Regulation of stimulus-dependent hippocampal acetylcholine release by okadaic acid-sensitive phosphoprotein phosphatases

Isolated nerve endings (synaptosomes) from rat hippocampas were used to characterize the influence by serine/threonine-specific phosphoprotein phosphatase (PP) inhibitors on acetylcholine release. Brief exposure to low concentrations of selective PP inhibitors (okadaic acid and calyculin A) caused a concentration-dependent attenuation of stimulus-dependent (calcium-evoked or potassium-evoked) [ 3H]acetylcholine ([ 3H]ACh) release, while having no effect on the rate of basal transmitter efflux. In view of the observed potencies for okadaic acid and calyculin A (pseudo-IC 50) values near 3 nM), these data indicate that Type 1 (PP1) or Type 2A (MA) enzymes play a permissive role in exocytotic [ 3H]ACh release. In contrast, the absence of any measurable effect by sodium ortho-vanadate argues against a similar influence by tyrosine-specific phosphoprotein phosphatases. While the neuronal substrate(s) responsible for PP regulation of [ 3H]ACh release are unknown, the underlying mechanism clearly differs from that through which muscarinic autoreceptors act since inhibition by okadaic acid and oxotremorine (an autoreceptor agonist) are additive and the former is not blocked by the muscarinic receptor antagonist atropine. Based upon these results, we conclude that dephosphorylation steps catalyzed by okadaic acid-sensitive PP represent an important regulatory mechanism for stimulus-dependent transmitter release in septo-hippocampal cholinergic neurons.