Binding Of Angiotensin Research Articles

Binding of the antagonist [ [formula omitted]]candesartan to angiotensin II AT 1 receptor-tranfected Chinese hamster ovary cells

Binding of the non-peptide angiotensin II AT 1 antagonist [ 3 H ](2-ethoxy-1-[(2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1 H-benzimidazoline-7-carboxylic acid ([ 3 H ]candesartan) to human angiotensin II AT 1 receptor-transfected Chinese hamster ovary (CHO-AT 1) cells was inhibited to the same extent by angiotensin II and non-peptide angiotensin II AT 1 antagonists. No binding was observed in control CHO-K 1 cells. Dissociation was slow (k −1=0.0010±0.0001 min −1) after removal of the free [ 3 H ]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT 1 antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin II responses also recovered more rapidly in the presence of 2- n-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [ 3 H ]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant ( K d=51±8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor.

Erythropoietin upregulates angiotensin receptors in cultured rat vascular smooth muscle cells.

Plasma renin is not elevated in recombinant human erythropoietin (rhEPO)-induced hypertension but angiotensin converting enzyme inhibitors reduce blood pressure in both human and animal studies. Since rhEPO elevates renin and angiotensinogen messenger RNAs in angiotensin II target tissues such as the aorta, we explored the actions of rhEPO on renin-angiotensin system-related gene transcription of cultured rat vascular smooth muscle cells. To separate direct actions of rhEPO from those mediated secondarily by potential activation of the renin-angiotensin system, vascular smooth muscle cells were cultured with rhEPO and enalapril to inhibit the angiotensin converting enzyme and losartan to inhibit angiotensin II type 1 receptors. Vascular smooth muscle cells cultured with rhEPO (6-8 units/ml) demonstrated elevations (40-120%) in messenger RNAs of the renin-angiotensin system (renin, angiotensinogen, angiotensin receptor types 1 and 2) and increased levels of several messenger RNAs known to respond to angiotensin II (transforming growth factor-beta, insulin-like growth factor-II, epidermal growth factor, c-fos and platelet-derived growth factor). In contrast, cells cultured in the presence of rhEPO and enalapril or losartan showed elevations of messenger RNA for only the two types of angiotensin II receptor. This increase was higher than that obtained when cells were cultured with rhEPO or either antagonist alone. The increase in specific binding of angiotensin II to cells cultured in the presence of rhEPO and enalapril or rhEPO and losartan paralleled the changes in receptor messenger RNA. rhEPO exerts its primary action on vascular smooth muscle cells via an increase in angiotensin receptor messenger RNA, resulting in a parallel increase in angiotensin II receptor expression. We suggest that increased receptor expression secondarily mediates the expression of other renin-angiotensin system messenger RNAs, which leads to angiotensin II-responsive gene transcription. The elevation in angiotensin II receptors, as observed in response to rhEPO, may provide a mechanism by which other forms of renin-dependent hypertension are initiated.

Angiotensin IV receptor-mediated activation of lung endothelial NOS is associated with vasorelaxation.

The hexapeptide angiotensin (ANG) IV, a metabolic product of ANG II, has been reported to play a functional role in the regulation of blood flow in extrapulmonary tissues. Here, we demonstrate that ANG IV-specific (AT4) receptors are present in porcine pulmonary arterial endothelial cells (PAECs) and that the binding of ANG IV to AT4 receptors can be blocked by its antagonist divalinal ANG IV but not by the ANG II-, AT1-, and AT2-receptor blockers [Sar1,Ile8]ANG II, losartan, and PD-123177, respectively. ANG IV significantly increased endothelial cell constitutive nitric oxide synthase (ecNOS) activity (P < 0.05) as well as cellular cGMP content (P < 0. 001). Western blot analysis revealed that ecNOS protein expression was comparable in control and ANG IV-stimulated cells. Divalinal ANG IV but not [Sar1,Ile8]ANG II, losartan, or PD-123177 inhibited the ANG II- and ANG IV-stimulated increases in ecNOS activity and cGMP content in PAECs. Incubation in the presence of N-nitro-L-arginine methyl ester (L-NAME) or methylene blue but not of indomethacin significantly diminished ANG IV-stimulated as well as basal levels of cGMP (P < 0.001). Similarly, in situ studies with precontracted porcine pulmonary arterial rings showed that ANG IV caused an endothelium-dependent relaxation that was blocked by L-NAME or methylene blue. Collectively, these results demonstrate that ANG IV binds to AT4 receptors, activates ecNOS by posttranscriptional modulation, stimulates cGMP accumulation in PAECs, and causes pulmonary arterial vasodilation, suggesting that ANG IV plays a role in the regulation of blood flow in the pulmonary circulation.

