Cosmological Higgs-Axion Interplay for a Naturally Small Electroweak Scale.

Recently, a new mechanism to generate a naturally small electroweak scale has been proposed. It exploits the coupling of the Higgs boson to an axionlike field and a long era in the early Universe where the axion unchains a dynamical screening of the Higgs mass. We present a new realization of this idea with the new feature that it leaves no sign of new physics at the electroweak scale, and up to a rather large scale, 10^{9} GeV, except for two very light and weakly coupled axionlike states. One of the scalars can be a viable dark matter candidate. Such a cosmological Higgs-axion interplay could be tested with a number of experimental strategies.

On the interaction between ultrasound waves and bubble clouds in mono- and dual-frequency sonoreactors.

Since the last decades, extensive work have been done on the numerical modeling of mono-frequency sonoreactors, we here consider the modeling of dual-frequency sonoreactors. We first present the basic features of the CAMUS code (CAvitating Medium under UltraSound), for mono-frequency excitation. Computation at low, medium and high frequency are presented. Extension of the numerical tool CAMUS is also presented: Caflisch equations are modified to take into account the dual-frequency excitation of the sound. We consider 28-56, 28-100 and 28-200 kHz sonoreactors. Fields of cavitation bubble emergence are quite different from the ones under mono-frequency. Study of spatio-temporal dynamics of cavitation bubbles in a 28-56 kHz sonoreactor is also considered. Taking into account the pressure field induced by the dual-frequency wave propagation, we compute the Bjerknes force applied on the cavitation bubble that is responsible for the bubble migration. A two phase flow approach allows to compute the bubble migration.

Spatio-temporal dynamics of cavitation bubble clouds in a low frequency reactor: comparison between theoretical and experimental results.

The propagation of ultrasound through a liquid induces the growth of inceptions and germs into bubbles. In a low frequency reactor, fragmentary transient bubbles emerge due to the acoustic driving. They violently collapse in one cycle and fragment into many smaller bubbles than in turn cavitate. This violent collapse is responsible for the mechanical effects of ultrasounds effects. The latter bubbles gather in a ball-shaped cloud and migrate to pressure antinodes. During their migration, their nonexplosive collapses mainly contribute to activate chemical reactions by producing OH. radicals. Mathematical modelling is performed as a new approach to predict the bubbles field. Through numerical simulation, we determinate emergence sites of mechanically active cavitation bubbles. Calculus are compared with aluminium foil degradation. The modelling of bubble migration allow us to have an insight on the privileged sites of the chemical reactions. Validation of the modelling is made through direct comparison with chemiluminescence photo. All experiments and computations are made in a 28.2 kHz sonoreactor.

Numerical simulation of cavitation bubble dynamics induced by ultrasound waves in a high frequency reactor.

The use of high frequency ultrasound in chemical systems is of major interest to optimize chemical procedures. Characterization of an open air 477 kHz ultrasound reactor shows that, because of the collapse of transient cavitation bubbles and pulsation of stable cavitation bubbles, chemical reactions are enhanced. Numerical modelling is undertaken to determine the spatio-temporal evolution of cavitation bubbles. The calculus of the emergence of cavitation bubbles due to the acoustic driving (by taking into account interactions between the sound field and bubbles' distribution) gives a cartography of bubbles' emergence within the reactor. Computation of their motion induced by the pressure gradients occurring in the reactor show that they migrate to the pressure nodes. Computed bubbles levitation sites gives a cartography of the chemical activity of ultrasound. Modelling of stable cavitation bubbles' motion induced by the motion of the liquid gives some insight on degassing phenomena.

Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products.

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.

Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation.

