losartan-potassium and 8-phenyltheophylline

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.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Carcinoma, Hepatocellular; Cell Hypoxia; Cyclic AMP; Erythropoietin; Humans; Liver Neoplasms; Receptors, Purinergic; Theophylline; Tumor Cells, Cultured

The present studies were undertaken to assess the direct effects of N6-cyclohexyladenosine (CHA), a stable adenosine analogue, on erythropoietin (Ep) secretion in hepatocellular carcinoma cells (Hep 3B). Ep levels in the medium of low density Hep 3B cells treated with CHA in concentrations of 10(-5) and 5 x 10(-5) M for 20 h under hypoxic conditions (1% O2) were significantly higher than that of hypoxic controls. In addition, CHA at the same concentrations produced significant increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels in Hep 3B cells after 1-h incubation under hypoxic conditions when compared with hypoxic controls. Dibutyryl cAMP (10(-5), 10(-4) M) also caused significant increases in Ep secretion when compared with control hypoxic cells. On the other hand, 8-phenyltheophylline, an adenosine receptor antagonist, significantly inhibited the stimulatory effects of CHA on both Ep secretion and cAMP accumulation in the Hep 3B cell cultures in response to hypoxia. These data suggest that Ep secretion may be regulated by adenosine receptor-coupled activation of adenylyl cyclase and the generation of cAMP.

Topics: Adenosine; Anaerobiosis; Bucladesine; Carcinoma, Hepatocellular; Cell Line; Cell Survival; Cyclic AMP; Erythropoietin; Humans; Hypoxia; Kinetics; Liver Neoplasms; Theophylline