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.
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Australia.
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Mustafa
S Y
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Chai
F A
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Mendelsohn
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Moeller
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Albiston
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D006031:Glycosylation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006454:Hemoglobins / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D009447:Neuroblastoma; D010446:Peptide Fragments / Q000494:pharmacology; D011869:Radioligand Assay; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
C090830:AT4 receptor; D003432:Cross-Linking Reagents; D006454:Hemoglobins; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D011945:Receptors, Angiotensin; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C074309:LVV-hemorphin-7; D013936:Thymidine