Characterization of angiotensin II receptor subtypes in the rat spleen.

Quantitative autoradiography was used to determine the subtype of ANG receptors in the red pulp of the rat spleen. The AT1 antagonist DuP 753 competed for ANG binding with high affinity; binding was abolished by dithiothreitol. The AT2 competitor CGP 42112 A showed lower affinity, and the AT2 competitor PD 123177 did not affect binding at 10(-5) M. These data indicated the presence of only AT1 receptors. AT1 receptor number was similar in immature (2 weeks old) and adult (8 weeks old) rats. Binding was sensitive to guanine nucleotides, suggesting an association with G-proteins. Angiotensin II, at a dose of 10(-7) M, stimulated inositol phosphate formation 33% over control values in spleen from 8-week-old rats. This effect was significantly blocked by 10(-5) M DuP 753. We suggest a possible role of AT1 receptors in the regulation of splenic volume, blood flow, and lymphocyte function.     

Identification of angiotensin II receptor subtypes

We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.     

Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monoclonal antibodies in the rat adrenal gland

Angiotensin II (Ang II), a major regulator of cardiovascular function and body fluid homeostasis, mediates its biological actions via two subtypes of G protein-coupled receptors, termed AT(1) and AT(2). The primary goal of this study was to raise monoclonal anti-peptide antibodies specific to angiotensin AT(1)- and AT(2)-receptor subtypes and to Ang II itself and using these monoclonal antibodies to determine the intraadrenal localization of AT(1) and AT(2) receptors and Ang II in male adult rats. Immunocytochemistry unambiguously demonstrates a regional colocalization of Ang II and angiotensin II receptors in the adrenal gland. The novel antibodies localized Ang II and the AT(1) receptors to the zona glomerulosa of the cortex and to the medulla whereas AT(2) receptors were limited to the medulla. The specificity of immunostaining was documented by pre-adsorption of the antibody with the immunogenic peptide. Our data underscore that AT(1) appears to mediate most of the physiological actions of Ang II in adrenal. Western blot analysis of rat adrenal protein extracts using AT(1) antibody showed a predominant 73-kDa band and a weaker 97-kDa immunoreactive band corresponding to glycosylated forms of the AT(1) receptor. Immunostaining with anti-AT(2) yielded one major immunoreactive band of 73-kDa size and one additional fainter band of 120 kDa. These antibodies may prove of value in unraveling the subcellular localization and intracellular effector pathways of AT(1) and AT(2).     

Identification of two subtypes in the rat type I angiotensin II receptor.

A rat adrenal cDNA library was screened by colony hybridization using a rat cDNA fragment of type I angiotensin II receptor (AT1A) previously isolated from the kidney. Two cDNA clones were identified, designated as AT1B, to have a nucleotide sequence highly homologous to and yet distinct from AT1A. The amino acid sequence of AT1B consists of 359 amino acid residues and has 96% identity with AT1A. No conspicuous difference in the ligand binding characteristics was observed between AT1A and AT1B. The mRNA for AT1B was expressed in many tissues as is the case with AT1A, and most abundantly expressed in the adrenal glands in the Sprague-Dawley rats. The existence of two subtypes in the rat type I angiotensin II receptor might explain the diverse actions of angiotensin II in various tissues.     

Characterization of the angiotensin II receptor

We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.     

Preliminary biochemical characterization of two angiotensin II receptor subtypes

Two angiotensin II receptor subtypes (A and B) are described from rat and human tissues. They have been characterised using specific peptidic and non-peptidic ligands with affinities differing by 1000 fold or more. These subtypes are present in adrenal glomerulosa of both species. Human uterus contains only subtype A, whereas both subtypes are found in rat uterus. Vascular smooth muscle cells in culture express only subtype B. Dithio-threitol totally inhibits binding to subtype B, but enhances the affinity to subtype A. There is a good correlation between the affinities of the selected agonists and antagonists for the two subtypes in the various tissues tested which is a usual requirement for receptor classification.     

Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat.

A simplified and sensitive method for measuring expression levels of type-1 angiotensin II (AT1) receptor subtypes, AT1A and AT1B, was established. The two receptor cDNAs were co-amplified and measured by polymerase chain reaction using primers based on the corresponding receptor subtype genes. Both AT1A and AT1B mRNAs were widely expressed in the rat tissues including adrenal gland, kidney, heart, aorta, lung, liver, testis, pituitary gland, cerebrum and cerebellum. AT1A mRNA was predominantly expressed in the rat tissues examined except adrenal gland and pituitary gland where AT1B mRNA was predominantly expressed. Sodium depletion did not change mRNA levels of AT1A and AT1B in the all tissues. However, both AT1A and AT1B mRNA levels in the heart and aorta were down-regulated by treatment with AT1 specific antagonist, TCV 116. In contrast, AT1B mRNA in the adrenal gland was mainly reduced by the treatment. These results suggest that the expression level of AT1B mRNA in the adrenal gland depends on the activity of the renin-angiotensin-aldosterone system (RAAS) and both receptor subtypes mediate contraction and hypertrophy of the smooth and cardiac muscles via the RAAS.     

