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Vol. 53, Issue 4, 569-596, December 2001
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada
Abstract
I. Introduction
II. The Blood-Brain Barrier and the Choroid Plexus
III. Brain Parenchyma
A. Astrocytes
B. Microglia
C. Oligodendrocytes and Neurons
IV. Methods to Quantitate Drug Transport into/out of the Central Nervous SystemIn Vivo and In Vitro Methods
A. In Vivo Models to Study Drug Transport across the Blood-brain Barrier and the Choroid Plexus
B. In Vitro Models to Study Drug Transport in the Brain
V. Drug Transport Mechanisms in the Brain
A. Organic Cation Transport Systems
B. Organic Anion Transport Systems
C. Nucleoside Transport Systems
D. Efflux Transport Systems
1. P-Glycoprotein.
2. Multidrug Resistance Protein Family.
VI. Summary
Acknowledgments
References
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Abstract |
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Drug transport in the central nervous system is highly regulated not only by the blood-brain and the blood-cerebrospinal fluid barriers but also in brain parenchyma. The novel localization of drug transporters in brain parenchyma cells, such as microglia and astrocytes, suggest a reconsideration of the present conceptualization of brain barriers as it relates to drug transport. That is, the cellular membranes of parenchyma cells act as a second "barrier" to drug permeability and express transporters whose properties appear similar to those localized at the conventional brain barriers. This review will focus on the molecular characteristics, localization, and substrate specificities of several classes of well known membrane drug transporters (i.e., the organic cation, organic anion, nucleoside, P-glycoprotein, and multidrug resistance proteins) in the brain. Comparisons to similar transporters localized within the peripheral system and clinical implications of the functional expression of specific drug transport families will be discussed when appropriate. Nutrient and neurotransmitter transporters, whose characteristics have been reviewed extensively elsewhere, will not be considered in this review.
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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).
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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).
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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).
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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
; Johanson et al.,
1990; Garner and Brown, 1992). The basolateral side is lined with
Na+/H+ antiporters,
Cl/HCO; 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).
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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.
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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).
A. Astrocytes
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).
B. Microglia
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
(5
1,
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).
C. Oligodendrocytes and Neurons
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).
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IV. Methods to Quantitate Drug Transport into/out of the
Central Nervous System In Vivo and In Vitro Methods |
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A. In Vivo Models to Study Drug Transport across the Blood-brain Barrier and the Choroid Plexus
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).
B. In Vitro Models to Study Drug Transport in the Brain
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.
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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. Organic Cation Transport Systems
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 pKa 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.
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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 Km 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 (Km = 2.5 mM) saturable system in OCT3 cDNA transfected HeLa cells (Kekuda et
al., 1998). OCT3-specific methyl-4-phenylpyridinium uptake activity
(apparent Km = 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 Km 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
Km 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
Km 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 (Km = 16-50 µM) with greater affinity than by i) BBB
(Km = 225-445 µM); ii) apical
membranes of the renal proximal tubule
(Km = 100 µM); and iii) the small
intestine (Km = 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
Km 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
(Km = 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.
B. Organic Anion Transport Systems
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; 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
(Km 330 µM) and the enkephalin
analog,
[D-Pen(2),D-Pen(5)]enkephalin
(Km ~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).
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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).
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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
(Km = 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 (Km = 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-deficient mice
[mdr1a(
/)] exhibit significantly increased brain concentrations of a variety of drugs, including digoxin, a cardiac glycoside (Mayer et
al., 1996; Schinkel et al., 1996; Kim et al., 1998). Interestingly, the
digoxin concentration continued to increase in the brains of the
mdr1a(/) over a long time period despite diminishing blood levels,
suggesting a possible active uptake system for digoxin along the BBB
(Mayer et al., 1996). It is now established that this system is the
novel multispecific OAT (Oatp2) that possesses an extremely high
affinity for digoxin (Km = 0.24 µM)
and has been cloned from rat brain. This transporter is localized along
both the luminal and abluminal membranes of the BBB (Noe et al., 1997;
Gao et al., 1999) (Fig. 5). Moreover, uptake studies in conditionally
immortalized mouse brain capillary endothelial cells (TM-BBB4) that
express Oatp2 show that dehydroepiandrosterone sulfate is a substrate
for this transporter (Km = 34.4 µM)
(Asaba et al., 2000). Recently, the first human OATP (now called
OATP-A), which was cloned from liver, 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 (Km = 330 µM) and
[D-Pen(2),D-Pen(5)]enkephalin
(Km ~202 µM), the latter also
transported by rat Oatp2 at the BBB (Kakyo et al., 1999).
