HEPG2 AND SK-N-MC - 2 HUMAN MODELS TO STUDY ALPHA-2-ADRENERGIC RECEPTOUS OF THE ALPHA-2C SUBTYPE

It is now clearly established that alpha-2 adrenergic receptors can be subdivided in three pharmacological subtypes (alpha-2A, alpha-2B and alpha-2C) encoded by distinct genes (alpha(2)C10, alpha(2)C2 and alpha (2)C4, respectively, in humans). Whereas the study of the regulation o f the human alpha-2A adrenergic receptor and of the promoter region of the alpha(2)C10 gene has being greatly helped by the availability of the colon carcinoma cell line HT29, the study of the other human recep tor subtypes has thus far been limited to homologous desensitization/d own-regulation in transfected cells, because of the lack of human cell ular models constitutively expressing alpha-2B or alpha-2C adrenergic receptors. Several human cell lines were thus screened, in an attempt to find such models. Radioligand binding studies with [H-3]RX821002 an d [H-3]MK912, reverse transcription-polymerase chain reactions and RNa se mapping experiments with pairs of primers and riboprobes specific f or each subtype demonstrated that the hepatoma cell line HepG2 and the neuroblastoma cell line SK-N-MC possess alpha-2 adrenergic receptors of the alpha-2C subtype. However, whereas HepG2 expresses exclusively alpha-2C receptors (55 +/- 7 fmol of [H-3]MK912 binding sites/mg of pr otein), SK-N-MC expresses both alpha-2A and alpha-2C subtypes in fairl y similar amounts (20 +/- 8 and 23 +/- 3 fmol of [H-3]MK912 binding si tes/mg of protein, respectively). The study of the inhibition of H-3-l abeled antagonist binaing by UK14304 demonstrated that a fraction of t he receptor population was coupled to pertussis toxin-sensitive G-prot eins, which were identified as G(i2) and G(i3) by immunoblotting. The alpha-2 agonist was, moreover, able to decrease forskolin-stimulated c AMP production by 47% in HepG2 and 23% in SK-N-MC, demonstrating that inhibition of adenylyl cyclase is one of the primary mechanisms of sig nal transduction in both cell lines. HepG2 and SK-N-MC are the first h uman cell lines unquestionably shown to natively express alpha-2C adre nergic receptors. The discovery of these two models may be useful for future study of the regulation of alpha(2)C4 gene expression in cells of different origins and investigation of the reciprocal regulation of alpha-2A and alpha-2C subtype in single cells.