Anesthesiology Research Division, Departments of 1 Anesthesiology and 3 Pharmacology and 2 Division of Nephrology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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The function of the apical
Na+-K+-2Cl
cotransporter in mammalian choroid plexus (CP) is uncertain and
controversial. To investigate cotransporter function, we developed a
novel dissociated rat CP cell preparation in which single, isolated
cells maintain normal polarized morphology. Immunofluorescence
demonstrated that in isolated cells the
Na+-K+-ATPase,
Na+-K+-2Cl
cotransporter, and aquaporin 1 water channel remained localized to the
brush border, whereas the
Cl
/HCO
3
(anion) exchanger type 2 was confined to the basolateral membrane. We
utilized video-enhanced microscopy and cell volume measurement
techniques to investigate cotransporter function. Application of 100 µM bumetanide caused CP cells to shrink rapidly. Elevation of
extracellular K+ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling was
blocked completely by Na+ removal
or by addition of 100 µM bumetanide. Exposure of CP cells to 5 mM
BaCl2 induced rapid swelling that
was inhibited by 100 µM bumetanide. We conclude that the CP
cotransporter is constitutively active and propose that it functions in
series with Ba2+-sensitive
K+ channels to reabsorb
K+ from cerebrospinal fluid to blood.
potassium transport; cerebrospinal fluid secretion; cerebral edema; intracranial hypertension; bumetanide
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INTRODUCTION |
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MAINTENANCE OF CEREBROSPINAL fluid (CSF) volume and composition is crucial for normal function of the nervous system. The bulk of the CSF is produced by the choroid plexus (CP), which lines the ventricles in the brain. CP epithelial cells play diverse roles in regulating brain function and metabolism. They supply neurons and glia with micronutrients, remove waste products and toxins from the nervous system, and provide a pathway for neuroendocrine communication within the brain (13, 31).
One of the most important functions of CP cells is secretion of the CSF
and control of its ionic composition. The mechanisms and regulation of
CSF secretion are incompletely understood. In CP cells, unlike most
epithelia, the
Na+-K+-ATPase
is located on the apical membrane, facing the CSF (22). The
Na+-K+-ATPase
maintains a low intracellular Na+
concentration. Secondary active transport of
Na+ occurs at the basolateral cell
membrane and appears to be mediated in part by a
Na+/H+
exchanger (24-26). A
Cl/HCO
3
exchanger is also located at the basolateral membrane (1, 17). It has
been proposed that these two exchangers transport NaCl and osmotically
obliged water into the CP cell (13, 14). At the apical membrane,
Na+ is extruded into the CSF via
the
Na+-K+-ATPase.
Cl
transport into the CSF
is thought to be mediated in part by an apical anion channel (6, 7,
10).
A number of studies have implicated the
Na+-K+-2Cl
cotransporter as having an important role in the secretion and
regulation of CSF ionic composition, but the precise function of the
cotransporter is uncertain and controversial. In most models of CP
function, the cotransporter has been postulated to be localized to the
basolateral membrane and is thought to play a central role in CSF and
NaCl secretion (12, 13, 30). Such a model is consistent with the
well-established function and localization of the cotransporter in
numerous other secretory epithelia (8). There are, however, no
unequivocal data to support this model in the CP. Administration of the
loop diuretics bumetanide and furosemide has been shown to either
inhibit or have no effect on CSF secretion in several animal models
(12). Interpretation of these discrepant findings is obscured by a
variety of significant methodological concerns (12, 17). Studies of
isolated tissues have shown clear effects of loop diuretics on CP ion
transport (2), but the membrane localization of the cotransporter
cannot be deduced from these experiments.
Based on their studies of isolated rat CP, Keep and co-workers (19, 20) proposed that the cotransporter is localized to the apical cell membrane and that it is operating in a reverse mode to secrete salt and water into the CSF. Delpire and co-workers have recently examined the distribution of the cotransporter in the brain. These investigators cloned the ubiquitous isoform of the cotransporter, NKCC1 (3), and demonstrated that it was expressed abundantly in the nervous system (29). The bulk of the signal in the brain is derived from the CP. Immunofluorescence studies of the CP demonstrated that the cotransporter is expressed predominantly, if not exclusively, on the apical cell membrane (29). The abundant apical expression of the cotransporter raises important questions regarding its role in CP function.
