Characterization of the AT(4) receptor in a human neuroblastoma cell line (SK-N-MC)

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.