Choline uptake across the ventricular membrane of neonate rat
choroid plexus
Alice R.
Villalobos1,
Judith T.
Parmelee2, and
J. Larry
Renfro1
1 Department of Physiology and
Neurobiology, University of Connecticut, Storrs 06269; and
2 Manchester Community-Technical
College, Manchester, Connecticut 06045
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ABSTRACT |
The uptake of
[3H]choline from the
cerebrospinal fluid (CSF) side of the rat neonatal choroid plexus was
characterized in primary cultures of the choroidal epithelium grown on
solid supports. Cell-to-medium concentration ratios were ~5 at 1 min
and as high as 70 at 30 min. Apical choline uptake was facilitated; the
Km was ~50
µM. Several organic cations (e.g., hemicholinium-3 and N1-methylnicotinamide)
inhibited uptake. The reduction or removal of external
Na+ or the addition of 5 mM LiCl
had no effect on uptake. However, increasing external
K+ concentration from 3 to 30 mM
depolarized ventricular membrane potential (
70 to
15 mV)
and reduced uptake to 45% of that for the control. Treatment with 1 mM
ouabain or 2 mM BaCl2 reduced uptake 45%, and intracellular acidification reduced uptake to ~90%
of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled to
Na+ influx but is sensitive to
plasma membrane electrical potential.
organic cation transport; cerebrospinal fluid-blood barrier; cholinergic metabolism
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INTRODUCTION |
THE NEUROTRANSMITTER of cholinergic systems, ACh, is
synthesized from acetyl-CoA and choline, and the availability of the latter is rate limiting in this synthesis. Although choline is liberated through the metabolism of membrane phospholipids, it is not
synthesized de novo at a significant rate in the brain (34). The
choline incorporated into the central nervous system (CNS) is derived
primarily from peripheral metabolism and traverses the blood-brain
barrier by facilitated diffusion in proportion to changes in blood
choline concentration (7). Despite physiological fluctuations in blood
choline levels (10-50 µM) and arteriovenous differences
(
2 to
4 µM), cerebrospinal fluid (CSF) choline
concentration remains virtually constant (~6 µM; Ref. 15).
According to the current model for the homeostatic regulation of
choline availability in the CNS, the two prevailing mechanisms
responsible for removal of excess choline from the brain are
1) high-affinity reuptake of choline
by presynaptic neurons and incorporation into membrane phospholipid and
2) active efflux of choline from
brain to blood across the epithelial cell layer of the choroid plexus
(CP) (21). Na+-coupled choline
reuptake by presynaptic neurons is well characterized; however, the
cellular transport mechanisms and modulation of the active transport of
choline across the CSF-blood barrier remain poorly understood.
Ventriculocisternal perfusion studies of adult animals have
demonstrated that the net clearance of choline from ventricular CSF
exceeds the rate of "washout" by bulk flow of CSF from the ventricles through the arachnoid sinuses. Furthermore, clearance of
radiolabeled choline from ventricular CSF is reduced in a
concentration-dependent manner by unlabeled choline or other quaternary
ammoniums (see, e.g., Refs. 1, 13, and 18). In vitro studies have also demonstrated carrier-mediated accumulation of choline by isolated CP
(1, 8). Together, these observations are the basis for the supposition
of active mediated efflux of excess choline and other organic cations
from the CNS across the CSF-blood barrier. The components of
transepithelial choline transport by CP are 1) uptake from CSF across the
ventricular (apical) plasma membrane, 2) intracellular transport across
the cell, and 3) efflux across the
vascular-side (basolateral) plasma membrane into the blood compartment.
However, because of the complex organization of epithelial and vascular
tissue, the small size of this tissue, and the anatomical location of the CP, particularly in mammalian species, most in vivo and
in vitro techniques limit direct access to the intact epithelium.
Consequently, the experimental characterization of the energetics and
polarities of carriers that mediate the transport of choline and other
organic cations from CSF to blood has been limited and the role of
choroidal choline transport in central cholinergic homeostasis remains
poorly understood.
The objective of the present study was to characterize directly the
energetics of choline uptake from CSF across the ventricular membrane
of CP by using a primary culture system for the neonatal choroidal
epithelium. This methodology yields differentiated, confluent
epithelial monolayers that maintain morphological and functional
polarization similar to that of intact tissue (33). Choroidal
epithelial cells plated on an impermeable substratum with the
ventricular (apical) membranes exposed at the free surface were used to
characterize the coupling of apical choline transport to transmembrane
ion gradients (e.g., K+,
Na+, and
H+) and membrane electrical
potential. The data presented here demonstrate that carrier-mediated
choline uptake across the ventricular membrane of neonatal CP is
electrogenic and is not directly coupled to Na+ transport.
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MATERIALS AND METHODS |
Animals and tissue harvest.
Three- to five-day-old Sprague-Dawley rats were anesthetized under
hypothermic conditions before decapitation and removal of the brain.
For each preparation, lateral, third, and fourth plexuses from a total
of 30-36 neonatal rats were removed and held in chilled
DMEM/Ham's F-12 medium (DMEM/F-12) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml).
