(Received for publication, October 5, 1994; and in revised form, November 15, 1994)
From the
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.
Precise control of cerebrospinal fluid (CSF) ()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.
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
1 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
subunit of
Na
,K
-ATPase (molecular mass
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
subunit after phosphorylation in vitro by protein kinase
C and of Na
,K
-ATPase
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
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.