Specific Binding Sites For Angiotensin Research Articles

Gene knockout of insulin-regulated aminopeptidase: Loss of the specific binding site for angiotensin IV and age-related deficit in spatial memory

The AT 4 ligands, angiotensin IV and LVV-hemorphin 7, elicit robust effects on facilitating memory by binding to a specific site in the brain historically termed the angiotensin AT 4 receptor. The identification of the AT 4 receptor as insulin-regulated aminopeptidase (IRAP) is controversial, with other proteins speculated to be the target(s) of these peptides. In this study we have utilized IRAP knockout mice to investigate IRAP in the brain. We demonstrate that the high-affinity binding site for angiotensin IV is absent in IRAP knockout mice brain sections in parallel with the loss of IRAP immunostaining, providing irrefutable proof that IRAP is the specific high-affinity binding site for AT 4 ligands. However, our characterization of the behavioural phenotype of the IRAP knockout mice revealed a totally unexpected finding. In contrast to the acute effects of IRAP inhibitors in enhancing memory, deletion of the IRAP gene resulted in mice with an accelerated, age-related decline in spatial memory that was only detected in the Y maze paradigm. Moreover, no alterations in behaviour of the IRAP knockout mice were observed that could assist in elucidating the endogenous substrate(s). Our results highlight the importance of analysing the behavioural phenotype of knockout mice across different ages and in distinct memory paradigms.

A globin fragment, LVV-hemorphin-7, induces [3H]thymidine incorporation in a neuronal cell line via the AT4 receptor.

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.

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

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

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.

Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells

This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37°C allowed the calculation of a kinetic K d of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37°C for 90 min indicated the presence of a single high-affinity site ( K d = 3.87 ± 0.60 nM), saturable and abundant ( B max = 9.64 ± 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1–7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2′-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-( N α-benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 μM. This binding site is, therefore, distinct from angiotensin II receptors, AT 1 and AT 2. Addition of the divalent cations Mg 2+, Mn 2+ or Ca 2+ to the incubation buffer resulted in 90–95% inhibition of the [ 125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites ( B max = 17.8 ± 2.5 pmol/mg protein), with no change in affinity ( K d = 5.7 ± 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTPγS, had no effect on [ 125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.

Characterization of a binding site for angiotensin IV on bovine aortic endothelial cells

We have characterized a specific binding site for angiotensin IV on bovine aortic endothelial cell membranes. Pseudo-equilibrium studies at 37°C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity ( K d=0.71; average of two experiments that yielded values of 0.71 and 0.72 nM). The binding site is saturable and relatively abundant with a maximal binding capacity of 0.59 pmol/mg protein (average of two experiments that yielded values of 0.39 and 0.78 pmol/mg of protein). Non-equilibrium kinetic analyses at 37°C revealed a calculated K d of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site is pharmacologically distinct from the classic angiontensin receptors AT 1 or AT 2. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar 1, Val 5, Ala 8]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2′-( H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl)methyl]-3 H-imidazo [4,5-β]pyridine H 2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacety)4,5,6,7-tetrahydro-1 H-imidazo[4,5- c]pyridine-6-carboxylic acid). A few unrelated hormones (bradykinin, [Arg 8]vasopressin, endothelin-1, atrial natriuretic factor, isoproterenol and adrenocorticotropic hormone) were unable to inhibit any 125I-angiotensin IV binding. The affinities of different structural analogues of angiotensin IV revealed that the N-terminal position is critical for receptor recognition and the C-terminal proline is also important. GTPγS and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg 2+ and Ca 2+ were shown to diminish the binding of 125I-angiotensin IV. Cross-linking of 125I-angiotensin IV to bovine aortic endothelial cell membranes in the presence of disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a major band of 186±12 kDa. The presence in high concentration of this angiotensin binding site an aortic endothelial cells suggests the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.

