Comparison of the Forms of the Dopamine D 2 Receptor Expressed in GH 4 C 1 Cells

Abstract

Abstract

The effects of dopamine (DA) on prolactin (PRL) secretion, phosphoinositide metabolism, cytosolic calcium concentrations ([Ca²⁺]₁), and cAMP production in GH₄C₁ cells expressed either the short (GH₄ZR₇ cells) or long (GH₄I₁₂ cells) form of the rat DA D₂ receptor were compared in this study. The GH₄C₁ cell line is derived from the rat anterior pituitary gland and lacks functional DA receptors. The GH₄ZR₇ and GH₄I₁₂ cell lines have been transfected with either the short or the long form of the D₂ receptor, respectively. In this study, the functional coupling of these receptors to both basal and stimulated PRL secretion and to common second messenger systems was examined. Both cell types expressing DA receptors exhibited similar saturable binding to the D₂ antagonist [³H]spiperone (K_d GH₄ZR₇ = 96 ± 8 pM, K_d GH₄I₁₂ = 107 ± 49 pM). In GH₄ZR₇ cells, 1 and 10 μM DA inhibited basal PRL secretion by 37% and 58%, respectively. In GH₄I₁₂ cells, 1 and 10 μM DA inhibited basal PRL secretion by 63% and 54%, respectively. In GH₄ZR₇ cells, 10 μM DA completely reversed the stimulatory effects of 1-100 nM thyrotropin-releasing hormone (TRH), and 1 μM DA attenuated the stimulatory effects of 10 and 100 nM TRH. Interestingly, GH₄I₁₂ cells were not responsive to TRH. In both cell lines, the inhibitory effects of DA were blocked by the specific D₂ antagonist, eticlopride. The stimulatory effects of TRH on [Ca²⁺], were dose dependent and could be blocked (at least in GH₄ZR₇ cells) by prior treatment of the cells with 1 μM DA. The ability of dopamine to block the TRH-mediated increase in [Ca²⁺], was attenuated by eticlopride. DA (1 μM) had no effect on resting [Ca²⁺], in either cell line expressing DA receptor. TRH (100 nM) maximally stimulated total inositol phosphate (IP) accumulation to values approximately three times greater than controls in GH₄C₁ and GH₄ZR₇ cells only. DA had no effect on basal or TRH-stimulated IP accumulation in any of the cell lines. DA (1 and 10 μM) inhibited cAMP production by 40% and 39%, respectively, in GH₄ZR₇ cells. Similarly, in GH₄I₁₂ cells, DA (1 and 10 μM) inhibited cAMP production by 48% and 60%, respectively. These data indicate that both the long and short forms of the D₂ receptors play similar roles when expressed in GH₄C₁ cells. However, maximal inhibition of PRL in these cell lines is approximately 20% less than that of normal lactotrophs.