In Vitro Activation of Human Adrenergic Receptors and Trace Amine-Associated Receptor 1 by Phenethylamine Analogues Present in Food Supplements

Abstract

Pre-workout supplements are popular among sport athletes and overweight individuals. Phenethylamines (PEAs) and alkylamines (AA) are widely present in these supplements. Although the health effects of these analogues are not well understood yet, they are hypothesised to be agonists of adrenergic (ADR) and trace amine-associated receptors (TAARs). Therefore, we aimed to pharmacologically characterise these compounds by investigating their activating properties of ADRs and TAAR1 in vitro. The potency and efficacy of the selected PEAs and AAs was studied by using cell lines overexpressing human ADRα_1A/α_1B/α_1D/α_2a/α_2B/β₁/β₂ or TAAR1. Concentration-response relationships are expressed as percentages of the maximal signal obtained by the full ADR agonist adrenaline or the full TAAR1 agonist phenethylamine. Multiple PEAs activated ADRs (EC₅₀ = 34 nM-690 µM; E_max = 8-105%). Almost all PEAs activated TAAR1 (EC₅₀ = 1.8-92 µM; E_max = 40-104%). Our results reveal the pharmacological profile of PEAs and AAs that are often used in food supplements. Several PEAs have strong agonistic properties on multiple receptors and resemble potencies of the endogenous ligands, indicating that they might further stimulate the already activated sympathetic nervous system in exercising athletes via multiple mechanisms. The use of supplements containing one, or a combination of, PEA(s) may pose a health risk for their consumers.

Keywords: adrenaline; adrenergic receptors; alkylamine analogues; food supplements; phenethylamine analogues; trace amine-associated receptor 1.

Figure A1

(A1) Gene expression of human trace amine-associated receptor 1 (hTAAR1) as fold change in GAPDH in untransfected HEK293T cells and in hTAAR1-transfected HEK293T cells. Data are presented as mean (± SEM) of 2–3 independent experiments which were run in duplicate. (A2) cAMP production in cells as percentage of maximal signal obtained by phenethylamine (PEA) in hTAAR1-transfected HEK293T cells exposed to PEA (triangles) or RO5166017 (squares) and in untransfected HEK293T cells exposed to PEA (diamonds). Data are presented as mean (±SEM) of 2–3 independent experiments which were run in duplicate.

Figure A2

Concentration–response relationship (mean ± SD) of adrenaline (closed circles) that activated adrenergic receptor (ADR) α_2B in chem-1 cells. Data are presented as percentages of maximal fluorescent Ca²⁺ response obtained by adrenaline (n = 8).

Figure A3

Concentration–response relationships (mean ± SD) of alkylamine analogues 1,3-dimethylamylamine (closed bar), dimethylbutylamine (vertical striped bar), dimethylaminoethanol (open bar) and dimethylhexylamine (checkered bar) that activated human trace amine-associated receptor 1 (TAAR1) in HEK293T cells transfected with this receptor at a concentration of 300 µM. The data are presented as percentages of the maximal intracellular cAMP level obtained by phenethylamine (n = 3).

Figure 1

Molecular structures of (a) well-known activators of sympathetic nervous system: adrenaline, noradrenaline and dopamine, (b) phenethylamines and (c) alkylamines present in nutritional supplements.

Figure 2

Concentration–response relationships (mean ± SD) of compounds that activated human adrenergic receptor (ADR) α_1A (a), α_1B (b) and α_1D (c) in chem-1 cells overexpressing these receptors. Data are presented as percentages of maximal fluorescent Ca²⁺ response obtained by adrenaline (n = 7–9).

Figure 3

Concentration–response relationships (mean ± SD) of compounds that activated human adrenergic receptor (ADR) α_2A in chem-1 cells. Data are presented as percentages of maximal fluorescent Ca²⁺ response obtained by adrenaline (n = 8).

Figure 4

Concentration–response relationships (mean ± SD) of compounds that activated human adrenergic receptor (ADR) β₁ (a) and β₂ (b) in chem-1 cells overexpressing these receptors. Data are presented as percentages of maximal fluorescent Ca²⁺ response obtained by adrenaline (n = 8).

Figure 5

Concentration–response relationships (mean ± SD) of (a) phenethylamine, tyramine, methyltyramine and higenamine, (b) phenethylamine, p-synephrine, hordenine, dimethylphenethylamine and halostachine, and (c) phenethylamine, β-methylphenethylamine, p-octopamine and isopropyloctopamine, which activated human trace amine-associated receptor 1 (TAAR1) in HEK293T cells stably transfected with this receptor. Data are presented as percentages of maximal intracellular cAMP level obtained by phenethylamine (n = 3–5).