Interaction of D₃ preferring agonist (-)-N⁶-(2-(4-(biphenyl-4-yl)piperazin-1-yl)ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (D-264) with cloned human D₂L, D₂S, and D₃ receptors: potent stimulation of mitogen-activated protein kinases and G protein-coupled inward rectifier potassium channels

Abstract

This study aims to determine the effect of the novel D(3) dopamine receptor agonist, D-264, on activation of D(3) and D(2) dopamine receptor signal transduction pathways and cell proliferation. AtT-20 neuroendocrine cells stably expressing human D(2S), D(2L), and D(3) dopamine receptors were treated with D-264 and the coupling of the receptors to mitogen-activated protein kinase (MAPK) and G protein-coupled inward rectifier potassium (GIRK) channels was determined using Western blotting and whole-cell voltage clamp recording, respectively. D-264 potently activated MAPK signaling pathway coupled to D(2S), D(2L), and D(3) dopamine receptors. The activation of MAPK was more pronounced than the reference agonist quinpirole and was longer lasting. D-264 also activated GIRK channels coupled to D(2S), D(2L), and D(3) receptors. In addition, D-264 dose-dependently induced cell proliferation in AtT-D(2L) and AtT-D(3) cells. These results indicate that D-264 robustly activates GIRK channels and MAPK coupled to D(2) and D(3) dopamine receptors in AtT-20 cells. D-264 is also a potent inducer of cell proliferation.

Figure 1

D-264 activates D₂/D₃-MAPK signaling pathway. (A) Representative Western blots showing 100 nM D-264-induced phosphorylation of p44/p42 MAPK proteins in AtT20 cells stably expressing human D_2S, D_2L or D₃ dopamine receptors. Cells were treated with vehicle (−) or 100 nM D-264 (+) for 5 minutes. (B) Cumulative data showing the relative increase of phospho MAPK in D_2S, D_2L and D₃ expressing AtT-20 cells following a 5 minute, 100 nM D-264 treatment. The levels of D-264-induced phosphorylated MAPK proteins were normalized to the levels of total MAPK proteins and were compared to vehicle treated cells (which was set to 1 for each cell line). *, P<0.05, statistically significant, two-tailed Student's t-test. Statistical comparison was between MAPK activation with and without agonist treatment in each cell line separately. The experiments were repeated three independent times.

Figure 2

D-264 induces persistent activation of the D₂/D₃-MAPK signaling pathway. Representative Western blots showing 100 nM D-264 (left panel) and 200 nM quinpirole (QP) (right panel) induced phosphorylation of p44/p42 MAPK proteins in AtT20 cells stably expressing human D_2S, D_2L or D₃ dopamine receptors. Cells were pretreated for 1 minute with control SES (No pretreatment) and either 100 nM D-264 or 200 nM quinpirole (Yes pretreatment), washed for 30 minutes and subsequently treated with control SES (−) and 100 nM D-264 or 200 nM quinpirole (+) for 5 minutes.

Figure 3

D-264, but not quinpirole, induces persistent activation of the D₂/D₃-MAPK signaling pathway. Cumulative data showing the ratio of phosphorylated to total MAPK induced by 100 nM D-264 or 200 nM quinpirole (QP) without (−) and with (+) pretreatment in AtT20 cells stably expressing human D_2S, D_2L or D₃ dopamine receptors. Cells were pretreated for 1 minute with control SES (No pretreatment)and either 100 nM D-264 or 200 nM quinpirole (Yes pretreatment), washed for 30 minutes and subsequently treated with control (−) and 100 nM D-264 or 200 nM quinpirole (+) for 5 minutes. The levels of D-264-induced P-p44/p42 MAPK proteins were normalized to the levels of total-p44/p42 MAPK proteins. The experiments were repeated three independent times. *, #, ψ, P<0.05, statistically significant, ANOVA, post-hoc Holms test. *, comparing control (−/−) to samples that received 2^nd treatment only (−/+). #, comparing control (−/−) to samples that were only pretreated (+/−). ψ, comparing samples that received pretreatment only (+/−) to those that received both treatments (+/+).

Figure 4

D-264 activates D₂/D₃-GIRK channel signaling pathway. Representative voltage clamp recordings from parental AtT20 cells (A) or AtT-20 cells stably expressing the human D_2S (B), D_2L (C and D) or D₃ (E and F) dopamine receptors. All drugs were applied in 30 mM K⁺ external solution (open rectangle). Parental AtT-20 cells do not respond to 300 nM D-264 (black rectangle) or 300 nM quinpirole (cross hatch). D-264 only activates the GIRK currents in AtT-20 clones expressing the various D₂-like dopamine receptor subtypes (B–F). Current traces were obtained in whole cell voltage clamp recording mode with the cells held at −65 mV. The cells were sequentially treated with SES (5 mM K⁺), 30 mM K⁺ external solution (open rectangle) and various doses of agonists in 30 mM K⁺ external solution. The time and duration of agonist application (typically- 60s) are indicated by the rectangles. Following each agonist application, the cells were treated with the 30 mM K⁺ external solution during washout (open rectangle). To test the integrity of the seal during the washout in 30 mM K⁺ external solution, following the D-264 treatment, the external solution was switched to SES (5 mM K⁺); the reduction in external K⁺ causes a rapid decrease in the inward current (for example, see panel D), suggesting that slow deactivation of GIRK currents induced by D-264 was not due to a loss of seal integrity.

Figure 5

Dose-dependent activation of D₂/D₃ induced GIRK response by D-264. Cumulative data showing GIRK responses induced by 10, 100 and 300 nM D-264 in AtT-20 cells stably expressing the human D_2S (A), D_2L (B) or D₃ (C) dopamine receptors. Current traces were obtained in whole cell voltage clamp recording mode with the cells held at −65 mV. The peak current value (pA) at −65 mV for each drug concentration was normalized to whole cell membrane capacitance (pF) to normalize differences due to cell size. The dashed line in each graph represents the mean GIRK response (in pA/pF) induced by 300 nM quinpirole in each cell line. In each cell line, the magnitude of GIRK response elicited by the three different doses of D-264 are significantly different (P<0.05, ANOVA, post-hoc SNK test).

Figure 6

Dose dependent effect of D-264 on proliferation of AtT-D_2L and AtT-D₃ cells. Cell viability was measured by MTT assay. Cell viability increased dose dependently with peak cell viability was observed at 1 nM for D_2L and 0.001 nM. Reference quinpirole exhibited the highest effect for D₃ receptor at 0.0001 nM. Bars represent means ± S.E.M. of cell viability values against control expressed as percentages of control for n = 3–4. ANOVA with Tukey's multiple comparison test was used to detect the differences among the treatments and control *P < 0.001.