The effect of brilliant black BN on agonist antagonist interactions at the human adenosine-A1 and -A3 receptors


Lauren May , Stephen Briddon, Stephen Hill. Institute of Cell Signalling, University of Nottingham, Nottingham, United Kingdom

The synthesis of XAC-BY630, a novel xanthine amine congener (XAC) derivative which incorporates the BODIPY 1 fluorophore has been described previously (Briddon et al, 2004). Brilliant Black BN is commonly used in experiments involving calcium-sensitive fluorescent dyes (e.g. Fluo 4AM) to reduce the signal from extracellular dye (Roth et al., 2006). This study investigated the effect of Brilliant Black BN on the ability of the agonist 5’-N-ethyl carboxamide (NECA) and the antagonists XAC and XAC-BY630 to bind to the human adenosine-A1 and -A3 receptors. Intracellular calcium mobilisation was measured using a fluorescence plate reader (FLEXstation, Molecular Devices). CHO cells (expressing either the human A 1 or A 3 receptors) were grown to confluency in 96-well black-walled plates and incubated at 37°C for 45 minutes in 100 μL DMEM containing 10% FCS, 2.5 mM probenecid, 2.3 μM Fluo 4AM and 0.023% pluronic acid. Cells were then washed twice with PBS and incubated in 100 μL HEPES-buffered saline containing 2.5 mM probenecid in the presence or absence of antagonist and/or Brilliant Black BN at 37°C for 30 minutes. Fluorescence was then measured in the presence of increasing concentrations of NECA. At both the adenosine-A1 and -A3 receptors, in the absence of Brilliant Black BN, the concentration-response relationship of the adenosine receptor agonist, NECA, was rightward shifted in the presence of 1 μM XAC-BY630 and 1 μM XAC. When repeated in the presence of Brilliant Black BN, the potency of 1 μM XAC-BY630 and 1 μM XAC was decreased in a concentration-dependent manner such that virtually no antagonism was observed in the presence of 500 μM Brilliant Black BN (Table 1). In summary, Brilliant Black BN caused a concentration-dependent decrease in the antagonism mediated by XAC-BY630 and XAC at both the adenosine-A1 and -A3 receptors.

Table 1: The potency (LogEC50) NECA in the presence or absence of adenosine receptor antagonists and/or Brilliant Black. Values are mean ± S.E.M. of 3-10 experiments conducted in triplicate.



Briddon, S.J. et al. (2004) P.N.A.S., 101: 4673-4678
Roth A.L. et al (2006) J. Biol Chem. 281: 20809-20816