Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements.

Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate.

Internalization of the rat AT1a and AT1b receptors: pharmacological and functional requirements.

The capacity of the angiotensin II (AngII) agonist [Sar1]AngII, the antagonist [Sar1-Ile8]AngII and the non-peptidic antagonist DuP753 to undergo receptor internalization were studied in Chinese hamster ovary cells expressing rat AngII type 1a or 1b receptors (AT1a or AT1b) or a mutant of AT1a (Asn74) unable to couple G-protein. In this expression system, the ligand-induced internalization of rat AT1a and AT1b are similar. Moreover, peptidic ligands, either the agonist or antagonist, induce a significant internalization of AT1 receptors, but the non-peptidic antagonist DuP753 is far less potent. Finally, the normal internalization of the mutant Asn74 demonstrates that receptor activation and G-protein coupling are not required for AT1a internalization.

Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling.

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in beta 2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagine or by glutamic acid. When expressed in CHO cells, the two mutants and the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(125I)4]angiotensin II [where Sar is sarcosine and Tyr(125I) is monoiodinated tyrosine] and the other agonists: ([Sar1]angiotensin II > angiotensin II > angiotensin III >> angiotensin I). However, the Asn74 mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affinity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmacological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosphate production and increase cytosolic Ca2+ concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II was able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74 residue of the AT1a receptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling cascade.

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

[Is angiotensin II a growth factor?]. / L'angiotensine II est-elle un facteur de croissance?

Angiotensin II is an octapeptide resulting from the enzymatic cascade of the renin-angiotensin system and involved in vasoconstriction and aldosterone secretion. The extensive use of converting enzyme inhibitors recently suggested that angiotensin II may have a specific action on growth of its target tissues. Cellular models confirm that angiotensin II is able to produce in vitro a cellular hypertrophy of many cell types. Nevertheless a controversy was developed on the real possibility for angiotensin II to act on cell division. Some cells, such as adrenocortical cells, present a clear induction of their division by angiotensin II, but contradictory results were obtained on vascular smooth muscle cells. The mechanism by which angiotensin II induces hypertrophy of its target tissues, is largely unknown but may involve a direct action on proto-oncogene synthesis, or an indirect action on growth factor secretion. The nature of the angiotensin II receptor involved in these mechanisms has to be identified.

Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions.

The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII.

NK-1 receptors are the only class of tachykinin receptors found on mouse cortical astrocytes.

Extending our previous studies, our results indicate that cultured cortical astrocytes from the mouse possess only NK-1 receptors coupled to phospholipase C. An excellent correlation was found in the potency of tachykinins and selective analogs at inhibiting 125I-BHSP binding and at stimulating phospholipase C activity, their rank order being that of NK-1 receptors. No binding sites could be found with ligands of NK-2 or NK-3 receptors. No additive effect could be shown with NK-2 or NK-3 agonists when phospholipase C activity was estimated with high concentrations of NK-1 agonists. C- or N-terminal SP fragments did not modify SP- or [Pro9]SP-evoked responses.

Major histocompatibility complex class II (Ia-like) antigens on chicken embryo fibroblasts: effect of transformation by Rous sarcoma virus.

In this study, three monomorphic monoclonal antibodies to chicken MHC class II molecules (B-L) were tested for reactivity in normal and RSV-transformed embryo fibroblasts. The immunocytochemical staining, the cell-bound ELISA assay, and the immunoprecipitation analysis showed that all three antibodies reacted with the B-L (Ia-like) molecules on normal cells of different genotypes. Conversely, the expression of these antigens was not detected in fibroblasts cultured from feather follicles of adult birds. The level of expression of B-L molecules as well as the class II specific RNA increased consistently after transformation of the cells by the SR-RSV and infection with the avian leukosis virus RAV-1. Analysis of genomic DNA by the Southern blot technique, performed after digestion with several restriction endonucleases, showed that the restriction pattern of B-L genes was not altered in cells transformed by Rous sarcoma virus.

Inhibition of HLA class I antigen and mRNA expression induced by Rous sarcoma virus in transformed human fibroblasts.