Angiotensin IV AT4-receptor system in the rat kidney.

Angiotensin IV, [[des-Asp1,Arg2]ANG II or ANG-(3-8)], has been shown to preferentially bind to a novel angiotensin binding site (AT4 receptor). The cellular location and function of this receptor in the rat kidney is unknown. Autoradiography localized AT4 receptors to the cell body and apical membrane of convoluted and straight proximal tubules in the cortex and outer stripe of the outer medulla. ANG IV (0.1 pM-1 microM) elicited a concentration-dependent decrease in transcellular Na+ transport (as measured by proximal tubule O2 consumption rates) in fresh suspensions of control or nystatin-stimulated (bypasses rate-limiting step of apical Na+ entry) rat proximal tubules. The inhibitory effect of 1 pM ANG IV was unaltered by either 1 microM losartan (AT1-receptor antagonist) or 1 microM PD-123319 (AT2-receptor antagonist) and yet was abolished by 1 microM divalinal-ANG IV (AT4-receptor antagonist) or ouabain pretreatment. These results demonstrate that the kidney AT4-receptor system is localized to the proximal tubule and suggests that one potential biological role of this system is in the regulation of Na+ transport by inhibiting a ouabain-sensitive component of Na(+)-K(+)-adenosinetriphosphatase activity in the rat.

Angiotensin II- and IV-induced changes in cerebral blood flow: Roles of AT 1 AT 2, and AT 4 receptor subtypes

Our laboratory has previously reported the discovery of a unique angiotensin binding site (termed AT 4) specific for angiotensin IV (AngIV) in cultured vascular endothelial and smooth muscle cells. The present investigation employed laser-Doppler flowmetry to examine the effect of angiotensin II (AngII) and AngIV stimulation of these receptors on cerebral microcirculation in anesthetized Sprague–Dawley rats. Internal carotid artery infusion of AngII at a low dose (0.1 pmol min −1 ) revealed a 23% reduction in cerebral blood flow (CBF), while the infusion of AngIV increased CBF in a dose-dependent fashion with the highest dose (100 pmol min −1 ) resulting in an elevation of 30%. In a second experiment separate groups of rats were pre-treated with the AT 1 receptor subtype antagonist DuP 753 (Losartan), the AT 2 receptor subtype antagonist PD123177, or a newly synthesized AT 4 receptor subtype antagonist Divalinal-AngIV (Divalinal), followed by AngII or AngIV for the purpose of determining which angiotensin receptor subtype is responsible for mediating these AngII- and AngIV-induced responses. Pre-treatment with Losartan completely blocked subsequent AngII-induced reductions in CBF, while both PD123177 and Divalinal failed to inhibit this response. In contrast, significant increases in CBF were measured due to AngIV stimulation following pre-treatment with Losartan and PD 123177, while Divalinal abolished this AngIV-induced response. These results suggest that AngII and IV play opposite roles in cerebral microcirculation, i.e., the AT 1 receptor subtype mediates AngII-induced reductions in CBF, while the AT 4 receptor subtype regulates increases in CBF.

