Characterization of the AT4 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 AT4 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 AT4 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 AT4 receptor: 125I]‐Ang IV and 125I]‐Nle1‐Ang IV bind specifically to the SK‐N‐MC cell membranes (Kd = 0.6 and 0.1 nm) in a saturable manner (Bmax= 1.2 pmol/mg of protein). AT4 receptor ligands, Nle1‐Ang IV, Ang IV and LVV‐haemorphin 7 (LVV‐H7), compete for the binding of [125I]‐Ang IV or [125I]‐Nle1‐Ang IV to the SK‐N‐MC cell membranes with rank order potencies of Nle1‐Ang IV > Ang IV > LVV‐H7 with IC50 values of 1.4, 8.7 and 59 nm ([125I]‐Ang IV) and 1.8, 20 and 168 nm ([125I]‐Nle1‐Ang IV), respectively. The binding of [125I]‐Ang IV or [125I]‐Nle1‐Ang IV to SK‐N‐MC cell membranes was not affected by the presence of GTPγS. Both Ang IV and LVV‐H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT4 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 AT4 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 AT4 receptor.