Choroid Plexus: Synergistic Regulation by Serotonin/Protein Kinase C and Isoproterenol/cAMP-PK/PP-1 Pathways

Background: The ion pump Na+,K+-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K+ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-I into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-I pathway in the regulation of Na+,K+-ATPase. Materials and Methods: The state of phosphorylation of Na+,K+-ATPase was measured by deternining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K+-ATPase. Results: The serotonin-mediated increase in Na+,K+ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the ,B-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Twodimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-i, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. Conclusions: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-i, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K+-ATPase at a protein kinase C phosphorylation site. Introduction The cerebrospinal fluid (CSF) provides the brain with a chemically stable environment necessary Address correspondence and reprint requests to: Dr. Gilberto Fisone, Dept. of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden. Phone: +46-8-728 73 75; Fax: +46-8-34 95 44 for normal neuronal function. Secretion of CSF occurs primarily at the level of the choroid plexus and is dependent on the activity of the ion pump Na+,K+-ATPase. Na+,K+-ATPase catalyzes the transport of Na+ across the choroidal cell with a consequent movement of chloride and bicarbonate ions into the ventricle. This in G. Fisone et al.: Na+,K+ -ATPase Phosphorylation in Choroid Plexus 259 turn results in the creation of an osmotic gradient responsible for the diffusion of water into the ventricular cavity and the production of CSF (1,2). Although several hormones and neurotransmitters have been implicated in the regulation of the volume and composition of CSF (3-5), little is known about the mechanisms by which such regulation occurs. It has been shown that stoichiometric phosphorylation of Na+,K+ -ATPase by protein kinase C results in a 50% inhibition of activity (6). More recently, it was shown that in the choroid plexus, phorbol 12, 13-dibutyrate inhibited the activity of Na+,K+ -ATPase by about 30% and serotonin inhibited the activity by about 15 %. It was calcu1ated that these inhibitions were associated with a stoichiometry of phosphorylation of 0.6 and 0.3, respectively (7). These effects appeared to be mediated via activation of protein kinase C (7) and could be responsible for the decrease in CSF secretion observed after systemic administration of serotonin (5) or of its precursor, 5-hydroxytryptophan (8). Previous studies performed using renal preparations indicate that protein phosphatase-l (PP-l) is also involved in the regulation of Na+,K+ -ATPase activity (9). The choroid plexus is highly enriched in two phosphoproteins which act as regulators of PP-l activity, a dopamineand cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and protein phosphatase inhibitor-I (I-I) (10). Phosphorylation catalyzed by cAMPdependent protein kinase (cAMP-PK) on a single threonyl residue converts DARPP-32 and I-I into potent inhibitors of PP-I (11). Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-I is enhanced by treatments that activate cAMP-PK (12). In the present study, we have examined the possible involvement of the cAMP-PK/PP-l cascade in the control of the state of phosphorylation of Na+,K+ -ATPase in the rat choroid plexus. Materials and Methods 32P-Labeling and Pharmacological Treatment of Choroid Plexus Male Sprague-Dawley rats (200-250 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 NaHC03, 1.5 mM CaCI2, 1.5 mM MgS04, 0.25 mM KH2P04t 10 mM glucose) equilibrated with 95% O2/5% CO2 (v/v). After 15 min, medium was replaced with the same amount of fresh buffer containing 2.5 mCi of 32P_or_ thophosphoric acid (DuPont NEN; specific activity 8500-9120 Ci/mmol) and the tissue was incubated for 60 min. The radioactive buffer was then removed, and 32P-labeled choroid plexuses were washed twice with 2 ml of fresh buffer and incubated for an additional 10 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. Assay of Na+,K+ -ATPase Phosphorylation in