[Increase in erythropoietin secretion mediated by adenosine A2 receptors]

The present study was undertaken to assess the adenosine receptor regulation of erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep3B) in response to hypoxia. In vitro cultures of Hep3B cells with N6-cyclohexyladenosine (CHA) at a concentration of 10(-5)M produced significant increases in cyclic AMP accumulation (142.43 +/- 13.31 pmole/10(6) cells) after 1 hr and Ep secretion (29.83 +/- 1.69 mU/ml) after 20 hr when compared with their respective hypoxia controls (cAMP: 3.05 +/- 0.26 pmole/10(6) cells, Ep: 19.41 +/- 1.41 mU/ml). The stimulatory effects of CHA on both Ep secretion and cyclic AMP accumulation were significantly inhibited by 8-phenyltheophylline (8-PT) at a concentration of 5 x 10(-7). No significant change in cell growth was observed at the CHA and 8-PT concentrations used in these experiments employing a spectrophotometric method. Incubation with 8-bromo cyclic AMP (10(-4)M) in response to hypoxia also produced a significant enhancement of Ep secretion (30.74 +/- 0.50 mU/ml) when compared with hypoxia controls (22.69 +/- 0.23 mU/ml), whereas no significant increase occurred in a normoxic atmosphere. CHA inhibited specific binding of [3H]5'-N-ethylcarboxamideadenosine (100 nM) to Hep3B cell membrane preparations in a dose-dependent manner in the displacement experiments and IC50 was 7.72 x 10(-6)M. These results indicate that Ep secretion is stimulated by adenosine membrane A2 receptors which are linked to adenylyl cyclase activation.