Cells from various human nonlymphoreticular neoplasms show reduced HLA class I antigen expression. In this report, a system of human fibroblasts transformed by an avian retrovirus has been employed to investigate the mechanism of this phenomenon. Rous sarcoma virus has been used to transform in vitro human dermal fibroblasts, and clonal cell lines have been established from these cultures. In all the clones studied the integration of the provirus induced a reduction of cell-surface HLA-A, -B, -C framework antigen and beta 2-microglobulin expression when compared to levels for the respective parental fibroblasts. The reduction was correlated with a diminished intracellular synthesis of these molecules. Uninfected cells derived from an osteogenic sarcoma exhibited a reduced expression comparable to that of dermal diploid fibroblasts obtained from the same donor and transformed by Rous sarcoma virus. RNA gel blot analysis of total cellular RNA and of poly(A)+ cytoplasmic RNA showed a markedly decreased amount of HLA class I transcripts in the transformed cells. Southern blot study of genomic DNAs digested with several restriction endonucleases showed that the banding patterns of the HLA genes were not altered in the cells harboring the Rous sarcoma provirus. Our data are consistent with the hypothesis that the Rous sarcoma provirus that does not seem to be linked to the major histocompatibility complex class I gene superfamily may negatively control HLA gene expression.

Integration and expression of provirus in human cells transformed by avian sarcoma virus.

Previously, human diploid fibroblasts from some donors infected in vitro by avian sarcoma virus (ASV) were transformed and found, by electron microscopy, to produce small numbers of virus particles that were infectious by bioassay; also, a line of human osteosarcoma cells infected with ASV developed additional characteristics of transformation and released a small number of infectious virus particles. In this study the complete proviral sequence was shown to be integrated in the genome of these cells. The env-related proteins gp85 and gp37 and the gag-related proteins pr76, pr60, and p19 can be detected in cytoplasmic extracts of ASV-infected human cells. Comparable amounts of pp60v-src were found in human and avian cells infected with ASV. The associated kinase activity in infected human cells was dramatically increased as compared to that of uninfected controls; the enzyme had the same cation and substrate requirements as those from ASV-transformed avian cells. Replicating particles from infected human cells were purified and were significantly modified compared to those from avian hosts as shown by a) higher specific gravity, b) the presence of RSV gag-related but not env-related antigens, and c) the fact that the virus-associated reverse transcriptase preferred the divalent cations Mn2+ and Fe2+ over Mg2+.

Reduced expression of MHC class I antigens in chicken fibroblasts transformed by Rous sarcoma virus.

Monoclonal monomorphic as well as polyclonal antibodies against H-B class I (B-F) antigen were used to evaluate the expression of these molecules on normal and RSV-transformed chick embryo fibroblasts. The results indicate that chicken fibroblasts transformed in vitro by the Schmidt-Ruppin strain Rous sarcoma virus of subgroup B (SR-RSV-B) present reduced H-B class I antigen expression as compared to uninfected cells of the same inbred strain. This quantitative reduction was observed at the cell membrane and in whole cell lysates. Chick embryo fibroblasts infected with avian leukosis virus RAV-1 show that the levels of H-B class I antigens were indistinguishable from the levels measured on the control cells. Normal embryo fibroblasts derived from the inbred lines B4/B4 express higher levels of H-B antigens than normal cells carrying the B12/B12 genotype.

[Transformation of diploid human fibroblasts and viral production after infection by avian retroviruses]. / Transformation de fibroblastes diploïdes humains avec production virale après infection par des rétrovirus aviaires.

Among the cultures established from skin biopsies of 128 donors, two were transformed by RSV and produced virus. One out of 6 cellular lines developed from six human sarcomas was likewise transformed and also produced virus.

[Morphology of ribosomal RNA in chicken fibroblasts]. / Morphologiè des ARN ribosomiques de fibroblastes de poulets.

It is shown with the electron microscope that the 28 S RNA component of the ribosomal RNA extracted from Chicken fibroblasts contains secondary structures which are not present in the 18S component.

Transformation in vitro of glial hamster cells by Rous sarcoma virus.

Cell lines from the brains of inbred CF hamster embryos were established in vitro. The morphology of the cells in the light and electron microscopes was that of glial cells, and the cells contained the nervous system-specific protein S-100. Infection with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup B, resulted in foci of transformation. The transformed cells were virogenic and upon intracerebral and sc inoculations into young hamsters, they developed into histologically typical gliomas.

[Susceptibility to avian tumor viruses: determination of chick phenotypes]. / Susceptibilité aux virus oncogènes aviaires: détermination du phénotype de Poussins.

The genetic cellular susceptibility to avian sarcoma viruses (RSV) of subgroups A, B, C and E has been determined in one week old Chicks. Fibroblasts from pin feathers were grown in vitro and tested by focus formation after infection. This technique will allow, for the first time, the study of the influence of the host phenotypes (susceptibility or resistance to the different subgroups of RSV) upon the immune response to a given virus.