Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.
Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Wintersgill
H P
HP
Department of Cardiovascular Studies, University of Leeds, UK.
Warburton
P
P
Bryson
S E
SE
Ball
S G
SG
Balmforth
A J
AJ
D000804:Angiotensin II / Q000502:physiology*; D000805:Angiotensin III / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry; D000242:Cyclic AMP / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D011869:Radioligand Assay; D011945:Receptors, Angiotensin / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D014316:Tritium; D014407:Tumor Cells, Cultured
D011945:Receptors, Angiotensin; D014316:Tritium; D000804:Angiotensin II; D000805:Angiotensin III; D000242:Cyclic AMP