On the basis of the patterns of conserved amino acid sequence, the angiotensin II type 2 (AT(2)) receptor belongs to the family of serpentine receptors, which relay signals from extracellular stimuli to heterotrimeric G proteins. However, the AT(2) receptor signal transduction mechanisms are poorly understood. We have measured AT(2)-triggered activation of purified heterotrimeric proteins in urea-extracted membranes from cultured COS-7 cells expressing the recombinant receptor. This procedure removes contaminating GTP-binding proteins without inactivating the serpentine receptor. Binding studies using [(125)I] angiotensin (Ang) II revealed a single binding site with a K(d)=0.45 and a capacity of 627 fmol/mg protein in the extracted membranes. The AT(2) receptor caused a rapid activation of alpha(i) and alpha(o) but not of alpha(q) and alpha(s), as measured by radioactive guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. Activation required the presence of activated receptors, betagamma, and alpha subunits. As a first step aimed at developing an in vitro assay to examine AT(2) receptor pharmacology, we tested a battery of Ang II-related ligands for their ability to promote AT(1) or AT(2) receptor-catalyzed G(i) activation. Two proteolytic fragments of Ang II, Ang III and Ang1-7, also promoted activation of alpha(i) through the AT(2) receptor. Furthermore, we found that [Sar(1),Ala(8)]Ang II is an antagonist for both AT(1) and AT(2) receptors and that CPG42112 behaves as a partial agonist for the AT(2) receptor. In combination with previous observations, these results show that the AT(2) receptor is fully capable of activating G(i) and provides a new tool for exploring AT(2) receptor pharmacology and interactions with G-protein trimers.

Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.

Spatial control of actin polymerization during neutrophil chemotaxis.

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis.

Persistent directional movement of neutrophils in shallow chemotactic gradients raises the possibility that cells can increase their sensitivity to the chemotactic signal at the front, relative to the back. Redistribution of chemoattractant receptors to the anterior pole of a polarized neutrophil could impose asymmetric sensitivity by increasing the relative strength of detected signals at the cell's leading edge. Previous experiments have produced contradictory observations with respect to receptor location in moving neutrophils. To visualize a chemoattractant receptor directly during chemotaxis, we expressed a green fluorescent protein (GFP)-tagged receptor for a complement component, C5a, in a leukemia cell line, PLB-985. Differentiated PLB-985 cells, like neutrophils, adhere, spread, and polarize in response to a uniform concentration of chemoattractant, and orient and crawl toward a micropipette containing chemoattractant. Recorded in living cells, fluorescence of the tagged receptor, C5aR-GFP, shows no apparent increase anywhere on the plasma membrane of polarized and moving cells, even at the leading edge. During chemotaxis, however, some cells do exhibit increased amounts of highly folded plasma membrane at the leading edge, as detected by a fluorescent probe for membrane lipids; this is accompanied by an apparent increase of C5aR-GFP fluorescence, which is directly proportional to the accumulation of plasma membrane. Thus neutrophils do not actively concentrate chemoattractant receptors at the leading edge during chemotaxis, although asymmetrical distribution of membrane may enrich receptor number, relative to adjacent cytoplasmic volume, at the anterior pole of some polarized cells. This enrichment could help to maintain persistent migration in a shallow gradient of chemoattractant.

Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II.

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.

The angiotensin II binding site on Mycoplasma hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

Angiotensin II (AngII) binding sites were characterized on rat pheochromocytoma cells (PC-12) which are known to express exclusively the type-2 (AT2) AngII receptor. Interestingly, we found that, on confluent PC-12 cells, only partial inhibition of 125I-AngII binding was achieved when cells were incubated with a saturating concentration of PD-123 319 (an AT2 selective ligand) suggesting the presence of an atypical binding site. In binding experiments, AngII exhibited high affinity for this atypical binding site with a dissociation constant (Kd) of 16 nM. Moreover, bacitracin potently inhibited PD-123 319-resistant 125I-AngII binding with an IC50 half-maximal inhibitory concentration of 44 microM. Enzyme immunoassay revealed that the cells were contaminated with Mycoplasma hyorhynis. Contaminated PC-12 cells were photolabeled with 125I-[p-benzoylPhe1]AngII and covalently labeled proteins were subjected to polyacrylamide gel electrophoresis followed by autoradiography. Under these conditions, two distinct labeled species of 140 kilodaltons (kDa) and 95 kDa were detected. Deglycosylation of the 140 kDa-labeled AT2 receptor with glycopeptidase-F (PNGase-F) resulted in a 35 kDa protein whereas the 95 kDa band was not affected by digestion with the endoglycosidase. Thus, our results show that the AngII binding site on M. hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

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.

Identification of angiotensin II-binding domains in the rat AT2 receptor with photolabile angiotensin analogs.