Discrimination of two angiotensin II receptor subtypes with a selective agonist analogue of angiotensin II, p-aminophenylalanine6 angiotensin II

Angiotensin II receptor binding sites in rat liver and PC12 cells differ in their affinities for a nonpeptidic antagonist, DuP 753, and p-aminophenylalanine6 angiotensin II. In liver, which primarily contains the sulfhydryl reducing agent-inhibited type of angiotensin II receptor, which we refer to as the AII alpha subtype, DuP 753 displays an IC50 of 55 nM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 8-9 microM. In PC12 cells, which primarily contain the angiotensin II receptor type whose binding affinity is enhanced by sulfhydryl reducing agents (AII beta), DuP 753 displays an IC50 in excess of 100 microM, while p-aminophenylalanine6 angiotensin II displays an IC50 of 12 nM. p-Aminophenylalanine6 angiotensin II binding affinity in liver is decreased in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) suggesting that this analogue is an agonist.     

Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium

CGP 42 112 A and DuP 753 block [125I]-angiotensin II binding in rabbit ventricular myocardial membranes in a clearly biphasic manner, indicating the existence of high- and low-affinity sites for these highly selective agents. Assays using concentrations of either agent large enough to prevent high-affinity binding show that their respective high-affinity sites are distinct, and each corresponds to the low-affinity site of the other. The two receptor subsets, present in nearly equal proportions, are also distinguishable by their different sensitivities to dithiothreitol. These findings afford strong evidence for the existence of two distinct angiotensin II receptors in rabbit myocardium, corresponding to the A and B subtypes recently described in adrenals.     

Differential loss in function of angiotensin II receptor subtypes during tissue storage

In vitro receptor autoradiography was performed on rat brain and kidney sections stored frozen at -20 degrees C for extended time periods (17, 40, 64, 121, 183, 251, and 333 days). The results indicate that prolonged tissue storage has a differential effect upon 125I sar1ile8 angiotensin II binding to AT1 and AT2 receptor sites. Binding at AT1 receptor rich tissues studied (renal medulla, renal cortex, anterior pituitary, ventral hippocampus, spinal trigeminal nucleus, and nucleus of the solitary tract) shows a first order exponential decay pattern. The logarithmic linear regression slope (log(e) specific binding versus time), is significantly different from zero (p<0.05) in all AT1 rich tissues except for nucleus of the solitary tract (p=0.086). There is no detected loss of 125I sar1ile8 angiotensin II binding at the AT2 prominent regions in the superior colliculus, medial geniculate nucleus, and the inferior olivary nucleus. The half lives of AT1 receptors are highly variable, ranging from 36 days in the anterior pituitary to 442 days in the nucleus of the solitary tract, and this might be related to variable stability of AT1A and AT1B receptors. These observations should be taken into account when assessing and comparing AT1 and AT2 receptor subtype densities.     

Localization of angiotensin II receptor subtypes in the rabbit adrenal and kidney

The localization of subtypes of the angiotensin II receptor has been determined by autoradiographic techniques using iodinated angiotensin II and two nonpeptide antagonists that exhibit selective affinities. DuP 753 specifically displaces type 1 sites (AII-1) and PD123177 inhibits only type 2 sites (AII-2). The rabbit adrenal cortex contains predominately AII-1 sites and the few AII-2 sites that are present are nonuniformly distributed. In the rabbit kidney, the fibrous outer sheath contains exclusively AII-2 sites whereas the glomeruli of the renal cortex and the renal medulla exhibit only AII-1 sites.     

