Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5

Abstract

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 B max values of 1269 ± 365 and 4190 ± 1055 fmol/mg protein and affinities (K d) 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 (K d) of 6.7 ± 1 nM and a B max value of 315 ± 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT 1 receptor antagonist. DuP 753, all had a high affinity for the receptor (K 1 values in the nanomolar range), but the selective angiotensin AT 2 ligands. PD 123177 and p-aminophenylalaninc 6 AII, had low affinity (K i values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT 1 rcceptor 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 IC 50 of 4.2 ± 0.9 mM. The coupling of the AT 1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca 2+] i). This increase in [Ca 2+] i was unaffected by PD 123177 (100 μ) but was abolished by DuP 753 (100 μM). Furthermore. AII (100 nM) did not inhibit forskohn (0.1–10 μM) stimulated cyclic AMP formalion. PLC-PRF- 5 cells express large numbers of AII receptors which are of the AT 1 receptor subtype. Stimulation of the receptor increases [Ca2+] i indicating coupling to phospholipase C. However, no evidence for coupling via G i to inhibition of adenylyl cyclase was found. This homogeneous cell line, expressing large amounts of the angiotensin II receptors linked to phospholipase C, will serve as a useful, simple system to study the regulation of the angiotensin AT i receptor subtype.