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT2), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of 125I-[Bpa1]AngII.AT2 receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of 125I-[Bpa1]AngII within amino acids 3-30 of AT2 receptor. Treatment of 125I-[Bpa8]AngII.AT2 receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT2 receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of 125I-[Bpa8]AngII within residues 129-138 of AT2 receptor. Our results indicate that the amino-terminal tail of the AT2 receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT2 receptor interacts with the carboxyl-terminal end of AngII.

Analysis of the role of N-glycosylation in cell-surface expression and binding properties of angiotensin II type-2 receptor of rat pheochromocytoma cells.

We previously demonstrated that the AT2 receptor is a glycoprotein containing N-linked oligosaccharide side chains and that the marked disparity between the sizes of AT2 receptors from different tissues was related to different degrees of N-glycosylation. In the present study, we used an inhibitor of N-glycosylation, tunicamycin, as well as an endoglycosidase, glycopeptidase-F, to examine the contribution of carbohydrate moieties to the ligand-binding properties, cell-surface expression and apparent molecular mass of AT2 receptors of rat pheochromocytoma cells (PC-12 cells). Photoaffinity labelling of cell-surface AT2 receptors revealed that PC-12 cells grown in the presence of tunicamycin expressed, in addition to the previously described 140 kDa receptor, lower-molecular-mass receptors of 63 kDa, 47 kDa and 32 kDa. Lectin affinity chromatography revealed that the 63 kDa and the 47 kDa receptors are partially glycosylated and that the 32 kDa receptor is completely deglycosylated. Competitive binding experiments were carried out on tunicamycin-treated cells that expressed predominantly the 63 kDa or the 47 kDa receptors. Both receptor forms exhibited a high affinity for angiotensin II, although a slight decrease (of about 2-fold) was consistently observed on tunicamycin-treated cells as compared with control cells. Endoglycosidase digestion of AT2 receptors of PC-12 cells also yielded smaller receptor forms of 47 kDa and 32 kDa. Similarly, angiotensin II showed a high but slightly decreased binding affinity (of about 2-fold) for deglycosylated membranes as compared with control membranes. In conclusion, the stepwise action of tunicamycin suggests the presence of at least three N-linked oligosaccharide side chains on the AT2 receptor of PC-12 cells. These oligosaccharide side chains have a minor contribution to the affinity of the receptor. Interestingly, glycosylation is not an essential requirement for the expression of AT2 receptor at the surface of PC-12 cells.

The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization.

The NPXnY motif is involved in the internalization process of several types of receptors, including lipoprotein receptors and G protein-coupled receptors. We replaced Tyr302 with either phenylalanine or alanine in the NPLFY site of the human angiotensin II receptor type 1 and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments revealed that COS-7 cells transfected with either the wild-type or mutant receptors expressed approximately the same amount of high affinity binding sites (Bmax 70,000 sites/cell and Kd approximately 2 nM). Photoaffinity labeling of both native and mutant receptors revealed apparent molecular masses of 110 kDa. Incubation of transfected cells with 0.2 nM [125I]Ang II at 37 degrees revealed an efficient internalization of the wild-type receptor and the mutant receptors, although the mutant receptors were internalized at a slower rate. Interestingly, however, the transmembrane signaling was severely impaired in transfected cells expressing mutant receptors. No significant production of inositol-1,4,5-trisphosphate was observed when these cells were challenged for 3 min with a concentration of angiotensin II as high as 1 microM. This is in contrast to the dose-dependent stimulation of inositol-1,4,5-trisphosphate production in cells expressing the wild-type receptor. Thus, our results show that the Tyr302 in the NPXnY motif of the human angiotensin II receptor type 1 is not essential for agonist binding properties or for internalization of the receptor but plays an important role in transmembrane signaling.

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 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (Kd = 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 degree C revealed a calculated Kd of 59 pM (average of two experiments that yielded values of 67 and 50 pM). The binding site displays a high affinity for angiotensin receptors AT1 or AT2. An analysis of specificity showed that the binding site displays a high affinity for angiotensin IV, low affinities for angiotensin II, [Sar1, Val5, Ala8]angiotensin II and does not recognize L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'-(1 H-tetrazole-5-yl)[1,1'-biphenyl]-4-yl)methyl]-3H-imidazo[4, 5-beta]pyridine H2O) 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). A few unrelated hormones (bradykinin, [Arg8] 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. GTP gamma S and polyvinyl sulfate did not affect the binding, suggesting that the receptor is not coupled to a G-protein. The divalent cations Mg2+ and Ca2+ 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 on aortic endothelial cells suggest the existence of a novel mechanism involved in the control of vascular tone or vascular permeability.