Characterization of angiotensin II receptors in the rat fetus

The presence of AII receptors during early and late embryonic development was studied by binding of 125I[Sar1, Ile8] AII to whole mouse blastocysts and membrane-rich fractions from rat conceptuses, 7 to 21 days in gestation. In early mouse embryos there was no detectable binding under a variety of experimental conditions. However, in late gestation rat fetuses, specific and high affinity binding was observed, with a concentration of sites similar in membranes from whole and eviscerated fetuses. Using less than 100 micrograms of membrane protein, binding was time and temperature dependent, maintaining equilibrium from 30 to 120 min at 23 degrees C and it was enhanced by addition of Mg+2 up to 5 mM, EGTA 2 mM and dithiothreitol up to 2.5 mM. Scatchard analysis of the binding data indicated Kd values ranging between 0.7 and 0.9 nM. Binding was first detectable at day 10 (14.3 +/- 2.3 fmol/mg), increasing to 104 +/- 16, 2,625 +/- 168, 5,993 +/- 152 and 5,902 +/- 92 by days 12, 15, 18, and 21 of gestational age, respectively. Since the functional significance of these binding sites depends on the availability of the agonist ligand, acid extracts from eviscerated 10-day-old fetuses were analyzed for the presence of AII. Measurement of AII by radioimmunoassay revealed immunoreactive AII-like material (845 pg/g of tissue), with an elution pattern identical to that of AII standard in a Sephadex G-50 column. This material was bioactive, as demonstrated by its ability to displace 125I[Sar1, Ile8]AII from adrenal glomerulosa membranes, an effect which was abolished by pretreatment of the extract with AII antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)     

Changes in expression of angiotensin receptor subtypes in the rat aorta during development

Quantitative autoradiography was used to characterize angiotensin AT1 and AT2 receptors, in the rat aorta at three developmental ages; embryonic day 18 (E18), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of E18 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT2 (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT1 was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT2 subtype was also present (28%). In pre- and postnatal rats, [125I]Sar1-angiotensin II binding to AT1 receptors was sensitive to GTP gamma S whereas binding to AT2 receptors was not. AT2 receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature.     

The specificity of angiotensin II receptor binding in rat brain

125I-angiotensin II (125I-AII) binding was examined in the hypothalamic-thalamic-septal-midbrain (HTSM) region of HLA-Wistar rats in the presence of CNS-active agents. Angiotensin I, II, and III and saralasin competed for 125 I-AII binding, whereas structurally unrelated peptides such as arginine and lysine vasopressin, oxytocin, LHRH, TRH, bradykinin, and substance P did not. In contrast, ACTH and neurotensin exhibited a weak, dose-dependent competition for 125 I-AII binding. The relative potencies of AII, AI, neurotensin and ACTH were 100:1:0.1:0.05, respectively. Neurotensin and ACTH competition was not additive with AII suggesting interaction at shared binding sites. Most importantly, a wide variety of other CNS active agents such as methyldopa, naloxone, catecholamines, clondidine, and reserpine, failed to inhibit 125 I-AII binding, thus further defining the specificity of the CNS AII receptor.     

Intracrine action of angiotensin II in mesangial cells: subcellular distribution of angiotensin II receptor subtypes AT1 and AT2

Biological effects of angiotensin II (AngII) such as regulation of AngII target genes may be triggered by interaction of AngII with intracellular AngII receptor types 1 and 2 (AT₁ and AT₂), defined as intracrine response. The aim of this study was to examine the presence of AT₁ and AT₂ receptors in nuclear membrane of human mesangial cells (HMCs) and evaluate the possible biological effects mediated by intracellular AT₁ through an intracrine mechanism. Subcellular distribution of AT₁ and AT₂ was evaluated by immunofluorescence and by western blot in isolated nuclear extract. Endogenous intracellular synthesis of AngII was stimulated by high glucose (HG). Effects of HG were analyzed in the presence of candesartan, which prevents AngII internalization. Both receptors were found in nuclear membrane. Fluorescein isothiocyanate (FITC)-labeled AngII added to isolated nuclei produced a fluorescence that was reduced in the presence of losartan or PD-123319 and quenched in the presence of both inhibitors simultaneously. HG induced overexpression of fibronectin and increased cell proliferation in the presence of candesartan, indicating an intracrine action of AngII induced by HG. Results showed the presence of nuclear receptors in HMCs that can be activated by AngII through an intracrine response independent of cytoplasmic membrane AngII receptors.

     

Identification of the angiotensin II receptor in rat mesenteric artery.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.     

Nodose ganglionectomy reduces angiotensin II receptor binding in the rat brainstem

Angiotensin II (Ang II) receptor binding sites in the dorsomedial medulla of intact and unilaterally nodose ganglionectomized rats were identified and characterized using 125I-sarcosine,isoleucine Ang II. This radioligand bound saturably and with high affinity to rat brain homogenates and to sections of rat brainstem. Specific (1 microM angiotensin II displaceable) binding of 125I-sarcosine,isoleucine Ang II was displaced by angiotensin analogues with a potency order similar to that described for angiotensin II receptors. Unilateral nodose ganglionectomy caused a reduction in Ang II receptor binding in the medial solitary tract nucleus, dorsal motor nucleus of the vagus, and area postrema ipsilateral to the lesioned ganglion. This observation suggests that Ang II receptors in the dorsomedial medulla may be located on axon terminals of vagal afferents and cell bodies of vagal efferents.