Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells

Abstract

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.

Fig. 1

[³H]XAC saturation curve in DDT₁ MF-2 smooth muscle cell membranes. Experiments were performed by incubating [³H]XAC with membranes (20–40 µg/assay tube) in the absence (specific binding) and presence (nonspecific binding) of 10 µM(R)-PIA. This is representative of five similar experiments performed in duplicate.

Fig. 2

(R)-PIA competition curves versus [³H]XAC. Membranes were incubated with [³H]XAC (1.5 nM) and various concentrations of (R)-PIA in the absence or presence of Gpp(NH)p (100 µM). Specific [³H]XAC binding was 0.23 ± 0.04 and 0.31 ± 0.06 pmol/mg of protein for control and Gpp(NH)p-treated membranes, respectively. Results are presented as the averaged data from five independent experiments of each treatment group. Competition curves were analyzed by a curve-fitting program (Scafit) according to a two-state model (27, 28).

Fig. 3

[¹²⁵I]APNEA saturation curve. Experiments were performed using 20–40 µg of DDT₁, MF-2 cell membranes incubated with [¹²⁵I]APNEA in the absence (total binding) and presence (nonspecific binding) of 10 µM (R)-PIA. This is a representative of three similar experiments, each performed in duplicate. B_max and K_d values for this representative experiment were 0.90 pmol/mg of protein and 1.4 nM, respectively.

Fig. 4

Agonist competition curves versus [¹²⁵I]APNEA. Competition curves were derived by incubating [¹²⁸l]APNEA (~0.5 nM) with membranes (as in Fig. 3) and different concentrations of the competitors. Specific binding in the absence of competitors averaged ~0.21 pmol/assay tube. This figure is representative of three similar experiments. The curves were fitted by a curve-fitting program (27, 28).

Fig. 5

Photoaffinity labeling of A₁, adenosine receptors in DDT₁, MF-2 cell membranes. Cell membranes were incubated with [¹²⁵I]AZPNEA (0.5 nM) in the absence (control) or presence of (R)-PIA, (S)-PIA, or XAC. Incubations were for 1 hr at 37 °, following which membranes were centrifuged, washed, and exposed to UV light. The photolabeled receptor was resolved on 12% acrylamide SDS-PAGE and subjected to autoradiography. The migration profile of iodinated molecular weight standards is indicated on the left.

Fig. 6

Inhibition of adenylate cyclase activity in DDT₁, MF-2 cell membranes by adenosine analogs. A, Adenylate cyclase assays were performed as described in Experimental Procedures using 10 µM isoproterenol and various concentrations of (R)-PIA. Isoproterenol-stimulated adenylate cyclase in the absence of inhibitor (control) was 32.8 ± 6.1 pmol/min/mg of protein, compared with basal activity of 3.7 ± 0.5 pmol/min/mg of protein. Results are expressed as the averaged data from eight independent experiments. B, Adenylate cyclase activity was assayed in the presence of isoproterenol (10 µM) and various concentrations of inhibitors. The IC₅₀ values (nM) for (R)-PIA, NECA, and (S)-PIA for inhibiting adenylate cyclase were 62 ± 10, 538 ± 189, and 750 ± 250, respectively (mean ± SE of four independent experiments).

Fig. 7

Attentuation of (R)-PIA mediated inhibition of adenylate cyclase by XAC and pertussis toxin. A, Adenylate cyclase assays were performed in the presence of isoproterenol (10 µM) and either reaction buffer or XAC (1 µM) diluted in buffer. Adenylate cyclase activity in absence of (R)-PIA (control) was 32.1 and 34.5 pmol/min/mg of protein for buffer and XAC, respectively. B, DDT₁, MF-2 cells in culture were treated with Dulbecco’s modified Eagle’s medium or Dulbecco’s modified Eagle’s medium plus pertussis toxin (100 ng/ml) for 18 hr. Control adenylate cyclase activity [in the absence of (R)-PIA] averaged 23.7 and 24.6 pmol/min/mg of protein.

Fig. 8

¹²⁵I-PAPA-APEC saturation curve in DDT₁ MF-2 cell membranes. Membranes (~60 µg) were incubated with different concentrations of the radioligand for 45 min at 37 °, in the absence (total binding) or presence (nonspecific binding) of theophylline (10 mM). This curve is representative of five similar experiments. Curves were modeled by a computer program, as previously described (27, 28). B_max and K_d values for this representative experiment were 0.17 pmol/mg of protein and 2.9 nM, respectively.

Fig. 9

Competition of various ligands with A₂ adenosine receptors. A, Membranes were incubated with 1.5 nM concentrations of the radioligand in the presence of various concentrations of NECA or (R)PIA. The data presented were analyzed by computer modeling (27, 28) and represent the mean of five independent experiments with standard errors at each point of less than 10%. Specific ¹²⁵I-PAPA-APEC binding in the absence of inhibitors was 30.1 fmol/mg of protein. B, Labeling was performed as described in Experimental Procedures using ~0.75 nM ¹²⁵I-azido-PAPA-APEC, in absence or presence of NECA (3.3 nM), (R)-PIA (3.3 nM), or theophylline (10 mM). The labeled A₂ adenosine receptor migrates with M_r ~42,000 on a 14% polyacrylamide gel. The migration of labeled standards is indicated on the left. Experiments were repeated three times.

Fig. 10

Activation of adenylate cyclase by adenosine analogs in DDT₁ MF-2 membranes. Assays were performed as described in Experimental Procedures, for 10 min at 30 °. Basal adenylate cyclase activity averaged 3.0 ± 0.6 pmol/mg/min. Data are presented as the mean ± standard error of three independent experiments.