Acute repeated intracerebroventricular injections of angiotensin II reduce agonist and antagonist radioligand binding in the paraventricular nucleus of the hypothalamus and median preoptic nucleus in the rat brain.

Angiotensin II (Ang II) stimulates water and saline intakes when injected into the brain of rats. This arises from activation of the AT1 Ang II receptor subtype. Acute repeated injections, however, decrease the water intake response to Ang II without affecting saline intake. Previous studies provide evidence that Ang II-induced water intake is mediated via the classical G protein coupling pathway, whereas the saline intake caused by Ang II is mediated by an ERK 1/2 MAP kinase signaling pathway. Accordingly, the different behavioral response to repeated injections of Ang II may reflect a selective effect on G protein coupling. To test this hypothesis, we examined the binding of a radiolabeled agonist ((125)I-sarcosine(1) Ang II) and a radiolabeled antagonist ((125)I-sarcosine(1), isoleucine(8) Ang II) in brain homogenates and tissue sections prepared from rats given repeated injections of Ang II or vehicle. Although no treatment-related differences were found in hypothalamic homogenates, a focus on specific brain structures using receptor autoradiography, found that the desensitization treatment reduced binding of both radioligands in the paraventricular nucleus of the hypothalamus (PVN) and median preoptic nucleus (MnPO), but not in the subfornical organ (SFO). Because G protein coupling is reported to have a selective effect on agonist binding without affecting antagonist binding, these findings do not support a G protein uncoupling treatment effect. This suggests that receptor number is more critical to the water intake response than the saline intake response, or that pathways downstream from the G protein mediate desensitization of the water intake response.

Biased agonism of the angiotensin II type 1 receptor.

G protein-coupled receptors (GPCRs) can be activated by multiple ligands and exhibit the capacity to couple to numerous intracellular signal transduction pathways. This property allows GPCRs to be modulated by biased agonists that selectively activate specific subsets of GPCR-regulated cellular signaling proteins. The angiotensin II type 1 receptor (AT1R) is a GPCR that endogenously binds to the peptide ligand angiotensin II. More recently it has been demonstrated that a modified peptide, [Sar1I-le4-Ile8]-angiotensin II (SII) acts as a biased agonist towards the AT1R. SII binds to the AT1R without promoting heterotrimeric G protein-coupling, but serves to link the receptor to the beta-arrestin-dependent activation of the mitogen activated protein kinase pathway. The present mini-review summarizes current knowledge regarding the role of biased agonists in stimulating biased AT1R signaling.

Association of the novel non-AT1, non-AT2 angiotensin binding site with neuronal cell death.

We have discovered a non-AT(1), non-AT(2) angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

Differential participation of angiotensin II type 1 and 2 receptors in the regulation of cardiac cell death triggered by angiotensin II.

BACKGROUND: The Angiotensin II (Ang II) type 1 (AT(1)R) and type 2 (AT(2)R) receptors are increased in the heart following myocardial infarction and dilated cardiomyopathy, yet their contribution at a cellular level to compensation and/or failure remains controversial. METHODS: We ectopically expressed AT(1)R and AT(2)R in cultured adult rat cardiomyocytes and cardiac fibroblasts to investigate Ang II-mediated cardiomyocyte hypertrophy and cardiac cell viability. RESULTS: In adult rat cardiomyocytes, Ang II did not induce hypertrophy via the AT(1)R, and no effect of Ang II on cell viability was observed following AT(1)R or AT(2)R expression. In adult rat cardiac fibroblasts, Ang II stimulated cell death by apoptosis via the AT(1)R (but not the AT(2)R), which required the presence of extracellular calcium, and induced a rapid dissipation of mitochondrial membrane potential, which was significant from 8 h. CONCLUSIONS: We conclude that Ang II/AT(1)R triggers apoptosis in adult rat cardiac fibroblasts, which is dependent on Ca2+ influx.

Human brain contains a novel non-AT1, non-AT2 binding site for active angiotensin peptides.

