Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT₂ subtype. However, displacement of ¹²⁵I-Angll with the AT₂ nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT₂ receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound ¹²⁵I-Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ～80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT₂ receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT₁-selective antagonist Losartan. However, whereas the nonpeptidic AT₂-selective antagonist PD-123319 completely displaced the binding of ¹²⁶I-Angll from peak I in a monophasic fashion (IC₅₀= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD-123319 was much less effective in displacing ¹²⁵I-Angll from peak III (IC₅₀= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in ¹²⁵I-Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT₂ receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated ¹²⁵I-Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT₂ receptors.     

Down-Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E-115 Cells

Abstract: Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either ¹²⁵I-AngII or the high-affinity antagonist ¹²⁵I-[Sarc¹,Ile⁸]-AngII. This apparent down-regulation was dose related with an ED₅₀ of 1 nM, and maximal decreases of ～90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT₁ receptor subtype during the first 5 min, followed by a decline in both AT₁ and AT₂ receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc¹, Thr⁸]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells. These results suggest that differentiated N1E-115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.     

The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT₁ receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B₁ and B₂ bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT₁-B₂ receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT₁ agonist, [Sar¹,Ile⁴,Ile⁸]-AngII, but not the neutral AT₁ antagonist, losartan, inhibits endogenous B₂ receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar¹,Ile⁴, Ile⁸]-AngII require the AT₁ receptor and result from arrestin-dependent co-internalization of AT₁-B₂ heterodimers. BRET₅₀ measurements indicate that AT₁ and B₂ receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar¹,Ile⁴,Ile⁸]-AngII blunts B₂ receptor activation of G_q/11-dependent intracellular calcium influx and G_i/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar¹,Ile⁴,Ile⁸]-AngII has no effect on B₂ receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar¹,Ile⁴,Ile⁸]-AngII promotes internalization of AT₁-B₂ heterodimers. Thus, [Sar¹,Ile⁴,Ile⁸]-AngII exerts lateral allosteric modulation of B₂ receptor signaling by binding to the orthosteric ligand binding site of the AT₁ receptor and promoting co-sequestration of AT₁-B₂ heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT₁ receptor ligands may translate into different pharmacologic actions.     

Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations

Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT₁) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the G_q/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT₁ receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT₁ receptor, the N111G-AT₁ receptor (constitutively active and biased for the G_q/11 pathway), and the D74N-AT₁ receptor (biased for the β-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT₁, N111G-AT₁, and AngII-N111G-AT₁ receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT₁ receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT₁ receptor. The biased agonist [Sar¹,Ile⁸]AngII also showed a preference for the ground state of the WT-AT₁ receptor compared with AngII. These results suggest that activation of the G_q/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor.     

Receptor Interactions of Abbott-81988, A Highly Potent,Non-Peptide Angiotensin-II Antagonist Selective for Type-1 Receptors

Abstract

Abbott-81988 (A-81988) was selected from a series of related compounds as a highly potent and selective antagonist of angiotensin receptors. In the rabbit aorta, A-81988 exhibited a _pA₂ of 10.12 (± 0.08) vs. angiotensin-II, for type 1 receptors (AT₁), and the antagonism appeared competitive. These results agreed with radioligand assays in which A-81988 inhibited the binding of [¹²⁵l]-Sar¹ –lle⁸—Angiotensin-II to rat liver membranes with a _pK_I of 9.12 (± 0.63). A-81988 was selective for AT₁ receptors based on its lack of activity at other sites, such as aortic α 1 receptors. Moreover, A-81988 lacked affinity for AT₂ receptors of bovine cerebellar membranes or for α or β adrenergic receptors in binding assays. A-81988 lowered blood pressure significantly in vivo in renal artery-ligated rats at doses of 0.3 mg/kg administered either i.v. or p.o. The compound was rapidly and almost completely absorbed from the duodenum of anesthetized rats and demonstrated very low first-pass metabolism in the rat liver. These properties of selectivity toward and potency for antagonizing AT₁ receptors, activity in lowering blood pressure in experimental animals, and favorable pharmacokinetic properties indicate that A-81988 should be a useful antihypertensive agent in man.     

