©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Na,K-ATPase in the Choroid Plexus
REGULATION BY SEROTONIN/PROTEIN KINASE C PATHWAY (*)

(Received for publication, October 5, 1994; and in revised form, November 15, 1994)

Gilberto Fisone (§) Gretchen L. Snyder Jessica Fryckstedt (1) Michael J. Caplan (2) Anita Aperia (1) Paul Greengard

From the  (1)Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, the Department of Pediatrics, St. Göran's Children's Hospital, Karolinska Institute, S-112 81 Stockholm, Sweden, and the (2)Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the choroid plexus, the ion pump Na,K-ATPase regulates the production of cerebrospinal fluid. We now report that incubation of choroid plexus with an activator of protein kinase C, phorbol 12,13-dibutyrate, strongly stimulates the phosphorylation of Na,K-ATPase and inhibits its activity. Similar effects were obtained with serotonin, which in the choroid plexus stimulates phosphoinositide turnover, thereby activating protein kinase C. Serotonin (10 µM) increased by about 10-fold the amount of phosphorylated Na,K-ATPase and significantly reduced its activity. Two-dimensional peptide mapping showed comigration of Na,K-ATPase phosphorylated by either phorbol 12,13-dibutyrate or serotonin in intact cells and by protein kinase C in vitro. These results demonstrate that first messengers can regulate the activity of Na,K-ATPase through a mechanism involving protein phosphorylation. Moreover, they provide a plausible mechanism for the demonstrated ability of serotonin to decrease cerebrospinal fluid production.


INTRODUCTION

Precise control of cerebrospinal fluid (CSF) (^1)volume and composition is necessary for normal neuronal function and is essential in maintaining a physiologic brain volume and intracranial pressure. The choroid plexus represents the primary source of CSF. In this tissue, the ion pump Na,K-ATPase is responsible for the translocation of Na across the apical membrane of the epithelial cells (1, 2) into the cerebral ventricles and accounts for the bulk of CSF formation(3, 4) . Suppression of the activity of Na,K-ATPase by ouabain results in a decrease of 70-80% in the rate of intraventricular CSF secretion(4) . Systemic administration of the neurotransmitter serotonin (5) or of its precursor, 5-hydroxytryptophan(6) , is known to decrease the secretion of CSF. Considering the high density of serotonin 5-HT receptors in the epithelial cells of the choroid plexus (7, 8) and the robust phosphoinositide hydrolysis initiated by their activation(9, 10) , it seemed plausible that serotonin might regulate CSF formation by a direct action on the choroid epithelium, via this signal transduction pathway. Stimulation of phosphatidylinositol turnover leads to the activation of protein kinase C, and protein kinase C has been shown to phosphorylate purified Na,K-ATPase and to inhibit its activity(11) . Therefore, we have examined the possibility that in the choroid plexus, serotonin, via protein kinase C, regulates the state of phosphorylation and activity of Na,K-ATPase and thereby controls CSF production.


EXPERIMENTAL PROCEDURES

P Labeling and Pharmacological Treatment of Rat Choroid Plexus

Male Sprague-Dawley rats (200-350 g) were killed by decapitation, and the choroid plexus was dissected from the lateral cerebral ventricles. Choroid plexus from one rat per treatment condition was incubated at 30 °C in 2 ml of Krebs bicarbonate buffer (124 mM NaCl, 4 mM KCl, 26 mM NaHCO(3), 1.5 mM CaCl(2), 1.5 mM MgSO(4), 0.25 mM KH(2)PO(4), 10 mM glucose), oxygenated with 95% O(2)/5% CO(2) (v/v). After 15 min, medium was replaced with the same amount of fresh buffer containing 2.5 mCi of [P]orthophosphoric acid (DuPont NEN; specific activity 8500-9120 Ci/mmol) and the tissue was incubated for 60 min. The radioactive buffer was then removed, and P-labeled choroid plexuses were washed twice with 2 ml of fresh buffer and incubated for an additional 2-60 min in the presence or absence of drugs, as described. After drug treatment, the buffer was removed and the tissue was rapidly frozen in liquid nitrogen and stored at -70 °C until assayed.