Cell culture.
Epithelial cells were isolated from CP tissue by enzymatic dispersion
as described previously in detail (33). Briefly, tissue was suspended
in dissociation buffer (in mM: 137 NaCl, 2.7 KCl, 0.7 Na2HPO4, 5.6 glucose, 10 HEPES; pH 7.4) with 5 U protease and 1,500 kU/ml DNAse I. The tissue-enzyme mixture was
incubated at 37°C and intermittently triturated over a 20-min
period. Aliquots of cells were filtered through 100-µm nylon mesh,
and the filtrate was centrifuged and washed twice with DMEM/F-12. Cells
were suspended in antibiotic-supplemented DMEM/F-12 with 10% Nu-Serum
IV and plated for 2-3 h (37°C, 95% air-5%
CO2). Unattached cells were then
aspirated, centrifuged, and suspended in MEM, with
D-valine substituted for
L-valine, containing 10%
Nu-Serum IV, triiodo-L-thyronine (1.5 µM), prostaglandin E1 (100 ng/ml), forskolin (10 µM), and epidermal growth factor (50 ng/ml).
Cells were plated at a density of 4.5 × 105
cells/cm2 in individual wells of
96-well tissue culture plates (0.32 cm2/well). Cells were maintained
at 37°C in a humidified 95% air-5% CO2 atmosphere. Unattached cells
were removed ~72 h postplating, and the initial plating medium was
replaced with maintenance medium (i.e., DMEM/F-12 with 5% Nu-Serum IV
and growth promoters). The medium was subsequently changed every
2-3 days.
Choline uptake studies.
On day
7 the apical uptake of radiolabeled
substrate was assayed. Cells were rinsed and preincubated with
artificial CSF (aCSF; in mM: 118 NaCl, 3 KCl, 0.7 Na2HPO4,
18 NaHCO3, 2 urea, 0.8 MgCl2, 1.4 CaCl2, 12 glucose, 20 Tris-HEPES;
pH 7.4) for 1 h at 37°C. To initiate uptake, the preincubation
buffer was replaced with 150 µl aCSF containing 10 µM
[3H]choline (0.075 µCi/150 µl); concentrations of inorganic and organic ions in
experimental uptake buffers were altered to test the effects of
external ions on the uptake of choline. Cells were incubated at
37°C (95% air-5% CO2) for
0-180 min. To terminate uptake, the radioactive buffer was removed
and the cells were immediately rinsed three times with 150 µl chilled
isotope-free aCSF that contained 5 mM unlabeled choline
chloride; this also removed all residual radioactive
isotopes from the transport well. Within the transport well, cells were
solubilized in 100 µl 1 N NaOH for 1 h and neutralized with 1 N HCl.
Two 50-µl aliquots of the solubilized cell suspension were
transferred to individual scintillation vials, and dpm were determined
by liquid scintillation. A third 50-µl aliquot of the cell suspension
was retained for determination of protein by a Bio-Rad microassay, with
BSA as a standard. The uptake of radiolabeled isotope was calculated as
picomoles of
[3H]choline per
milligram of protein. Cell-to-medium choline concentration ratios were
calculated from the amount of substrate radioactivity per microliter of
intracellular water and uptake buffer specific activity. Cell volume
was calculated from total protein by using the conversion factors 3.85 × 10
7 mg protein/cell
and 3.75 × 10
6 µl
water/cell (33).
Intracellular electrical potential recordings.
The whole cell patch-clamp recording method was used to study the
effects of external K+
concentration on resting intracellular electrical potential in cells
plated on glass coverslips (5 × 105
cells/cm2). Glass
microelectrodes with tip diameters of ~0.5 µm and resistances of
5-10 M
were used. Electrodes were filled with a solution
containing (in mM) 130 KCl, 1 MgCl2, 1 CaCl2, 10 EGTA, and 10 HEPES-HCl
(pH 7.4). Intracellular potential was measured with a patch-clamp amplifier. Data were acquired and analyzed with a Pulse-Pulse Fit data
acquisition system (HEKA, Lambrecht, Germany). Potential was recorded
for individual cells incubated for 30 min in aCSF containing 118 mM
NaCl and 3 mM KCl (i.e., control) and for cells bathed in aCSF
containing 91 mM NaCl and 30 mM KCl (i.e.,
high-K+ CSF).
Chemicals.
[3H]choline chloride
(53 mCi/mmol) was obtained from DuPont NEN Research Products
(Wilmington, DE). Tissue culture medium, growth promoters, and
antibiotics were obtained from Sigma Chemical (St. Louis, MO). Nu-Serum
IV was obtained from Collaborative Biochemical (Bedford, MA). All other
chemicals were obtained from commercial sources and were of the highest
grade available.
Statistical analysis.
Unless otherwise noted, cellular uptake was assayed in triplicate for
cells from at least three separate culture preparations (i.e.,
n = 3). Data are presented as means ± SE. Control and experimental means were compared by Student's
t-test for paired observations and
were deemed to be significantly different at
P < 0.05.