As first documented by Pappenheimer et al. (1961), active transporters
eliminate organic anions from the brain, thus preventing the buildup of
potentially toxic compounds. In vivo and in vitro kinetic studies have
suggested the presence of efflux transport pathways for organic anions
in the BBB and blood-CSF barrier. CP was shown to mediate the removal
of organic anions from the CSF into the blood for their subsequent
elimination by liver or kidney. This has been established by the
observations that organic anions are eliminated from CSF after
intraventricular administration and/or during ventriculocisternal
perfusion and that the respective organic anions are accumulated in the
isolated CP (Suzuki et al., 1997). This includes neurotransmitter
metabolites [i.e., 5-hydroxyindole acetic acid (from serotonin) and
homovanillic acid (from dopamine) (Neef et al., 1967; Cserr and Van
Dyke, 1971; Forn, 1972)] as well as various anionic compounds [i.e.,
2,4-dichlorophenoxy acetic acid, methotrexate, salicylate, and
benzylpenicillin (Rubin et al., 1968; Lorenzo and Spector, 1973;
Pritchard, 1980; Suzuki et al., 1987)]. It has also been suggested
that an apical Oatp1, an energy-dependent and probenecid-sensitive
transport system, mediates the uptake of 17-estradiol
17-D-glucuronide (Km = 3.4 µM) in isolated rat CP (Nishino et al., 1999). Similar kinetic constant for oatp1 transport of this substrate has been reported in
cRNA-injected oocytes and cDNA-transfected mammalian cells (Meier et
al., 1997). When microinjected into cerebral cortex, the rapid and
saturable elimination of p-aminohippurate from the brain was
observed (Kakee et al., 1997). These results suggest that these efflux
transport properties are consistent with transport by the OAT family,
especially OAT3. The uptake of the anion 2,4-dichlorophenoxy acid by
bovine CP brush-border membrane vesicles and intact bovine and rat
plexus tissues involved a glutarate/organic anion exchanger (Pritchard
et al., 1999) (Fig. 5). These results are similar to those reported for
p-aminohippurate uptake by renal basolateral membrane
vesicles (Shimada et al., 1987; Pritchard, 1988). The apical
localization of an organic anion/dicarboxylate exchanger was
demonstrated by the expression of OAT1-green fluorescent protein constructs in rat CP (Pritchard et al., 1999), in direct contrast to
the basolateral localization of this same construct in rat proximal
tubules (Sweet et al., 1999). In addition, the nucleoside analog
didanosine, was shown to be transported from CSF into the blood by a
probenecid-sensitive OAT system across the rat CP, whereas zidovudine
is recognized, but not transported, by this system (Galinsky et al.,
1990; Wong et al., 1993; Masereeuw et al., 1994; Takasawa et al.,
1997a). Moreover, the presence of transporters for cellular extrusion
on the basolateral membrane would account for the efficient
transcellular transport of organic anions across the CP from CSF to the
blood side. Recently, it has been established that MRP homologs
(MRP1/MRP2) are responsible for the cellular extrusion of organic
anions from various organs (Keppler and Konig, 1997; Muller and Jansen,
1997; Suzuki and Sugiyama, 1998) including the CNS via the BBB
(Kusuhara et al., 1998) (see Section V.D.).
In summary, as described for the OCT systems, a vectorial transfer of organic anions is mediated by a concerted action of members of the OAT family and ATP-binding cassette (ABC) family of multispecific transporters. The polar localization of these transporters may complement their function in the transport of organic anions into and/or out of the CSF. Although localization and transport mechanisms are unclear in the brain, several multispecific OAT systems, predominantly OAT3, and to a lesser extent Oatp1, Oatp2, and OAT1 have been identified at the CP and BBB. Future immunohistochemical and transport analysis of these OAT systems, and those yet to be identified, will reveal the cellular localization and transport regulation of these carriers and elucidate more precisely their physiological role in the CNS.
C. Nucleoside Transport Systems
Purine and pyrimidine nucleosides and their metabolic products are
the precursors of the nucleic acids, DNA and RNA, and participate in
numerous biological brain processes. For example, the nucleoside adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels (Bender et al., 1980; Thampy and Barnes, 1983). In general, nucleosides are synthesized endogenously via de novo synthetic pathways (Carver, 1999). However, a number of tissues including brain are deficient in de
novo nucleotide synthetic pathways and rely on the salvage of exogenous
nucleosides to maintain nucleoside pools and to meet their metabolic
demands (Fox and Kelley, 1978). Therefore, the brain is dependent on a
continuous and balanced supply of purine and pyrimidine nucleoside
constituents from both synthesis in situ and the blood (Santos et al.,
1968; Tremblay et al., 1976; Kraupp and Marz, 1995).
Nucleosides and their analogs form the basis of a wide variety of
clinical agents that are used in the treatment of brain cancers,
cardiac disorders, parasitic, and viral diseases (Paterson et al.,
1981; Daval et al., 1991; Perigaud et al., 1994). The purine
nucleoside, adenosine, exerts significant cardiac effects and is used
clinically in the treatment of cardiac arrhythmias (Brady et al.,
1996). Nucleoside analogs (i.e., zidovudine, lamivudine, didanosine,
and abacavir) are currently used in the treatment of patients with HIV
infection (Beach, 1998). Most nucleosides and their analogs exert their
biological activity intracellularly, but due to their hydrophilic
nature do not readily permeate the lipid bilayer. Therefore, the uptake
or release of nucleosides and/or nucleoside analogs in mammalian cells
is mediated by multiple distinct transporters (Cass, 1995; Cass et al.,
1998). Currently, eight functionally distinct nucleoside transporters
have been identified (Table 3). The
classification of nucleoside transport proteins is based on functional
and pharmacological characteristics including transport mechanism
(e = equilibrative, c = concentrative), sensitivity to nitrobenzylmercaptopurine riboside (s = sensitive, i = insensitive), and selectivity sequence
of permeant molecules.
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Equilibrative processes are widely distributed among mammalian cells
and tissues and exhibit broad permeant selectivity (Plagemann et al.,
1988; Gati and Paterson, 1989; Belt et al., 1993; Griffiths et al.,
1997a,b; Crawford et al., 1998). The two bidirectional, equilibrative
systems, es/ENT1 and ei/ENT2, have similar
kinetic properties, but differ markedly in their sensitivity to
nitrobenzylmercaptopurine riboside (Plagemann and Wohlhueter, 1980;
Belt, 1983; Belt and Noel, 1985). ENT1 and ENT2 have been isolated from
both human and rat placenta (Griffiths et al., 1997b; Yao et al.,
1997). Both equilibrative transporters are inhibited by low
concentrations (0.1-100 nM) of dipyridamole and dilazep (Plagemann et
al., 1988). The es transporter is inhibited at low
concentrations (
1 nM) (Belt, 1983) as a direct result of the
interaction of nitrobenzylmercaptopurine riboside with high affinity
binding sites (Kd = 0.1-1 nM) (Cass et al., 1974; Jarvis and Young, 1980) whereas the ei
transporter is not affected by nitrobenzylmercaptopurine riboside up to
>10 µM (Jarvis and Young, 1980; Belt, 1983; Belt and Noel, 1985).