AIMS: To determine whether the novel non-AT1, non-AT2 binding site for angiotensins recently discovered in rodent brains occurs in the human brain. MAIN METHODS: Radioligand binding assays of (125)I-sarcosine(1), isoleucine(8) angiotensin II binding were carried out in homogenates of the rostral pole of the temporal cortex of human brains containing 0.3 mM parachloromercuribenzoate (PCMB), 10 microM losartan to saturate AT1 receptors, 10 microM PD123319 to saturate AT2 receptors, with or without 10 microM angiotensin II to define specific binding. Competition binding assays employed a variety of angiotensin peptides, specific angiotensin receptor antagonists, several neuropeptides and an endopeptidase inhibitor to determine pharmacological specificity for this binding site. KEY FINDINGS: The novel non-AT1, non-AT2 binding site was present in similar amounts in female and male brains: Bmax 1.77+/-0.16 and 1.52+/-0.17 fmol/mg initial wet weight in female and male brains, respectively. The K(D) values, 1.79+/-0.09 nM for females, and 1.53+/-0.06 nM for males were also similar. The binding site shows pharmacological specificity similar to that in rodent brains: sarcosine(1), isoleucine(8) angiotensin II>angiotensin III>angiotensin II>angiotensin I'angiotensin IV>angiotensin 1-7. Shorter angiotensin fragments and non-angiotensin peptides showed low affinity for this binding site. SIGNIFICANCE: The presence in human brain of this novel non-AT1, non-AT2 binding site supports the concept that this binding site is an important component of the brain angiotensin system. The functional significance of this binding site, either as a novel angiotensin receptor or a highly specific angiotensinase remains to be determined.

Variety of angiotensin receptors in 3T3-L1 preadipose cells and differentiated adipocytes.

A local paracrine acting angiotensin (ANG) system of preadipocytes and mature adipocytes is involved in metabolic effects and tissue differentiation. The present study reports on the investigation of binding affinities for various angiotensin receptors including their relevance in 3T3-L1 adipocytes and preadipocytes and 3T3-442A preadipocytes. Competitive binding studies using both 125I-ANG II and its more stable analogue 125I-SARILE for investigating AT1/AT2 binding sites in 3T3-L1 preadipocytes reveal a biphasic competition curve with KDs at a low and high nanomolar range. By using the AT2 receptor selective ligand 125I-CGP4112A the presence of high affinity AT2 binding sites in preadipocytes was observed. High nonspecific binding and a low receptor number is characteristic for all these experiments. An AT4 binding site (binding site for ANG IV) exists in 3T3-L1 and F442A preadipocytes and adipocytes with a high nanomolar KD. This low binding affinity was confirmed by a biological assay, the IRAP assay (=insulin regulated aminopeptidase assay). IRAP is associated with the AT4 receptor, which is a binding site at the luminal part of membrane bound IRAP. The curves for competition binding and for inhibition of IRAP activity are superimposable with respect to angiotensin IV. In conclusion, AT1 and AT2 binding sites are present in preadipocytes. AT2 receptor binding affinities are shown in preadipocytes for the first time. The description of a non-AT1/AT2 binding site with low affinity remains speculative albeit of high interest because antidiabetic and obesity related effects of angiotensin peptides and sartanes as antagonists are observed at these high concentrations. Local concentrations of ANG II and their degradation products may be extremely high. The low amounts of AT1 and AT2 binding sites emphasize the relevance of other binding sites in adipose tissue development and metabolic effects. The AT4 binding site seems to be one of the predominant receptors in adipose cells. Other degraded, but still bioactive peptides like ANG III, IV and ANG(1-7), activating receptors not influenced by ANG II, could be of importance.

Effects of losartan on angiotensin receptors in the hypertrophic rat heart.

The effects of losartan on angiotensin receptors in hypertrophic rat hearts were studied. The study was prompted by inconsistent findings of either an increase or decrease in the mRNA of the AT1 receptor in the hearts of cardiac hypertrophic rats treated with losartan, and a paucity of information on the effects of losartan on functional angiotensin receptors in the heart. Losartan, administered i.p. to aortic coarcted rats, dose-dependently attenuated the cardiac hypertrophy. Significant effect was observed with a dose of 2.72 micromol/kg/day for four days. Hypertrophy was accompanied by an increase in [125I]-Sar1-Ile8-angiotensin II binding sites (due mainly to an increase in AT2 binding) and AT2 receptor protein in cardiac ventricles of aortic coarcted rats. Treatment with effective anti-hypertrophic doses of losartan dose-dependently downregulated the [125I]-Sar1-Ile8-angiotensin II binding sites, constitutive AT1 receptor protein, and the over expressed AT2 receptor protein. It was suggested that the anti-cardiac hypertrophic action of losartan resulted from its ability to suppress the expression of both the basal and enhanced cardiac angiotensin receptors. This raises the question as to whether such drastic action could form the therapeutic basis for the use of losartan in cardiac pathologies.