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RESULTS |
Time-dependent accumulation of choline across the apical membrane.
The apical uptake of 10 µM
[3H]choline by cells
grown on solid substratum was time dependent through 180 min (Fig.
1). The uptake was linear
through 30 min [sample coefficient of correlation (r) = 0.9918]. At
the earliest sample (1 min) total cellular choline uptake was 481 ± 5.5 pmol/mg protein; the mean cell-to-medium concentration ratio was
~5. Even at cell-to-medium concentration ratios of 395, steady state
was not reached (by 180 min). The total cellular accumulation of
choline could reflect both concentrative transport across the apical
membrane into the cytosolic compartment and intracellular sequestration
of substrate, as has been shown for other organic cations in this
culture system (24). Although choline metabolism was not directly
examined in this culture system, others have shown that in CP the
metabolic conversion of choline is minimal (see, e.g., Ref. 18).

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Fig. 1.
Time-dependent uptake and accumulation of choline across apical
membrane of primary cultures of choroidal epithelium. Cells plated on
impermeable supports were incubated for 1-180 min at 37°C in
artificial cerebrospinal fluid (CSF; 20 mM Tris-HEPES; pH 7.4) with 10 µM [3H]choline
chloride (triplicate measurements in 2 separate culture preparations).
Conc., concentration.
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Kinetics of apical choline uptake.
The kinetic parameters of the initial rate of choline uptake across the
apical membranes of cultured cells were determined by tracer
displacement (23). The 5-min uptake of 5 µM
[3H]choline in the
presence of 0-5 mM unlabeled choline was assayed (Fig.
2). On the basis of the assumption that
unlabeled choline competitively inhibits the uptake of
[3H]choline, the
initial rate of
[3H]choline uptake
(V) was expressed as a function of
the external concentration of unlabeled choline (CHOL):
V = (Vmax × [3H]CHOL)/(Km + [3H]CHOL + CHOL),
where Vmax is the
maximal rate of uptake,
[3H]CHOL is the
concentration of
[3H]choline, and
Km is the
Michaelis-Menten constant for choline. As the external concentration of
unlabeled choline increased, total cellular
[3H]choline uptake
progressively decreased, asymptotically approaching zero. Apical
[3H]choline uptake was
inhibited 98% by 5 mM unlabeled choline. Thus a saturable carrier
mediates uptake. The calculated mean Vmax ± SE was
4,671 ± 841 pmol · mg
protein
1 · 5 min
1; the mean
Km for choline ± SE was 49 ± 5 µM (n = 4). With consideration of technical limitations, it appears that
a nonsaturable process such as simple diffusion or the nonspecific
high-affinity binding of a radioisotope accounts for a minimal fraction
of total apical [3H]choline
uptake.

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Fig. 2.
Effects of increasing external concentration of unlabeled choline on
initial rate of apical uptake of 5 µM
[3H]choline by primary
cultures of choroidal epithelium. Cells plated on impermeable supports
were incubated at 37°C for 5 min in artificial CSF with 5 µM
[3H]choline and
0-5 mM choline chloride. Uptake was measured in triplicate in 4 separate culture preparations. However, data shown are from a
representative experiment in which calculated
Km and maximal
rate of uptake for mediated choline uptake were 51 µM and 3,878 pmol · mg
protein 1 · 5 min 1, respectively
(triplicate measurements).
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Cis-inhibitory effects of organic
cations on apical choline uptake.
To assess the general specificity of the apical choline carrier, the
uptake of [3H]choline
(10 µM) at 30 min in the presence of several endobiotic and
xenobiotic organic cations was assayed (Fig.
3). Increasing unlabeled choline to 5 mM
inhibited uptake 95% (3,387 ± 277 vs. 128 ± 19 pmol · mg
protein
1 · 30 min
1); 1 mM choline was
nearly as effective. At comparable test concentrations, two other
prototypic organic cations,
N1-methylnicotinamide
(NMN) and tetraethylammonium (TEA), reduced uptake 20-50%. At 1 mM, tetrapentylammonium (TPeA) inhibited uptake ~95%.
Hemicholinium-3 (HC-3) and quinacrine inhibited uptake >90%.

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Fig. 3.
Inhibitory effects of various organic cations on apical choline uptake
by primary cultures of choroidal epithelium plated on an impermeable
support are shown. Cultures were incubated at 37°C for 30 min in
artificial CSF (20 mM Tris-HEPES; pH 7.4) with 10 µM
[3H]choline chloride
and a 0 or 0.05-5 mM concentration of given test compound. Uptake
is expressed as mean percentage of control (i.e., no inhibitor) ± SE
(n = 3). * Significantly less
than control uptake (P < 0.05).
HC-3, hemicholinium-3; NMN,
N1-methylnicotinamide;
TEA, tetraethylammonium; TPeA, tetrapentylammonium.
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Trans effects of organic cations on
apical choline uptake.
Cis inhibition of apical choline
uptake by various agents suggested that uptake was carrier mediated.
