Effects of 5'-N-ethylcarboxamideadenosine (NECA) on erythropoietin production.
The present studies were undertaken to assess the effects of 5'-N-ethylcarboxamideadenosine (NECA), an adenosine analogue, on erythropoietin (Epo) production. NECA (0.05 and 0.1 mumol/kg i.v.) produced significant increases in serum Epo levels (368.8 +/- 56.1 and 384.6 +/- 45.9 mU/ml, respectively) in exhypoxic polycythemic mice after a four hour exposure 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 (< 1.24 mU/kg kidney). Theophylline (20 mg/kg i.p.), an adenosine receptor antagonist, 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 (> or = 10(-6) M) for 20 hours under hypoxic conditions (1% O2) produced significant increases in medium levels of Epo when compared with hypoxia controls. 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 atmosphere also had significantly higher cAMP levels than that of hypoxia controls. Scatchard analyses of [3H]NECA binding to membrane preparations of hepatocellular carcinoma cells showed low affinity binding sites with a dissociation-constant (Kd) of 0.44 microM and a binding capacity of 863 fmol/mg protein. These findings suggest that the increase in Epo production in response to NECA under hypoxic conditions can be attributed, at least in part, to stimulation of adenosine A2 receptors which is coupled to adenylyl cyclase activation.
Nakashima
J
J
Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana.
Ohigashi
T
T
Brookins
J W
JW
Beckman
B S
BS
Agrawal
K C
KC
Fisher
J W
JW
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000096:biosynthesis* / Q000097:blood; D005260:Female; D000860:Hypoxia / Q000097:blood; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008815:Mice, Inbred Strains; D009994:Osmolar Concentration; D011086:Polycythemia / Q000097:blood; D013806:Theophylline / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000378:metabolism
D004921:Erythropoietin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D019830:Adenosine-5'-(N-ethylcarboxamide); D013806:Theophylline; D000241:Adenosine