Evidence of essential disulfide bonds in angiotensin II binding sites of rabbit hepatic membranes. Inactivation by dithiothreitol

Radiolabelled angiotensin II binds to a single class of high-affinity binding sites on purified rabbit hepatic membranes. The binding is specific, reversible and saturable. Displacement studies using angiotensin and various analogs of angiotensin II disclosed a structure-activity profile similar to that found in physiologically relevant angiotensin II receptor sites. Treatment of membranes with the reducing agent, dithiothreitol, cause a significant decrease in the affinity of angiotensin II binding sites for the native ligand. This effect is mimicked by a 15-fold higher concentration of the monosulfhydryl derivative, 2-mercaptoethanol. Kinetic studies also indicated that dithiothreitol increases the rate of dissociation of bound ligand from the membrane without significantly affecting the association rate. In contrast, treatment of membranes with the metal chelators, ethylenediaminetetracetic acid (EDTA) and ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetracetic acid (EGTA), does not affect the binding of radiolabeled angiotensin II. Furthermore, dithiothreitol inhibited the binding of angiotensin II to a solubilized partially purified preparation of angiotensin II-binding protein from the same tissue and also increased the dissociation of bound angiotensin II. This indicates that the effect of the sulfhydryl reagents on the membrane binding sites is the result of a direct alteration of the binding sites rather than a gross modification of the structure of the membrane.     

Cyclic insulin-regulated aminopeptidase (IRAP)/AT4 receptor ligands.

The angiotensin IV receptor (AT4 receptor) is the insulin-regulated aminopeptidase enzyme (IRAP, EC 3.4.11.3). This membrane-spanning enzyme belongs to the M1 family of zinc-dependent metallo-peptidases. It has been proposed that AT4 receptor ligands exert their physiological effects by binding to the active site of IRAP and thereby inhibiting the catalytic activity of the enzyme. The biological activity of a large series of linear angiotensin IV analogs was previously disclosed. Herein, the synthesis and biological evaluation of a series of angiotensin IV analogs, encompassing macrocyclic ring systems of different sizes, are presented. It is demonstrated that disulfide cyclizations of angiotensin IV can deliver ligands with high IRAP/AT4 receptor affinity. One ligand, with an 11-membered ring system (4), inhibited human IRAP and aminopeptidase N (AP-N) activity with similar potency as angiotensin IV but was considerably more stable than angiotensin IV toward enzymatic degradation. The compound provides a promising starting point for further optimization toward more drug-like derivatives. The cyclic constrained analogs allowed us to propose a tentative bioactive conformation of angiotensin IV and it seems that the peptide adopts an inverse gamma-turn at the C-terminal.     

Characterization of the angiotensin II receptor in primary cultures of rat hepatocytes. Evidence that a single population is coupled to two different responses

The angiotensin II receptor of cultured rat hepatocytes was characterized using [3H]angiotensin II as radioligand. Binding at 23 degrees C was rapid (t1/2 = 0.65 min) with equilibrium being reached in 10-12 min. At this time, binding was completely reversible after 20 min (t1/2 = 3.5 min), indicating negligible internalization of the ligand. Analysis of the saturation binding curve showed one population of binding sites with an apparent KD of 8.6 nM and a Bmax of 35 fmol/mg of protein. The time courses of association and dissociation were also consistent with one class of binding sites with an apparent kinetically derived KD of 7.7 nM. The order of potency of different agonists and antagonists to increase cytosolic Ca2+ or phosphorylase a or inhibit the effects of angiotensin II on these parameters was the same as for their mimicry or reversal of angiotensin II inhibition of glucagon-induced cAMP accumulation, and was well correlated with their order of potency to inhibit angiotensin II specific binding. Treatment of cultured hepatocytes with dithiothreitol caused a time- and concentration-dependent inhibition of angiotensin II binding and corresponding alterations of angiotensin II effects on phosphorylase and cAMP. It also inhibited the actions of other hormones on phosphorylase. These results indicate that hepatocytes contain a homogeneous population of angiotensin II receptors that are coupled to two different biological effects apparently mediated by different G-proteins.     

Solubilization and characterization of an angiotensin II binding protein from liver

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II. Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high-affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high-affinity binding sites (Kd = 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure-activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non-physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68 000 by polyacrylamide gel electrophoresis.     