This possibility was further examined by assaying the mediated uptake
of 10 µM [3H]choline
(i.e., in the absence of HC-3 and in the presence of 500 µM HC-3) by
cells preincubated with unlabeled choline, TEA, or NMN (Fig.
4). Mediated choline uptake was stimulated
~40% after preincubation with each test compound. For example, in
cells preloaded with unlabeled choline, the uptake of
[3H]choline increased
from 3,547 ± 164 to 5,517 ± 117 pmol/mg protein.

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Fig. 4.
Trans effects of organic cations on
apical choline uptake by primary cultures of choroidal epithelium
plated on an impermeable support and preincubated at 37°C for 60 min in artificial CSF without test compound or with 10 mM unlabeled
choline chloride, 5 mM TEA bromide, or 10 mM NMN chloride. After a
triple rinse with chilled artificial CSF, cells were incubated at
37°C for 30 min with artificial CSF containing 10 µM
[3H]choline chloride
and 0 or 500 µM HC-3. Uptake is expressed as mean percentage ± SE
of uptake in absence of HC-3 by cells preincubated without test
compound (n = 3).
* Significantly different from control
(P < 0.05). Regardless of
preincubation treatment, HC-3 significantly inhibited
[3H]choline uptake
(P < 0.05).
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Na+
dependence of apical choline uptake.
The energetic coupling of choline uptake across the ventricular plasma
membrane to gradients of inorganic ions was examined. The 30-min apical
uptake of [3H]choline
(10 µM) in response to changes of extra- or intracellular concentrations of Na+,
K+, and
H+ was monitored. Studies of the
isolated adult CP have suggested that tissue accumulation and CSF
clearance of choline may be Na+
dependent (8, 18, 19). The Na+
dependence of choline uptake in CP was examined directly by observing a
cultured choroidal epithelium incubated with various external concentrations of Na+ (i.e.,
isosmotic replacement of Na+ with
mannitol; Fig. 5). The reduction of
external Na+ from 140 to 20 mM or
the complete removal of external
Na+ failed to alter uptake. The
addition of 5 mM LiCl in the presence of high external
Na+ concentration (135 mM) also
had no effect. These data indicated that apical choline uptake is not
directly coupled to Na+ influx
across the ventricular membrane.

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Fig. 5.
Effects of external Na+ and
Li+ on apical choline uptake by
primary cultures of choroidal epithelium plated on impermeable supports
and incubated at 37°C for 30 min in artificial CSF with 10 µM
[3H]choline and
various concentrations of Na+ and
Li+. Control CSF contained ~140
mM Na+ (in mM: 118 NaCl, 0.7 Na2HPO4,
18 NaHCO3, 20 Tris-HEPES; pH 7.4).
Low-Na+ CSF contained ~20 mM
Na+; NaCl was isosmotically
replaced with mannitol. In
Na+-free CSF, NaCl and
NaHCO3 were isosmotically replaced
with mannitol and
Na2HPO4
was isosmotically replaced with
K2HPO4;
KCl concentration was adjusted to maintain total
K+ concentration at 3 mM. LiCl (5 mM) was added to control CSF containing 113 mM NaCl. Uptake is
expressed as mean percentage of uptake ± SE in presence of control
Na+ concentration; mean control
uptake: 3,583 ± 224 pmol · mg
protein 1 · 30 min 1. Uptake was not
significantly altered in any of the three experimental conditions
(P > 0.25;
n = 3).
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K+ dependence
of apical choline uptake.
Other in vitro studies have suggested that choline transport by CP is
K+ dependent (see, e.g., Refs. 8
and 18). To examine directly the
K+ dependence of ventricular
choline uptake by CP, apical
[3H]choline uptake was
assayed in the presence of various external concentrations of
K+, as well as of ouabain, a
Na+-K+-ATPase
inhibitor, and Ba2+, a nonspecific
K+ channel blocker (Fig.
6). A 10-fold increase in external
K+ concentration (3 vs. 30 mM KCl)
significantly depolarized the intracellular potential (
70 mV ± 5.1 mV, n = 7 cells, vs.
15 mV ± 4.8 mV, n = 6;
P < 0.0001) and reduced choline
uptake to ~45% of control. In the presence of 3 mM
K+, treatment of cells with
ouabain (1 mM) or Ba2+ (2 mM
BaCl2) reduced choline uptake
~50 and ~35%, respectively.

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Fig. 6.
Effects of external K+, ouabain,
and Ba2+ on apical choline uptake
by primary cultures of choroidal epithelium plated on impermeable
supports and incubated at 37°C for 30 min in artificial CSF with 10 µM [3H]choline and
various concentrations of K+.
Control CSF contained 3 mM KCl and 118 mM NaCl;
high-K+ CSF contained 30 mM KCl
and 88 mM NaCl. Effects of 1 mM ouabain and 2 mM
Ba2+
(BaCl2) were tested in presence
of control concentrations of KCl and NaCl. Cells were preincubated with
either agent for 30 min before initiation of uptake, when preincubation
buffer was replaced with control CSF containing
[3H]choline and
ouabain or BaCl2. Uptake is
expressed as mean percentage of uptake ± SE
(n = 3) in presence of control
concentrations of KCl and NaCl and no test agent (i.e., control); mean
control uptake: 3,359 ± 303 pmol · mg
protein 1 · 30 min 1. * Significantly
different from control (P < 0.007).