Unlike the ubiquitous equilibrative systems, concentrative NT processes
have only been identified in selected mammalian cells: macrophages
(Plagemann and Aran, 1990), choroid plexus (Wu et al., 1992), microglia
(Hong et al., 2000), leukemia cells (Crawford et al., 1990),
splenocytes (Darnowski et al., 1987), intestinal cells (Vijayalakshmi
and Belt, 1988), and renal brush-border membrane vesicles (Williams et
al., 1989). These transporters, with the exception of cgs/N6
and cs/N5 systems, are insensitive to
nitrobenzylmercaptopurine riboside and dipyridamole at concentrations
up to 10 µM (Plagemann et al., 1988; Vijayalakshmi and Belt, 1988).
Evidence suggests that six subclasses of concentrative transporters
exist that display a complex pattern of overlapping substrate
selectivity (Cass, 1995; Flanagan and Meckling-Gill, 1997). Due to
overlapping broad substrate specificities, a complex phenotype can
result whereby one nucleoside may be simultaneously transported by more
than one process within one cell or tissue. The nucleosides are
transported against a concentration gradient into the cell, coupled
with the movement of Na+ down a concentration
gradient (Jarvis et al., 1989). These transporters use the
physiological Na+-gradient (~140 mM
out > 5-10 mM in)
generated by the ubiquitous Na+/K+-ATPase (Cass, 1995).
Thus, these processes are described as inwardly directed
Na+/nucleoside symporters (Lee et al., 1988).
Concentrative transporters mediate the nucleoside flux against their
concentration gradient by Na+ or
K+ cotransport mechanisms. The major types of
Na+ nucleoside cotransport systems can be
classified based on functional studies, including primarily substrate
selectivity. The N1 (or cif) system is selective for purines, with
guanosine and formycin B being the commonly used substrates. The N2 (or
cit) transporter is selective for pyrimidines, and thymidine is the
usual test substrate. Both types have been cloned from humans and rats
(hCNT1/rCNT and hSPNT1/SPNT for N2 and N1, respectively) and when
heterologously expressed, they display a 1:1
Na+/nucleoside-coupling ratio (Cass et al.,
1998). The N3 (or cib) transporter has a 2:1
Na+/nucleoside stoichiometry and is broadly
selective for both purines and pyrimidines. The fourth type, N4, has
similar N2 stoichiometry and selectivity but guanosine and adenosine
are also transported. The less characterized N5 (or cs) transporter is
nitrobenzylmercaptopurine riboside-sensitive and exhibits selectivity
for adenosine and formycin B (Cass, 1995).
There are large differences in the functional expression of NT
depending on cell type. For instance, erythrocytes express only a
single NT system, es (Woffendin and Plagemann, 1987),
whereas both es and ei are present in BeWo human
choriocarcinoma cells (Boumah et al., 1992) and murine L1210 leukemia
cells and IEC-6 intestinal epithelial cells, (Vijayalakshmi and Belt,
1988; Crawford et al., 1990). In addition, NT activities within the
same cell type vary with changes in growth state (Jakobs et al., 1990;
Meckling-Gill et al., 1993), differentiation (Sokoloski et al., 1991;
Jones et al., 1994), and neoplastic transformation (Meckling-Gill and Cass, 1992). For example, induction of monocytic or neutrophilic differentiation of HL-60 human leukemia cells leads to a decrease in
es NT activity and an increased
Na+-dependent NT activity (Lee et al., 1991;
Sokoloski et al., 1991; Goh et al., 1993).
Several NT systems have been identified in the BBB and blood-CSF
barrier (Fig. 6). Early in vivo studies
revealed that at the BBB, purine ribonucleosides cross from blood to
brain by facilitated diffusion, whereas pyrimidine deoxyribonucleosides
did not show appreciable uptake into the brain (Cornford and Oldendorf,
1975; Spector and Berlinger, 1982; Spector and Huntoon, 1983). Further experiments using cultured endothelial cells and isolated brain capillaries demonstrated the expression of cit/N2,
es and ei NT systems at the BBB (Kalaria and
Harik, 1986; Thomas and Segal, 1997). The low BBB transport of
deoxyribonucleosides suggests that the measured saturable entry of
these solutes occur predominantly through the CP. In vitro studies
using isolated rabbit CP have identified several NT systems including
an es, ei, and a concentrative N3 type
transporter mediating the transport of both ribonucleosides and
deoxyribonucleosides at the CP (Spector, 1982; Kalaria and Harik, 1986;
Wu et al., 1992, 1994). In vivo, both N3 and ei systems have
been shown to transport nucleosides from blood to CSF (Spector, 1982).
The N3 transporter identified in rabbit CP tissue slices is selective
for naturally occurring purine and pyrimidine ribo- and
deoxyribonucleosides (i.e., guanosine, inosine, formycin B, uridine,
cytidine; IC50 range = 5-23 µM), base
modified nucleoside analogs (i.e., 5-fluorouridine,
2-chlororadenosine; IC50 range = 12-24 µM), but not for synthetic nucleoside analogs (i.e.,
zidovudine, zalcitabine) substituted on the ribose ring (Wu et al.,
1992, 1994). Similarly, Wu and colleagues (1992) have found that the N3
NT system in rabbit CP tissue slices transports both uridine (Km = 18 µM) and thymidine
(Km = 13 µM). Sodium-stimulated
hypoxanthine uptake also occurs in rabbit CP slices (Washington and
Giacomini, 1995).
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Studies using rat and guinea pig whole brain membrane preparations have
demonstrated the presence of a nitrobenzylmercaptopurine riboside-sensitive NT system, which specifically recognizes adenosine as its preferred endogenous substrate (Geiger et al., 1985; Jarvis and
Ng, 1985). The wide distribution of ENT1 or es systems in human and rat adult brains has been verified using RT-PCR, Northern blot, and in situ hybridization methods (Anderson et al., 1999a). Similarly, ei NT transcript expression has been recently
detected in several regions of the rat brain suggesting that adenosine levels in brain is achieved by multiple transport processes (Anderson et al., 1999b). However, the actual membrane localization of these facilitative and active NT systems has not been investigated.
