the first and second domains and a large intracellular hydrophobic loop between the sixth and seventh domains (Koepsell, 1998). Although the extracellular loop contains several glycosylation sites, the intracellular loop has a number of potential phosphorylation sites (Fig. 4). The exact membrane topology of this system remains to be fully characterized.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Organic cation transport systems in the CP. The proposed topology for rat OCT1 is shown. P, phosphorylation sites. In the CP, several studies have reported the removal of organic cations (i.e., tetraethylammonium, choline) from the CSF into the blood by a variety of mechanisms (i.e., pH dependent, electrogenic). Only the OCT systems that have been experimentally localized to a specific side of the CP epithelium are presented. Arrows indicate the direction of transport. Adapted from Koepsell, 1998.

OCT1 was originally cloned from rat kidney (Grundemann et al., 1994), followed by the isolation of the mouse, human, and rabbit homologs (Schweifer and Barlow, 1996; Gorboulev et al., 1997; Zhang et al., 1997; Terashita et al., 1998). Similarly, OCT2 was cloned from rat kidney by homolog screening (Okuda et al., 1996). Subsequently, the human and porcine homologs were isolated and characterized (Gorboulev et al., 1997; Grundemann et al., 1997). Northern blot analysis has shown that both OCT1 and OCT2 are expressed primarily in the kidney and liver and, to a smaller extent, in the intestine (Grundemann et al., 1994; Okuda et al., 1996; Gorboulev et al., 1997; Zhang et al., 1997). Both of these transporters recognize a variety of endogenous and exogenous organic cations as substrates and exhibit considerable overlap in substrate specificity. Several cationic neurotoxins and monoamine neurotransmitters are accepted as substrates by OCT1 and OCT2 (Martel et al., 1996; Gorboulev et al., 1997; Zhang et al., 1997; Busch et al., 1998). In particular, the polyspecific, electrogenic OCT2 present in human neurons has been reported to mediate the transport of monoamine neurotransmitters dopamine, norepinephrine, serotonin, histamine, and the antiparkinsonian drugs, amantadine and memantine (Busch et al., 1998). In voltage-clamp experiments with rOCT1-expressing Xenopus oocytes, tracer flux of dopamine, serotonin, noradrenaline, histamine, and acetylcholine induced saturable currents with K_m values ranging from 20 to 100 µM (Busch et al., 1996). Although not detectable by Northern analysis, RT-PCR studies indicate that OCT1 and OCT2 may be expressed in the brain, but at very low levels (Gorboulev et al., 1997; Grundemann et al., 1997). A third organic cation transporter, OCT3, was cloned more recently from rat placenta and appears to be ubiquitously expressed (Kekuda et al., 1998). Moreover, OCT3 appears to be expressed more abundantly in the mammalian brain than either OCT1 or OCT2. For example, in situ hybridization studies demonstrated that OCT3 is widely expressed in several different brain regions, including the hippocampus, cerebellum, and cerebral cortex (Wu et al., 1998a). Accumulation studies utilizing tetraethylammonium demonstrated a low affinity (K_m = 2.5 mM) saturable system in OCT3 cDNA transfected HeLa cells (Kekuda et al., 1998). OCT3-specific methyl-4-phenylpyridinium uptake activity (apparent K_m = 91 µM) in a human retinal pigment epithelial cell line indicates that various cationic neurotoxins and neurotransmitters are OCT3 substrates. The order of affinity was amphetamine > desipramine > metamphetamine > dopamine > serotonin (IC₅₀ range = 42-970 µM) (Wu et al., 1998a). The transport characteristics and steroid (i.e., -estradiol) sensitivity provide strong evidence for the molecular identity of OCT3 as an extraneuronal monoamine transporter (uptake₂) (Wu et al., 1998a).

The H⁺ driven organic cation transporters OCTN1 and OCTN2, were cloned originally from human fetal liver and placenta, respectively (Tamai et al., 1997; Wu et al., 1998b). OCTN1 is predominantly expressed in kidney, trachea, bone marrow, fetal liver, and in several human cancer cell lines but not in adult liver (Tamai et al., 1997). Northern blot analysis revealed that the expression of OCTN2 occurs mainly in heart, placenta, skeletal muscle, kidney and pancreas, with weak signals observed in brain, lung, and liver (Wu et al., 1998b). The regional brain distribution of this transporter remains to be established. When expressed in HeLa cells, OCTN2 mediates the transport of a number of classical organic cation transporter substrates including tetraethylammonium and methyl-4-phenylpyridinium (Wu et al., 1998b). Recently it has been shown that OCTN2 can transport carnitine in a Na⁺-dependent manner in HEK293 cells (Tamai et al., 1998). Three other cDNA clones, NLT (cloned from rat liver) RST, and NKT (cloned from mouse kidney) exhibit significant sequence homology to the OCT transporter family. The transport functions of these three clones in the brain remains to be examined (Simonson et al., 1994; Lopez-Nieto et al., 1997; Mori et al., 1997).