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Effects of intracellular acidification on apical choline uptake.
The mediated transport of organic cations, including quaternary
ammoniums, by H+-coupled exchange
has been well characterized for the renal proximal tubule (25).
Transport of the organic base cimetidine was shown to be pH sensitive
in CP (35). The possibility that ventricular uptake of choline was
mediated by H+ exchange was tested
with choroidal cells subjected to
NH4Cl pulse acidification. In the
control condition cells were preincubated for 15 min with standard aCSF
and then incubated for 30 min in aCSF containing
[3H]choline. To
acidify the intracellular pH, cells were preincubated with 30 mM
NH4Cl in
HCO
3-free CSF and then incubated in
Na+- and
HCO
3-free CSF containing
[3H]choline and no
NH4Cl.
Na+ was isosmotically replaced
with mannitol for these experiments. The effectiveness of this
technique to reduce intracellular pH in this culture system was
previously demonstrated with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM, which is converted intracellularly to the free acid, BCECF,
a fluorescent pH indicator. A 30 mM
NH4Cl pulse and the subsequent
removal of NH4Cl and external
Na+ reduce intracellular pH by
~1 (32). Intracellular acidification reduced mediated apical choline
uptake ~90% (Fig. 7). A preliminary experiment assayed choline uptake in cells that were incubated with
control aCSF (containing Na+ and
HCO
3) after the
NH4Cl pulse and, thereby, allowed
to regulate intracellular pH (26). Total choline uptake was the same as
that in cells never subjected to the
NH4Cl pulse (control: 5,375 ± 945 pmol · mg
protein
1 · 30 min
1; recovered: 6,026 ± 379 pmol · mg
protein
1 · 30 min
1; triplicate
measurements in a single culture preparation). Furthermore, uptake was
equally inhibited by 500 µM HC-3 (control: 658 ± 52 pmol · mg
protein
1 · 30 min
1; recovered: 543 ± 24 pmol · mg
protein
1 · 30 min
1). These preliminary
results suggest that decreased choline uptake after intracellular
acidification was not the result of cellular deterioration.

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Fig. 7.
Effects of intracellular acidification on apical choline uptake by
primary cultures of choroidal epithelium plated on impermeable
supports. Control: cells were preincubated for 15 min at 37°C with
control artificial CSF before 30-min uptake period (37°C), and
uptake was initiated by replacement of preincubation buffer with
control artificial CSF containing 10 µM
[3H]choline and 0 or
500 µM HC-3. Acidification: cells were preincubated for 15 min with
30 mM NH4Cl in
HCO 3-free CSF and immediately rinsed
(3 times) with Na+- and
HCO 3-free CSF containing no
NH4Cl; the 30-min uptake period
(37°C) was initiated with application of
Na+- and
HCO 3-free CSF containing 10 µM
[3H]choline and 0 or
500 µM HC-3. All buffers contained 20 mM HEPES-Tris (pH 7.4). In
NH4Cl buffer,
NaHCO3 was isosmotically replaced
with mannitol and NaCl concentration was reduced to 88 mM.
Na+- and
HCO 3-free CSF was similar in
composition to Na+-free CSF
described in Fig. 5. Uptake is expressed as mean percentage ± SE of
uptake without HC-3 in cells subjected to control conditions; mean
control uptake in absence of HC-3 was 4,785 ± 464 pmol · mg
protein 1 · 30 min 1;
n = 3. Choline uptake was
significantly reduced after intracellular acidification
(* P < 0.004); HC-3
significantly inhibited choline uptake in control and acidified
conditions (P < 0.005).
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DISCUSSION |
The present direct characterization of the first step in the
transepithelial absorption of choline across the neonate CSF-blood barrier, i.e., uptake of choline across the ventricular (apical) membrane of the choroidal epithelium, showed that choline is
accumulated by a saturable, electrogenic transport process. Unlike the
mediated reuptake of choline by presynaptic cholinergic neurons,
choline uptake is not directly coupled to
Na+ influx across the apical
membrane of CP. Instead, uptake is energetically driven by the
K+ diffusion potential across the
apical membrane, as indicated by the decreased choline uptake after
either direct or pharmacological perturbations of the
K+ gradient. Thus the
K+ dependence of apical choline
uptake may not involve a direct physical coupling to the
K+ flux but rather an energetic
coupling of uptake to the negative intracellular potential dictated
predominantly by the electrodiffusive efflux of
K+ across the plasma membrane.
Mediated uptake of choline across the ventricular membrane of
cultured choroidal epithelium.
Ventriculocisternal clearance studies have demonstrated that choline
absorption across the CSF-blood barrier is an active process (1, 18).