Immunoprecipitation of Na,K-ATPase

P-Labeled choroid plexuses were sonicated in 1 ml of lysis buffer (20 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% bovine serum albumin (BSA); pH 8.0) containing 50 mM NaF to block phosphatase activity and the following protease inhibitors: 1 mM EGTA, 25 mM benzamidine, 100 µM phenylmethylsulfoxide, 20 µg/ml chymostatin, 20 µg/ml pepstatin A, 5 µg/ml leupeptin, and 5 µg/ml antipain (Peptide International). Aliquots of the homogenate (20 µl) were used for determination of total P incorporation into trichloroacetic acid-precipitated proteins. Ten mg of preswollen Protein A-Sepharose CL-4B (Pharmacia Biotech Inc.) were added to each tube and the samples mixed for 30 min at 4 °C. The Sepharose beads and associated, nonspecifically adsorbed proteins were removed by centrifugation for 10 s at 15,000 rpm in a tabletop microcentrifuge. The supernatants were mixed for 2 h at 4 °C with 15 µl of mouse ascites fluid containing a monoclonal antibody specific for the alpha1 isoform of Na,K-ATPase (12) (the only isoform expressed in the choroid epithelium; (13) ), followed by a 1-h incubation with 15 µl of affinity-purified rabbit anti-mouse antibody (Cappel, 1 mg/ml). The samples were then transferred to Eppendorf tubes containing 10 mg of preswollen Protein A-Sepharose beads and incubated for 1 h at 4 °C. The beads were collected by centrifugation and washed once with 1 ml of lysis buffer; three times with 1 ml of a buffer containing 20 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.2% BSA (pH 8.0); three times with 1 ml of a buffer containing 20 mM Tris/HCl, 500 mM NaCl, 0.5% Triton X-100, 0.2% BSA (pH 8.0); and once with 1 ml of a buffer containing 50 mM Tris/HCl (pH 8.0). After the final wash, the beads were resuspended in 50 µl of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (50 mM Tris/HCl, 10% glycerol, 2% SDS, 10% 2-mercaptoethanol, 0.01% bromphenol blue, pH 6.8), vortexed, and centrifuged. The recovered proteins were separated by SDS-PAGE on 7.5% acrylamide gels(14) . Gels were dried, and P incorporation into Na,K-ATPase was quantified using a PhosphorImager 400B and ImageQuant(TM) software from Molecular Dynamics. Individual values of P incorporation into Na,K-ATPase were corrected for the total P incorporation measured for each sample.

Phosphorylation of Purified Na,K-ATPase

Phosphorylation of purified rat kidney Na,K-ATPase (15) was carried out for 30 min at 22 °C in a reaction volume of 100 µl containing 50 mM Hepes, pH 7.5, 10 mM MgCl(2), 1.2 mM CaCl(2), and 2 µg of Na,K-ATPase. The final concentration of protein kinase C in the reaction mixture was 100 µg/ml. Reactions were initiated by the addition of 100 µM [-P]ATP (6 times 10^5 cpm/nmol), and terminated after 30 min by the addition of SDS-PAGE sample buffer. Samples were subjected to SDS-PAGE(14) .

Two-dimensional Phosphopeptide Mapping

Pieces containing P-labeled Na,K-ATPase alpha1 subunit purified from rat renal cortex or immunoprecipitated from choroid plexus were excised from dried gels, washed with two changes of 10% acetic acid/30% methanol, three changes of 50% methanol, and lyophilized. One ml of 50 mM NH(4)HCO(3), pH 8.0, containing trypsin (Calbiochem; 100 µg/ml), was added to the dried gel pieces, and the mixture was incubated at 37 °C for 20 h. The supernatants were removed, and the gel pieces were washed with an additional 0.5 ml of 50 mM NH(4)HCO(3). The pooled supernatants were lyophilized. Dried samples were resuspended in electrophoresis buffer (10% acetic acid, 1% pyridine, pH 3.5) and spotted 10 cm from the right and 4 cm from the bottom on thin-layer cellulose sheets (20 times 20 cm, Eastman Kodak). Phosphopeptides were separated by electrophoresis at 400 V for 60 min in the first dimension (left, positive), followed by chromatography in the second dimension in a buffer containing pyridine:1-butanol:water:acetic acid (10:15:12:3, v/v/v/v). Dried sheets were subjected to autoradiography.