Intracranial hypertension and cerebral edema are common and very
serious clinical problems. Administration of drugs that reduce CSF
secretion is a standard approach for reducing brain swelling (12). An
understanding of the precise mechanisms and regulation of CSF secretion
therefore has significant clinical importance. However, detailed
cellular and molecular investigations of solute and fluid
transport in the mammalian CP are difficult, due to the complicated
morphology of the tissue and the small quantities that can be isolated
and studied. To circumvent these difficulties, we developed a novel
isolated CP cell preparation that maintains a normal polarized
morphology and distribution of transport proteins. These cells are
amenable to detailed study using optical and electrophysiological techniques. In the present investigation, we demonstrate that the
Na+-K+-2Cl
cotransporter is constitutively active, that it functions to reabsorb
NaCl and KCl from the CSF, and that it is the major pathway for
concentration gradient-driven K+
uptake in the CP.
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MATERIALS AND METHODS |
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Isolation of single, polarized rat CP epithelial cells. The method for isolating single, polarized CP cells was similar to that described by Torres et al. (35) for the Necturus gallbladder. Briefly, 3- to 7-wk-old Sprague-Dawley rats were anesthetized by ether inhalation. After decapitation of the rats, CP were dissected from the lateral and fourth ventricles. Isolated CP were washed with Hanks' balanced salt solution (HBSS) and then treated with 1 mg/ml collagenase IV (Sigma Chemical, St. Louis, MO) and 1 mg/ml protease XIV (Sigma) in HBSS for 60 min at room temperature. The epithelium was disrupted, and single cells suspensions were produced by gentle pipetting. Suspensions were centrifuged at 300 g for 5 min, and pellets were resuspended in artificial CSF (aCSF; see Table 1). The pH and gas content of the aCSF in which the cells were suspended was kept constant by continuously blowing a stream of 5% CO2-95% air over the surface of the medium. Cells were utilized for experiments within 6 h after isolation.
Several criteria were utilized in selecting isolated cells for experimentation. Cells that were obviously swollen and cells that had blebbing membranes, intracellular vacuoles, or disrupted microvilli were discarded.Antibodies.
Rabbit polyclonal antibodies against a carboxy-terminal region of the
mouse bumetanide-sensitive
Na+-K+-2Cl
cotransporter (mNKCC1) were produced as described previously by Kaplan
et al. (18). Polyclonal antibodies to the rat
Na+-K+-ATPase
1-subunit were purchased from
Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies to the
mouse aquaporin 1 water channel and the mouse anion exchanger type 2 (AE2) were generous gifts of Dr. Dennis Brown (Massachusetts General
Hospital, Boston, MA) and Dr. Seth Alper (Beth Israel Hospital, Boston,
MA), respectively.
Immunofluorescence. Isolated CP cells were attached to glass coverslips coated with Cel-Tak (Collaborative Biomedical Products, Bedford, MA) and fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. After fixation, cells were permeabilized with 0.075% saponin in PBS for 10 min. Permeabilized cells were then exposed for 30 min at room temperature to a blocking solution containing 0.075% saponin and 0.2% BSA in PBS and incubated overnight at 4°C with primary antibodies diluted in PBS containing 0.075% saponin and 0.2% BSA. After a wash in PBS, cells were incubated for 90 min at room temperature with indocarbocyanine-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) diluted 1:600 in PBS containing 0.2% BSA. The cells were then washed with PBS and mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA).
Cells were visualized using a Nikon Eclipse E800 microscope equipped with a Nikon Plan Apo ×100 objective lens [1.4 numerical aperture (NA)] and an Optronics DEI-750 color charge-coupled device (CCD) camera (Optronics Engineering, Goleta, CA). Montages were generated from digitized images using Adobe PhotoShop 4.0 and printed with a Tektronix Phaser 450 color printer (Tektronix, Wilsonwill, OR).Measurement of relative cell volume changes. Video-enhanced differential interference contrast (DIC) microscopy (33) was used to measure relative volume changes in isolated, polarized CP cells. Briefly, cells were attached to the poly-L-lysine-coated coverslip bottom of a bath chamber (model R-26G, Warner Instrument, Hamden, CT) that was mounted onto the stage of a Nikon TE 300 inverted microscope. The bath chamber was perfused continuously at room temperature with experimental solutions gassed with 5% CO2-95% air. The compositions of various solutions used in these studies are shown in Table 1.
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Statistics. Data are reported as means ± SE (n = number of cells, number of CP). Statistical significance was assessed using Student's t-test.
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RESULTS |
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Isolated CP cells maintain functional polarity. Reuss and co-workers (34, 35) have shown that certain epithelial cells maintain morphological and functional polarity when isolated from their native tissues. Figure 1 demonstrates clearly that CP cells behave in a similar fashion. A distinct apical pole, marked by a prominent brush border, and a basolateral pole are readily visible by DIC microscopy in fixed (Fig. 1, left) and living cells (not shown).
