Angiotensin-II-binding sites on hepatocyte nuclei.
Angiotensin-II (Ang II) stimulates gene expression and cell growth in several cell types. Studies that have shown localization of Ang II to nuclei of myocytes and hepatic nuclear Ang II binding suggest that these actions may be mediated by nuclear receptors. We characterized Ang II binding to rat liver nuclei, which were free of plasma membrane based on enzyme analysis and electron microscopy. At 18 C, specific binding of 0.1-0.3 nM [125I]Ang II to nuclei and nuclear envelopes reached equilibrium by 2 h. Unlabeled Ang II inhibited [125I]Ang II binding to nuclei with an IC50 of 1.4 +/- 0.2 nM (+/- SE; n = 6). In half of the nuclear preparations, a lower affinity site (IC50, 50.4 +/- 23.6 nM), which accounted for 7-32% of specific Ang II binding, was detected by Scatchard analysis. Results similar to these were obtained with nuclear envelopes. Other Ang peptides competed for binding in the rank order: Ang III (IC50, 2.1 nM) greater than Ang I (IC50, 33) greater than [Des-Phe8]Ang II (IC50, 362) greater than [Des-Asp1-Des-Arg2]Ang II (IC50, 736). Losartan (DuP 753), an AT1 receptor antagonist, inhibited binding (IC50, 10.9 +/- 0.9 nM), whereas the AT2 receptor antagonist PD123177 did not. The pH optimum for binding to nuclear envelopes was 7, with binding more sensitive to low (5 and 6) than high (8 and 9) pH. Nonhydrolyzable GTP analogs accelerated displacement of bound [125I]Ang II by 10(-5) M Ang II. Differences were noted in pH sensitivity, time course, binding affinity for Ang I, II, and III, and rate of dissociation between nuclei or nuclear envelopes and plasma membrane Ang II binding. These results suggest that nuclear envelopes have a G-protein-coupled Ang II-binding site, which belongs to the AT1 class of Ang II receptors, with properties different from the plasma membrane receptor.
Booz
G W
GW
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Conrad
K M
KM
Hess
A L
AL
Singer
H A
HA
Baker
K M
KM
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D011945:Receptors, Angiotensin; D000804:Angiotensin II; D016244:Guanosine 5'-O-(3-Thiotriphosphate)