A specific binding site recognizing a fragment of angiotensin II in bovine adrenal cortex membranes

We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37°C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity ( K d = 0.24 ± 0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37°C revealed a calculated kinetic K d of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT 1 or AT 2. Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3–7) (Val-Tyr-Ile-His-Pro) > angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) ≥ angiotensin II-(4–7) (Tyr-Ile-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > angiotensin II-(1–6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4–8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3–6) (Val-Tyr-Ile-His), angiotensin II-(4–6) (Tyr-Ile-His), L-158,809 5,7-dimethyl-2-ethyl-3-[2′-(1 H-tetrazol-5-yl)[1,1′-biphenyl]-4-yl)methyl]-3 H- imidazo[4,5- β]pyridine H 2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)4,5,6,7-tetrahydro-1 H-imidazo[4,5- c]pyridine-6- carboxylic acid). The divalent cations Mg 2+ and Ca 2+ were shown to diminish the binding of 125I-angiotensin IV to bovine adrenal cortex membranes. The presence of a high concentration of this binding site in the adrenal cortex suggests the possibility that it may play an important, yet unrecognized role, in the renin-angiotensin system.

Presence of Angiotensin II Immunoreactivity in the Ovary of the Rainbow Trout, Oncorhynchus mykiss

Renin-angiotensin system is present in mammalian ovarian follicular fluid and follicles. The demonstration of specific binding sites for angiotensin II in the follicular structures of rats suggests that angiotensin II may be related to the regulation of steroid biosynthesis in the mammalian ovary. Little is known about the presence and the action of angiotensin II in nonmammalian ovaries. An immunocytochemical investigation of angiotensin II in developing ovarian follicles of trout has been carried out. The specific antiserum was raised against (Val5)-angiotensin II of the trout. There was strong immunoreactivity in the ooplasm of endogenous vitellogenic follicles. Angiotensin II immunoreactive material was present in the follicular cells, but not within oocytes at the end of vitellogenesis. Immunocytochemically angiotensin II-like material in the ovary of rainbow trout fluctuates during the cycle of development.

Renal angiotensin receptor mapping in obese spontaneously hypertensive rats.

Obese spontaneously hypertensive rats (SHR) develop nephropathy with severe proteinuria, but lean littermates do not develop renal disease. Intrarenal angiotensin has been suggested to contribute to nephropathy in other experimental models. We examined the regulation of angiotensin receptors as a reflection of target tissue response to possible changes in the renin-angiotensin system. We visualized angiotensin receptors in kidneys of 6-8-month-old obese SHR and their lean littermates. Both obese and lean rats were hypertensive as determined by tail-cuff or by direct measurement. Histologic studies showed early glomerular sclerosis in obese but not lean rats. Autoradiographic visualization of angiotensin receptor binding sites in both obese and lean SHR showed glomeruli and medullary rays having the highest levels of binding with additional diffuse labeling in cortex and outer medulla. In obese rats, binding was reduced relative to lean littermates, particularly in the medulla, while intense binding in glomeruli was preserved. Loss of receptors did not reflect tissue damage, since the medulla showed no pathological changes. Biochemical assays of the binding of subtype-selective antagonists to 125I-angiotensin sites in intact sections showed that both losartan-sensitive and PD 123319-sensitive sites were decreased in nephrotic obese rats. We conclude that specific binding sites for angiotensin are decreased in obese SHR with early glomerular sclerosis, suggesting that angiotensin receptors may be regulated by pathogenic processes in this model of renal disease.

Intrarenal localization of angiotensin II specific binding in rat fetuses.

Specific binding sites for angiotensin II were localized in the developing rat kidney (18th day of pregnancy and immediately before birth) by autoradiography using [125I]-ileu-5-angiotensin II either perfused in vivo through the fetal aorta or added in vitro to frozen sections in an incubation mixture. Specific binding was localized in the walls of the afferent and efferent arterioles, in the intraglomerular cells and in the peritubular arterioles of the subcapsular cortical zone. The immunohistochemical analysis, carried out on receptors saturated with unlabelled angiotensin II perfused through the mother's aorta, confirmed the autoradiographical localization. Antisera against ileu-5-angiotensin II were used in the indirect immunofluorescence technique and in the PAP method. Immunolocalization of angiotensin II was also found in the proximal tubule and in the thick ascending limb of Henle's loop.