Preliminary evidence suggests that active NT systems are also present
in brain parenchyma. In cultured human astrocytes, Gu et al. (1996)
reported the presence of a concentrative uptake system for adenosine.
Although these transporters have yet to be fully characterized, the NT
inhibitors dipyridamole, nitrobenzylmercaptopurine riboside, and
dilazep biphasically inhibited the inhibitor-sensitive component of
adenosine transport (IC50 = 1.3, 0.7, and 3.3 nM, respectively). More recently, Sinclair et al. (2000) identified a
nitrobenzylmercaptopurine riboside-insensitive transporter (rENT2) as
the primary functional NT transporter in rat C6 glioma cells.
In addition to NTs that transport nucleosides into the brain, there
also exists a saturable carrier system for the efflux of nucleosides
out of the brain (Spector, 1980, 1985; Suleiman and Spector, 1982;
Spector and Huntoon, 1983; Wu et al., 1993). An equilibrative NT
system, sensitive to nitrobenzylmercaptopurine riboside, is responsible
in part for the elimination of formycin B and uridine from the rat and
rabbit CSF, respectively, in vitro (Wu et al., 1992 and 1993).
Similarly, the efflux of thymidine out of the CSF in intraventricularly
perfused rabbits was partially nitrobenzylmercaptopurine
riboside-sensitive and saturable with a
Km of 17.8 µM (Thomas et al., 1997).
The involvement of NT systems in the transport of nucleoside analog
drugs, such as zidovudine, stavudine, and didanosine has been
demonstrated in several cell systems. This includes intestinal transport of stavudine by N3- and N2-type NT systems (Waclawaski and
Sinko, 1996), Na+-dependent hCNT2 transport of
didanosine (Ritzel et al., 1998) and rCNT1 transport of zidovudine
(Huang et al., 1994; Yao et al., 1996). Interestingly, nucleoside
transporters identified at the BBB and blood-CSF barrier (i.e.,
Na+-dependent NT system N3) are not involved in
the transport of nucleoside analog drugs (Terasaki and Pardridge, 1988;
Wu et al., 1994). Our laboratory has found evidence for NT-mediated
transport within brain parenchyma. A sodium-dependent NT responsible
for the uptake of thymidine was characterized in rat microglia using a
continuous cell line (MLS-9 cells) (Hong et al., 2000). The Na+/nucleoside-coupling stoichiometry was found
to be 2:1 and the standard inhibitor for equilibrative NT,
nitrobenzylmercaptopurine riboside, did not inhibit thymidine uptake.
The Km (44 µM) and Vmax (273 pmol/mg/min) for thymidine
uptake in this system were similar to
Na+-dependent NTs in bovine renal and
brush-border membrane vesicles, respectively (Williams and Jarvis,
1991; Gutierrez and Giacomini, 1993). Furthermore, various purine and
pyrimidine nucleosides inhibited thymidine uptake in a
concentration-dependent (IC50 = 30-40 µM) and
competitive manner (Ki = 38-45 µM).
These properties are remarkably similar to the broad substrate
selectivity of a cib (N3) type of NT (Wang et al., 1997b).
Zidovudine was found to be a potent noncompetitive inhibitor for
thymidine uptake in microglia (IC50 = 0.6 µM)
suggesting that it only acts as an inhibitor of the system and not as a substrate.
We have also characterized a novel electrogenic
zidovudine/H+-dependent transporter (Hong et al.,
2001) in microglia. Kinetic analysis of zidovudine uptake by confluent
monolayers of MLS-9 cells showed a saturable low-affinity system
(Km = 1 mM) sensitive to membrane
potential and pH. Specificity studies revealed that zidovudine uptake
was sensitive to organic cations (verapamil, mepiperphenidol,
quinidine, cimetidine, and
N1-methylnicotinamide;
IC50 values in the range of 156 to 200 µM) but
was unable to transport the standard OCT substrate, tetraethylammonium. The system was also insensitive to classic organic anions (benzyl penicillin, salicylic acid, p-aminohippuric acid) and
probenecid, a standard OAT inhibitor. Furthermore, nucleosides
(thymidine, cytidine, guanosine, and adenosine), standard nucleoside
analog inhibitors (dipyridamole, dilazep, and
6-(4-nitrobenzylthio-9--D-ribofuranosylpurine), and nucleoside analogs (lamivudine, abacavir, didanosine, and zalcitabine) did not inhibit zidovudine uptake by microglia. Although the zidovudine system in microglia has some specificity features of an
organic cation transporter, it involves a carrier, distinct from other
cloned OCT systems that is novel in its sensitivity to pH and membrane
potential. This system may play a significant role in the transport of
other weak organic cation substrates and metabolites in brain
parenchyma (Hong et al., 2001).
D. Efflux Transport Systems
1. P-Glycoprotein.
P-gp is a 170-kDa plasma membrane,
energy-dependent efflux pump that belongs to the ABC superfamily of
transporters (Ling, 1997). Originally discovered in Chinese hamster
ovary cells selected for colchicine resistance, these cells exhibited
broad cross-resistance to a number of naturally occurring structurally
diverse antineoplastic agents including anthracyclines, vinca
alkaloids, and taxanes (Juliano and Ling, 1976). Consequently, this
phenomenon was termed multidrug resistance (MDR) (Biedler and Riehm,
1970; Kessel and Bosmann, 1970).; Roninson et al., 1986). Although the MDR1 protein is involved in
the MDR phenotype (Gros et al., 1986; Ueda et al., 1987), the protein
encoded by the human MDR2 gene functions as a phosphatidyl translocase
in the liver (Ruetz and Gros, 1994). In addition to the human P-gp
homologs, hamster pgp1, 2, and 3 (Gerlach et al., 1986) and mouse
mdr1a, 1b, and 2 (Gros et al., 1986) also exist (Table
4). Similar to their human counterparts, murine P-gp encoded by mdr1a/b confers the MDR phenotype (Gros et al.,
1986; Ueda et al., 1987; Schinkel et al., 1994, 1995) whereas the mdr2
gene product serves in the transport of hepatic phospholipid into the
bile (Smit et al., 1993).