In vitro choline accumulation by CP isolated from several species also
indicates a saturable process(es) (see, e.g., Refs. 1 and 8). In the
present study, choline accumulated across the apical membrane of a
cultured neonatal CP epithelium in a time-dependent manner and the
total cell-to-medium concentration ratios at all time points
exceeded unity, suggesting a metabolically dependent process (Fig. 1).
Developmental studies of isolated CP from rabbits have demonstrated
that the capacity to accumulate choline increases approximately
fourfold as the animal matures (20). However, the present 30-min
cell-to-medium choline ratios for neonate rat CP at the same external
choline concentration (10 µM) are comparable to tissue-to-medium
ratios reported for isolated CP of adult rats (19).
These present data indicate that the initial uptake of choline from CSF
into the choroidal epithelial cell is a carrier-mediated membrane
transport process (Figs. 2 and 3). Previous studies indicated that the
transport capacity for choline in CP increases with age (20) and may
vary among species (19). However, the
Km of the rat
neonatal CP apical carrier for choline (~50 µM) closely
approximates the reported
Km for adult
choline clearance from CSF in vivo (~16 µM; Ref. 18) and that for
choline accumulation by the adult rabbit CP in vitro (~40 µM; Ref.
1). On the basis of these collective kinetic data on CSF clearance and
tissue accumulation, choline is transported with greater affinity
across the CSF-blood barrier than it is across the blood-brain barrier
(Km
225-445 µM; Ref. 7). Although the affinity of this CP choline
carrier is much lower than that of the choline carrier in cholinergic neurons
(Km
1-3
µM; Ref. 12), it is similar to that of the choline carrier in the
apical membranes of the renal proximal tubule
(Km
100 µM;
Ref. 36) and small intestine
(Km
150 µM; Ref. 27). More importantly, these kinetic data support the proposed role of CP in central cholinergic homeostasis. The rate of the
carrier-mediated removal of choline from CSF would be most responsive
to changes in CSF choline concentration within a range whose upper
limit is less than the experimentally determined
Km. The physiological range of CSF choline concentration is 3-10 µM (15). Thus it appears that the CP choline transporters, working in
series or in parallel, have affinities appropriately set to respond to
normal or pathophysiological increases in CSF choline concentrations.
Prior evaluation of the specificity of choline transport by CP has been
limited. NMN, an endogenous quaternary ammonium that is transported
across the CSF-blood barrier and that inhibits the transport of organic
cations and bases by CP (see, e.g., Refs. 28, 30, and 33), also
inhibits CSF clearance of choline and its accumulation by isolated CP
in a concentration-dependent manner (1, 16, 18). In a cultured
choroidal epithelium, NMN also inhibited apical choline uptake in a
concentration-dependent manner (Fig. 3). However, hexamethonium,
another cationic substrate transported by CP (28, 30), did not alter
uptake, even at concentrations 3 orders of magnitude greater than that
of radiolabeled choline (see Fig. 3). Nevertheless, choline uptake was
inhibited by several other organic cations that are known substrates of carriers for choline and other organic cations in neuronal and epithelial tissues, including the present CP culture system. Uptake was
inhibited by HC-3, a high-affinity inhibitor of choline transport by
presynaptic neurons and the renal proximal tubule, liver, intestine, and placenta (12, 17, 27, 31, 36), as was the clearance of choline from
ventricular CSF (9). Tetraalkylammoniums TEA and TPeA
also reduced choroidal uptake of choline; their relative inhibitory
potencies at 1 mM (TPeA > TEA) qualitatively paralleled those
observed for transport by apical membranes of the renal proximal tubule
(36). However, the reduction of uptake of 10 µM choline across the
intact choroidal epithelial cell by these tetraalkylammoniums at 1 mM
may be due in part to reduced K+
channel conductance (see, e.g., Ref. 4). Quinacrine, which shares a
common apical carrier with TEA in CP (24), was among the most potent
inhibitors tested; however, other possible effects of this agent have
not been characterized. Collectively, these kinetic and inhibitor data
indicate that mediated choline uptake across the ventricular membrane
of a cultured choroidal epithelium is partially mediated by an organic
cation carrier with a substrate specificity profile qualitatively
consistent with that described previously for choline transport in
intact CP and other epithelial tissues.
Na+
dependence of ventricular choline uptake.
Several studies have indicated that choline absorption across the
CSF-blood barrier of the adult rabbit is energetically coupled in some
way to the
Na+-K+-ATPase
at the apical pole of the choroidal epithelium (see, e.g., Ref. 33).
Lindvall-Axelsson and colleagues (19) have demonstrated that treatment
of adult rabbits and rats with steroid hormones reduced CP
Na+-K+-ATPase
activity, an effect accompanied by attenuated CSF formation in vivo and
decreased choline accumulation by CP isolated from treated animals.