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The CP
Na+-K+-2Cl
cotransporter is constitutively active and reabsorbs solute from the
CSF.
Cell volume is held constant under steady-state conditions by a precise
matching of rates of solute influx and efflux across the plasma
membrane. Alterations in the activity of either solute influx or efflux
pathways can result in net gain or loss of solute and osmotically
obliged water, with resultant cell swelling or shrinkage. Experimental
manipulation of transport pathway activity and quantification of
associated cell volume changes can therefore provide insight into the
nature and regulation of transporters and channels (5, 33).
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K+ efflux
from CP cells is mediated in part by
Ba2+-sensitive
K+ channels.
If the
Na+-K+-2Cl
cotransporter is constitutively active and moving
K+ into the cell, then
K+ must exit via other transport
pathways for cell volume to remain stable. In certain epithelia, there
is a tight coupling between the activity of
K+ channels and the
Na+-K+-2Cl
cotransporter (37). We assessed the role of
K+ channels in CP
K+ transport by exposing cells to
5 mM BaCl2. As shown in Fig.
4A, exposure to BaCl2 caused CP cells
to swell 35 ± 9% (measured 5 min after
Ba2+ addition) at an initial rate
of 7.3 ± 2 %/min (n = 13,6). Removal of BaCl2 caused the
cells to shrink back toward their original volume (Fig.
4A). These findings indicate that
K+ channels mediate net
K+ efflux from CP cells.
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DISCUSSION |
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Reabsorption of NaCl and KCl from the CSF by the apical
Na+-K+-2Cl
cotransporter.
A number of in vivo and in vitro studies have suggested an important
role for the
Na+-K+-2Cl
cotransporter in CP function. Most models of CP transport have localized the cotransporter to the basolateral membrane and have proposed that it plays a central role in the secretion of NaCl and
water into the brain interstitium (12, 13, 15). The postulated
basolateral localization is consistent with the function and
localization of the cotransporter in a variety of other secretory epithelia (8).
Role of K+
channels in CP
K+ transport.
If K+ is taken up into CP cells
via the
Na+-K+-2Cl
cotransporter, it must also exit at a similar rate for cell volume to
remain stable. Under normal conditions,
K+ typically exits cells via
K+ channels and/or the
K+-Cl
cotransporter. We assessed the role of channels in
K+ efflux by exposing CP cells to
5 mM BaCl2, a selective
K+ channel blocker.
Ba2+ caused CP cells to swell
rapidly (Fig. 4). Both the initial rate and maximal swelling were
inhibited ~65% by 100 µM bumetanide (Fig.
4B), indicating that
K+ taken up by the cotransporter
exits the cell, at least in part, via
K+ channels. Ouabain completely
inhibited Ba2+-induced swelling
(Fig. 4B). This inhibition most
likely reflects inhibition of pump-mediated
K+ uptake, as well as inhibition
of the
Na+-K+-2Cl
cotransporter, which is expected to occur as intracellular
Na+ concentration rises during
exposure of CP cells to ouabain.
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Clinical implications of CSF solute reabsorption through the apical
Na+-K+-2Cl
cotransporter.
Intracranial hypertension is a serious and life-threatening consequence
of traumatic brain injury and a variety of disease states. Systemic
administration of furosemide is commonly used to treat intracranial
hypertension. The effect of furosemide is widely believed to be due to
inhibition of CSF secretion via inhibition of a basolateral NaCl
transporter in the CP, as well as to a furosemide-induced diuresis and
increased whole body fluid loss (12). Experimental studies in a variety
of animal models, however, have failed to show any effect of furosemide
on CSF secretion (12). Furthermore, the recent studies of Plotkin et
al. (29) demonstrating an apical localization of the
Na+-K+-2Cl
cotransporter, as well as the findings of this investigation, indicate
that there is no sound rationale for attempting to
directly inhibit CP CSF secretion via systemic
administration of furosemide. We propose that if furosemide inhibits
CSF secretion in patients with intracranial hypertension, it does so by
indirect and nonspecific mechanisms.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants NS-30591 (to K. Strange), HL-49251 (to E. Delpire), and DK-36803 (to S. C. Hebert).
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FOOTNOTES |
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E. Delpire is an Established Investigator of the American Heart Association.
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: K. Strange, Vanderbilt University Medical Center, Dept. of Anesthesiology, 504 Oxford House, 1313 21st Ave. South, Nashville, TN 37232-4125.
Received 25 June 1998; accepted in final form 21 August 1998.
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