[3H]Dopamine Depletion from Osmotically Defined Storage Sites: Effects of Reserpine, 53 mMKC1, andd-Amphetamine

Abstract

A crude synaptosome-containing fraction (P2'') prepared from rat striatal slices incubated with [3H]dopamine was exposed to hypoosmotic conditions and rapidly subjected to Millipore filtration. P2''-associated [3H]dopamine trapped on the filters was defined as hypoosmotic resistant, whereas P2''-associated [3H]dopamine that washed through the filters was defined as hypoosmotic sensitive. EM examination of sections prepared from a P2'' pellet that was exposed to hypoosmotic conditions revealed extensive synaptosomal lysis. [3H]Dopamine accumulation and retention by the hypoosmotic-resistant fraction were reduced by reserpine. The proportional distribution of [3H]dopamine between hypoosmotic-resistant and -sensitive fractions was measured following in vitro exposure of the preloaded P2'' fraction to reserpine, 53 mM KCl, and d-amphetamine. Each of these treatments resulted in a time-dependent loss of [3H]dopamine from the loaded P2'' fraction without eliciting an alteration in the proportional distribution of [3H]dopamine between hypoosmotic-resistant and -sensitive fractions. Release induced by reserpine and d-amphetamine was independent of extrasynaptosomal Ca2+, whereas 53 mM KCl-induced release was dependent on exotrasynaptosomal Ca2+. Dopamine may be rapidly equilibrated between osmotically defined storage compartments, and thus specific compartmental depletion of loaded [3H]dopamine cannot be identified on the basis of osmotic lability.