Comparison of the Binding and Functional Actions of Angiotensin Agonists in Clone 9 Cells: Additional Evidence for Angiotensin II Receptor Heterogeneity

Abstract

The effects of the angiotensin-II (All) agonists and antagonists on both ¹²⁵I-SARILE binding and phosphoinositol (PI) accumulation in clone 9 cells were examined. Clone 9 cells, which are derived from rat liver, have been shown to respond to All agonists with an increase in PI accumulation which is inhibitable by Sar₁, Ile₈-AII (SARILE) and DUP-753 but not PD-123319, suggesting that they possess the AT₁ subtype of All receptor. The present results confirmed these properties. The order of potency of AII agonists was AII> AIII> AI. Clone 9 cells also possessed binding sites for ¹²⁵I-SARILE. The majority of these were AT₁ type receptors, although a small number of AT₂ receptors may also have been present. The order of potency of All agonists in inhibiting ¹²⁵-SARILE binding was All » AIII> = AI. The difference in rank order of potency between the functional and binding assays was due to AIII being much less potent in the binding assay than the functional assay. Since the potency of AIII relative to AII was lower than that at either AT₁ or AT₂ subtypes of AII receptor, these data suggest that an additional subtype, with selectively low affinity for AIII, exists.     

Intracerebroventricular Administration of AT1 Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II

Abstract: Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT₁) receptors in cultured cells and brain. In both liver epithelial WB and neuro-blastoma N1E-115 cells AT₁ antisense oligomers substantially decreased AT₁ receptor density, whereas angiotensin type 2 (AT₂) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT₁ antisense oligomers in rats decreased AT₁ receptor density in hypothalamic-thalamic-septal tissue, and AT₂ receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra-cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.     

Requirement of membrane-proximal amino acids in the carboxyl-terminal tail for expression of the rat AT1a angiotensin receptor

A series of deletion mutants was created to analyze the function of the membrane-proximal region of the cytoplasmic tail of the rat type 1a (AT_1a) angiotensin receptor. In transiently transfected COS-7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar¹,Ile⁸]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II-induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine³⁰⁹ and adjacent lysine residues as significant determinants of AT_1a receptor expression.     

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT₁ receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT₁ receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT₁ receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar¹-Ile⁴-Ile⁸]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT₁ receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT₁ receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.     

Receptor interactions of the position 4 side chains of angiotensin II analogues: Importance of aromatic ring quadrupole

A triad of interacting group (TyrOH? His$ \underline\ominus$O₂C) in angiotensin II (ANG II) has been postulated to create the tyrosinate anion pharmacophore (tyanophore) responsible for receptor activation/triggering (Biochim. Biophys. Acta 1991, 1065, 21). In the present study we investigated the effects on bioactivity of substituting the Tyr⁴ residue in [Sar¹]ANG II with other anionic or electronegative amino acids, and with a number of aromatic amino acids lacking a hydroxyl group. [Sar¹ Nva(δ-OH)⁴]ANG II, [Sar¹ Nva(δ-OCH₃)⁴]ANG II, [Sar¹ Met⁴]ANG II, [Sar¹ Gln⁴]ANG II, [Sar¹ Glu⁴]ANG II and [Sar¹ DL -Alg⁴]ANG II had agonist activities in the rat isolated uterus assay of 4, 3, 19, 10, > 0.1 and > 0.1%, respectively, of that of ANG II. [Sar¹ Nal⁴]ANG II, [Sar¹ Pal⁴]ANG II, [Sar¹ DL -Phg(4′-F)⁴]ANG II, [Sar¹ Phe(4′-F)⁴]ANG II, [Sar¹ Phe(F₅)⁴]ANG II and [Sar¹ His⁴]ANG II had agonist activities of 4.5, 7, < 0.1, 0.2, 1 and 0.6%, respectively. All peptides investigated were devoid of measurable antagonist activity except [Sar¹] Phe(4′-F)⁴ ANG II (pA₂ = 7.7). These findings illustrate that anionic or electronegative aliphatic side chains replacing tyrosinate at position 4 can partially activate the angiotension receptor. For ANG II analogues containing an aromatic amino acid other than Tyr at position 4, ligand binding and agonist activity are not dependent on the electronegativity or dipole moment of the aromatic ring, or on the ability of the 4′ ring substituent to accept a proton. Modelling based on ab initio calculations of aromatic ring multipoles illustrate that the apparent binding affinity (PA₂) of ANG II analogues is associated with a perpendicular electrostatic interaction of the position 4 aromatic ring with a receptor-based group. In addition, intramolecular interactions providing for the conformation of the ligand as it approaches its receptor appear to have a role in determining agonist vs antagonist activity.     