Determination of the Activity of Na,K-ATPase

Male Sprague-Dawley rats (150-200 g) were anesthetized with Inactin-Byk (BykGulden, Konstanz, Germany), 80 mg/kg, intraperitoneally. After a midline incision, a catheter was inserted into the aorta and the rats perfused with a modified Hank's solution (137 mM NaCl, 5 mM KCl, 0.33 mM Na(2)HPO(4), 0.44 mM KH(2)PO(4), 1 mM CaCl(2), 0.8 mM MgSO(4), 1 mM MgCl(2), 10 mM Tris/HCl, 0.05% collagenase, and 0.1% BSA; pH 7.4). The choroid plexus was removed from the lateral ventricle and incubated at 35 °C for 10 min in 10 ml of perfusion solution containing 1 mM sodium butyrate and bubbled with oxygen. After rinsing, the tissue was transferred to a solution of the same composition, except that collagenase and BSA were omitted and CaCl(2) concentration was reduced to 0.25 mM. Segments of the middle part of the choroid plexus were dissected out and their length measured. In preliminary experiments, total as well as ouabain-sensitive ATPase activities were found to correlate linearly with the length of the segments. Choroid plexus segments were incubated for 30 min at 22 °C in the presence or absence of drugs, as described. Na,K-ATPase activity was determined as described previously(16) .


RESULTS AND DISCUSSION

P-Labeled choroid plexuses were incubated for 10 min at 30 °C in the presence of a direct activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu), or serotonin. An antibody specific for the alpha1 isoform of Na,K-ATPase (12) was used to immunoprecipitate Na,K-ATPase. Increases in the amount of P incorporated into a protein corresponding to the alpha subunit of Na,K-ATPase (molecular mass approx 95 kDa) were observed with both PDBu and serotonin as compared to control, unstimulated samples. The results of a typical experiments are shown in Fig. 1. In a series of five experiments, 5 µM PDBu caused a 16 ± 4-fold and 10 µM serotonin caused a 7 ± 2-fold (mean ± S.E.) increase in phosphorylation; 100 nM PDBu stimulated phosphorylation by about half of that observed with 5 µM PDBu (data not shown).


Figure 1: Effect of serotonin and PDBu on the phosphorylation of Na,K-ATPase in rat choroid plexus. Phosphorylation of Na,K-ATPase was measured as incorporation of radioactivity into the protein after stimulation of P-labeled choroid plexuses for 10 min at 30 °C in the presence of either 5 µM PDBu or 10 µM serotonin (5-HT). A, autoradiogram obtained after immunoprecipitation of Na,K-ATPase and separation by SDS-PAGE, showing the incorporation of P into Na,K-ATPase. B, quantitative analysis of the autoradiogram shown in A. The results are expressed as percentage of control.



The increase in phosphorylation of the 95-kDa protein induced by 10 µM serotonin was measured as a function of time. In each of three experiments, it reached a maximum at about 5 min of incubation. A representative experiment is shown in Fig. 2.


Figure 2: Effect of serotonin on Na,K-ATPase phosphorylation in choroid plexus as a function of incubation time. Phosphorylation of Na,K-ATPase was measured as incorporation of radioactivity into the protein after stimulation of P-labeled choroid plexuses with 10 µM serotonin for 2-60 min at 30 °C. The results are expressed as percentage of control.



Two-dimensional phosphopeptide mapping was employed to verify that the P-labeled protein immunoprecipitated from choroid plexuses was protein kinase C-phosphorylated Na,K-ATPase. Thus, tryptic cleavage of purified Na,K-ATPase (15) phosphorylated in vitro using [-P]ATP and protein kinase C, and of the P-labeled protein immunoprecipitated from choroid plexuses stimulated with either PDBu or serotonin, reproducibly generated two major radioactive peptides, which were found to comigrate when subjected to two-dimensional phosphopeptide mapping (Fig. 3).


Figure 3: Autoradiograms of two-dimensional phosphopeptide maps of Na,K-ATPase alpha subunit after phosphorylation in vitro by protein kinase C and of Na,K-ATPase alpha subunit immunoprecipitated from PDBu or serotonin-stimulated choroid plexus. Left panel: A, purified Na,K-ATPase phosphorylated with protein kinase C in the presence of 100 µM [-P]ATP; B, Na,K-ATPase immunoprecipitated from P-prelabeled choroid plexus incubated with PDBu; C, a combination of A plus B. Right panel: D, purified Na,K-ATPase phosphorylated with protein kinase C in the presence of 100 µM [-P]ATP; E, Na,K-ATPase immunoprecipitated from P-prelabeled choroid plexus incubated with serotonin; F, a combination of D plus E. A, B, and C were obtained in a different experiment from D, E, and F.