Action and binding of the endothelin antagonist bosentan on astrocytes of cultured rat central nervous system. Electrophysiological and autoradiographic studies

Our autoradiographic studies demonstrate that astrocytes in explant cultures of rat central nervous system possess binding sites for the first orally active, mixed, nonpeptide endothelin receptor antagonist [ 3H]bosentan. Binding of [ 3H]bosentan was inhibited by unlabelled bosentan and endothelin-1 at high concentrations, suggesting specific binding of the antagonist. Electrophysiological studies have revealed that bosentan reversibly blocked the depolarizations by endothelin but not by angiotensin II, indicating that the antagonist specifically antagonizes the action of endothelin on the glial membrane. This is consistent with biochemical studies from other laboratories demonstrating that bosentan did not interfere with binding of angiotensin II. The availability of bosentan, a potent and selective endothelin receptor antagonist should help to elucidate the role of endothelin on astrocyte function.

Tissue-Specific Regulation of Type 1 Angiotensin II Receptor mRNA Levels in the Rat

Most of the biological effects of the renin-angiotensin system are mediated by the binding of angiotensin II (Ang II) to the type 1 Ang II (AT1) receptor, the predominant receptor subtype present after fetal life. To study tissue-specific regulation of the expression of the AT1 receptor in the rat, we altered activity of the renin-angiotensin system by feeding rats a low (0.07% NaCl), normal (0.3% NaCl), or high (7.5% NaCl) salt chow for 14 days; infusing Ang II (200 ng/kg per minute IP) or vehicle for 7 days; and administering an angiotensin-converting enzyme inhibitor (captopril, 100 mg/dL in the drinking water) or vehicle for 7 days. Renin, angiotensinogen, and total AT1 receptor mRNA levels were measured by slot-blot hybridization with cRNA probes, and AT1 receptor subtypes (A and B) were measured by reverse transcription-polymerase chain reaction in the presence of a cRNA internal standard. Plasma renin concentration and renal renin, renal and hepatic angiotensinogen, and hepatic AT1 receptor mRNA levels were all inversely related to salt intake; in contrast, renal AT1 receptor mRNA levels were significantly lower in rats fed low salt, a difference that was exclusively due to a decrease in the AT1A subtype. This difference did not appear to be mediated by a change in the circulating levels of Ang II, because Ang II infusion reduced plasma renin concentration and renal renin mRNA with no effect on either angiotensinogen or AT1 receptor mRNA levels in kidney or liver, renal Ang II receptor density (determined by in situ autoradiography) decreased, presumably via a posttranscriptional mechanism. Similarly, inhibition of Ang II generation with captopril increased plasma renin concentration and renal renin mRNA levels without altering renal or hepatic angiotensinogen mRNA or renal AT1 receptor mRNA levels. Thus, AT1 receptor gene expression is regulated in a tissue-specific manner that is distinct from other components of systemic and local renin-angiotensin systems and that appears to be mediated by a mechanism other than through changes in the circulating levels of Ang II.

Molecular recognition of peptide and non-peptide ligands by the extracellular domains of neurohypophysial hormone receptors

This study was designed to ascertain whether the extracellular loops of vasopressin/oxytocin receptors bind ligands and, if so, to locate the molecular determinants of this ligand-receptor interaction. Ligand-binding studies were employed using a rat liver V1a vasopressin receptor preparation and both peptide and non-peptide receptor ligands. Synthetic peptides corresponding to defined regions of the extracellular surface of the neurohypophysial hormone receptors recognized radioligands. These receptor mimetics inhibited the binding of radioligands to the V1a receptor with apparent affinities (pKi) ranging from 3.1 to 6.75. The same mimetics had no effects on the binding of angiotensin II to the rat AT1 receptor, indicating specificity for V1a receptor ligands. A mimetic peptide (DITYRFRGPDWL) of the first extracellular loop (ECII) of the V1a vasopressin receptor also inhibited vasopressin-stimulated, but not angiotensin II-stimulated, glycogen phosphorylase in isolated rat hepatocytes. In contrast, scrambled ECII mimetics displayed greatly reduced affinity for vasopressin. In addition, the role of peptide side-chain versus main-chain atoms in the binding of ligands by vasopressin receptors was addressed using retro-inverso peptide mimetics. Our findings indicate a precise orientation of the extracellular receptor surface (particularly the ECII domain) which facilitates the initial 'capture' of both peptide and non-peptide ligands. Moreover, the data indicate that the main-chain atoms of both a major binding-site determinant in the first extracellular loop of the receptor and the neurohypophysial hormones contribute significantly to the ligand-receptor interaction. These findings also suggest that soluble receptor-binding domains have therapeutic potential.