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) (Fig.
7). Mutations in one or both
nucleotide-binding consensus sequences results in failure to confer
MDR, suggesting that both ATP-binding sites are required for substrate
transport (Rothenberg and Ling, 1989). Three putative glycosylation
sites are located within the first extracellular loop of the protein and do not appear to be involved in the transport of substrates. Studies with tunicamycin treatment, which blocks N-linked
glycosylation, did not demonstrate altered drug sensitivity in human
MDR cells (Beck and Cirtain, 1982). In addition, P-gp is phosphorylated at several sites (Hamada et al., 1987) by cAMP-dependent protein kinase
A and by protein kinase C (Mellado and Horwitz, 1987; Chambers et al.,
1992). Treatment with phorbol ester
12-O-tetradecanoylphorbol-13-acetate, which stimulated P-gp
phosphorylation, resulted in increased drug resistance and decreased
drug accumulation in some multiresistant cell lines (Fine et al.,
1988), whereas in the presence of protein kinase inhibitors, there
appeared to be increased anticancer drug accumulation within some cells
(Bates et al., 1993). These results suggest that P-gp activity may be
modulated by phosphorylation.
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). Studies using mdr1a/b
gene knockout mice show that P-gp deficient mice are viable, fertile, and healthy compared to wild type, but exhibit significantly elevated drug levels, particularly in the brain (Schinkel et al., 1994; Schinkel, 1997).
The exact mechanism by which P-gp extrudes substrates from the cell
cytoplasm remains unresolved. Substrates may bind to a cytoplasmic
region of P-gp, resulting in an energy-dependent conformational change
that shuttles the drug to the outside of the plasma membrane. This
"classical" model of a transporter resembles a typical
substrate-enzyme interaction. Alternatively, recent evidence suggests
that P-gp may act as a "flippase" where membrane-bound drug is
relocated between inner and outer lipid leaflets of the plasma membrane (Higgins and Gottesman, 1992; Sharom, 1997). This model is consistent with the mdr2 protein, which acts as a flippase for phosphatylcholine (Romsicki and Sharom, 2001). Whether P-gp transports substrates by a
combination of several different methods remains to be elucidated.
In the periphery, P-gp is found on the apical surface of intestinal and
renal epithelia, pancreas, the secretory glands in the endometrium of
pregnant mice, biliary canalicular membranes of hepatocytes, and on the
luminal side of the blood-testis barrier (Fojo et al., 1987; Thiebaut
et al., 1987, 1989; Cordon-Cardo et al., 1989; Croop et al., 1989;
Borst et al., 1993; Schinkel, 1997). In the brain, P-gp expression has
primarily been investigated at the barriers. Although P-gp is expressed
on the apical side of the CP epithelia (Rao et al., 1999), the location
of P-gp along the brain endothelial microvessels remains debatable.
Immunohistochemistry studies and luminal membrane isolation have
localized P-gp to the luminal surface of the brain endothelium
(Sugawara et al., 1990; Jette et al., 1993; Beaulieu et al., 1997;
Drion et al., 1997). However, P-gp has also been identified at the
abluminal surface of endothelial cells on neighboring astrocyte foot
processes (Golden and Pardridge, 1999). The primary P-gp isoform
detected in brain microvessels is mdr1a whereas mdr1b mRNA is the main isoform detected in brain parenchyma (Regina et al., 1998). The level
of P-gp and specific homologs expressed also varies depending on the
isolation procedure. Regina et al. (1999) reported the variability in
P-gp levels between immortalized endothelial cell lines and isolated
brain microvessels. It also appears that a down-regulation of mdr1a,
and an up-regulation of the P-gp isoform mdr1b occurs in rat brain
endothelia cultures (Barrand et al., 1995; Regina et al., 1998).
Finally, P-gp is expressed in primary cultures of rat brain astrocytes
but at lower levels when compared with primary endothelial cultures
(Declèves et al., 2000). This weak expression in astrocytes
correlated with a weak functional activity as various P-gp modulators
produced only a slight effect on the uptake of the P-gp-specific
substrate colchicine. Thus, if expressed at the BBB on astrocyte foot
processes, the higher expression of P-gp at the endothelial level would
be more important at limiting the entry of xenobiotics into the brain
than at the astrocyte level (Declèves et al., 2000).
Data from our laboratory suggest that P-gp is also expressed and
functional in brain microglia (Lee et al., 2001). Using a continuous
rat brain microglia cell line (MLS-9), immunocytochemistry studies
reveal the location of P-gp along the nuclear envelope and plasma
membrane of microglia. In primary microglia cultures, RT-PCR analysis
detected both mdr1a and mdr1b, whereas in the MLS-9 cells only mdr1b
gene was expressed. This was corroborated by Western blot analysis; a
single band with a molecular weight of 170 to 180 kDa, similar to those
reported for P-gp in other cell lines, was detected (Doige and Sharom,
1992; Regina et al., 1998). Functional studies using the known P-gp
substrate, digoxin, demonstrated that digoxin accumulation in microglia
was significantly increased in the presence of various P-gp inhibitors
(verapamil, quinidine, cyclosporin A, PSC 833), protease inhibitors
(saquinavir, ritonavir, indinavir), and sodium azide, a metabolic
inhibitor. Digoxin accumulation was not increased in the presence of
standard MRP inhibitors such as sulfinpyrazone, indomethacin, and
probenecid. These results provide the first evidence for the functional
expression of P-gp in microglia and imply that entry of pharmacological
agents may be prevented within the brain parenchyma as well as the BBB.