Although OCT mechanisms in the kidney and liver are well characterized, experimental assessment of the transport mechanisms in the brain, such as via the CP, is limited by the small size, complex morphology, and anatomic inaccessibility of the CP epithelia. Some transport properties of the CP OCT have been obtained using in situ ventriculocisternal perfusion (Miller and Ross, 1976; Lanman and Schanker, 1980), preparations of isolated CP (Tochino and Schanker, 1965a,b), and apical membrane vesicles (Whittico et al., 1990). However, these techniques do not provide direct access to both interfaces of the intact CP epithelium and information on the energetics and polarities of the OCT carriers across the CSF-blood barrier remains incomplete. A notable tissue related difference between the OCT systems is that in the kidney, the transporter functions in the secretory direction (i.e., from blood to urine) whereas in the CP, the transporter functions in the absorptive direction (i.e., from CSF to blood). Carrier-mediated transepithelial absorption of both endogenous and xenobiotic organic cations (e.g., choline, N¹-methylnicotinamide, tetraethylammonium, cimetidine, serotonin, and norepinephrine) from CSF has been demonstrated both in vivo and in vitro using isolated CP tissue slices and ventriculocisternal perfusion techniques (Schanker et al., 1962; Tochino and Schanker, 1965b; Hug, 1967; Barany, 1976; Miller and Ross, 1976; Lanman and Schanker, 1980; Suzuki et al., 1985, 1986).

Apical tetraethylammonium uptake by rat cultured CP epithelial cells was a pH dependent saturable process with an apparent K_m of 315 µM (Villalobos et al., 1997) (Fig. 4). This affinity constant is similar to that reported for tetraethylammonium in both basolateral (160 µM, Ullrich et al., 1991; 280 µM, Brandle et al., 1992) and luminal (192 µM, Wright et al., 1995) membranes of the kidney. However, a K_m of 900 µM was estimated from tetraethylammonium inhibition of cimetidine transport by isolated CP in vitro (Suzuki et al., 1986). The apical tetraethylammonium uptake by cultured CP epithelium was markedly reduced (40%) by other organic cations, such as choline, mepiperphenidol, but not by the organic anion p-aminohippurate (Villalobos et al., 1997). This sensitivity of tetraethylammonium transport in cultured CP cells to quaternary ammonium compounds and its insensitivity to the organic anion p-aminohippurate corroborates previous studies of OCT transport across the intact CSF-blood barrier and in isolated CP (Schanker et al., 1962; Tochino and Schanker, 1965a; Hug, 1967; Miller and Ross, 1976; Lanman and Schanker, 1980).

Interestingly, transepithelial absorption of the organic cation cimetidine from rat CSF and CP uptake in vitro are inhibited by the organic anions benzylpenicillin and salicylic acid but not by tetraethylammonium and other quaternary ammonium compounds (Suzuki et al., 1985, 1986, 1988). Furthermore, cimetidine inhibits transport of organic anions but poorly inhibits quaternary ammonium transport. The lipophilic organic bases quinidine and quinine are potent inhibitors of cimetidine transport in isolated rat plexus tissue and bovine ventricular brush-border membrane vesicles (Suzuki et al., 1986; Whittico et al., 1990). The transport kinetics of cimetidine, either by isolated rat CP, or by isolated bovine CP vesicles (proven to be pH driven in this system) were similar with apparent K_m values of 53 and 58 µM, respectively. These data suggest that cimetidine is transported across the CP apical membrane by a different mechanism than the brush-border membrane of the kidney, with a lower affinity and higher capacity (Gisclon et al., 1987). Electrogenic apical uptake of choline across the ventricular membrane of neonate rat CP has been demonstrated (Villalobos et al., 1999) (Fig. 4). Choline appears to be transported across the CSF-blood barrier (K_m = 16-50 µM) with greater affinity than by i) BBB (K_m = 225-445 µM); ii) apical membranes of the renal proximal tubule (K_m = 100 µM); and iii) the small intestine (K_m = 150 µM) (Aquilonius and Winbladh, 1972; Cornford et al., 1978; Lanman and Schanker, 1980; Saitoh et al., 1992; Wright et al., 1992).

