2-[2-[4-[2-[2-[ 1,3-Dihydro- 1,1-bis (4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5'- N-ethylcarboxamidoadenosine (FITC-APEC): A Fluorescent Ligand For A2a-Adenosine Receptors

Abstract

The fluorescein conjugate, FITC-APEC (2-[2-[4-[2-[2-[1,3-dihydro-l,l-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine), is a novel ligand derived from a series of functionalized congeners that act as selective A_2a-adenosine receptor agonists. The binding of FITC-APEC to bovine striatal A_2a,-adenosine receptors measured by fluorescence techniques was saturable and of a high affinity, with a B_max, of 2.3 ± 0.3 pmol/mg protein and K_D of 57 ± 2 nM. The K_D value estimated by fluorescence was consistent with the K_i (11 ± 0.3 nM) obtained by competition studies with [³H]CGS 21680. Additionally, the B_max, value found by FITC-APEC measurement was in agreement with B_max, values obtained using radioligand binding. FITC-APEC exhibited rapid and reversible binding to bovine striatum. The potencies of chemically diverse A_2a-adenosine receptor ligands estimated by inhibition of FITC-APEC binding were in good agreement with their potencies determined using radioligand binding techniques (r = 0.97, P = 0.0003). FITC-APEC binding was not altered by purine derivatives that do not recognize A_2a-adenosine receptors. These findings demonstrate that the novel fluorescent ligand FITC-APEC can be used in the quantitative characterization of ligand binding to A_2a-adenosine receptors.

Keywords: A2a-adenosine receptors; Fluorescence; bovine striatum; receptor binding.

Fig. 1

Synthesis of the fluorescent A_2a-adenosine receptor ligand, FITC-APEC.

Fig. 2

Association and dissociation of FITC-APEC binding to bovine striatal membranes. Tissues were prepared and assayed as described under Materials and Methods. Association of FITC-APEC (50 nM) binding was performed by varying the incubation period (1–130 rain). Equilibrium was attained within 60 rain and maintained throughout the 130-rain duration. A 90-min incubation period was selected for use in subsequent experiments. Association data (■, solid line) represent means (mean % specific binding) from triplicate samples of two separate determinations. The rate constants were estimated using the equation k₁ = (k_obs – k₋₁/LT, where L_T is the total FITC-APEC concentration. The k_obs and k₁ estimates for FITC-APEC were 0.029 min ⁻¹ and 7.47 × 106 M⁻¹ min⁻¹, respectively. Dissociation of bound FITC-APEC was performed as described under Materials and Methods. The dissociation rate constant k₋₁ was 0.345 min⁻¹ Dissociation data (□, dotted line) represent means (mean % specific binding) from triplicate samples of two separate experiments. The K_D determined by nonequilibrium methods (K_D = k₋₁/k₁) was 46 nM.

Fig. 3

Plot of a saturation isotherm constructed from equilibrium saturation data of FITC-APEC binding to bovine striatal membranes. The concentration of FITC-APEC was varied from 16 to 248 nM. Nonspecific binding was defined by the presence of 2-CADO (10 μM). Specific binding of FITC-APEC (50 mM) determined by fluorescence measurement was approximately 20% of the total binding (specific binding of [³H]CGS 21680 (6 nM) was determined to be approximately 50% of total binding). The isotherm is representative of three separate determinations. The K_D and B_max estimates for specific FITC-APEC binding determined using saturation isotherms were (mean ± SE) 57 ± 2 nM and 2.3 ± 0.3 pmol/mg protein, respectively. Data analysis was performed using GraphPAD (ISS Software, San Diego, CA). Inset: Representative Scatchard plot of saturation data.

Fig. 4

Inhibition of specific FITC-APEC binding by A_2a-adenosine receptor ligands. Inhibition of FITC-APEC binding (50 nM) to bovine striatal membranes was assessed as described under Materials and Methods. K₁ values for inhibition of FITC-APEC binding in descending order of potency were as follows: NECA, 4.1 ± 0.1 nM (■); DPMA, 13 ± 3 nM (△, dotted line); 2-CADO, 34 ± 14 nM (•); and CV 1808, 131 ± 62 nM (□). Data (mean % specific binding) represent triplicate samples from at least three experiments. The competition curves demonstrate mass action displacement of FITC-APEC binding by the four compounds.

Fig. 5

Correlation between the potencies of A_2a-adenosine receptor ligands as inhibitors of FITC-APEC and radioligand binding. The K₁ (nM) values assessed by inhibition of FITC-APEC binding (ordinate) by A_2a-adenosine receptor ligands were related to K₁ values found using [³H]NECA binding (abscissa). FITC-APEC inhibition data (means) were obtainted from the values in Fig. 4 and Table I. [³H]NECA inhibition data were from the report by Bruns et al. [23]. The following compounds were used in the graph: NECA, DPMA, 2-CADO, CHA, R-PIA, CV 1808, and theophylline. The correlation coefficient for inhibition of FITC-APEC binding versus [³H]NECA binding was r = 0.97 (P = 0.0003).