CONCOMITANT PROTEIN-PHOSPHORYLATION AND ENDOGENOUS ACETYLCHOLINE-RELEASE INDUCED BY NICOTINE - DEPENDENCY ON NEURONAL NICOTINIC RECEPTORS AND DESENSITIZATION

1. Nicotine stimulated two Ca2+-dependent processes in rat frontal cor tex synaptosomes: the phosphorylation of an 88-kDa protein band and th e release of endogenous ACh.(3) Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptasomal plasma m embrane induced by the drug. Changes in the state of phosphorylation o f the 80-kDa band (presumed to contain synapsin I) were correlated wit h changes in the release of ACh as follows, from 2 to 4. 2. Blockade o f predominant, nerve terminal P-type Ca2+ channels with omega-agatoxin -IVA, did not prevent nicotine from stimulating ACh release. In contra st, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylat ion induced by either nicotine or veratridine. Taken together, these d ata suggest that, upon nicotine stimulation, Ca2+ enters nerve termina ls through two distinct pathways. The first, via Ca2+ channels, is nec essary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotin e-induced phosphorylation and release, is the neuronal nAChR itself. 3 . Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosp horylation. This shows that diminished release is associated to decrea sed phosphorylation of the 80-kDa protein band, most likely as a conse quence of nicotine-promoted nAChR desensitization. 4. Augmented ACh re lease and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in othe r neurons, increases intracellular Ca2+ (Cholewinski et al., 1993), th us promoting desensitization of ACh release.