Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

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Abstract

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.

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    The cells were treated with 80 μM of forskolin or 100 μM of SKF38393, and changes in tau phosphorylation state were studied by Western blotting, using antibodies directed against tau phosphorylated at serine 214, a site known to be phosphorylated via PKA (Liu et al., 2004). These time and doses were chosen based on previous published studies in which activation of PKA by forskolin or SKF38393 were performed (Sidhu and Fishman, 1990; Brami-Cherrier et al., 2002; Liu et al., 2004; Zhang et al., 2006). Note that a mouse monoclonal antibody against the phosphorylated and non-phosphorylated form of (total) tau (tau 5) was also used.

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Present address: Department of Pediatrics, Georgetown University Medical Center, 3700 Reservoir Road, Washington, D.C. 20007.

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