Invited Symposium: Molecular and Cellular Analysis of Dopamine and Serotonin Transporters



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Role of the 5-HT Transporter in the Drug-Induced 'Carrier-Dependent' Release

Contact Person: Tiziana Mennini (tiziana@irfmn.mnegri.it)

Materials and Methods

Male CRL:CD(SD)BR rats were used. Crude synaptosomal pellets (P2), prepared from brain cortices, were preincubated for 15 min at 37 C with 60 nM [3H]5-HT in Krebs-Henseleit buffer with the following composition (mM): NaCl (125); KCl (3); CaCl2 (1.2); MgSO4 (1.2); NaH2PO4 (1); NaHCO3 (22); glucose (10), gassed with 95% O2 and 5% CO2, pH 7.2-7.4. Synaptosomes were then layered onto 0.65 microm filters in a 16-chamber superfusion apparatus held thermostatically at 37 C.
Superfusion was started (t=0 min) at a rate of 0.5 ml/min with standard medium; after 42 min equilibration period, fractions were collected every 2 min until t=60 min.
The filters were put into scintillation vials and counted for radioactivity, as the fractions.
The releasing agents were present in the superfusion medium for 3 min from t=47 to t=50 min.
When used, 1 microM citalopram was present from t=40 to t=60 min. In some experiments, synaptosomes were superfused from t=40 to t=60 min with a calcium-free medium containing 3 mM EGTA. When used, 250 microM pargyline was present from the beginning of superfusion (t=0).
The fractional release rate (FRR) was calculated as 100 times the amount of radioactivity released into each 2-min fraction over the total radioactivity present on the filter at the start of that fraction.
The overflow (%) was calculated as the difference between the FRR in the presence (t=48-56) and absence of the drug (mean t=44-48 and t=56-60) (see fig. 2). The content of unmetabolized [3H]5-HT in the superfusate was determined after organic extraction of [3H]5-HIAA, as previously described (Gobbi et al., 1992).

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Gobbi, M; Dalla Valle, F; Ciapparelli, C; Mennini, T; (1998). Role of the 5-HT Transporter in the Drug-Induced 'Carrier-Dependent' Release. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/simantov/gobbi0727/index.html
© 1998 Author(s) Hold Copyright