Ligand binding and functional properties of human angiotensin AT1 receptors in transiently and stably expressed CHO-K1 cells.

Chinese Hamster Ovary Cells (CHO-K1) were transiently and stably transfected to express the human angiotensin AT(1) receptor. Cell surface receptor expression was maximal 2 days after transient transfection. Their pharmacological and signalling properties differed from stably expressed receptors. Receptor reserve was significant in the transient cells but not in stable cells, explaining the higher potency of angiotensin II and the lower degree of insurmountable inhibition by candesartan in the transient cells. [Sar(1)Ile(8)]angiotensin II (sarile) is a potent angiotensin AT(1) receptor antagonist for the stable cells but is a partial agonist, producing 19% of the maximal response by angiotensin II, in transient cells. Internalization of [(3)H]angiotensin II and [(125)I]sarile (i.e., acid-resistant binding) was more pronounced in stable cells. CHO-K1 cells were also transiently transfected with the enhanced green fluorescence-AT(1) receptor gene. Confocal microscopy revealed rapid internalization induced by angiotensin II and sarile but not by candesartan. The above disparities may result from differences in receptor maturation and/or cellular surrounding.

Selective silencing of angiotensin receptor subtype 1a (AT1aR) by RNA interference.

Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT1a-, AT1b-, and AT2-receptors. Because of a high degree of similarity among these G-protein-coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT1a receptor (AT1aR) subtype. A dsRNA, AT1 47, was found to be highly selective and efficient in reducing the levels of AT1aR subtype. Transfection of AT1aR-expressing CHO cells with dsRNA AT1 47 resulted in an 80% decrease in the AT1aR expression. In contrast, dsRNA AT1 47 showed no significant effects on both AT1bR and AT2R subtypes. Thus, AT1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.

Papel de la agiotensina II en el desarrollo aterosclerótico: efecto de su bloqueo / Role of angiotensin II in the development of atherosclerosis: effect of blockade

La angiotensina II, el principal efector del sistema renina-angiotensina, ejerce un papel importante en la génesis y en las complicaciones de la aterosclerosis. La angiotensina II estimula la producción de especies reactivas de oxígeno en el vaso que desempeñan un papel clave en la disfunción endotelial y en la oxidación de las lipoproteínas de baja densidad (LDL). Asimismo, este péptido participa en la inducción de la respuesta inflamatoria en la pared vascular mediante la producción de moléculas de adhesión y citoquinas quimiotácticas y proinflamatorias. La angiotensina II, además, estimula la proliferación y migración de células de músculo liso y modula el cambio fenotípico de las mismas dando lugar a un aumento de la síntesis de la matriz extracelular. Finalmente, la angiotensina II también participa en las complicaciones de la aterosclerosis al favorecer la ruptura de la placa y trombogenicidad de la misma. En consecuencia, el sistema renina-angiotensina desempeña un papel clave en la patofisiología de la aterosclerosis, por lo que su bloqueo ejercerá un efecto beneficioso sobre el desarrollo aterosclerótico previniendo las alteraciones trombóticas asociadas a él (AU)

The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities.

BACKGROUND: Endogenous intratubular angiotensin II (Ang II) supports an autocrine tonic stimulation of NaCl absorption in the proximal tubule, and its production may be regulated independently of circulating Ang II. In addition, endogenous Ang II activity may be regulated at the brush border membrane (BBM), by the rate of aminopeptidase A and N (APA and APN) activities and the rate of Ca2+-independent phospholipase A2 (PLA2-dependent endocytosis and recycling of the complex Ang II subtype 1 (AT1) receptor (AT1-R). The aim of the present study was to look for subcellular localization of AT1-R, and APA and APN activities in the medullary thick ascending limb of Henle (mTAL), as well as search for an asymmetric coupling of AT1-R to signal transduction pathways. METHODS: Preparations of isolated basolateral membrane (BLMV) and luminal (LMV) membrane vesicles from rat mTAL were used to localize first, AT1-R by 125I-[Sar1, Ile8] Ang II binding studies and immunoblot experiments with a specific AT1-R antibody, and second, APA and APN activities. Microfluorometric monitoring of cytosolic Ca2+ with a Fura-2 probe was performed in mTAL microperfused in vitro, after apical or basolateral application of Ang II. RESULTS: AT1-R were present in both LMV and BLMV, with a similar Kd (nmol/L range) and Bmax. Accordingly, BLMV and LMV preparations similarly stained specific AT1-R antibody. APA and APN activities were selectively localized in LMV, although to a lesser extent than those measured in BBM. In the in vitro microperfused mTAL, basolateral but not apical Ang II induced a transient increase in cytosolic [Ca2+]. CONCLUSIONS: Besides the presence of basolateral AT1-R in mTAL coupled to the classical Ca2+-dependent transduction pathways, AT1-R are present in LMV, not coupled with Ca2+ signaling, and co-localized with APA and APN activities. Thus, apical APA and APN may play an important role in modulating endogenous Ang II activity on NaCl reabsorption in mTAL.

