Angiotensin II Binding Sites Research Articles

Nuclear-delimited Angiotensin Receptor-mediated Signaling Regulates Cardiomyocyte Gene Expression

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [alpha-(32)P]UTP (e.g. 36.0 +/- 6.0 cpm/ng of DNA control versus 246.4 +/- 15.4 cpm/ng of DNA Ang-II, 390.1 +/- 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 +/- 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFkappaB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFkappaB mRNA levels at saturating concentrations with approximately 2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca(2+)] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca(2+) release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

The impact of ANG II and IV on INS-1 cells and on blood glucose and plasma insulin

The impact of angiotensin (ANG) for peripheral, global effects is well known. Local ANG systems including that of the insulin-releasing β cell are not well investigated. In insulin-secreting cell line (INS-1), AT1 and AT4 receptors for ANG II and IV were demonstrated by Western blots. Only small amounts of ANG II-binding sites of low affinity were observed. ANG II and SARILE displaced binding of 125I-ANG II. ANG II and IV as well as their non-degradable analogs SARILE and Nle-ANG IV increased the glucose-induced insulin release in a bell-shaped way; the maximum effect was at ∼1 nM. The increase was antagonized by 1 µM losartan or 10 µM divalinal (AT1 and AT4 receptor antagonists, respectively). The insulin release was accompanied by a 45Ca2+ uptake in the case of ANG II and ANG IV. Divalinal abolished the effect of ANG IV and Nle-ANG IV on this parameter. ANG IV reduced the increase in blood glucose during a glucose tolerance test with corresponding, albeit smaller effects on plasma insulin. Using confocal laser scanning microscopy, transfected insulin-regulated aminopeptidase (IRAP) with AT4 receptors was shown to be accumulated close to the nucleus and the cytosolic membrane, whereas GLUT4 was not detectable. IRAP was inhibited by ANG IV. In conclusion, AT1 and AT4 receptors may be involved in diabetic homeostasis. Effects are mediated by insulin release, which is accompanied by an influx of extracellular Ca2+. The impact of ANG IV/IRAP agonists may be worth being used as antidiabetics.

The use of heat-induced hydrolysis in immunohistochemistry on angiotensin II (AT 1) receptors enhances the immunoreactivity in paraformaldehyde-fixed brain tissue of normotensive Sprague–Dawley rats

The research on components of the renin–angiotensin system delivered a broad image of angiotensin II-binding sites. Especially, immunohistochemistry (IHC) provided an exact anatomical localization of the AT 1 receptor in the rat brain. Yet, controversial results between in vitro receptor autoradiography and IHC as well as between immunohistochemical studies using various antisera started a vehement discussion concerning specificity and cross-reactivity of these antisera. In particular the magnocellular subdivision of the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) provided controversial results on the localization of AT 1 receptors. Both areas are known for angiotensin II-induced release of vasopressin (VP) and oxytocin (OXT). To evaluate the significance of the appropriate method of antigen retrieval and its relevance for the detection of AT 1 receptors we performed IHC on AT 1 receptors in paraformaldehyde-fixed and paraffin-embedded brain tissue of Sprague–Dawley rats using either the detergent Triton X-100 or microwave oven heating. This study demonstrates that heat-induced hydrolysis enhances the quality and quantity of immunoreactivity (IR) in IHC on AT 1 receptors. In the organum vasculosum lamina terminalis and in the parvocellular subdivisions of the PVN we report a distribution of AT 1-like-IR similar to that observed with other methods. However, in addition, we provide evidence that distinct AT 1-like-IR is also localized in few magnocellular neurons of the PVN and in few parvocellular neurons of the dorsal SON but not in magnocellular neurons of the SON. Moreover, parallel IHC indicates that few magnocellular OXT- or VP-releasing neurons of the PVN as well as parvocellular OXT-releasing neurons of the SON do also contain AT 1 receptors.

Up-regulation of the Angiotensin II Type 1 Receptor by the MAS Proto-oncogene Is Due to Constitutive Activation of Gq/G11 by MAS

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT(1)) receptor in CHO-K1 cells has been reported to increase the number of [(3)H]angiotensin II-binding sites, although MAS does not bind [(3)H]angiotensin II. In HEK293 cells stably expressing AT(1) receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT(1) receptor-CFP amounts and [(3)H]angiotensin II binding levels. The time course of AT(1) receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [(35)S]guanosine 5'-O-(thiotriphosphate) onto the phospholipase Cbeta-linked G protein Galpha(11). Protein kinase C (PKC) inhibitors and the selective Galpha(q)/Galpha(11) inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT(1) receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT(1) receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT(1) receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT(1) receptor levels reflects the constitutive capacity of MAS to activate Galpha(q)/Galpha(11) and hence stimulate PKC-dependent phosphorylation of the AT(1) receptor.