2. Multidrug Resistance Protein Family.
A second efflux
transport protein subfamily, which belongs to the ABC protein
superfamily and can confer MDR, is the MRP. Thus far the mammalian MRP
family consists of seven proteins ranging from 1325 to 1545 amino acids
(Borst et al., 1999; Konig et al., 1999a). All MRPs contain two
transmembrane domains of six -helices each (P-gp-like core)
connected to a cytoplasmic linker (Lo) region (Fig. 7). In addition MRP1, -2, -3, and -6 contain up to six additional membrane-spanning helices (TMDo) at the
NH2 terminus (Lautier et al., 1996; Borst et al.,
1999; Klein et al., 1999). Although this extra N-terminal domain is not
required for drug transport, the linker region (Lo) is absolutely
necessary to maintain the protein transport properties (Gao et al.,
1996; Bakos et al., 1998). MRP1, -2, and -3 appear to have overlapping
substrate specificities, but differ with respect to kinetic properties
(Keppler et al., 1999). Most cells appear to express multiple MRP
family members, with high levels of one MRP generally dominating
(Keppler et al., 1999). While MRP2, -3, and -6 are found mainly in the
liver and kidney, and MRP4 is found in high concentrations in the
prostate, MRP1 and -5 appear to be ubiquitous, and both proteins are
expressed in the brain (Klein et al., 1999). Within polarized cells
(e.g., kidney and liver) MRP2 is the only homolog located in the apical membrane (similar to P-gp), MRP1, -3, and -5 are all routed to the
basolateral membrane (Flens et al., 1996; Schaub et al., 1997; Konig et
al., 1999b; Kool et al., 1999). The general features of the MRP
homologs are summarized in Table 5.
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; Leier et al., 1996; Loe et al., 1996a), steroid glucuronides
(Jedlitschky et al., 1996; Loe et al., 1996b), and GSH disulfide (Leier
et al., 1996). Thus, MRP1 may also play a role in the regulation of
intracellular redox potential, flux of ions (Jirsch et al., 1993; Rappa
et al., 1999), inflammatory mediation, and elimination of potentially
toxic endo- and xenobiotics (O'Brien and Tew, 1996; Deeley and Cole,
1997). Furthermore, MRP1 transport of heavy metal oxyanions such as
sodium arsenate and antimony potassium tartrate suggests that MRP1 may play a protective role against environmental toxins.
Several MRP knockout mouse models have been developed recently (Lorico
et al., 1997; Rappa et al., 1999). As murine MRPs possess greater than
85% homology with human MRPs, the transgenic mouse model is a powerful
tool for further understanding the physiological role of these
proteins. The absence of MRP does not appear to alter the viability,
fertility, and/or biochemical profile of MRP knockout mice versus
wild-type mice (Rappa et al., 1999). Some caution must be used however
when utilizing murine models for characterization of substrate and for
extrapolation to the human MRP protein. Differences have been
identified for example in the transport of anthracyclines by murine and
human MRP. On the other hand, these differences can be exploited by
researchers to further characterize the specific MRP domains required
for substrate transport (Stride et al., 1999).
MRP2 (cMOAT) appears to play an important role in the biliary excretion
of various GSH and glucuronide conjugates including bilirubin
glucuronide (Konig et al., 1999a). Furthermore, a splice mutation of
the human MRP2 gene results in development of hyperbilirubinemia, also
known as the Dubin-Johnson Syndrome (Paulusma et al., 1997; Kajihara et
al., 1998; Wada et al., 1998; Toh et al., 1999). Conversely, MRP3,
which is located in the basolateral membrane of hepatocytes, may be
involved in bile acid uptake from the gut (Borst et al., 2000). The
physiological functions of the remaining MRP family members is
currently unknown, however recent studies demonstrating the ability of
MRP4 and 5 to transport nucleotide analogs suggest these two MRP
homologs may be involved in nucleotide/nucleoside transport in vivo
(Schuetz et al., 1999; Jedlitschky et al., 2000; Wijnholds et al.,
2000). Thus, the substrate specificity of MRP appears to be quite
broad. Many MRP substrates are amphiphilic anions with at least one
negatively charged group; however not all amphiphilic anions are
transported (Jedlitschky et al., 1996). For example, the anthracyclines
daunorubicin and doxorubicin cannot enhance the transport of
LTC4 and direct transport of these agents is
unaffected in the presence of GSH (Loe et al., 1998).
To date, the highest affinity substrate of MRP1, as determined through
membrane vesicle transport studies, has been identified as the
GSH-conjugated leukotriene LTC4, with a
Km of approximately 100 nM
(Jedlitschky et al., 1996; Loe et al., 1996a). Other efficiently transported substrates of MRP1 include the anthracyclines doxorubicin and daunorubicin, the vinca alkaloids vincristine and vinblastine, and
the epipodophyllotoxin etoposide. The anthracyclines and vinca alkaloids are not direct substrates for MRP (Jedlitschky et al., 1996)
but rather are only transported efficiently in the presence of
physiologically relevant concentrations of GSH. Two mechanisms for
transport of MRP substrates have been suggested, i.e., anionic compounds are transported directly, whereas some cationic and neutral
compounds may be cotransported in the presence of GSH. GSH itself does
not appear to be a direct substrate for the MRP transporter. Studies
characterizing the transport properties of MRP have been difficult in
intact cells due to the concurrent expression of P-gp and OATP
transporters in most cells. Although several potent inhibitors of P-gp
exist (PSC833, cyclosporin A), identifying specific and potent
inhibitors of MRP has been more difficult. One inhibitor identified to
date is the leukotriene antagonist MK-571, a potent inhibitor
(Ki = 0.6 µM) of
LTC4 transport in membrane vesicles (Leier et
al., 1994). LY329146 (structural analog of the estrogen receptor
modulator raloxifene) also inhibits LTD4
transport into HL60/ADR cells with an IC50 = 0.8 µM (Norman, 1998). Both indomethacin and the isoflavanoid genestein
alter intracellular concentrations of GSH and are therefore very useful as specific inhibitors of MRP1 in vitro (Draper et al., 1997). Finally,
transport studies of MRP substrates have been further aided by the
development of specific antibodies for MRP. The monoclonal antibodies
QCRL2-4 recognize distinct intracellular epitopes specific to MRP1 and
have been shown to inhibit transport of several MRP1 substrates
including vincristine, daunorubucin, and aflatoxin-B1 in inside-out
vesicles (Loe et al., 1996b, 1997, 1998). Unfortunately, specific
inhibitors for each MRP homolog do not exist, therefore caution must be
used in the interpretation of results when utilizing inhibitors in
whole cell systems that contain multiple isoforms of MRP.