Angiotensin Receptors in Myometrium and Myometrial Vessels from Uteri of Women during the Follicular and Luteal Phases of the Menstrual Cycle and in Late Pregnancy

1. Receptors for angiotensin II were identified and characterized in membrane fractions from myometrial samples obtained at non-pregnant hysterectomy in women of reproductive age. Specific binding was also measured in paired samples of vascular and "vessel-free' myometrium. 2. A single class of high-affinity receptor sites was identified in the total myometrial preparations (n = 10), with a median equilibrium dissociation constant (Kd) of 0.122 nmol/l (range 0.065-0.465 nmol/l) and concentration of receptor sites (Bmax) of 149 fmol/mg protein (range 105-435 fmol/mg). Receptor characterization studies using losartan (an angiotensin type 1 receptor antagonist) and PD123177 (an angiotensin type 2 receptor antagonist) showed the myometrium to contain almost entirely type 2 receptors. 3. The median Kd and Bmax for specific angiotensin II binding in isolated myometrial vessel homogenates were 0.086 nmol/l (range 0.060-0.433) and 260 fmol/mg protein (range 197-737 fmol/mg) respectively. Vascular specific binding density was always higher in dissected out myometrial vessels than in paired vessel-free myometrium (median 372 compared with 120 fmol/mg protein; n = 20; P < 0.001). The specific binding in the paired samples was strongly correlated (r = 0.8904, P < 0.0001). 4. The specific binding of 125I-labelled angiotensin II in "vessel-free' myometrium was higher in samples from the follicular phase than in samples from the luteal phase (median 144 compared with 37 fmol/mg; P < 0.02). A similar trend was found in the vessels themselves, but this failed to reach statistical significance (459 compared with 225 fmol/mg; P = 0.051). 5. It is suggested that the down-regulation of the angiotensin type 2 receptors in the luteal (secretory) phase is a preparation for pregnancy, removing an inhibitory factor to uterine growth and vascularization and allowing greater prostacyclin synthesis.

Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells

Recent studies have been reported indicating that angiotensin II may potentiate neointimal formation. In the present study, we examined the antagonistic effect of tranilast on angiotensin II. Losartan was used as the reference compound. First, tranilast inhibited the angiotensin II-induced contraction of rabbit aortic strips in a noncompetitive manner (pD′ 2 = 3.7), whereas it had little effect on the contraction induced by noradrenaline or endothelin-1. Second, tranilast inhibited the binding of 125I-labeled angiotensin II to angiotensin AT1 receptors in rat liver membranes with an IC 50 value of 289 μM. Finally, functional antagonism of tranilast (100 and 300 μM) was demonstrated by its blockade of angiotensin II (10 −8M)-induced 45Ca 2+-efflux from human vascular smooth muscle cells (VSMC). However, tranilast (30–300 μM) exerted no influence on PDGF-induced formation of inositol triphosphates which cause an increase in [Ca 2+] i in human VSMC. The antagonistic activity of tranilast towards angiotensin II may be involved in part in preventing restenosis after percutaneous transluminal coronary angioplasty (PTCA).