Action of brain cathepsin B,cathepsin D,and high-molecular-weight aspartic proteinase on angiotensins I and II

The action of three previously isolated electrophoretically homogeneous brain proteinases—cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), and high-molecular-weight aspartic proteinase (M_r=90K; EC 3.4.23.−)—on human angiotensins I and II has been investigated. The products of enzymatic hydrolysis have been identified by thin-layer chromatography on Silufol plates using authentic standards and by N-terminal amino acid residue analysis using a dansyl chloride method. Cathepsin D and high-molecular-weight aspartic proteinase did not split angiotensin I or angiotensin II. Cathepsin B hydrolyzed angiotensin I via a dipeptidyl carboxypeptidase mechanism removing His-Leu to form angiotensin II, and it degraded angiotensin II as an endopeptidase at the Val³-Tyr⁴ bond. Cathepsin B did not split off His-Leu from Z-Phe-His-Leu. Brain cathepsin B may have a role in the generation and degradation of angiotensin II in physiological conditions. Special Issue dedicated to Dr. Eugene Kreps.     

Angiotensin II receptor type 1 (AT1) selective nonpeptidic antagonists--a perspective

Hypertension is a major risk factor for human morbidity and mortality through its effects on target organs like heart, brain and kidneys. More intensive treatment for the effective control of blood pressure significantly reduces the morbidity and mortality. The renin angiotensin system (RAS) is a coordinated hormonal cascade of major clinical importance in the regulation of blood pressure. The principal effector peptide of RAS is angiotensin II, which acts by binding to one of the two major angiotensin II receptors AT(1) and AT(2). Angiotensin II through AT(1) receptor mediates vast majority of biologically detrimental actions. Nonpeptidic angiotensin II (AT(1)) antagonists are the most specific means to block the renin angiotensin enzymatic cascade available presently. Majority of AT(1) antagonists are based on modifications of losartan structure, the first clinically used AT(1) antagonist. In this review, a comprehensive presentation of the literature on AT(1) receptor antagonists has been given.     

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.     

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.     

Heme-hemopexin: a 'chronosteric' heme-protein

Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed beta-propeller domains, resembling two thick disks that lock together at a 90 degrees angle. The heme is bound between the two beta-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, (*)NO, and cyanide by detaching His213; however, O(2) induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates (*)NO/O(2) and (*)NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a 'chronosteric' heme-protein.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

Influence of tissue fixation on the binding of (125)I-angiotensin receptor ligands in the rat,mouse and rabbit kidney

Aldehyde fixatives are often used to preserve tissue morphology and thereby aid in the identification of cellular structures expressing a target of interest. However, the effect of fixatives on target detection methods is unpredictable and it is currently unknown whether tissue fixation would allow the accurate detection of angiotensin AT(4) receptors in the kidney. In vitro receptor autoradiography on tissues fixed with 4% paraformaldehyde and 0.5% glutaraldehyde (+/-20% sucrose) had differing effects on the density of (125)I-AT(4) receptor ligand binding without affecting the tissue distribution of ligand binding in the rat and mouse kidney, whereas an increased expression of specific (125)I-AT(4) receptor ligand binding was found in the medulla region of the rabbit kidney. In contrast, such tissue fixation conditions dramatically decreased the renal binding of (125)I-angiotensin II receptor ligands, and altered the distribution of such ligand binding, in all three species. These results suggest that the method of tissue fixation and processing should be used cautiously in angiotensin receptor density measurements but can provide an accurate representation of kidney AT(4) receptor distribution only in the rat and mouse.     

Mechanisms and sites of action of newer angiotensin agonists and antagonists in terms of activity and receptor.

From the myotropic and vasopressor activities of the numerous analogs of angiotensin II, it has been determined that the phenyl group of position 8 possesses the information for biologic response while the aromatic side groups in positions 4 and 6, the guanido group in position 2 and the C-terminal carboxyl are involved in binding to the receptor site. Removal of a side group of the C-terminal phenyalanine yields peptides that bind to the receptor. While many of these have low agonist properties, all have antagonist properties. Modifications in the aromatic side groups affect conformation of the octapeptide. This change may relate to receptor binding but sufficient data are not yet available to determine a correlation pattern. A proposed conformation for angiotensin is given as well as an artist's concept of angiotensin II binding to its membrane receptor utilizing the groups known to be involved in binding. Both angiotensin II and III [des-Asp] angiotensin II stimulate the biosynthesis and release of aldosterone from adrenal glomerulosa cells. Sufficient data are not yet available to determine whether the conversion of angiotensin II to angiotensin III is neccessary for the steroidogenesis activity.     