125I[Sar(1)Ile(8)] angiotensin II has a different affinity for AT(1) and AT(2) receptor subtypes in ovine tissues.

Iodinated angiotensin II (Ang II) and its analogues are often assumed to have equal affinities for AT(1) and AT(2) receptor subtypes. However, using saturation and competition binding assays in several tissues from pregnant, nonpregnant, and fetal sheep, we found the affinity of 125I[Sar(1)Ile(8)] Ang II for Ang II receptors was different (P<0.05) between tissue types. The dissociation constants (Kd) and half maximal displacements of [Sar(1)Ile(8)] Ang II (Sar IC(50)) were directly related (P<0.05) to proportions of AT(1) receptors, and inversely related (P<0.05) to proportions of AT(2) receptors in tissues from all groups combined, in tissues from individual groups (pregnant, nonpregnant or fetal), and in some individual tissues (uterine arteries and aortae). This suggests that 125I[Sar(1)Ile(8)] Ang II has a different affinity for AT(1) and AT(2) receptors in ovine tissues. The Kds of 125I[Sar(1)Ile(8)] Ang II for "pure" populations of AT(1) and AT(2) receptors were 1.2 and 0.3 nM, respectively, i.e. affinity was four-fold higher for AT(2) receptors. We corrected the measured proportions of the receptor subtypes using their fractional occupancies. In tissues which contained at least 10% of each receptor subtype, the corrected proportions were significantly altered (P<0.05), even in some tissues, to the extent of being reversed.

Angiotensin II receptor subtype distribution in the rabbit brain.

Previous work has reported that the distribution of AT(1) binding sites in the rabbit brain is similar to that in the rat, but AT(2) binding sites are confined to the septum and cerebellum of the rabbit brain. This receptor autoradiographic study was designed to enhance the detection of angiotensin II binding sites by using greater radioligand concentrations, and to survey the midbrain in more detail than in previous studies. Tissue sections from five rabbit forebrains, three midbrains, and three hindbrains were incubated with 520 pM (125)I-sar(1)ile(8) angiotensin II. The results confirm abundant AT(1) binding in regions involved in cardiovascular and drinking regulation: the nucleus of the solitary tract, ventrolateral medulla, subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and several hypothalamic structures. Novel AT(1) binding sites were discovered in the pituitary, retrorubral field, periolivary region, dorsolateral nucleus of the lateral lemniscus, dorsal raphe, and laterodorsal tegmental nuclei. The distribution of AT(1) binding was similar to the distribution of monoaminergic neurons. AT(2) binding was moderately dense and well visualized in the cerebellum. In contrast to the rat, AT(2) binding was not detected in the inferior olive of the rabbit, but lobe 9 of the cerebellum exhibited a banding pattern of AT(2) binding reminiscent of the pattern of neuronal projections from the inferior olive. It is possible that AT(2) protein is observed at different stages of axonal transport between the inferior olive and the cerebellum in the two species. Our results did identify new AT(2) binding sites in the superior colliculus and cerebral cortex, but it is clear that AT(2) binding in the rabbit brain is weak and is not as widely distributed as in the rat.

Elevated AT1 receptor protein but lower angiotensin II-binding in adipose tissue of rats with monosodium glutamate-induced obesity.

