Tolcapone

PC12 cells were from the American Type Culture Collection (ATCC, Manassas, VA; PC12 cells catalog no. CRL-1721); F12K cell culture medium from Gibco Life Technologies (Grand Islands, NY); tolcapone (to block catechol-O-methyltransferase) from Orion Pharma (Espoo, Finland); DOPAL standard from Santa Cruz Biotechnology, Inc. (Dallas, TX); and Cys-DA standard from the NIMH Chemical Synthesis and Drug Supply Program (No. C-805).
Non-adherent, non-differentiated cells PC12 cells were kept frozen in liquid nitrogen until passaged for experiments. The cells were grown in F12K medium with 15% horse serum and 2.5% fetal bovine serum and incubated at 37 °C in an atmosphere of 5% carbon dioxide. The medium was replaced several times per week and cells passaged once per week.
At 24 hours prior to plating for experiments, the cells were centrifuged and the medium was replaced with medium containing 10 mM tolcapone. After the 24 hours, the cells were collected, suspended in the same medium, and counted (Cellometer, Nexcelom Bioscience, Lawrence, MA). About 500,000 cells/well were plated in 12-well plates. The experiments began after 24 hours of incubation in tolcapone-containing medium.

Human recombinant AS was purchased from Calbiochem (La Jolla, CA, USA), mutant A53T-AS from rPeptide (Bogart, GA, USA). DA, L-DOPA, Dithiothreitol, benomyl, and Dulbecco’s Modified Eagle’s medium (DMEM) were from Sigma (St. Louis, MO, USA). DOPAL was from Santa Cruz Biotech (Dallas, TX, USA). All these reagents were dissolved in Type 1 water. Mouse anti-AS antibody and cell culture medium were from Invitrogen (Camarillo, CA, USA). Human glioblastoma (U118MG (ATTC^R HTB-15^Trade;), non-adherent PC12 cells, and F-12K medium were from ATTC (Manassas, VA, USA) and human oligodendrocytes (MO3.13) from Cellutions Biosystems Ins (Burlington, Ontario, Canada). The aldehyde reductase inhibitor AL-1756 was a gift from Alcon Laboratories, Fort Worth, TX. Tolcapone was from Orion Pharma (Espoo, Finland). benomyl, AL-1576, and Tolcapone were dissolved in DMSO and stored at −20 °C. Co-incubation of PC12 cells with glial cells was done using ThinCert membranes from Greiner Bio-One North America Inc. (Monroe, NC, USA). For Western blotting NuPage LDS Sample Buffer (4X) (Invitrogen, ThermoFisher Scientific, Waltham, MA) containing sodium dodecyl sulfate (SDS) was used.

Rat pheochromocytoma PC12 cells and human glioblastoma cells were from the American Type Culture Collection (ATCC, Manassas, VA; PC12 cells catalog no. CRL-1721, glioblastoma cells catalog no. HTB-15). The cell culture media, F12K and DMEM, were from Gibco Life Technologies (Grand Islands, NY); tolcapone (to block catechol-O-methyltransferase) from Orion Pharma (Espoo, Finland); and dopamine, norepinephrine, DOPAC, DOPET, and DHPG from Sigma. 6F-Dopamine was obtained in solution (about 1.35 mM) from the NIH PET Department after having passed quality control for i.v. administration to humans. DOPAL standard was synthesized in the laboratory of and provided by Dr. Kenneth L. Kirk (NIDDK).