Antidepressants Differentially Regulate Intracellular Signaling from α1-Adrenergic Receptor Subtypes In Vitro

Abstract

Currently utilized antidepressants have limited effectiveness and frequently incur undesired effects. Most antidepressants are thought to act via the inhibition of monoamine reuptake; however, direct binding to monoaminergic receptors has been proposed to contribute to both their clinical effectiveness and their side effects, or lack thereof. Among the target receptors of antidepressants, α1‑adrenergic receptors (ARs) have been implicated in depression etiology, antidepressant action, and side effects. However, differences in the direct effects of antidepressants on signaling from the three subtypes of α1-ARs, namely, α1A-, α1B- and α1D‑ARs, have been little explored. We utilized cell lines overexpressing α1A-, α1B- or α1D-ARs to investigate the effects of the antidepressants imipramine (IMI), desipramine (DMI), mianserin (MIA), reboxetine (REB), citalopram (CIT) and fluoxetine (FLU) on noradrenaline-induced second messenger generation by those receptors. We found similar orders of inhibition at α1A-AR (IMI < DMI < CIT < MIA < REB) and α1D‑AR (IMI = DMI < CIT < MIA), while the α1B-AR subtype was the least engaged subtype and was inhibited with low potency by three drugs (MIA < IMI = DMI). In contrast to their direct antagonistic effects, prolonged incubation with IMI and DMI increased the maximal response of the α1B-AR subtype, and the CIT of both the α1A- and the α1B-ARs. Our data demonstrate a complex, subtype-specific modulation of α1-ARs by antidepressants of different groups.

Keywords: G-protein-coupled receptor; alpha1-adrenergic receptor subtypes; antagonist; antidepressants; citalopram; desipramine; imipramine; inositol phosphate; mianserin; second messenger.

Figure 1

The creation and functional characterization of cell lines stably expressing the α1A- and α1B-adrenergic receptor subtypes in PC12 cells. Expression cassettes of pcDNA3.1+ plasmids used to express human (A) α1A- and (B) α1B- adrenergic receptors. (C) Metabolic activity of untransfected and receptor-expressing lines. Second messenger response of (D) α1A- and (E) α1B-adrenergic receptor-expressing cells to stimulation with the agonists noradrenaline (black) and phenylephrine (green). Second messenger response to a submaximal (EC₉₀) noradrenaline concentration in the presence of the antagonists (F) prazosin and (G) WB4101. P_CMV—CMV promoter; hAlpha1A/B-AR—human α1A- and α1B-adrenergic receptors; P_SV40—SV40 promoter; NeoR—neomycin resistance gene; BGH pA—bovine growth hormone polyA; SV40 pA—SV40 polyadenylation site. Data are shown as the mean ± SEM, n = 2–4 wells.

Figure 2

Saturation curves of [³H]prazosin binding in the PC-12 cell lines expressing α1A-AR (A,D) and α1B‑AR (B,E) and in the ChemiSCREEN™ Ready-to-Assay™ α_1D cells (C,F). Specific binding (A–C) was determined by subtracting the amount bound in the presence of 10 μM prazosin for [³H]prazosin from the total radioactivity bound per milligram protein (D–F). Each point represents the mean ± SEM of quadruplicate determinations from four experiments.

Figure 3

Inhibitory effects of antidepressants on noradrenaline-stimulated second messenger activation. Cells were incubated with varying concentrations of selected antidepressant drugs (imipramine (A), desipramine (B), reboxetine (C), mianserin (D), citalopram (E), and fluoxetine (F)) and noradrenaline at submaximal concentrations (EC₉₀). Data are expressed as a percentage of noradrenaline-stimulated second messenger accumulation (inositol phosphate synthesis in PC-12 cell lines expressing α1A- and α1B-AR and calcium ion influx from ChemiSCREEN™ Ready-to-Assay™ α_1D cells). Each value represents the mean ± S.E.M. of at least three independent experiments conducted in duplicate.

Figure 4

Timelines of experiments testing prolonged (24 h (A) and 120 h (B)) preincubation of cells expressing α1-AR subtypes with antidepressants. AD—antidepressant drug; NA—noradrenaline.

Figure 5

Dose–response curves for noradrenaline and the α1A-, α1B- and α1D-adrenergic receptor subtypes’ second messenger responses after a 24 h preincubation with the chosen antidepressant drugs. The graphs show the responses of cells after preincubation with the antidepressant imipramine (IMI, (A–C)), desipramine (DMI, (D–F)), mianserin (MIA, (G–I)), reboxetine (REB, (J–L)), citalopram (CIT, (M–O)) or fluoxetine (FLU, P–S) (green solid lines on the respective graphs), or the responses of control cells (black dotted lines on all graphs). The effects were tested by measuring the inositol phosphate production by cells expressing α1A-AR (A,D,G,J,M,P) or α1B-AR (B,E,H,K,N,R), or by measuring the noradrenaline-induced calcium ion influx of ChemiSCREEN™ Ready-to-Assay™ α1DA-AR-expressing cells (C,F,I,L,O,S). Each value represents the mean ± S.E.M. of at least four independent experiments conducted in duplicate.

Figure 6

Dose–response curves for noradrenaline and the α1A-, α1B- and α1D-adrenergic receptor subtypes’ second messenger responses after a 120 h preincubation with the chosen antidepressant drugs. The graphs show the responses of cells after preincubation with the antidepressant imipramine (IMI, (A–C)), desipramine (DMI, (D–F)), mianserin (MIA, (G–I)), reboxetine (REB, (J–L)), citalopram (CIT, (M–O)) or fluoxetine (FLU, (P–S)) (green solid lines on the respective graphs), or the responses of control cells (black dotted lines on all graphs). The effects were tested by measuring inositol phosphate production by cells expressing α1A-AR (A,D,G,J,M,P) or α1B-AR (B,E,H,K,N,R), or by measuring the noradrenaline-induced calcium ion influx of ChemiSCREEN™ Ready-to-Assay™ α1DA-AR-expressing cells (C,F,I,L,O,S). Each value represents the mean ± S.E.M. of at least four independent experiments conducted in duplicate.

Figure 7

Effects of incubation with the chosen antidepressants on the metabolic activity of cells expressing the α1A-, α1B- or α1D- adrenergic receptor subtypes. The cells were incubated with the chosen antidepressants for 30 min (A), 24 h (B) or 120 h (C). IMI—imipramine; DMI—desipramine; MIA—mianserin; REB—reboxetine; CIT—citalopram; FLU—fluoxetine or PBS (dashed line). Data are shown as the mean ± SD; n = 3–4 independent experiments.