Identification of atypical (non-AT 1, non-AT 2) angiotensin binding sites with high affinity for angiotensin I on IEC-18 rat intestinal epithelial cells

Specific high-affinity ( Kd = 3.4 nM) binding sites for 125I-labelled angiotensin I ([ 125I]Ang I) were identified on an epithelial cell line (IEC-18) derived from the rat small intestine. The sites, which also have high affinity for Ang II, are insensitive to both AT 1- and AT 2-specific angiotensin receptor antagonists, The rank order of potency with which various angiotensin peptides inhibited[ 125I]Ang I binding to the cells (Ang I ≥ Ang II > Ang(1–7) > [Sar 1,Ile 8]-Ang II > Ang(3–8) > Ang III) also distinguishes these sites from AT 1 and AT 2 angiotensin receptors.

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.

Mutagenesis and the Molecular Modeling of the Rat Angiotensin II Receptor (AT1)

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

Autoradiographic identification of brain angiotensin IV binding sites and differential c-Fos expression following intracerebroventricular injection of angiotensin II and IV in rats

A unique angiotensin binding site specific for the hexapeptide, angiotensin II(3–8) (AngIV), has been previously reported by our laboratory in the guinea pig brain and is presently described in the rat brain. This angiotensin receptor subtype has been termed AT 4 and is prominently distributed in cerebral cortex, piriform cortex, hippocampus, habenulae, colliculi, septum, periaqueductal gray, several thalamic nuclei, the arcuate nucleus of the hypothalamus and cerebellum. In the second part of the present investigation, separate groups of rats received i.c.v. injections of angiotensin II (AngII), AngIV or artificial cerebrospinal fluid (aCSF) and were euthanized 2 h later for the purpose of evaluating for brain c-Fos expression. After i.c.v.-injected AngIV, Fos-like immunoreactivity was present in the hippocampus and piriform cortex. This immunoreactivity was unaffected by i.c.v. pretreatment with the AT 1 angiotensin receptor antagonist DuP 753 (losartan) or the AT 2 receptor ligand PD123177 but was blocked by the AT 4 angiotensin receptor antagonist, divalanal-AngIV. I.c.v. injection of AngII resulted in Fos-like immunoreactivity in the dorsal third and lateral ventricles, subfornical organ, lateral hypothalamus and amygdala. Pretreatment with losartan or PD123177 significantly interfered with this AngII-induced immunoreactivity while divalanal-AngIV did not. These results indicate that in both guinea pig and rat brains the AT 4 receptor has a distribution different than that previously reported for AT 1 and AT 2 receptor subtypes. The c-Fos expression results suggest that different brain neuronal pathways are activated by i.c.v. injection of AngII and AngIV.

Glucocorticoids but not mineralocorticoids modulate endothelin-1 and angiotensin II binding in SHR vascular smooth muscle cells.

Both glucocorticoids and mineralocorticoids are involved in circulatory homoeostasis and blood pressure control. In recent years direct effects of both steroid classes on vascular smooth muscle cells (VSMC) have been reported. We have thus examined the effects of RU 28362, a pure glucocorticoid agonist, and aldosterone, the physiologic mineralocorticoid, on the binding to VSMC from spontaneously hypertensive rats (SHR) of two key vasoactive peptides, endothelin-1 and angiotensin II. Binding of angiotensin II rose, and that of endothelin-1 declined, in a time- and dose-dependent fashion with maximal effects observed at 24 h and half-maximal effects for each at 2-3 nM RU 28362. Scatchard analysis showed that for both endothelin-1 and angiotensin II, RU 28362 alters receptor number but not affinity; competition studies with receptor-selective ligands (BQ123, S6C, DuP753 and PD123319) show that glucocorticoids specifically elevate (X2) AT-1 receptors and specifically lower (to approximately 30%) levels of ETA receptors. Treatment of VSMC with the antiglucocorticoid RU 38486 reversed the effect of glucocorticoids on endothelin-1 and angiotensin II binding, confirming the Type II (glucocorticoid) receptor mediated effect of the glucocorticoids. Aldosterone (100 nM) also lowers endothelin-1 binding and increases angiotensin II binding in VSMC; that this effect reflects aldosterone occupancy of classical glucocorticoid receptors is shown by the blockade of the aldosterone effect by an equal concentration (100 nM) of RU 38486--i.e. there is no evidence for an action of aldosterone via mineralocorticoid receptors. We interpret our results as evidence for a complex modulation of receptors for vasoactive peptides in VSMC by glucocorticoid but not mineralocorticoid hormones.

Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization.

Phosphorylation of serine and threonine residues in the carboxyl-terminal region of many G-protein-coupled receptors directs the rapid uncoupling from signal transduction pathways. In Chinese hamster ovary cells, we have stably expressed a truncated mutant of the angiotensin II (AT1A) receptor devoid of the carboxyl-terminal 45 amino acids, encompassing 13 serine/threonine residues. One clone, designated TL314 to indicate truncation after leucine 314, expressed a single class of angiotensin II receptors with a dissociation constant of 1.08 nM and a receptor density of 560 fmol/mg of protein (approximately 75,000 receptors/cell). A nonhydrolyzable analog of GTP accelerated the angiotensin II-induced dissociation of [125I]angiotensin II from TL314 plasma membranes 3.6-fold, indicating G-protein coupling. In TL314 cells, angiotensin II stimulated the release of intracellular calcium and the induction of mitogen-activated protein kinase activity, the level of which were comparable with the full-length AT1A receptor. The AII-stimulated calcium response was rapidly desensitized in both full-length and truncated AT1A receptors. Interestingly, angiotensin II-induced endocytosis of the truncated receptor was almost completely inhibited, suggesting that a recognition motif within the carboxyl-terminal 45 amino acids of the AT1A receptor promotes sequestration. Thus, truncation of the AT1A receptor after leucine 314 inhibits agonist-induced internalization without affecting the capacity of the expressed protein to adopt the correct conformation necessary for high affinity binding of angiotensin II, coupling to G-proteins, and activation of signal transduction pathways. The rapid desensitization and refractoriness of the angiotensin II-induced calcium transient in the TL314 cell line, in which putative carboxyl-terminal phosphorylation sites are absent, suggests that the mechanism of AT1A receptor desensitization differs from that of other prototypical G-protein-coupled receptors.

Non-peptide Angiotensin Agonist: FUNCTIONAL AND MOLECULAR INTERACTION WITH THE AT1 RECEPTOR

Non-peptide ligands for peptide receptors for the G-protein-coupled type are generally antagonists, except in the opiate system. Recently, it was observed that a subset of biphenylimidazole derivatives surprisingly possessed angiotensin-like activity in vivo. In COS-7 cells transfected with the rat AT1 receptor a prototype of these compounds, L-162,313 stimulated phosphoinositide hydrolysis with an EC50 of 33 +/- 11 nM. The maximal response to the compound was 50% of that of angiotensin II in COS-7 cells but only 3% in stably transfected Chinese hamster ovary cells. The agonistic effect of L-162,313 was blocked by the AT1-specific antagonist L-158,809 and was not observed in untransfected cells. In Chinese hamster ovary cells, L-162,313 also acted as an insurmountable antagonist of the angiotensin stimulated phosphoinositide hydrolysis. In contrast to previously tested non-peptide ligands, L-162,313 bound with reasonably high affinity to the Xenopus laevis AT1 receptor. In the human receptor, the binding of L-162,313 was found to be unaffected by point mutations in transmembrane segments III and VII, which impaired the binding of biphenylimidazole antagonists. Substitutions in the extracellular domains of the human and rat receptor, which impaired the binding of angiotensin II, did not affect the binding of L-162,313. It is concluded that a subset of biphenylimidazole compounds can act as high affinity partial agonists on the AT1 receptor. These compounds have molecular interactions with the receptor which appear to differ both from that of the structurally similar non-peptide antagonists and from that of their functional counterpart, the peptide agonist.

Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor

Binding of angiotensin II (Ang II) to its receptor type 1A (AT1A) is known to trigger its internalization. We studied the role of cytosolic segments of AT1A in the internalization, and obtained results indicating a functional role of the cytosolic carboxyl terminal tail of AT1A in the internalization. Deletion of 50 amino acids from the carboxyl terminus abolished the receptor internalization. Deletion mutants lacking 13 and 32 amino acid residues in the carboxyl terminal cytosolic region were internalized to the same extent as wild type AT1A; however, internalization of a mutant lacking the last 42 residues was partially suppressed. Thus, residues 310 through 327 were shown to be essential for the internalization. We propose that a short domain in the cytoplasmic tail (residues 310 to 327) may play a dominant role in the agonist-induced receptor internalization of AT1A. Our results also suggest that the molecular determinants of the AT1A receptor involved in receptor internalization are distinct from those participating in the desensitization process.

Novel angiotensin receptor subtypes in fowl

We reported previously that blood vessels of domestic fowl contain angiotensin (ANG) receptors on 1) endothelium, mediating vasorelaxation via endothelium-derived relaxing factor and guanosine 3',5'-cyclic monophosphate; 2) vascular smooth muscles, mediating neither relaxation nor contraction; and 3) presumably adrenergic nerve endings, transmitting vasopressor action via a release of norepinephrine. We aimed in the present study to determine fowl vascular ANG receptor subtypes and relate them to function. [Val5]ANG II (native fowl ANG II) increased mean arterial pressure of anesthetized, ganglion-blocker-treated fowl. The dose-pressor response curve for fowl ANG II was not altered by pretreatment (i.v.) with the ANG receptor subtype 1 (AT1) antagonist Dup-753 (losartan, 10 mg/kg) or the subtype 2 (AT2) antagonist PD-123319 (10 mg/kg). Furthermore, cumulative doses (1-20 mg/kg) of losartan or PD-123319 did not selectively inhibit ANG II-induced pressor responses. In reserpine- and prazosin-treated anesthetized fowl, [Val5]ANG II caused dose-dependent vasodepressor actions inhibited by neither losartan (10 mg/kg) nor PD-123319 (10 mg/kg). Likewise, [Val5]ANG II-induced vasorelaxation of fowl aortic rings in vitro was not inhibitable by PD-123319 or losartan (10(-5) M). Specific binding of 125I-labeled ANG II to the aortic endothelium was markedly displaced by ANG II, but not selectively by PD-123319 or losartan. Specific binding of 125I-ANG II ligand to the membrane fraction of aortic smooth muscles was displaced (50% inhibitory concentration) by [Val5]ANG II (3.3 x 10(-8) M) and slightly by PD-123319 (3.7 x 10(-5) M), but not by losartan or EXP-3174, an active metabolite of losartan.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Cardiac Fibroblasts Express an Angiotensin Receptor with Unusual Binding Characteristics Which Is Coupled to Cellular Proliferation

To assess the cellular localization of angiotensin receptors in human heart, we isolated human cardiac fibroblasts from explanted end-stage failing human hearts. As judged by an immunofluorescence assay, the isolated cells consisted to 96% of fibroblasts. Using receptor binding studies, we could identify a single angiotensin binding site on these cells, with a Kd of 0.6nM and a Bmax of 1.5 fmol/mg protein. Inhibiting concentrations 50 for Ang II and Ang I/II (1-7) were 40nM and 10nM, respectively. Stimulation with Ang II (100nM) and Aug I/II (1-7) (100nM) led to cellular proliferation which could not be inhibited by Losartan or PD 123319. These results suggest that human cardiac fibroblasts express an as yet unknown angiotensin receptor-subtype.