Angiotensin II Receptors in Human Preadipocytes: Role in Cell Cycle Regulation

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled[ Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

Angiotensin II receptors in human preadipocytes: role in cell cycle regulation.

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

The angiotensin II binding site on Mycoplasma hyorhynis is structurally distinct from mammalian AT 1 and AT 2 receptors

Angiotensin II (AngII) binding sites were characterized on rat pheochromocytoma cells (PC-12) which are known to express exclusively the type-2 (AT 2) AngII receptor. Interestingly, we found that, on confluent PC-12 cells, only partial inhibition of 125I-AngII binding was achieved when cells were incubated with a saturating concentration of PD-123 319 (an AT 2 selective ligand) suggesting the presence of an atypical binding site. In binding experiments, AngII exhibited high affinity for this atypical binding site with a dissociation constant ( K d) of 16 nM. Moreover, bacitracin potently inhibited PD-123 319-resistant 125I-AngII binding with an IC 50 half-maximal inhibitory concentration of 44 μM. Enzyme immunoassay revealed that the cells were contaminated with Mycoplasma hyorhynis. Contaminated PC-12 cells were photolabeled with 125I-[ p-benzoylPhe 1]AngII and covalently labeled proteins were subjected to polyacrylamide gel electrophoresis followed by autoradiography. Under these conditions, two distinct labeled species of 140 kilodaltons (kDa) and 95 kDa were detected. Deglycosylation of the 140 kDa-labeled AT 2 receptor with glycopeptidase-F (PNGase-F) resulted in a 35 kDa protein whereas the 95 kDa band was not affected by digestion with the endoglycosidase. Thus, our results show that the AngII binding site on M. hyorhynis is structurally distinct from mammalian AT 1 and AT 2 receptors.

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes.

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II-receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II-receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6.8 and 6.5, were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II-receptor complex migrating at pI 6.8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II-receptor complex profiles, indicating that the binding sites corresponding to pI 6.5 and pI 6.8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II-receptor complex isoform at pI 6.8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6.5.

Characterization of a specific binding site for angiotensin II in chicken liver.

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pKD of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pKD of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT1 and AT2. Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII(Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl] methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl] imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp1]AngII > [Suc1]AngII > or = [Sar1]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly1]AngII > [Cys1]AngII approximately [aminoisobutyryl1]AngII approximately [Ser1]AngII > > > [Sar1]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.

Down-regulation of angiotensin II receptors in hypertrophied human myocardium.

1. Specific [125I]-angiotensin II (AngII) binding in normal and hypertrophied human myocardial membranes was saturable and of high affinity. Low concentrations of unlabelled AngII and saralasin competed with [125I]-AngII for the binding sites in these tissues. Thus, saturable [125I]-AngII binding in human myocardium exhibited pharmacological specificity that characterized high affinity receptors for AngII. 2. There was little difference in the apparent dissociation constant (Kd) values for [125I]-AngII binding between normal and hypertrophied human myocardium, whereas the maximal number of binding sites (Bmax) was significantly (51%) lower in the hypertrophied group. Further, PD123177, a selective antagonist of the AT2 receptor subtype, showed three orders of magnitude higher affinity for [125I]-AngII binding sites in both normal and hypertrophied myocardium than losartan, a selective antagonist of the AT1 receptor subtype; the Hill coefficients for these drugs were close to one. 3. A significant decrease in Bmax and Kd values for (-)-[125I]-iodocyanopindolol binding between normal and hypertrophied human myocardium rarely occurred. 4. The present study suggests that both normal and hypertrophied human myocardium predominantly contains the AT2 receptor subtype and that these receptors are down-regulated in hypertrophied tissues.

Angiotensin-(1–7) binding at angiotensin II receptors in the rat brain

Angiotensin-(1–7) (Ang-(1–7)) is reported to be equipotent with angiotensin II (AII) in producing some central biological effects but the receptors responsible for these actions have not been defined. Three classes of receptor have been proposed: AT 1, AT 2, and a putative Ang-(1–7) selective receptor. This study specifically evaluates Ang-(1–7) competition at AII binding sites (AT 1 and AT 2) in the rat brain. 125I Sar 1 Ile 8 AII (269–312 pM) was used to conduct receptor autoradiographic binding assays in brain sections. Competition with Ile 5 AII and Val 5 AII was similar at nuclei in which either AT 1 or AT 2 receptor subtypes predominate ( K i = 11–18 nM). Ang-(1–7) competed 150-fold less effectively than native AII at AT 1 predominant brain nuclei ( K i = 2.4 μM). At brain regions where AT 2 receptors predominate, Ang-(1–7) showed a very low affinity ( K i = 104 μM) for the majority of the 125I Sar 1 Ile 8 AII binding sites (AT 2). A small proportion of 125I Sar 1 Ile 3 AII binding sites showed an affinity of 2.0 μM, presumably AT 1 receptors present in those brain regions. For biological responses where Ang-(1–7) is reported to be equipotent with AII, it is unlikely that these actions are mediated by the widely distributed AT 1 or AT 2 receptor subtypes which recognize 125I Sar 1 Ile 8 AII.

