EFFECTS OF 5'-N-ETHYLCARBOXAMIDEADENOSINE (NECA) ON ERYTHROPOIETIN PRODUCTION

The present studies were undertaken to assess the effects of 5'-N-ethy lcarboxamideadenosine (NECA), an adenosine analogue, on erythropoietin (Epo) production. NECA (0.05 and 0.1 mumol/kg i.v.) produced signific ant increases in serum Epo levels (368.8 +/- 56.1 and 384.6 +/- 45.9 m U/ml, respectively) in exhypoxic polycythemic mice after a four hour e xposure to hypoxia when compared with hypoxia controls (133.2 +/- 18.2 mU/ml). The hypoxic kidney Epo levels were 46.4 +/- 13.4 mU/kg kidney which were significantly higher than normoxic kidney Ep levels (< 1.2 4 mU/kg kidney). Theophylline (20 mg/kg i.p.), an adenosine receptor a ntagonist, significantly inhibited the stimulatory effects of NECA on serum Epo levels. In vitro cultures of an Epo producing hepatocellular carcinoma (Hep3B) cell line with NECA (greater-than-or-equal-to 10(-6 ) M) for 20 hours under hypoxic conditions (1% O2) produced significan t increases in medium levels of Epo when compared with hypoxia control s. Hepatocellular carcinoma cells treated with NECA at a concentration range of 10(-7) M to 5 x 10(-5) M for one hour in a hypoxic atmospher e also had significantly higher cAMP levels than that of hypoxia contr ols. Scatchard analyses of [H-3]NECA binding to membrane preparations of hepatocellular carcinoma cells showed low affinity binding sites wi th a dissociation-constant (K(d)) of 0.44 muM and a binding capacity o f 863 fmol/mg protein. These findings suggest that the increase in Epo production in response to NECA under hypoxic conditions can be attrib uted, at least in part, to stimulation of adenosine A2 receptors which is coupled to adenylyl cyclase activation.