The marked disparity between the sizes of angiotensin type 2 receptors from different tissues is related to different degrees of N-glycosylation.

We recently described the photoaffinity labeling and partial characterization of the angiotensin type 2 (AT2) receptor from human myometrium. In the present study, specific receptors for angiotensin II (AII) were also analyzed in a murine fibroblast cell line (R3T3) and a rat pheochromocytoma cell line (PC-12). Dose-displacement experiments with PD 123319 (an AT2-selective antagonist) completely inhibited 125I-AII binding, whereas L-158,809 (an AT1-selective antagonist) had no significant effect on 125I-AII binding, thus revealing that these two cell lines express exclusively AT2 sites. High yields of covalent and selective labelling of AT2 receptors from human myometrium, R3T3 cells, and PC-12 cells were obtained with the photosensitive analogue 125I-[Sar1,Val5,p-benzoyl-Phe8]AII. Gel permeation chromatography of Triton X-100-solubilized AT2 receptors from the three different sources revealed similar Stokes' radii of about 65 A. Interestingly, upon electrophoresis under reducing conditions, marked disparities were observed between the apparent molecular masses of AT2 receptors from the three different sources. As observed previously, AT2 receptors from human myometrium showed a molecular mass of 68 +/- 4.6 kDa. AT2 receptors from PC-12 cells showed a larger molecular mass of 113 +/- 12 kDa, whereas AT2 receptors from R3T3 cells showed a molecular mass of 91 +/- 7.8 kDa. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the molecular masses of the denatured AT2 receptors of human myometrium, R3T3 cells, and PC-12 cells were decreased by 54%, 66%, and 73%, to 31.3 +/- 1.6 kDa, 30.9 +/- 0.7 kDa, and 30.6 +/- 0.8 kDa, respectively. Kinetic studies with AT2 receptors from human myometrium revealed a complex, multiple-step process of deglycosylation involving at least three different sites of N-linked saccharides. These results suggest that the disparity in the sizes of AT2 receptors from different sources is mostly related to different degrees of N-glycosylation. They also imply that the AT2 receptor contains at least three asparagine-linked sites of glycosylation.

Photoaffinity labeling of subtype 2 angiotensin receptor of human myometrium.

Angiotensin II (AII) binding sites were characterized in human myometrium membrane preparations. The sites were saturable and of high affinity (Kd of 0.09 nM and Bmax of about 200 fmol/mg of protein). PD 123319 completely inhibited 125I-AII binding, with an IC50 of 30 nM, whereas L-158,809 (1 microM) had no significant effect on 125I-AII binding. These results indicate that human myometrium contains almost exclusively the AT2 receptor subtype. Association and dissociation studies performed with 125I-AII on human myometrium membranes revealed that AII had a very high affinity for AT2 receptors, with a Kd of 0.01 nM (association rate constant K1 = 1.056 x 10(12) mol-1 min-1; dissociation rate constant K2 = 0.003 min-1). The photoactivable AII analogue [Sar1, Val5, D-Phe8(N3)]AII displayed a high affinity for AT2 receptors (IC50 of 0.18 nM), but its radioiodinated form showed poor efficiency in photoaffinity labeling experiments. A newly synthesized photoactivatable analogue of AII, [Sar1, p-benzoyl-Phe8]AII, (AII-Bpa), also displayed a high affinity for AT2 receptors of human myometrium (IC50 of 0.3 nM). Photoaffinity labeling experiments were performed with 125I-AII-Bpa, and a high yield (70%) of covalent incorporation to human myometrium membranes was obtained upon photolysis. Covalently labeled receptors were solubilized, denatured, and subjected to polyacrylamide gel electrophoresis. Autoradiography of the polyacrylamide gel revealed a single band, of 68 kDa, and the labeling of this band was completely abolished in the presence of 1 microM PD 123319, indicating selective labeling of the AT2 receptor subtype. These results demonstrate that AII-Bpa is a very efficient tool for selective photoaffinity labeling of the AT2 receptor.