AT1 receptor characteristics of angiotensin analogue binding in human synovium

1. Angiotensin II (AII) reduces blood flow, modulates vascular remodelling and is a growth factor. Human inflammatory arthritides are characterized by synovial hypoperfusion, hypoxia and proliferation. We aimed to localize and characterize receptors for AII in human synovium. 2. We used quantitative in vitro receptor autoradiography with [125I]-(Sar1, Ile8)AII and [125I]-AII on human synovium from patients with chondromalacia patellae, osteoarthritis and rheumatoid arthritis. 3. [125I]-(Sar1, Ile8)AII and [125I]-AII bound to similar sites on synovial blood vessels, lining cells and stroma. Binding to microvessels (< 100 microns diameter) was more dense than to arteriolar media, and vascular binding was more dense than that to lining cells and stroma. 4. Microvessels and arterioles which displayed angiotensin converting enzyme-like immunoreactivity also displayed specific binding of [125I]-(Sar1, Ile8)AII. 5. Specific binding of [125I]-(Sar1, Ile8)AII to each structure was completely inhibited by 10 microM dithiothreitol and was inhibited by unlabelled ligands with the rank order of potency (Sar1, Ile8)AII > AII > losartan = SKF108566 > PD123319 indicating an AT1 subclass of angiotensin receptor. 6. GTP gamma S (1 microM) abolished specific binding of [125I]-AII and abolished the high affinity component of the binding inhibition curve for AII against [125I]-(Sar1, Ile8)AII, indicating G protein coupling. 7. The distribution of [125I]-(Sar1, Ile8)AII binding sites was similar in all disease groups and no significant differences in binding densities, affinities or specificities were observed between disease groups. 8. Locally generated AII may act on synovial AT1 receptors to modulate synovial perfusion and growth. Specific AT1 receptor antagonists should help elucidate the role of angiotensins in human arthritis.

Ontogenesis of the angiotensin II (ANGII) receptor system in the duck brain

The ontogenetic development of the central nervous angiotensin II (ANGII) receptor system in the duck was studied at embryonic days E20 and E27 and at postnatal days P3 and P14 by computerized semiquantitative autoradiography employing the receptor antagonist 125I[ 1Sar, 8Ile]ANGII as radioligand. For circumventricular structures involved in the sensing of brain-intrinsic (AV3V region) or blood-borne (subfornical organ, SFO) ANGII, binding sites for 125I[ 1Sar, 8Ile]ANGII were first detectable at E27, with a steady rise in binding density up to P14. The choroid plexus of the lateral (PCVL) and third (PCVIII) cerebral ventricles responsible for cerebrospinal fluid (CSF) production were endowed with maximal ANGII receptor densities at E20 with subsequent reduction to constant medium (PCVIII) or low (PCVL) values. Besides the choroid plexus, the magnocellular paraventricular nucleus (PVN) was the only structure presenting ANGII specific binding sites at E20, although at low density. As for the SFO and AV3V region, labelling of ANGII binding sites in the PVN increased continuously during development to high values at P14. Nuclear components of the limbic system (archistriatum, amygdala and habenular complex) did not reveal specific labelling by the radioligand at E20 with constant, moderate binding densities evaluated for E27, P3 and P14. In the duck brain, functionally related structures exhibited a homogenuous ontogenetic development of their ANGII receptor system.

Characterization of a functional angiotensin II receptor in Xenopus laevis heart.

High-affinity (104 +/- 18 pmol/l) and high-density (204 +/- 25 fmol/mg) angiotensin II (AII) binding sites have been identified in Xenopus laevis heart membranes. Competition binding of [125I]Sar1,Ile8 angiotensin (SIA) to these receptors by peptide analogs selective for the mammalian AII receptor subtypes AT1 and AT2 suggested that the amphibian AII binding sites were more closely related to the AT1 receptor subtype. Also in common with AT1 receptors, dithiothreitol and GTP gamma S inhibited [125I]SIA binding to Xenopus heart receptors, exhibiting IC50 values of 600 and 0.95 mumol/l, respectively. In addition, Xenopus oocytes injected with Xenopus heart mRNA were capable of mobilizing calcium when exposed to AII, demonstrating that Xenopus AII receptors are functionally linked to a second-messenger system similar to that coupled to mammalian AT1 receptors. However, in contrast to both AT1 and AT2 receptor subtypes, nonpeptide antagonists DUP 753 and SK&F 108566 (AT1 receptor selective) and PD123319 (AT2 selective) did not bind the Xenopus AII receptors, thus establishing that the amphibian receptors were pharmacologically unique. Together, these results demonstrate that Xenopus heart AII receptors are functionally similar to mammalian AT1 receptors but are pharmacologically distinct from both AT1 and AT2 receptors.