Others determined that ouabain, an inhibitor of
Na+-K+-ATPase,
decreases ventricular choline uptake by isolated plexuses of bullfrogs
and clearance of choline from CSF in rabbits (8, 18). The reduction of
apical choline uptake by ouabain found in the present study indicates
that the uptake of choline across the apical membrane of neonatal CP is
energetically coupled in some manner to
Na+-K+-ATPase
activity (Fig. 6). Uptake could be directly coupled to the
Na+ gradient that is generated and
maintained by the
Na+-K+-ATPase,
as is choline reuptake by presynaptic cholinergic neurons (12). For
isolated adult rabbit CP, Hamel et al. (11) report that complete
removal of external Na+, which
involved isosmotic replacement of 118 mM NaCl with LiCl, reduced
choline accumulation ~85%. However,
Li+ competes for the
K+-binding site on the
Na+-K+-ATPase
in CP (8); thus reduced choline accumulation in the presence of such
high Li+ concentrations may have
been secondary to disruptions in transmembrane gradients of both
Na+ and
K+. In our neonate CP system,
decreasing external Na+ from 140 to 20 mM significantly reduced apical
L-proline uptake, a
Na+-coupled electrogenic transport
mechanism (33). However, neither a similar reduction in external
Na+ nor complete removal of
external Na+ altered apical
choline uptake (Fig. 5). Furthermore, at 5 mM, a concentration known to
inhibit renal Na+-coupled
transporters (25) and to stimulate transepithelial choline transport in
the frog arachnoid membrane (8),
Li+ had no significant effect on
choline uptake in the present experimental CP system (Fig. 5). Thus
choline uptake across the apical membrane of the choroidal epithelium
of the neonate is not directly coupled to
Na+ influx.
K+ dependence
of ventricular choline uptake.
Several Na+-independent
electrogenic transport systems for choline in nonneuronal tissues have
been characterized. For example, choline transport across the apical
membranes of the renal proximal tubule, hepatocyte, and placenta is
mediated by Na+-independent
electrogenic facilitated diffusion (17, 31, 36). The electrodiffusion
of K+ across the apical membrane
ultimately dictates the negative membrane potential of the choroidal
epithelial cell (37, 39), and studies of isolated CP from adult
amphibians demonstrate that pharmacological manipulation of either the
K+ gradient or
K+ conductance across the
ventricular membrane depolarizes the membrane potential. A 10-fold
increase in ventricular K+
concentration depolarizes membrane potential (39). The addition of
ouabain to the ventricular compartment reduces CP tissue
K+ content and increases
ventricular (CSF) K+ concentration
(14, 29, 39), as well as depolarizing membrane potential (38, 39). In
the present CP culture system, increased external
K+ (3-30 mM KCl) or treatment
with ouabain reduced apical choline uptake to 45-50% of control
(Fig. 6), and by direct measurement it was confirmed that
increases in external K+ at the
membrane depolarized intracellular potential. In addition, choline
uptake was significantly reduced by
Ba2+, a nonspecific
K+ channel blocker shown to not
only inhibit K+ efflux from
isolated CP but also to induce marked depolarization of ventricular
membrane potential (14, 39). On the basis of single-channel recordings
from excised apical membranes of amphibian CP,
Ba2+-induced depolarization may
involve decreased K+ conductance
through Ca2+-activated channels as
a result of reduced channel-open probability and increased closed time
(4). In the renal proximal tubule, treatment with
Ba2+ also results in the
depolarization of membrane potential and decreased basolateral organic
cation uptake, an electrogenic facilitated diffusion
mechanism (25). Thus the marked reductions of apical choline uptake
after increased external K+ and
pretreatment with either ouabain or
Ba2+ are consistent with the
energetic coupling of choline transport to the
K+ diffusion potential across the
ventricular pole of the neonatal choroidal epithelial cell.
Apical choline uptake in the cultured choroidal epithelium was also
reduced by TEA and TPeA (Fig. 3), tetraalkylammoniums that directly
block K+ channels (see, e.g., Ref.
5). At 1 mM, TEA modestly inhibited choroidal apical choline uptake
(20%). At similar or higher concentrations, TEA does not alter choline
transport in microvillus membranes isolated from other epithelia (see,
e.g., Refs. 17 and 36). However, inhibition of uptake in the intact
choroidal cell may involve the concurrent block of
Ca2+-activated
K+ channels. Indeed, in patches
from the apical membrane of amphibian CP, TEA at the extracellular or
cytoplasmic face reduces single-channel current and channel-open
probability, albeit with greater potency at the extracellular face (4).
In membrane patches, the affinity of the binding site for TEA at the
external opening of the
Ca2+-activated
K+ channels is relatively high
[dissociation constant
(Kd)
230 µM; Ref. 4]; however, in intact epithelial cells
the affinity for extracellular TEA is greatly reduced (22). A similar
decreased sensitivity to TEA of K+
channels in membrane patches vs. those in intact tissue is also observed in colonic smooth muscle (5). In contrast to TEA, TPeA reduced
choline uptake ~95%. In apical membranes of the renal proximal
tubule at similar substrate and inhibitor concentrations, TPeA inhibits
choline transport ~60%; Wright et al. (36) have suggested that this
may involve interactions of TPeA at sites other than the
substrate-binding site of the choline transporter. Extracellular TPeA
is a potent inhibitor of maxi-K+
channels in colonic smooth muscle
(Kd
12 µM;
Ref. 5). Thus, in the choroidal epithelium, decreased choline uptake in
the presence of TPeA could involve reduced
K+ channel activity and the
subsequent depolarization of membrane potential, as well as a direct
inhibitory interaction with the choline carrier.
