In Vitro Evidence for an Intracellular Site of Angiotensin Action

Abstract

Abstract-To differentiate the relative effects of nuclear and cell surface angiotensin II (Ang II) receptors, we mutated the angiotensinogen cDNA by removing the signal sequence-encoding region to produce a nonsecreted form of angiotensinogen [Ang(ϪS)Exp]. Rat hepatoma cells (which produce renin and angiotensin-converting enzyme mRNAs) were stably transfected with Ang(ϪS)Exp/pSVL (or a corresponding control) expression plasmid, and mitotic indices were measured for stably transfected cell lines. Experimental clonal cell lines demonstrate an average of 33Ϯ4.4% (PϽ0.001) increase in percentage-labeled nuclei compared with control cell lines. The mitogenic effect is blocked by 10 Ϫ6 mol/L losartan and by 1 mol/L renin antisense phosphorothioate oligomers but not by 10 Ϫ6 mol/L candesartan. In addition, phenylarsine oxide, which blocks angiotensin receptor internalization, abolishes the losartan inhibitory effect, suggesting that after cell-surface receptor-mediated endocytosis, losartan blocks Ang II nuclear receptors. PDGF mRNA levels are elevated 2.2-fold in Ang(ϪS)Exp transfected cell lines; addition of anti-PDGF antibodies to the culture medium partially blocks the mitogenic effect of Ang(ϪS)Exp, while anti-Ang II antibodies have no effect. These results suggest that the Ang(ϪS)Exp growth effect is due, in part, to autocrine/paracrine stimulation by secreted PDGF after Ang II/Ang II receptor intracellular interactions. We further demonstrate that these cells produce the alternative renin transcript, renin 1A, which apparently lacks a signal sequence and is maintained intracellularly. Collectively, these studies of cultured cells suggest that some cell types may possess components of the renin-angiotensin system that permit intracellular processing of angiotensinogen to Ang II and that Ang II generated intracellularly may be mitogenic. Re and colleagues 1 showed that isolated rat liver and spleen nuclei specifically bound 125 I-labeled Ang II with high affinity. In these studies, Ang II competed effectively for radiolabeled tracer whereas Ang I did so less effectively and neurotensin not at all. Their studies extended earlier investigations that suggested that labeled Ang II localized to nuclear and mitochondrial regions of myocardial, brain, and smooth muscle cells. [5][6][7] Re and colleagues further demonstrated 125 I-Ang II binding to solubilized rat liver chromatin fragments, the existence of a discrete Ang II-binding nucleoprotein particle by deoxynucleoprotein gel electrophoresis, and direct effects of nuclear angiotensin on transcription. 2,8,9 In addition, Ang II immunoreactivity has been found associated specifically with euchromatin in cerebellum, liver, and adrenal tissue. 10 Erdmann and colleagues, 10 using immunogold staining, found Ang II immunoreactivity to be prominent in cerebellar neurons; the peptide was localized to nuclei and also to vesicle-like structures in cytoplasm. In some cell types such as endothelial and granule cells, the peptide was nearly exclusively present in the transcription...

Glia- and Neuron-specific Expression of the Renin-Angiotensin System in Brain Alters Blood Pressure, Water Intake, and Salt Preference

“…If translated, this mRNA would encode an intracellular (nonsecreted) and constitutively active form of the protein, suggesting the intriguing possibility of an intracellular pathway of angiotensin production in brain. Intracellular production of Ang-II has been proposed but remains controversial (42). The GFAP-hREN and SYN-hREN models described herein utilized a mutant renin that should essentially be constitutively active because of the cleavage of prorenin to renin by furin, a ubiquitous processing protease expressed in the brain (43).…”

Implications of intracrine hormone action for physiology and medicine

“…Nuclear angiotensin receptors were subsequently reported to be At-1-like in that they were blocked by losartan, and these reports showed receptors were present in association with both the nuclear membrane and, again, with chromatin. Confirmation that binding of angiotensin to isolated nuclei resulted in increased gene transcription was later provided by the observation that nuclear angiotensin upregulates renin, angiotensinogen, and platelet-derived growth factor (PDGF) transcription (9,10,12,47,48,(50)(51)(52)(53). Moreover, electron microscopic immunohistochemical techniques were used to demonstrate angiotensin II immunoreactivity located in association with the euchromatin of cerebellar neurons, hepatocytes, and adrenal cells from normal animals (13).…”

“…In particular, we have shown that the large isoform of PDGF was upregulated in the transformed cells, and this effect was blocked by losartan but not candesartan CV-11974. Although it frequently has been assumed that nonpeptide angiotensin II antagonists are not internalized after receptor binding, this has not actually been shown for losartan (9,10,33,52). Indeed, a recent study employing confocal microscopy demonstrated clear internalization of angiotensin II receptors following the administration of losartan (33).…”

Identification and characterization of a functional mitochondrial angiotensin system

“…Although many studies have focused on the impact of extracellular Ang II and its receptors, type 1 (AT 1 R) and type 2 (AT 2 R), on the cardiovascular system, others have reported that Ang II is also present in the intracellular compartment and can be released upon cell stretch to mediate cellular growth and/or apoptosis (1)(2)(3). Although many of the autocrine effects of this endogenous Ang store are believed to be mediated by plasma membrane Ang receptors, an intracellular RAS acting on nuclear Ang receptors has also been proposed (4,5).…”