There is also evidence for the transport of endogenous bioactive amines such as choline and thiamine across the BBB (Koepsell, 1998). An in vivo study showed that thiamine monophosphate is transported across the BBB into the brain by a saturable mechanism with a K_m of 2.6 to 4.8 µM, possibly by an organic cation transporter (Patrini et al., 1988). In cultured brain capillary endothelial cells, there is both saturable and nonsaturable uptake processes for choline (Sawada et al., 1999). The saturable process was energy-dependent (Galea and Estrada, 1992), sodium, and pH independent and could be inhibited by various OCT substrates and inhibitors (Sawada et al., 1999). Furthermore, in situ brain perfusion studies corroborate the in vitro data by demonstrating the presence of a sodium-independent transporter for choline uptake into the brain (K_m = 39-42 µM) (Murakami et al., 2000; Allen and Smith, 2001). These in vitro and in vivo results suggest that the choline transporter at the BBB is a member of the OCT family. The membrane location of these transporters remains to be elucidated.

The liver and kidney are organs central to the elimination of endogenous and exogenous organic anions, many of which are harmful to the body (Pritchard and Miller, 1993; Ullrich and Rumrich, 1993; Meier, 1995; Muller and Jansen, 1997). Several families of multispecific organic anion transporters have been identified, of which the two main families, i.e., the organic anion transporter polypeptide (oatp), and the organic anion transporter OAT will be discussed (Sekine et al., 2000).

To date, seven isoforms [oatp1, oatp2, oatp3, OAT-K1, OAT-K2, OATP, prostaglandin transporter (PGT), and the liver-specific transporter-1 (LST-1)] have been identified in the oatp family (Sekine et al., 2000) (Table 2). In the liver, oatp1 and oatp2 are multispecific organic anion carriers that transport structurally unrelated anionic compounds in a sodium-independent manner (Meier, 1995; Muller and Jansen, 1997; Noe et al., 1997; Kakyo et al., 1999a). Both are expressed in the brain. Oatp1, a bidirectional organic anion/HCO and/or organic anion/glutathione exchanger, is expressed at the apical membrane of the CP (Angeletti et al., 1997; Li et al., 1998) in contrast to its basolateral localization in the hepatocyte (Bergwerk et al., 1996). It possesses a broad substrate specificity and mediates the transport of bile salts, steroid hormones, and a variety of organic anions and cations (Sekine et al., 2000). However, whether oatp1 is responsible for the uptake or efflux of organic anions across the CP remains to be elucidated (Angeletti et al., 1997). Oatp2, cloned from rat brain, is expressed in liver, kidney, brain capillaries, and the basolateral membrane of the CP (Noe et al., 1997; Gao et al., 1999). It mediates the uptake of bile acids taurocholate, cholate, estrogen conjugates, ouabain, and digoxin (Noe et al., 1997; Asaba et al., 2000). Oatp3, isolated from rat retina and expressed in kidney and retina, was shown to transport thyroid hormones and taurocholate (Abe et al., 1998). OAT-K1 and OAT-K2 are both localized to the luminal membrane of the renal proximal tubule (Masuda et al., 1997, 1999). OAT-K1 is involved in the transport of methotrexate and folate whereas OAT-K2 transports hydrophobic organic anions such as taurocholate, methotrexate, folate, and prostaglandin E2 (Masuda et al., 1997, 1999). OATP is the cloned human liver organic anion carrier that transports bromosulfophthalein, cholate, taurocholate, glycocholate, taurochenodeoxycholate, and tauroursodeoxycholate in a sodium-independent manner (Kullak-Ublick et al., 1995). It is expressed in human lung, kidney, and testes. Recently, OATP was shown to be expressed along the BBB in cultured human brain endothelial cells (Gao et al., 2000). This transporter was found to transport two opioid peptides, deltorphin II (K_m 330 µM) and the enkephalin analog, [D-Pen(2),D-Pen(5)]enkephalin (K_m ~202 µM), the latter also transported by rat oatp2 at the BBB (Kakyo et al., 1999). On the basis of sequence homology, PGT and LST-1 are believed to be oatp isoforms, of which the latter may be important for bile clearance (Kanai et al., 1995; Kakyo et al., 1999b).

