Vol. 281, Issue 2, 983-991, 1997

HepG2 and SK-N-MC: Two Human Models to Study Alpha-2 Adrenergic Receptors of the Alpha-2C Subtype

Stéphane Schaak, Cécile Cayla, Régis Blaise, Françoise Quinchon and Hervé Paris

Institut National de la Santé et de la Recherche Médicale U.317, Institut Louis Bugnard, Toulouse, France

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 (₂C10, ₂C2 and ₂C4, respectively, in humans). Whereas the study of the regulation of the human alpha-2A adrenergic receptor and of the promoter region of the ₂C10 gene has being greatly helped by the availability of the colon carcinoma cell line HT29, the study of the other human receptor subtypes has thus far been limited to homologous desensitization/down-regulation in transfected cells, because of the lack of human cellular 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 [³H]RX821002 and [³H]MK912, reverse transcription-polymerase chain reactions and RNase mapping experiments with pairs of primers and riboprobes specific for 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 [³H]MK912 binding sites/mg of protein), SK-N-MC expresses both alpha-2A and alpha-2C subtypes in fairly similar amounts (20 ± 8 and 23 ± 3 fmol of [³H]MK912 binding sites/mg of protein, respectively). The study of the inhibition of ³H-labeled antagonist binding by UK14304 demonstrated that a fraction of the receptor population was coupled to pertussis toxin-sensitive G-proteins, which were identified as G_i2 and G_i3 by immunoblotting. The alpha-2 agonist was, moreover, able to decrease forskolin-stimulated cAMP production by 47% in HepG2 and 23% in SK-N-MC, demonstrating that inhibition of adenylyl cyclase is one of the primary mechanisms of signal transduction in both cell lines. HepG2 and SK-N-MC are the first human cell lines unquestionably shown to natively express alpha-2C adrenergic receptors. The discovery of these two models may be useful for future study of the regulation of ₂C4 gene expression in cells of different origins and investigation of the reciprocal regulation of alpha-2A and alpha-2C subtype in single cells.