Angiotensin II binding sites in the hamster brain: localization and subtype distribution

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [ 125I][sar 1, ile 8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT 2 subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [ 125I][sar 1, ile 8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT 1 binding sites in the hamster but AT 2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.

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 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.

Angiotensin II binding sites in frog kidney and adrenal

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [ 125I]-[Val 5]AII binds toa single class of binding sites with a dissociation constant (K d) of 548 ± 125 pM in glomeruli and 593 ± 185 pM in interrenal tissue ( n = 8). The corresponding maximal binding capacities (B max) were 2.48 ± 0.71 and 3.05 ± 1.02 fmol/mm 2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar 1]AII > human AII > [ 125I]-[Val 5]AII = [Val 5]AII = human AIII > > human AI . The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.

Analysis of Angiotensin II Receptor Subtypes in Individual Rat Brain Nuclei

Previous studies have used new angiotensin II (AII) receptor subtype selective compounds to localize AII receptor subtypes within discrete rat brain nuclei. The purpose of this autoradiographic study was to extend these preliminary findings and provide a comprehensive analysis of AII binding sites in 22 rat brain nuclei and the anterior pituitary, to include estimates of the binding affinity for 125I sar1 ile8 AII (125I SIAII) at each nucleus, and determine the fractional distribution of each subtype at each nucleus. Estimates of KD in separate experiments revealed that AT1 nuclei had a consistently higher affinity for 125I SIAII than AT2 nuclei (0.66 vs. 2.55 nM). Displacement of subsaturating concentrations of 125I SIAII by 10(-8)-10(-4) M DuP753 (selective for the AT1 subtype) or PD123177 (selective for the AT2 subtype) indicated that approximately half of the brain regions surveyed contained predominantly AT1 sites and half contained predominantly AT2 sites. Binding was partially displaced by both compounds in several regions and two site analyses were performed to estimate the distribution of subtypes within each nucleus. The data were then corrected for differential occupancy by 125I SIAII. Brain nuclei associated with cardiovascular or dipsogenic actions of AII, e.g., subfornical organ, organum vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and area postrema, contained pure, or almost pure, populations of AT1 receptors. The functions of AII in brain regions containing predominantly AT2 binding sites, e.g., thalamus, colliculi, inferior olive and locus ceruleus, remain undefined. Thus, AII binding sites in the rat brain have been differentiated into two subtypes with similar characteristics to those reported in peripheral tissues. However, the unexpected finding that they can be differentiated on the basis of their affinity for 125I SIAII raises questions concerning their coidentity with peripheral receptor subtypes.

Cloning and Functional Characterization of a Novelmas-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.

Angiotensin II-binding protein in adult and neonatal rat heart

An angiotensin II-binding activity has been identified in the 100 000 × g supernatant fraction of adrenal gland, kidney, liver, heart and brain of adult rat. The binding is specific for angiotensin II; it is of high affinity and completely dependent upon the presence of an organomercurial, p-chloromercuriphenylsulfonic acid. Reducing agents, on the other hand, cause a dissociation of bound ligand. Covalent cross-linking of [ 125I]-angiotensin II to the soluble fraction from rat heart followed by SDS-polyacrylamide gel electrophoresis and autoradiography indicated that the macromolecule that binds angiotensin II is most probably a protein with an apparent mass of 78 000 dalton. A comparison of the binding of angiotensin II to the 100 000 × g supernatant fraction from both neonatal (1–3-day-old) and adult (3-month-old) rat hearts revealed that angiotensin II binds with similar affinity and specificity, but the number of binding sites is 3-fold higher in the neonatal heart ( K D and B max were 10.4 ± 3.1 n m and 1.6 ± 0.4 pmol/mg protein for adult and 8.8 ± 2.9 n m and 4.9 ± 0.7 pmol/mg protein for neonatal heart, respectively). The membrane fraction prepared from neonatal rat heart similarly bound angiotensin II in a saturable manner and with high affinity ( K D 4.3 ± 0.5 n m and B max 146.4 ± 4.9 fmol/mg protein), but a similar membrane fraction prepared from adult rat heart failed to show any angiotensin II binding. These observations indicated that, in rat heart, there is a decrease of angiotensin II-binding sites, both soluble and membrane bound, with age. Hence, rat heart, at various stages of development, and myocytes prepared from it should provide a suitable system with which to study the developmental regulation of the angiotensin II-binding protein.