Angiotensin-Induced Changes in the Apparent Size of Rat Liver Angiotensin Receptors

Abstract

Angiotensin receptors from rat liver were labeled using four different ligands : (Sar¹-(³H)Tyr⁴)-Angiotensin II ((³H)SarAII); (Sar¹-(³H)Tyr⁴-IIe⁸) -Angiotensin II ((³H)SarIIeAII); (Sar¹-(¹²⁵I)Tyr⁴-(4′-N₃)Phe⁸)-Anglotensin II (IN₃AII); (Sar¹-(¹²⁵I)Tyr⁴-(4′N₃D-Phe)⁸)-Angiotensin II (IN₃DPheAII) (³H) SarAII and IN₃AII behaved like agonists and (³H) SarlleAII and IN₃DPheAII like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 ± 0. 1 nm (6) after labeling with (³H) SarAII) or with an antagonist (Stokes radii of 5. 5 ± 0. 1 (7), and 5.6 ± 0.1 nm (4) after labeling with (³H) SarIIeAII and IN₃DPheAII respectively). After covalent labeling with IN₃All anglotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of M_r 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component Involved in the coupling of the receptor to its primary effector.     

Effects of angiotensin II and its blockers Sar1-lle8-angiotensin II and DuP 753 on drinking in ducks in relation to properties of subfornical organ neurons

Properties of systemically applied angiotensin II in stimulating water intake of normally hydrated ducks were studied and the results compared with properties of angiotensin II-responsive neurons of the subfornical organ which are considered as targets for circulating angiotensin, II acting as a dipsogen. Following intravenous infusion of hypertonic saline (2000 mosmol·kg^-1 at 0.3 ml·min^-1 for 1 h), intravenous infusion of 0.3 ml·min^-1 isotonic saline with angiotensin II (200 ng·min^-1), starting 1 h later, stimulated drinking in each case at an angiotensin II plasma level of about 1400 pg·ml^-1. Without hypertonic priming, the same angiotensin II infusion did not stimulate drinking in each experiment; however, if effective, repeated infusions of ANGII induced stable dipsogenic responses. Angiotensin II infusions did not alter plasma levels of antidiuretic hormone. Sar¹-Ile⁸-angiotensin II, a non-selective angiotensin II antagonist, acted weakly as a partial agonist when injused at a dose 200-fold higher than angiotensin II and effectively blocked the dipsogenic action of angiotensin II; this corresponds to the inhibition of angiotensin II-induced excitation by Sar¹-Ile⁸-angiotensin II observed in duck subfornical organ neurons. DuP 753 (losartan), an angiotensin II antagonist specifically blocking AT₁ receptors in mammals, had equivocal effects on angiotensin II-induced drinking in ducks at rates 50- and 200-fold higher than angiotensin II, which corresponds to the weak inhibitory action of this compound on angiotensin II-induced neuronal excitation in the duck SFO. Blood pressure was only marginally elevated by the applied angiotensin II dose and Sar¹-Ile⁸-angiotensin II had no effect.Abbreviations ANGII angiotensin II - AVT arginine vasotocin - DuP 753 losartan - EDTA ethylene diamine tetra-acetic acid - HR heart rate - ICV intracerebroventricular - IV intravenous - MAP mean arterial pressure - SARILE Sar¹-Ile⁸-angiotensin II - SFO subfornical organ     