Although the exact mechanism by which GSH enhances MRP substrate
transport is currently unknown, MRP may contain bipartite binding sites
that would allow for direct binding of drug-GSH complexes or sequential
binding of GSH and drug (Loe et al., 1996a). It has been suggested that
one of these binding sites may have a high affinity for drug and low
affinity for GSH, whereas the second binding site is of the opposite
conformation (Borst et al., 1999). Whether GSH may elicit a
conformational change in the MRP protein that might favor transport of
selected compounds remains unknown (Loe et al., 1996a). GSH itself
appears to be a poor substrate of MRP and is transported only
marginally in the absence of a proper MRP substrate (Leier et al.,
1996). Thus, it appears there are two mechanisms for transport of MRP
substrates: anionic compounds are transported directly, whereas some
cationic and neutral compounds require the presence of GSH, likely via cotransport.
Enhancement of substrate transport (aflatoxin
B1-GSH, and vincristine) by GSH does not
appear to be a consequence of an alteration in the redox state of MRP
since other reducing agents (2-mercaptoethanol, dithiothreitol,
L-cysteine) have not been able to alter the transport of
these substrates in a similar manner (Loe et al., 1997). Whether GSH
(and possibly other unknown organic anions) and unconjugated substrates
interact molecularly for the purpose of cotransport is presently
unclear (Rappa et al., 1997). Data from studies undertaken in
GSH-depleted cells provide further evidence for the role of GSH in the
efflux of some unconjugated substrates. Rappa et al. (1997) demonstrate
that intracellular etoposide accumulation in wild-type embryonic stem
cells is increased significantly in the presence of
DL-buthionine sulfoximine, an irreversible inhibitor of
-glutamylcysteine (Rappa et al., 1997). Within double knockout stem
cells, BSO depletion had no effect. Furthermore, in double knockout
mice, Lorico et al. (1997) demonstrated that levels of GSH were
increased in all tissues, although only highly expressing cells
displayed significance (i.e., lung, colon, and muscle). -Glutamylcysteine activity was unchanged in the knockout mice, thus
making it unlikely that increased GSH synthesis, as opposed to
increased GSH efflux, was responsible for the observed GSH increases.
Within T14 HeLa cells, vincristine transport is increased markedly in
the presence of GSH. However, the precursor dipeptides cysteinylglycine
and -glutamylcysteine do not affect vincristine transport, thus it
appears the tripeptide is required for stimulation of vincristine
transport (Loe et al., 1998). Finally, overexpression of MRP leads to
decreased intracellular GSH levels in some cell lines (Loe et al.,
1998).
MRP expression and localization within the brain is only beginning to
be understood. High levels of MRP1 have been detected in the CP using
RT-PCR and Western blot techniques (Nishino et al., 1999). Protein and
mRNA levels of MRP1 in CP were 5-fold higher than that observed in
lung, a tissue known to express MRP1 in abundance (Nishino et al.,
1999). Using a combination of fluorescent confocal and electron
microscopy, Rao et al. (1999) determined the localization of MRP1 to be
primarily basolateral within cultured CP. In addition, the transport of
17-estradiol 17-D-glucuronide, both an MRP1 and an
OATP1 substrate, was probenecid-sensitive. Thus, transcellular
transport of organic anions across the CP may be synergistically
mediated via the combined effort of Oatp1 and MRP1 at the apical and
basolateral membranes, respectively.
Whereas MRP1 and MRP5 expression has been observed in rat, in mouse and
human brain endothelial cells (Kusuhara et al., 1998; Regina et al.,
1998; Homma et al., 1999; Nishino et al., 1999; Zhang et al., 2000)
similar to P-gp the level of expression is quite dependent on the
isolation procedure and cell systems utilized (Seetharaman et al.,
1998; Gutmann et al., 1999; Sugiyama et al., 1999). In rats, Regina et
al. (1998) noted that MRP1 is overexpressed in rat brain endothelial
cell cultures, with primary cultures having higher amounts compared
with immortalized cell lines. Furthermore, MRP1 is expressed in higher
amounts in brain homogenate, as opposed to isolated microvessels. This
finding is not surprising since recently MRP1 expression was detected
using RT-PCR in rat astrocyte cultures (Declèves et al., 2000).
