Antidepressant drugs affect dopamine uptake

The catecholamine hypothesis of depression postulates a deficiency of norepinephrine at functional receptor sites. The evidential basis derives from studies on the turnover and uptake of norepinephrine and the blockade of reuptake by tricyclic antidepressants El 3]. Serotonin systems may also be involved in antidepressant drug action [4, 5]. Relatively little attention has been paid to the possible role of dopamine in the pharmacotherapy of depression. Although Horn et al. [6] included some tricyclic antidepressants in their study on regional catecholamine uptake by rat brain synaptosomes, it is not known whether inhibition of dopamine uptake is a common property of antidepressants currently in clinical use. Our laboratory has been investigating the mode of action of antidepressant compounds. We have reported time-dependent changes in serotonin metabolism after chlorimipramine: rat brain serotonin increased 30 min after an i.p. injection of the drug and this effect was interpreted as reflecting re-uptake blockade [7]. Since brain dopamine levels are also increased by chlorimipramine (unpublished observation), we investigated the possible effect of this and other compounds from the family of antidepressants on dopamine uptake. We report here that a number of widely used antidepressants inhibit dopamine uptake by rat brain nuclei-free homogenates. For comparative purposes, four stimulants were included in the study. Male Sprague Dawley rats (150-200g, Madison, Wis.) were killed by decapitation and their brains immediately removed. Brains were homogenized (12 strokes, I min) in 10 vol. ice-cold 0.32 M sucrose in Tri-R glass homogenizers with Teflon pestles (clearance 0.009 to 0-011 in.), and centrifuged at 1000g for 10 min in a Sorvall RC 2-B centrifuge. The supernatant nuclei-free suspension was separated from the pellet, gently stirred to make a uniform suspension and reserved for uptake and protein assays. We used the method of Snyder and Coyle I-8] with minor modifications. The incubation mixture was agitated at 37 ° in 4-0ml Kreb~Henselei t bicarbonate buffer (pH 7.4) in 15-ml Corex tubes. The drugs tested (all at 10 ~ M) were added to the incubation mixture prior to the addition of tritiated dopamine (sp. act. 9-8 Ci/m-mole, New England Nuclear Corp.); preincubation was omitted. At the end of the 8-min incubation period, the samples were placed in ice-water for 2 min. The tubes were centrifuged at 60409 for 20 min in a Sorvall RC 2-B centrifuge to form the tissue pellet. The incubation mixture was aspirated and the pellet rinsed once with 4-0 ml ice-cold saline. Solubilization of the pel-. lets was performed by incubation with 1 ml Soluene-350 (Packard) at 50 ° for 30 min. The solutions were transferred to glass counting vials, 10 ml toluene fluor was added, and radioactivity was measured in an Isocap/300 scintillation counter (Searle Radiographics, Inc.). The protein content of 0.2ml aliquots of the synaptosomal suspensions was determined by the method of Lowry et al. [9]. The aim in this pilot study was to detect whether inhibition of dopamine uptake is a common property of a number of antidepressants. Preliminary data expressing the variation of uptake of aH-dopamine with increasing substrate concentrations (0-01 to 1 pM) when graphed revealed two consecutive saturable curves. These data were resolved into two straight lines in Lineweaver Burk plots, suggesting the existence of two components responsible for the accumulation of 3H-dopamine into synaptosomal suspensions from whole brain. One component operated at low substrate concentrations with a Km at 1 x 10 v M and another component at high concentrations of 3H-dopamine with a K,, at 3.3 × 10 7 M (Fig. 1). Snyder and Coyle E8] have previously reported two affinity systems for dopamine uptake by homogenates from all brain regions except for the striatum which possesses only one uptake system. The value reported by these authors for high affinity uptake was 0-8 x 10 -v M; the corresponding value for low affinity uptake was 1.4 × 10 ~' M. The different value for low affinity uptake is probably due to the