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Expression of Functional D2 Dopamine Receptors Following Differentiation of Y‐79 Human Retinoblastoma Cells

Y‐79 human retinoblastoma cells grown in serumfree medium in monolayer culture have previously been shown to undergo differentiation in response to dibutyryl cyclic AMP (Bt2cAMP). We report here that Y‐79 cells treated in this manner also express very high levels of functional D2 dopamine receptors. In control Y‐79 cells, cultured in suspension, D2 dopamine receptors, quantified via saturation analysis with the D2 antagonist [3H]methylspiperone, are expressed at a level of ∼3 fmol/106 cells (∼1,800 receptor sites/cell). Differentiation is initiated by attachment of the cells to the culture dish with poly‐D‐lysine and fibronectin and continued culture in serum‐free medium. After 8 days in serumfree culture, differentiation is further induced with continuous Bt2cAMP treatment. Using this differentiation protocol, D2 receptor levels increase up to a maximum of 30 fmol/106 cells (18,000 receptors/cell) on day 20, the limit of culture viability. Cultures of 15–17 days (7–9 days of Bt2cAMP treatment) expressing receptor levels of 15–20 fmol/106 cells are used for pharmacological and functional characterization of D2 dopamine receptors. The pharmacology of competition for [3H]methylspiperone binding to differentiated Y‐79 (dY‐79) cell membranes by a series of dopaminergic antagonists verifies the D2 receptor nature of this site, exhibiting appropriate affinities and the following rank order of potency: YM‐09151‐2 ∼ spiperone > domperidone ∼ (+)‐butaclamol ∼ fluphenazine > chlorpromazine > (–)‐sulpiride > (+)‐sulpiride > promethazine > (+)‐SCH 23390 ≫ (–)‐butaclamol. Inhibition of [3H]methylspiperone binding by dopaminergic agonists in dY‐79 cell membranes indicates the presence of multiple agonist affinity states, which can be converted to a homogeneous low‐affinity state by addition of guanine nucleotides. In addition, incubation with dopamine or other D2‐selective agonists results in an ∼50% reduction in forskolin‐stimulated cyclic AMP production in lysed dY‐79 cells, which can be blocked by D2‐selective antagonists. These data indicate that the D2 receptors expressed in these cells are indeed functional. The ability to induce high levels of expression of functional D2 dopamine receptors in a cultured human cell line should provide an excellent model system with which to examine the molecular events involved in the expression and regulation of this receptor system.