Age-related hypertrophy of adipose tissue has been associated with a significant decrease in the number of angiotensin II receptors. The aim of this study was to investigate the characteristics of angiotensin II receptors in hypertrophic adipose tissue in animal obesity model using rats postnatally treated with monosodium glutamate. Angiotensin II is known to induce hypertrophy in several tissues of the cardiovascular system and might do the same in fat tissue. The expression and binding properties of angiotensin II AT(1) receptors in epididymal fat tissue of adult rats were studied using membrane-binding, RT-PCR, and immunoblotting. The amount of AT(1) receptor mRNA did not differ significantly between obese and control rats. Despite that glutamate-treated rats displayed approximately 4-times more AT(1) receptor immunoreactive protein content in fat tissue cell membranes than the controls did. In contrast, binding experiments showed a significant (40.3 +/- 6.2 %) decrease of (125)I-Sar(1)-Ile(8)-angiotensin II-binding to fat tissue cell membranes in obese rats compared to controls. In conclusion, the present study provides evidence for the low binding properties associated with an accumulation of AT(1) receptor protein in cell membranes of the fat tissue of rats with glutamate-induced obesity. Discrepancies among angiotensin II-binding, AT(1) receptor protein, and AT(1) receptor mRNA levels indicate a possible defect in the receptor protein, which remains to be identified. The results obtained support a role of angiotensin II and AT(1) receptors in the pathogenesis of obesity.

Chimeric AT1/AT2 receptors reveal functional similarities despite key amino acid dissimilarities in the domains mediating agonist-dependent activation.

Chimeric AT1/AT2 angiotensin II (AngII) receptors in which the sixth and/or seventh transmembrane-spanning domains of the AT2 receptor were substituted into the AT1 receptor were used to investigate the activation mechanisms of the two receptor subtypes. Numerous reports have identified amino acid residues in the sixth and seventh transmembrane-spanning domains of the AT1 receptor involved in the intrareceptor activation mechanism following agonist binding. Many of these residues are not conserved in the AT2 receptor; the corresponding AT2 receptor residues are, in fact, disruptive of AngII-dependent activation when substituted into the AT1 receptor. Surprisingly, the chimeric AT1/AT2 receptors--which also lack these crucial AT1 residues--exhibited AngII-induced activation of phosphoinositide hydrolysis with efficacies and potencies similar to the wild-type AT1 receptor. Consistent with earlier reports, a AT1[Y292F] point mutant demonstrated greatly decreased agonist-induced activation of phosphoinositide hydrolysis. However, a AT1[Y292F/N295S] double-point mutant allowed for normal agonist-induced activation with a pharmacodynamic profile indistinguishable from the wild-type receptor. Despite amino acid dissimilarities, the same corresponding domains and even the same residue loci in both of the AngII receptor subtypes are equally able to mediate agonist-induced receptor activation. This suggests that these corresponding domains in the AT1 and the AT2 receptors are crucial to the activation mechanism, demonstrating greater structural flexibility than previously believed regarding AT1 receptor activation and supporting the possibility of a common activation mechanism for the two receptor subtypes.

Significance of exaggerated natriuresis after angiotensin AT1 receptor blockade or angiotensin- converting enzyme inhibition in obese Zucker rats.

1. Obese Zucker rats (OZR) were shown to be salt-sensitive in that they develop hypertension when placed on a high-salt diet. Because angiotensin (Ang) II is a major antinatriuretic factor, the present studies were undertaken to determine whether the characteristic of salt-sensitivity of OZR is associated with an enhanced antinatriuretic function of endogenous AngII. 2. The extent of AngII-mediated antinatriuresis was investigated in OZR and lean Zucker rats (LZR) using candesartan (100 microg/kg, i.v.), a selective angiotensin AT1 receptor antagonist, and ramipril (1 mg/kg, i.v.), an angiotensin-converting enzyme (ACE) inhibitor. The total number of AngII binding sites and their affinity were also assessed in renal cortical tubular membrane preparations of OZR and LZR using a specific radioligand-binding assay. Plasma renin activity was determined using a standard radioimmunoassay. 3. Both candesartan and ramipril produced substantially greater increases in urinary sodium excretion and urine flow in OZR and these effects were significantly greater than those observed in LZR. These observations suggest that basal antinatriuretic function of endogenous AngII is exaggerated in OZR. 4. The functional overexpression of AngII was not due to any alterations in the affinity or the total number of AngII binding sites in renal cortical tubular membranes. Higher plasma renin values in the OZR could have contributed to the phenomenon. 5. In conclusion, marked diuresis and natriuresis after AT1 receptor blockade and/or ACE inhibition suggest that the extent of endogenous AngII-mediated sodium transport under basal conditions is greatly augmented in OZR. It is proposed this phenomenon may be a contributing factor for the salt- sensitivity in the OZR.