Organic cation/H+ exchange
mechanisms mediate the transport of organic cations in several
epithelial tissues, including the renal proximal tubule, liver, and
intestine (25). Investigation of mediated transport of organic cations
by H+ exchange in CP has been
limited. Nonetheless, as shown in ATP-depleted slices and isolated
apical membrane vesicles of CP, accumulation of the xenobiotic
cimetidine is stimulated after intracellular acidification or
imposition of an outwardly directed
H+ gradient, respectively. Such
stimulation was taken as an indication of
H+-driven exchange for the organic
base (35). Likewise, in cultured neonatal CP, intracellular
acidification markedly stimulates mediated apical uptake of the organic
cation TEA (32). In contrast, apical choline uptake was reduced 90%
after intracellular acidification (Fig. 7). These data indicate that
choline uptake is not meditated by a proton exchange mechanism but may
indeed be a pH-sensitive process. On the basis of the observed
correlation of changes in intracellular pH with changes in
K+ conductance and membrane
potential in CP and other tissues, these data are consistent with
energetic coupling of choline uptake to membrane potential. In isolated
hepatocytes intracellular acidification induced by the
NH4Cl pulse technique is
associated with depolarization of the resting membrane potential (2).
Furthermore, in isolated hepatocytes and isolated CP, intracellular
acidification reduces the K+
permeability of the plasma membrane and intracellular alkalinization has the converse effect (2, 38). It has been suggested that the
depolarization of membrane potential and reduced
K+ permeability are due in part to
reduced activity of K+ channels.
In excised patches of the apical membrane of amphibian CP,
acidification of the cytoplasmic side decreases the open probability of
both maxi-K+ channels and
Ca2+-activated
K+ channels (4, 6). Indeed, for
maxi-K+ channels, cytoplasmic
acidification (pH 7.4 to 6.4) decreases open time and increases closed
time by factors of 10 and 2, respectively, and reduces
voltage-dependent activation (6).
In the present study, total cellular choline uptake did not reach
equilibrium, even at 180 min. Others have reported similar delays in
the equilibration of choline and other organic cations in isolated CP
(8, 30), as well as tissue-to-medium ratios that exceed predicted
values for electrical equilibrium (20, 30). Indeed, for this CP system
with a membrane potential of
70 mV, the predicted cell-to-medium
choline ratio would be 15, and 30-min values ranged from 40 to 70. Thus
facilitated electrodiffusive uptake of choline across the apical
membrane alone could not account for this unexpectedly high
accumulation. Metabolic transformation of substrate could increase the
total radioactivity within the cell over time. However, uptake by
isolated CP apparently involves little synthesis of ACh because of low
levels of choline acetyltransferase activity (11), and unlike renal or
hepatic clearance of choline from plasma, which involves metabolic
conversion to betaine (3), the clearance of exogenous choline from
ventricular CSF involves no metabolism of radiolabeled substrate (18).
Total cellular accumulation of choline may instead reflect
concentrative uptake across the apical membrane into the cytosol and
maintenance of the choline electrochemical diffusion potential by
subsequent intracellular sequestration of substrate, as has been shown
for the organic cations quinacrine and TEA in this culture system (24).
The sequestration of substrate from the cytosol into an intracellular
compartment is most likely metabolism dependent and could very well
contribute to the high tissue-to-medium ratios for organic cations
observed in this and other experimental CP systems. The possible role
of the intracellular compartmentation of substrate in the net
transepithelial transport of choline and other organic ions across the
CSF-blood barrier remains to be elucidated.
In conclusion, choline uptake across the ventricular plasma membrane of
primary monolayer cultures of the neonatal CP epithelium is a mediated
process directly dependent on the plasma membrane electrical potential
and factors that may alter that potential. Uptake is not directly
coupled to the Na+ flux across the
membrane. However, the energetic coupling of choline uptake to
Na+ transport at the apical pole
of the CSF-blood barrier may change as the animal develops. Better
characterization of the mechanisms of choline transport across this
brain barrier in the adult may yield greater insight into any such
developmental changes.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge the expert assistance of Dr. Joseph Lo
Turco and Eric Charych in measuring the intracellular electrical potential.
 |
FOOTNOTES |
This work was supported by National Science Foundation Grants
IBN9604070 and IBN9808616, National Institute of Environmental Health
Sciences Grant ES-07163-07, and National Institute of Neurological Disorders and Stroke Grant F32-NS-10475.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. R. Villalobos, Dept. of Physiology and Neurobiology, Univ. of Connecticut,
Box U-156, 3107 Horsebarn Hill Rd., Storrs, CT 06269-4156 (E-mail:
villalobos{at}oracle.pnb.uconn.edu).
Received 28 September 1998; accepted in final form 1 March 1999.
 |
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