The OAT family is primarily responsible for the elimination of organic anions from the kidney. While all four isoforms (OAT1, OAT2, OAT3, OAT4) are expressed in the kidney, a few are also expressed in the liver, brain, and placenta (Sekine et al., 2000). In general, these proteins possess 12 putative transmembrane domains, with large hydrophobic loops between the first and second, and the sixth and seventh domains (Sekine et al., 2000) (Fig. 5). N-Glycosylation sites are predicted on the hydrophobic loop between the first and second transmembrane domain (Kuze et al., 1999) as well as several phosphorylation sites on the loop between the sixth and seventh domains (Sekine et al., 2000).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Organic anion transport systems in the BBB and CP. The proposed topology for rat OAT1 is shown. P, phosphorylation sites. In addition to the transport of a variety of organic anions across the brain barriers, the active uptake of digoxin across the BBB and the secretion of estrogen conjugates and opioid peptides across the CP have been reported. Only the OAT systems that have been experimentally localized to a specific side of the BBB endothelium and CP epithelium are shown. Arrows indicate the direction of transport. Adapted from Sekine et al., 2000.

Organic anion transporters are categorized into three classes depending on their energy requirements: sodium-dependent OATs, sodium-independent facilitators or exchangers, and active OATs that require ATP. The active and sodium-independent OATs possess broad substrate specificity and are primarily involved in the secretion of organic anions in both kidney and liver. The sodium-dependent OATs on the other hand, have a narrow substrate specificity and play a major role in the reabsorption of essential anionic substances into the proximal tubules of the kidney (Sekine et al., 2000) (Table 2).

In the kidney, it is well established that anionic drugs and other xenobiotics are actively transported from the blood to the urine (Pritchard and Miller, 1993). The basolateral step is indirectly coupled to the sodium gradient by Na⁺/dicarboxylate cotransport, which maintains a large in > out gradient for the -ketoglutarate/organic anion exchange (Shimada et al., 1987; Pritchard, 1988, 1990). This exchanger (OAT1) has recently been cloned in rat (Sekine et al., 1997; Sweet et al., 1997) and human (Cihlar et al., 1999; Hosoyamada et al., 1999; Lu et al., 1999). OAT1, specifically expressed in kidney, is a multispecific organic anion/dicarboxylate exchanger that interacts with a variety of organic anions, i.e., p-aminohippurate (K_m = 14.3 µM), dicarboxylates, cyclic nucleotides, prostaglandin E, urate, antibiotics, nonsteroidal anti-inflammatory drugs, and diuretics (Sekine et al., 1997). OAT2 is a liver-specific organic anion transporter and accepts p-aminohippurate, salicylate and acetylsalicylate, prostaglandin E, and dicarboxylates as substrates (Sekine et al., 1998). Apical renal exit of organic anions is also carrier-mediated but is not well characterized and may involve either potential or exchange driven mechanisms (Pritchard and Miller, 1993).

In the brain, the expression of OAT1 is very low (Sekine et al., 1997). Recently, a new member, OAT3, was isolated from rat brain by RT-PCR cloning methods (Kusuhara et al., 1999). OAT3 mRNA is expressed in liver, brain, kidney, and eye. When expressed in Xenopus oocytes, it mediates the transport of p-aminohippurate (K_m = 65 µM) and cimetidine. Acidic metabolites of neurotransmitters (i.e., dopamine, epinephrine, norepinephrine, and serotonin) inhibited the uptake of estrone sulfate by OAT3 suggesting its role in the excretion/detoxification of endogenous anionic substrates from the brain (Kusuhara et al., 1999). OAT4, expressed in the placenta and kidney, is a novel member of the multispecific OAT family exhibiting approximately 38 to 44% amino acid sequence homology to the other members of the OAT family (Cha et al., 2000). It mediates the transport of estrone sulfate, dehydroepiandrosterone sulfate, and a variety of anionic compounds (i.e., bile salts, sulfobromophthalein, diuretics) in a sodium-independent manner.

Direct mechanistic information on organic anion systems along the blood-CSF barrier is sparse, owing in part to the small size and physical inaccessibility of the plexus and in part to gaps in our understanding of the mechanisms and driving forces mediating OAT processes (Pritchard and Miller, 1993). Although the molecular mechanisms responsible for CP transport are largely unexplored, one fundamental difference from excretory renal epithelia is evident: organic anions are transported into the blood, not extracted from it. Indeed, this reversal in function is reflected in other important ways, most notably in the unique apical distribution of Na⁺,K⁺-ATPase in CP, whereas it is basolateral in virtually all other epithelia (Quinton et al., 1973; Ernst et al., 1986; Villalobos et al., 1997).

In addition to the blood-CSF barrier, OAT systems have also been localized along the BBB. Studies show that P-gp-defici