A variety of evidence indicates that modulation of the activity of Na,K-ATPase can be achieved by controlling its state of phosphorylation(11, 17, 18) . Experiments in vitro have shown that protein kinase C (11, 19, 20) and cAMP-dependent protein kinase (11, 18, 20) are able to phosphorylate the catalytic alpha subunit of Na,K-ATPase, thereby inhibiting its activity(11) . Recently, Middleton et al. obtained evidence for protein kinase C-mediated regulation of Na,K-ATPase in an opossum kidney cell line (17) .

We examined the possibility that the treatments which increased the phosphorylation of Na,K-ATPase in the choroid plexus (i.e. incubation with PDBu or serotonin) were also able to affect the activity of this enzyme in intact cells. Microdissected segments of choroid plexus were used to determine the activity of Na,K-ATPase. Incubation of choroid plexus for 10 min at 30 °C in the presence of 5 µM PDBu resulted in a 28 ± 4% decrease in Na,K-ATPase activity (Table 1). Treatment of tissue in the presence of 10 µM serotonin reduced the activity of Na,K-ATPase by 13 ± 2% (Table 1). These changes are most likely of physiological relevance, since variations in CSF volume of more than 10% would result in symptomatic alterations of intracranial pressure.



PDBu had a stronger effect than serotonin in both modulating activity and increasing phosphorylation of Na,K-ATPase. The inhibition exerted by serotonin on Na,K-ATPase activity was similar to the reduction in CSF production of about 15%, ascribable to stimulation of 5-HT receptors(5) . Previous data showed that stoichiometric phosphorylation of purified Na,K-ATPase by protein kinase C resulted in a 50% inhibition of the activity of the ion pump(11) . Assuming the same relationship between phosphorylation of the pump and inhibition of activity in intact cells, we estimate the proportion of Na,K-ATPase phosphorylated following incubation of choroid plexus with PDBu or serotonin to be 56% and 26%, respectively.

In this study, evidence has been presented for regulation of the state of phosphorylation and activity of Na,K-ATPase by the first messenger serotonin in an intact tissue, i.e. the choroid plexus. Since the activity of Na,K-ATPase appears to represent the driving force for the secretion of CSF(3, 4) , the present results provide a model to explain the decrease in CSF production observed after systemic administration of serotonin (5) or of its precursor(6) . According to this model (Fig. 4), serotonin by binding to the 5-HT receptor located on choroid epithelial cells (7, 8) activates phospholipase C(9, 10) , leading to the formation of diacylglycerol and activation of protein kinase C. This results in the phosphorylation of Na,K-ATPase and the associated inhibition of its activity. Inhibition of the activity of Na,K-ATPase causes a decreased transport of Na across the choroidal cell, with a consequent reduction of the movement of chloride and bicarbonate ions into the ventricle. This results, in turn, in a decrease in the osmotic gradient responsible for the diffusion of water into the ventricular cavity and therefore a decrease in the production of CSF.


Figure 4: A schematic diagram of the proposed mechanism by which serotonin inhibits the secretion of CSF from choroid epithelial cells. For details see ``Results and Discussion.'' DAG, diacylglycerol; PKC, protein kinase C. The precise localization of 5-HT receptors on choroid epithelial cells has not yet been determined. In this figure, the 5-HT receptor has been arbitrarily assigned to the basolateral membrane.



Numerous hormones and neurotransmitters are known to regulate the secretion of CSF from the choroid plexus(21, 22, 23) . It is conceivable that their effects are mediated, at least in part, via modulation of Na,K-ATPase activity(3, 4) . It has been proposed that the choroid plexus receives a tonic serotonergic input from the raphe nuclei(24, 25, 26) . If so, the effects of other first messengers on CSF production would be superimposed on a background of serotonin-activated 5-HT receptors. Further studies should elucidate the relative importance and mechanism of action of the various first messengers that regulate the production of CSF.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant MH-40899. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of Fellowship B-PD 06633-301 from the Swedish Natural Science Research Council. To whom correspondence should be addressed: Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8787; Fax: 212-327-7888.

(^1)
The abbreviations used are: CSF, cerebrospinal fluid; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate.


ACKNOWLEDGEMENTS

We thank Dr. Angus C. Nairn for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.