Glial angiotensinogen regulates brain angiotensin II receptors in transgenic rats TGR(ASrAOGEN).

TGR(ASrAOGEN)680, a newly developed transgenic rat line with specific downregulation of astroglial synthesis of angiotensinogen, exhibits decreased brain angiotensinogen content associated with a mild diabetes insipidus and lower blood pressure. Autoradiographic experiments were performed on TGR(ASrAOGEN) (TG) and Sprague-Dawley (SD) control rats to quantify AT(1) and AT(2) receptor-binding sites in different brain nuclei and circumventricular organs. Dose-response curves for drinking response to intracerebroventricular injections of ANG II were compared between SD and TG rats. In most of the regions inside the blood-brain barrier [paraventricular nucleus (PVN), piriform cortex, lateral olfactory tract (LOT), and lateral preoptic area (LPO)], AT(1) receptor binding (sensitive to CV-11974) was significantly higher in TG compared with SD. In contrast, in the circumventricular organs investigated [subfornical organ (SFO) and area postrema], AT(1) receptor binding was significantly lower in TG. AT(2) receptors (binding sensitive to PD-123319) were detected at similar levels in the inferior olive (IO) of both strains. Angiotensin-binding sites sensitive to both CV-11974 and PD-123319 were detected in the LPO of SD rats and specifically upregulated in LOT, IO, and most notably PVN and SFO of TG. The dose-response curve for water intake after intracerebroventricular injections showed a higher sensitivity to ANG II of TG (EC(50) = 3.1 ng) compared with SD (EC(50) = 11.2 ng), strongly suggesting that the upregulation of AT(1) receptors inside the blood-brain barrier of TG rats is functional. Finally, we showed that downregulation of angiotensinogen synthesized by astroglial cells differentially regulates angiotensin receptor subtypes inside the brain and in circumventricular organs.

Effect of cortisol on fetal ovine vascular angiotensin II receptors and contractility.

The renin angiotensin system is important in the regulation of fetal blood pressure. This study investigated the expression of angiotensin AT(1) and AT(2) receptors in the ovine fetal heart, aorta and umbilical artery, and how these receptors are affected by cortisol. Cortisol infusion into the fetus has previously been shown to cause an increase in fetal blood pressure. We hypothesised that this effect of cortisol is mediated by upregulation of the angiotensin AT(1) receptor. Binding studies performed on tissues with intact endothelium demonstrated both receptor subtypes in the fetal aorta and right ventricle, although the latter contained mainly angiotensin AT(2) receptors. In contrast, only angiotensin AT(1) receptors were found in the umbilical artery. Cortisol infusion into fetuses (3 mg/day for 3-5 days) caused a physiological increase in plasma cortisol levels to 29+/-4 nM. This was associated with an increase in systolic pressure (57.8+/-1.7 vs. 52.2+/-1.5 mm Hg, P<0.05), but cortisol had no effect on the density or affinity of angiotensin receptors, nor on the in vitro contractile responses of carotid and umbilical arterial rings to 5-microM angiotensin II. In conclusion, this study has demonstrated differential expression of angiotensin AT(1) and AT(2) receptors in the different regions of the ovine fetal cardiovascular system and that the angiotensin AT(1) receptor is functional. The lack of any effect of low doses of cortisol on these receptors and on the contractility of isolated fetal vessels to angiotensin II suggests cortisol acts by other mechanisms to raise fetal arterial pressure.

Expression of renal and vascular angiotensin II receptor subtypes in children.

Angiotensin II (Ang II) AT1 receptors modulate most of the known physiological functions of Ang II in the kidney and cardiovascular structures. In contrast, the physiological role of AT2 receptors, which are abundantly expressed in fetal tissues, is not clearly defined. The changes that occur in the expression and distribution of AT2 receptors in the kidney and arteries during the first 2 years of life have not been studied. We have localized and characterized the expression of Ang II receptor subtypes, AT1 and AT2, in the kidney, interlobular arteries, thoracic aorta, and middle cerebral artery, in children during their first 2 years of life, using quantitative autoradiography. Renal glomeruli and middle cerebral arteries expressed exclusively AT1 receptors. In contrast, more than 80% of the Ang II receptors expressed in thoracic aorta and interlobular arteries belonged to the AT2 subtype. These findings demonstrate that the expression of Ang II receptor subtypes in different vascular structures in young children varies according to the tissue.

Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding.

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.