Sar1-p-benzoylphenylalanine-angiotensin, a new photoaffinity probe for selective labeling of the type 2 angiotensin receptor.

Previous photoaffinity labeling of angiotensin II (Ang) receptors with azidophenylalanine containing Ang analogs produced high yield labeling of a 60 kDa protein on bovine adrenocortical membranes. This preparation is mostly enriched in the type 1 Ang receptor (AT1) and AT1 selective ligands (L158,809) totally prevented labeling, therefore confirming the AT1 nature of the labeled protein. Our attempt to photolabel the type 2 Ang receptor (AT2) of human myometrium with [Sar1,D-Phe(N3)8]Ang was unsuccessful, revealing a high degree of photolabeling selectivity. An Ang analog, [Sar1,Bpa8]Ang (or BpaAng) was prepared containing the photosensitive amino acid p-benzoylphenylalanine (Bpa). This compound was a specific but non-competitive Ang antagonist on rabbit aorta with a pA2 of 8.5. It displayed good binding affinities for bovine adrenocortical membranes (Kd = 6.5 nM), a predominantly AT1 preparation, and for human myometrium membranes (Kd = 0.39 nM), a predominantly AT2 preparation. Photolabeling experiments with iodinated BpaAng showed that AT1 was not covalently labeled whereas AT2 was covalently labeled with high yield. Labeling specificity was verified with the AT2-selective ligand PD123319 and with the AT1-selective antagonist L158,809. Our results indicate that 125I-BpaAng is exclusively labeling AT2 sites. This compound should be a useful tool for further biochemical characterization of the AT2 binding site.

Modulation of angiotensin II binding affinity by allosteric interaction of polyvinyl sulfate with an intracellular domain of the DuP-753-sensitive angiotensin II receptor of bovine adrenal glomerulosa.

Angiotensin II (AII) is an important regulator of aldosterone secretion by adrenal glomerulosa cells. All interacts with a specific receptor coupled to a guanine nucleotide-binding protein that controls the activity of phospholipase C. Recently, novel All nonpeptide antagonists (DuP-753 and PD-123319) have been shown to discriminate between two subclasses of All receptors in many different tissues. Our studies confirmed that 125I-All specifically labeled two classes of binding sites for All in a membrane preparation of bovine adrenal glomerulosa cells. The first class (DuP-753 sensitive) represented approximately 85% of the total binding sites for All and possessed a high affinity (IC50 of 92.9 +/- 19.5 nM) for DuP-753. PD-123319 did not have any effect on 125I-All binding to this site. The second class of binding sites was more sensitive to PD-123319, with an IC50 of 6.9 +/- 3.7 nM, and had a much lower affinity for DuP-753 (IC50 around 10 microM). The two classes of receptors had different affinities for All. All showed an affinity around 2 nM for All type 1 receptor (AT1)(DuP-753 sensitive) and a higher affinity, around 0.3 nM, for All type 2 receptor (AT2) (PD-123319 sensitive). All-induced steroidogenesis was completely abolished in the presence of 3 microM DuP-753, indicating that this activity was mediated through a DuP-753-sensitive receptor. We also found that polyvinyl sulfate (PVS), a polyanion, could partly inhibit the binding of 125I-All to bovine adrenal glomerulosa cell membranes, with half-maximal efficiency at 17.3 +/- 8.2 nM. The inhibitory effect of PVS was selective for AT1. The inhibitory effect of PVS was due to a change in the affinity state of the receptor. Unexpectedly, PVS had no effect on All-induced steroidogenesis or on All binding to intact bovine adrenal glomerulosa cells. However, the inhibitory effect of PVS on All binding was recovered after permeabilization of cells. Direct interaction of polyanions with AT1 was suggested by the capacity of solubilized photoaffinity-labeled 125I-AT1 to adsorb to heparin-agarose gels. The adsorption of 125I-AT1 to heparin-agarose was inhibited by prior incubation of solubilized receptor with heparin or PVS. These results suggest that All-induced steroidogenesis is mediated by a DuP-753-sensitive receptor and that PVS decreases the affinity of this receptor by interacting with an intracellular domain (possibly the positively charged domain responsible for coupling with guanine nucleotide-binding proteins).