Metal Ion and Guanine Nucleotide Modulations of Agonist Interaction in G-Protein-Coupled Serotonin1A Receptors from Bovine Hippocampus

1. The serotonin type 1A (5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP-binding regulatory proteins (G-proteins). We have studied the modulation of agonist binding to 5-HT_1A receptors from bovine hippocampus by metal ions and guanine nucleotide.2. Bovine hippocampal membranes containing the 5-HT_1A receptor were isolated. These membranes exhibited high-affinity binding sites for the specific agonist [³H]OH-DPAT.3. The agonist binding is inhibited by monovalent cations Na⁺, K⁺, and Li⁺ in a concentration-dependent manner. Divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, on the other hand, show more complex behavior and induce enhancement of agonist binding up to a certain concentration. The effect of the metal ions on agonist binding is strongly modulated in the presence of GTP--S, a nonhydrolyzable analogue of GTP, indicating that these receptors are coupled to G-proteins.4. To gain further insight into the mechanisms of agonist binding to bovine hippocampal 5-HT_1A receptors under these conditions, the binding affinities and binding sites have been analyzed by Scatchard analysis of saturation binding data. Our results are relevant to ongoing analyses of the overall regulation of receptor activity for G-protein-coupled seven transmembrane domain receptors.     

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

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

     

Angiotensin II Receptor Type I-Regulated Anion Secretion in Cystic Fibrosis Pancreatic Duct Cells

The β-adrenergic (cAMP-dependent) regulation of Cl⁻ conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (I _SC ) technique. An increase in I _SC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC₅₀ at 3 μm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT₁ and AT₂ receptors, respectively. It was found that losartan (1 μm) could completely inhibit the AII-induced I _SC , whereas, PD123177 exerted insignificant effect on the I _SC , indicating predominant involvement of AT₁ receptors. The presence of AT₁ receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT₁ receptors. Confocal microscopic study demonstrated a rise in intracellular Ca²⁺ upon stimulation by AII indicating a role of intracellular Ca²⁺ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca²⁺ diminished the AII-induced I _SC . Treatment of the monolayers with a Cl⁻ channel blocker, DIDS, markedly reduced the I _SC , indicating that a large portion of the AII-activated I _SC was Cl⁻-dependent. AII-induced I _SC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the I _SC was mediated by apical Cl⁻ channels. Our study indicates an AT₁-mediated Ca²⁺-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas. Received: 17 June 1996/Revised: 14 November 1996     

Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes

We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT₁Rs) induced electrical remodeling, including inhibition of the transient outward potassium current I_to, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and I_to in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced I_to magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of I_to (I_to,fast and I_K,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K_0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on I_to and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar¹-Ile⁴-Ile⁸]A2), which stimulates receptor internalization without G protein activation, caused I_to reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT₁R mediated regulation of I_to in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.     

Ethanol Inhibits the Binding of Substance P to Rat Brain Cortex NK1 Receptors

The binding of ¹²⁵I-labeled substance P (SP) to rat brain cortex membranes has been studied Under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20–1000 pM) gave two components, one with a K _D value of 80 pM and another one with a K _D of 500 pM. The higher-affinity component is due to NK₁ receptors, as confirmed by the inhibition Of the SP binding by the rodent NK1 specific agonist [Sar₉ Met(O₂)₁₁]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK₁ receptors involved in this binding component. The ethanol effect disappeared at [EtOH] 0.17 mM.     

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

The bovine striatal dopamine D₁ receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [³H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a K_d of 0.95 and 1.1 nM and a B_max of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [³H]SCH-23390 binding exhibited a clear D₁ dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent thancis-flupentixol in displacing [³H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[³H]SCH-23390 competition studies revealed the presence of a highaffinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40–80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obrained from the agonist-treated membrane. In both the preparations, 100 M guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.