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.     

The adipose tissue renin-angiotensin system and metabolic disorders: a review of molecular mechanisms

The renin-angiotensin system (RAS) is classically known for its role in regulation of blood pressure, fluid and electrolyte balance. In this system, angiotensinogen (Agt), the obligate precursor of all bioactive angiotensin peptides, undergoes two enzymatic cleavages by renin and angiotensin converting enzyme (ACE) to produce angiotensin I (Ang I) and angiotensin II (Ang II), respectively. The contemporary view of RAS has become more complex with the discovery of additional angiotensin degradation pathways such as ACE2. All components of the RAS are expressed in and have independent regulation of adipose tissue. This local adipose RAS exerts important auto/paracrine functions in modulating lipogenesis, lipolysis, adipogenesis as well as systemic and adipose tissue inflammation. Mice with adipose-specific Agt overproduction have a 30% increase in plasma Agt levels and develop hypertension and insulin resistance, while mice with adipose-specific Agt knockout have a 25% reduction in Agt plasma levels, demonstrating endocrine actions of adipose RAS. Emerging evidence also points towards a role of RAS in regulation of energy balance. Because adipose RAS is overactivated in many obesity conditions, it is considered a potential candidate linking obesity to hypertension, insulin resistance and other metabolic derangements.     

The adipose tissue renin-angiotensin system and metabolic disorders: a review of molecular mechanisms

The renin-angiotensin system (RAS) is classically known for its role in regulation of blood pressure, fluid and electrolyte balance. In this system, angiotensinogen (Agt), the obligate precursor of all bioactive angiotensin peptides, undergoes two enzymatic cleavages by renin and angiotensin converting enzyme (ACE) to produce angiotensin I (Ang I) and angiotensin II (Ang II), respectively. The contemporary view of RAS has become more complex with the discovery of additional angiotensin degradation pathways such as ACE2. All components of the RAS are expressed in and have independent regulation of adipose tissue. This local adipose RAS exerts important auto/paracrine functions in modulating lipogenesis, lipolysis, adipogenesis as well as systemic and adipose tissue inflammation. Mice with adipose-specific Agt overproduction have a 30% increase in plasma Agt levels and develop hypertension and insulin resistance, while mice with adipose-specific Agt knockout have a 25% reduction in Agt plasma levels, demonstrating endocrine actions of adipose RAS. Emerging evidence also points towards a role of RAS in regulation of energy balance. Because adipose RAS is overactivated in many obesity conditions, it is considered a potential candidate linking obesity to hypertension, insulin resistance and other metabolic derangements.     

Angiotensin 'antipeptides': (-)messenger RNA complementary to human angiotensin II (+)messenger RNA encodes an angiotensin receptor antagonist

(-)mRNA complementary to human angiotensin II (+)mRNA encodes the 'antipeptide' Glu-Gly-Val-Tyr-Val-His-Pro-Val which is structurally related to angiotensin II. Angiotensin II 'antipeptide' (antiANG II) and the desglutamyl heptapeptide (antiANG III) are Type I antagonists which inhibit the contractile action of angiotensin at smooth muscle receptors by binding to a negative modulatory site on the angiotensin receptor which is distinct from the angiotensin binding site. These findings may illustrate that the inhibitory binding site on the angiotensin receptor exists to accomodate a naturally occurring inhibitor(s), which is encoded by the DNA strand complementary to that encoding angiotensin II.     

Asparatic acid 221 is critical in the calcium-induced modulation of the enzymatic activity of human aminopeptidase A

Aminopeptidase A (APA) plays an important role in the regulation of blood pressure by mediating angiotensin II degradation in the renin-angiotensin system. The Ca2+-induced modulation of enzymatic activity is the most characteristic feature of APA among the M1 family of aminopeptidases. In this study, we used site-directed mutagenesis for any residues responsible for the Ca2+ modulation of human APA. Alignment of sequences of the M1 family members led to the identification of Asp-221 as a significant residue of APA among the family members. Replacement of Asp-221 with Asn or Gln resulted in a loss of Ca2+ responsiveness toward synthetic substrates. These enzymes were also unresponsive to Ca2+ when peptide hormones, such as angiotensin II, cholecystokinin-8, neurokinin B, and kallidin, were employed as substrates. These results suggest that the negative charge of Asp-221 is essential for Ca2+ modulation of the enzymatic activity of APA and causes preferential cleavage of acidic amino acid at the N-terminal end of substrate peptides.