Bolus intravenous injection: 0.02 mg to 0.06 mg; Intravenous infusion: 5 mcg/min; Intramuscular 0.2 mg; Subcutaneous 0.2 mg; Intracardiac 0.02 mg;

Human embryonic kidney cells (HEK293) were cultured in Minimum Essential Medium (MEM) Earle’s (Biochrom AG) supplemented with 10% FBS (PAA Laboratories GmbH), 100 U/ml penicillin, and 100 mg/ml streptomycin (Biochrom AG) and 2 mM L-glutamine (Invitrogen) at 37C with 5% CO2. 48 well plates were coated with Poly-L-Lysine (Biochrom) and HEK293 cells were seeded with 37,500 cells per well. Transient transfection in triplicates with 84 ng DNA/well using metafectene according to manufactures instructions (Biontex, Munich, Germany) was performed 28 hours later. 40 hours after transfection cells were pre-incubated for 5 minutes with stimulation buffer containing of MEM Earle’s media and 1 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma). This stimulation buffer was used for all further steps. For ligand competition experiments cells were incubated with tyramine, 2-phenylethylamine or octopamine ranging from 6.7 nM to 6700 nM, diluted in stimulation buffer for 15 minutes followed by a stimulation with isoprenaline in concentrations ranging from 1 nM to 10000 nM for 30 minutes. All reactions were performed at 37C with 5% CO2 saturated air and stopped by aspirating medium.