CYCLIC AMP-MEDIATED ENHANCEMENT OF HIGH-AFFINITY CHOLINE TRANSPORT AND ACETYLCHOLINE SYNTHESIS IN BRAIN

Intracerebroventricular administration of N-6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cyclic AMP) to mice increased high-aff inity choline transport (HAChT) into synaptosomal preparations from th e hippocampus, striatum, and frontal cortex in a time-, dose-, and bra in region-dependent manner. Similar observations were made when the cy clic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator f orskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanth ine were administered. Inhibition of phosphatase 1 and 2A, with okadai c acid, increased basal choline transport and enhanced the response to db-cyclic AMP, The early increase of HAChT activity induced by db-cyc lic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent V-m ax without a change of the K-m for choline. Hemicholinium-3 (HC-3) bin ding was not altered when studied 1 h after db-cyclic AMP administrati on. In contrast, HC-3 binding and HAChT activity were both elevated wh en estimated 3 h after the treatment, and pretreatment with cyclohexim ide partially prevented the db-cyclic AMP-induced HAChT rise, As evide nce that enhanced HAChT is associated with a direct action of cyclic A MP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an incr ease of HAChT that was prevented by H-89. Choline acetyltransferase ac tivity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP additi on, Our studies show that cyclic AMP-mimetic compounds appear to modul ate the choline carrier by a dual mode: an early increase of the maxim al velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 bin ding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.