A [3H]amine congener of 1,3-dipropyl-8-phenylxanthine. A new radioligand for A2 adenosine receptors of human platelets

Abstract

A xanthine amine congener (XAC), an amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine, is an antagonist ligand for A2 adenosine receptors of human platelets. XAC inhibited 5'-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of adenylate cyclase activity with a KB of 24 nM. [3H]XAC exhibits saturable, specific binding with a Kd of 12 nM and a Bmax of 1.1 pmol/mg protein at 37 degrees C. [3H]XAC binding in platelets is the first example of labeling of A2 adenosine receptors in which the potencies of adenosine agonists and antagonists in inhibiting binding are commensurate with their potencies at these receptors in functional studies. Furthermore, [3H]XAC is the first antagonist radioligand with high affinity at A2 adenosine receptors.

Fig. 1

Stimulation of adenylate cyclase activity of human platelet membranes by NECA in the absence ( ) and presence of XAC (2 nM, ; 20 nM, ; 200 nM, ). Adenylate cyclase activity was determined at 37°C for 90 min. Right: Schild plot of the same data with the concentration ratio (CR) of the EC₅₀ values for NECA in the presence and absence of XAC vs the XAC concentration (slope, 1.05; r = 0.9993). EC₅₀ values for NECA: 0.255 μM in the absence and 0.27, 0.46 and 2.29 μM in the presence of 2, 20 and 200 nM XAC, respectively. Values are means of a typical experiment done in triplicate.

Fig. 2

Saturation of [³H]XAC binding to human platelet membranes. Specific ( ) and nonspecific ( ) binding were determined for 90 min at 37°C. Values are means of a typical experiment done in triplicate. Right: Scatchard plot of the same data. K_d, 12 nM; B_max, 1.1 pmol/mg protein.

Fig. 3

Competition for [³H]XAC binding to human platelet membranes. Binding of 1 nM [³H]XAC was measured for 90 min at 37°C. Slope factors: 0.94 for XAC ( ), 0.91 for NECA ( ), 0.97 for 8-phenyltheophylline ( ), 0.99 for R-PIA ( ) and 0.91 for theophylline ( ). Values are means of a typical experiment done in triplicate.