Corroborating the findings of Regina et al. (1998), Declèves et
al. (2000) observed a higher expression of MRP1 in primary astrocytes,
compared with primary brain endothelial cells. In this in vitro
astrocyte model, indomethacin, probenecid, and sulfinpyrazone increased
accumulation of the fluorescent probe fluorescein by 30 to 80% at
concentrations of 10 µM, 1 mM, and 2 mM, respectively (Declèves
et al., 2000). Sulfinpyrazone also increased the uptake of radiolabeled
vincristine within astrocytes. In an in vitro bovine endothelial cell
system, Gutmann et al. (1999) reported an up-regulated MRP protein
expression following culture of these endothelial cells for a period of
10 days. More recently, studies using RT-PCR (Zhang et al., 2000) have
found that in addition to MRP1, MRP4-6 are also expressed to a high degree within brain endothelial cells. Similarly, the newest member of
the MRP family MRP7 was detected in whole brain homogenate using RT-PCR
(Hopper et al., 2001). Recently we have characterized the functional
expression of MRP within the MLS-9 microglia cell (Dallas et al.,
2001). Using the MRP substrate vincristine, we observed increased
substrate accumulation in the presence of various MRP inhibitors
including MK571, genestein, and sulfinpyrazone. Vincristine uptake was
both energy- and glutathione-dependent, which suggests a functional
form or forms of MRP is/are located within the microglia cell line.
Furthermore, while the protease inhibitors saquinavir and indinavir
acted as potent inhibitors of vincristine transport, none of the
currently available nucleoside analogs drugs showed an appreciable
effect. Interestingly, RT-PCR studies have demonstrated the absence of
MRP1 within the MLS-9 cell line, whereas MRP1 was present in primary
microglia cells derived from the same species of rat (Lee et al.,
2001). Studies are now underway in our laboratory to clarify the
expression pattern of MRP within both the continuous cell line and
primary microglia using both RT-PCR and Western blotting.
The clinical implications of MRP protein expression in the brain are
apparent when one considers the prognosis of primary brain tumor
patients. The 5-year survival rate of patients with astrocytomas and
glioblastomas is extremely low, largely in part due to a high degree of
MDR and therefore therapeutic failure of anticancer agents (Mousseau et
al., 1993; Hosli et al., 1998). Furthermore, it now appears that MRP
may also play a role in resistance to other classes of drugs. For
example, accumulation of the MRP substrate calcein acetoxymethyl ester
is increased significantly in the presence of the anti-HIV-1 protease
inhibitors saquinavir, ritonavir, indinavir, and nelfinavir in the
MRP1-overexpressing cell line VM1-5 (Srinivas et al., 1998). Recently
Wijnholds et al. (2000) have characterized MRP5-related transport of
the anti-HIV nucleotide analog 9-(2-phosphonylmethoxyethyl)adenine in
polarized Madin-Darby canine kidney II (MDCKII) cells transfected with
MRP5 cDNA constructs. Reduced accumulation and enhanced efflux of the MRP substrate S-(2,4-dinitrophenyl)glutathione (DNP-GS) was
observed in the MRP5-overexpressing cells in the presence of the MRP
inhibitors. Whereas the MRP inhibitor sulfinpyrazone caused almost
complete inhibition of DNP-GS efflux, probenecid, indomethacin, and
dipyridamole had little effect. Sulfinpyrazone also decreased the
efflux 9-(2-phosphonylmethoxyethyl) adenine at low concentrations
(IC50 < 0.5 mM). The authors suggested that MRP5
may play a role in resistance of HIV-1 patients to nucleoside analog
drugs. Whether antiretroviral drugs are transported by MRP1 and/or -5 within the brain to an appreciable, and clinically relevant extent,
remains to be examined.
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VI. Summary |
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The brain is a dynamic and highly regulated organ compartmentalized by the BBB (cerebral endothelial cells), and the blood-CSF barrier (CP epithelial cells). Brain parenchymal cells (i.e., neuroglia and neurons) exist within this highly regulated environment and function in intimate interplay with one another. Each brain compartment possesses a specific and selective set of metabolic enzymes, receptor proteins, and secretory factors that serve to maintain the homeostatic environment that is necessary for normal function within that compartment. In addition, the localization and expression of various putative drug transporters in these barriers play a critical role in the influx/efflux of numerous xenobiotics and has an important impact on the overall pharmacokinetic/pharmacodynamic profile of drugs in the brain (i.e., distribution, pharmacological response, drug-drug interactions). Novel localization and functional expression of standard transporters (i.e., NT) as well as the efflux transporters (P-gp and MRP) in brain parenchyma suggest a reconsideration of the present conceptualization of brain barriers as it relates to drug transport. The cellular membranes of parenchyma cells, such as microglia and astrocytes, also act as barriers to drug permeability and express transporters whose properties appear similar to those localized to the conventional brain barriers (i.e., cerebral endothelial cells and CP epithelial cells). Much work is needed to fully characterize the drug transporters at the parenchymal barrier site to fully appreciate its role in clinical practice.
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Acknowledgments |
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This work is supported by a grant from the Canadian Foundation for AIDS Research, the Ontario HIV Treatment Network (OHTN), and the Positive Action Fund, AIDS Bureau, Ontario Ministry of Health.
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Footnotes |
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1 Address for correspondence: Dr. Reina Bendayan, Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, ON M5S 2S2, Canada. E-mail: r.bendayan{at}utoronto.ca
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Abbreviations |
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CNS, central nervous system; CSF, cerebrospinal fluid; P-gp, P-glcoprotein; MRP, multidrug resistance protein; BBB, blood-brain barrier; CP, choroid plexus; AIDS, acquired immunodeficiency syndrome; HIV-1, human immunodeficiency virus type 1; RT-PCR, reverse transcription-polymerase chain reaction; PGT, prostaglandin transporter; OAT, organic anion transport; OCT, organic cation transport; NT, nucleotide transport; GSH, glutathione; ECF, extracellular fluid; MDR, multidrug resistance; ABC, ATP-binding cassette.
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References |
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-estradiol 17-(-D-glucuronide) transport by multidrug resistance protein (MRP).
J Biol Chem
271:
9683-9689
an overview, in
Taurine: Functional Neurochemistry, Physiology and Cardiology (Paseantes-Morales H,
Martin DL,
Shain W anddel Rio R eds) pp 243-252,
Wiley-Liss Inc., New York.
0031-6997/01/5304-569-596$3.00
PHARMACOLOGICAL REVIEWS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
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