NA-ATPASE PHOSPHORYLATION IN THE CHOROID-PLEXUS - SYNERGISTIC REGULATION BY SEROTONIN(,K+)PROTEIN KINASE-C AND ISOPROTERENOL/CAMP-PK/PP-1 PATHWAYS/

Background: The ion pump Na+,K+-ATPase is responsible for the secretio n 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 high ly enriched In two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation cata lyzed by cAMP-dependent protein kinase on a single threonyl residue co nverts DARPP-32 and inhibitor-1 into potent inhibitors of protein phos phatase-1. Previous work has shown that in the choroid plexus, phospho rylation of DARPP-32 and I-1 is enhanced by isoproterenol and other ag ents that activate cAMP-PK. We have now examined the possible involvem ent of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K+-ATPase. Materials and Methods: The state of phosphorylation of Na+,K+-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from P-32 -prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the p rotein kinase involved in the phosphorylation of Na+,K+-ATPase. Result s: The serotonin-mediated increase in Na+,K+- ATPase phosphorylation i s potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isopr oterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at t he level of a protein kinase C phosphorylation site. Forskolin and iso proterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. Conclusions: The results present ed here support a model in which okadaic acid, forskolin, and isoprote renol achieve their synergistic effects with serotonin through phospho rylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatas e-1, and a reduction of dephosphorylation of Na+,K+-ATPase at a protei n kinase C phosphorylation site.