Regulation of angiotensinogen production by angiotensin II analogues.

Specific binding sites for angiotensin II (Ang II) were identified in a human hepatoma cell line, HepG2. Binding of [125I]-Sar1 Ang II to these cells showed a high-affinity site with a Kd of 2.4 +/- 0.2 nmol/l. This specific binding was not changed during the cell cycle and showed no alteration after 24 h of treatment with Sar1-Ang II (10(-8) mol/l). Exposure of HepG2 cells to the Ang II agonist Sar1-Ang II caused a dose-dependent decrease in angiotensinogen production. The maximal inhibitory effect was at a dose of 10(-6) mol/l Sar1-Ang II which elicited 67% inhibition of angiotensinogen production after 24 h (control: 2.015 +/- 0.5 micrograms angiotensinogen/mg DNA; Sar1-Ang II 10(-6) mol/l: 0.68 +/- 0.03 micrograms angiotensinogen/mg DNA). Fifty per cent inhibition was obtained at a dose of 10(-9) mol/l Sar1-Ang II. Angiotensin II had a less marked effect, showing maximal inhibition of 40%. This study shows that the HepG2 cells possess specific Ang II binding sites and that Ang II analogues induce a dose-dependent inhibition of angiotensinogen production in cell culture.

Autoradiographic localization and characterization of angiotensin II binding sites in the spleen of rats and mice

Specific binding sites for angiotensin II (Ang II) were localized in the red pulp of the spleen of rats and mice by quantitative autoradiography using 125I-Sar 1-Ang II as a ligand. In the rat, the binding was saturable and specific, and the rank order for Ang II derivatives as competitors of 125I-Sar 1-Ang II binding correlates well with their affinity for Ang II receptors in other tissues. Kinetic analysis in the rat spleen revealed a single class of binding sites with a K D of 1.11 nM and a B max value of 81.6 fmol/mg protein. Ang II binding sites were also localized on isolated rat spleen cells with similar affinity but with much lower B max, 9.75 fmol/mg protein. Ang II receptors were not detected in thymus sections from rats or mice, or on isolated rat thymocytes. The binding sites described here might represent a functional Ang II receptor with a role in the regulation of splenic volume and blood flow and in the modulation of the lymphocyte function.

Characterization of angiotensin II receptors in the anterior pituitary gland

Specific and high affinity binding sites for angiotensin II were demonstrated in the anterior pituitary gland by binding studies with [ 125I]iodoangiotensin II. The binding properties of the pituitary receptors were similar to those of angiotensin II receptors present in the adrenal gland. The concentration of binding sites in rat anterior pituitary (293 ± 50 fmoles/mg protein) was less than in the adrenal gland, but was much greater than in smooth muscle. Angiotensin II receptors were identified in the anterior pituitary tissue of mature and immature animals of both sexes, and in species including rat, rabbit and dog. No binding of angiotensin II was detected in posterior pituitary homogenates, or in GH 3 pituitary tumor cells. Collagenase-dispersed anterior pituitary cells also contained specific binding sites for angiotensin II, with equilibrium binding constant ( K a) of 3.6 × 10 9 M −1. The presence of specific high-affinity angiotensin II receptor in the anterior pituitary gland provides a mechanism by which angiotensin-like peptides could modulate the process of pituitary hormone secretion.

Angiotensin II binding to mammalian brain membranes.

High affinity binding sites for angiotensin II in bovine and rat brain membranes have been identified and characterized using monoiodinated Ile5-angiotensin II of high specific radioactivity. Degradation of labeled and unlabeled peptide by washed brain particulate fractions was prevented by adding glucagon to the final incubation medium and including a proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) in preincubation and incubation procedures. 125I-Angiotensin II binding can be studied using either centrifugation or filtration techniques to separate tissue-bound radioactivity. 125I-Angiotensin II binding to calf brain membranes is saturable and reversible, with a dissociation binding constant of 0.2 nM at 37 degrees. A similar binding constant is found in rat brain membranes. Analogues and fragments of angiotensin II compete for these brain binding sites with potencies which correlate with both their in vivo potencies and their binding inhibition protencies at adrenal cortex angiotensin II receptors. Angiotensin I is 1 to 2 orders of magnitude weaker than angiotensin II; the 3-8 hexapeptide and 4-8 pentapeptide are much weaker still. (desAsp1) angiotensin II (angiotensin III) is slightly more potent than angiotensin II, as are several antagonists of angiotensin II with aliphatic amino acids substituted at position 8. In calf brain 125I-angiotensin II binding is restricted almost exclusively to the cerebellum (cortex and deep nuclei). In rat brain, angiotensin II binding is highest in the thalamus-hypothalamus, midbrain, and brainstem, areas which are believed to be involved in mediating angiotensin II-induced central effects. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in rat and bovine brain and suggest a physiological role for angiotensin peptides in the central nervous system.

Properties of Angiotensin II Receptors in the Bovine and Rat Adrenal Cortex

Abstract Receptors for angiotensin II have been identified and characterized in bovine and rat adrenal cortex by binding studies with tritiated and monoiodinated angiotensin II. The angiotensin II binding sites of bovine adrenal cortex homogenate were enriched severalfold in a microsomal membrane fraction together with alkaline phosphatase, adenylate cyclase, 5'-nucleotidase and 21β-hydroxylase. Uptake of angiotensin II by adrenal cortex particles and adrenal cells was rapid, reaching equilibrium at 15 to 30 min in the presence of 0.2 to 1.5 nm angiotensin II. The equilibrium association constant of angiotensin II for bovine adrenal cortex receptors was 0.5 nm-1 at 22°, and that for rat adrenal particles was significantly higher. Dissociation of bound angiotensin II from adrenal particles and cells was also rapid, with initial half-time of 13 to 23 min. Angiotensin II released from adrenal cortex binding sites at low pH retained activity in subsequent binding studies, whereas angiotensin II in the incubation medium was rapidly inactivated. Degradation of angiotensin II by adrenal cortex particles was partially inhibited by addition of unrelated peptides including glucagon and insulin, by reducing agents such as dithiothreitol, and by reduced temperature. Fragments and analogues of angiotensin II showed binding-inhibition potencies which correlated with their biological activities in vivo. In particular, competitive antagonists, such as the (Sar1, Ala8) derivative of angiotensin II, inhibited angiotensin II binding in proportion to their antagonistic activity in vivo. This system provides a simple and rapid method for evaluation of the competitive binding activity of angiotensin agonists and antagonists in vitro. In contrast to angiotensin II, the decapeptide angiotensin I exhibited relatively low affinity for angiotensin II binding sites in competitive studies, and direct binding studies with monoiodinated 125I-angiotensin I showed considerably lower uptake than that of angiotensin II. The uptake of labeled angiotensin I and II by adrenal medulla homogenates was much lower than that of adrenal cortex particles, and again angiotensin II showed higher binding affinity than angiotensin I. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in bovine and rat adrenal cortex and suggest a plasma membrane location for the angiotensin II receptors.

Angiotensin receptors in smooth muscle cell membranes.

THE currently accepted hypothesis of the mechanism of action of peptide hormones affecting smooth muscle tone includes an initial obligatory step of binding to specific receptors in the cell plasma membranes. We have shown the presence of specific angiotensin receptors in microsomal membranes of rabbit aorta1. Here we report the isolation of a membrane fraction derived from the plasma membrane possessing specific binding sites for angiotensin, and the structural requirements of the angiotensin molecule for its binding.