DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A₂/prostaglandin H₂ (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [³H]SQ 29,548 (5 nM) in human platelets with k_d/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [³H]SQ 29,548 binding in platelets (K_d/slope factor:200 μM/0.86). [³H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (K_d: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (B_max: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC₅₀=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT₁) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT₁ receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT₁ receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT₁ receptor in a pure cell system, the recombinant human AT₁ receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT₁-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [K_D=0.15±0.02 nM] for [¹²⁵I][SAR¹, IIe⁸] angiotensin II (SIA). The pharmacological profile of ligands competing for [¹²⁵I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5-(-thio) triphosphate [GTPS]. Angiotensin II evoked a rapid efflux of⁴⁵Ca²⁺ from LhAT₁-D6 cells that was blocked by AT₁ receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT₁-D6 cell line provides a powerful tool to explore the human AT₁ receptor regulation.     

Model of the whole rat AT1 receptor and the ligand-binding site

We present a three-dimensional model of the rat type 1 receptor (AT₁) for the hormone angiotensin II (Ang II). Ang II and the AT₁ receptor play a critical role in the cell-signaling process responsible for the actions of renin–angiotensin system in the regulation of blood pressure, water-electrolyte homeostasis and cell growth. Development of improved therapeutics would be significantly enhanced with the availability of a 3D-structure model for the AT₁ receptor and of the binding site for agonists and antagonists. This model was constructed using a combination of computation and homology-modeling techniques starting with the experimentally determined three-dimensional structure of bovine rhodopsin (PDB#1F88) as a template. All 359 residues and two disulfide bonds in the rat AT₁ receptor have been accounted for in this model. Ramachandran-map analysis and a 1 nanosecond molecular dynamics simulation of the solvated receptor with and without the bound ligand, Ang II, lend credence to the validity of the model. Docking calculations were performed with the agonist, Ang II and the antihypertensive antagonist, losartan.      

The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT₁) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT₁ receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT₁ receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT₁, L81C-AT₁, A85C-AT₁, T88C-AT₁, and A89C-AT₁ mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT₁ receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT₁ receptor background. Indeed, mutant D74C-N111G-AT₁ became insensitive to MTSEA, whereas mutant L81C-N111G-AT₁ lost some sensitivity and mutant V86C-N111G-AT₁ became sensitive to MTSEA. Our results suggest that constitutive activation of the AT₁ receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.The octapeptide hormone angiotensin II (AngII)⁵ is the active component of the renin-angiotensin system. It exerts a wide variety of physiological effects, including vascular contraction, aldosterone secretion, neuronal activation, and cardiovascular cell growth and proliferation (1). Virtually all of the known physiological effects of AngII are produced through the activation of the AT₁ receptor, which belongs to the G protein-coupled receptor (GPCR) superfamily (2, 3). GPCRs possess seven transmembrane domains (TMD), which provide structural support for signal transduction. The AT₁ receptor interacts with the G protein G_q/11, which activates a phospholipase C, which in turn generates inositol 1,4,5-trisphosphate and diacylglycerol from the cleavage of phosphatidylinositol 4,5-bisphosphate (4, 5). Inositol 1,4,5-trisphosphate causes the release of Ca²⁺ from an intracellular store, whereas diacylglycerol activates protein kinase C.Like other GPCRs, the AT₁ receptor undergoes spontaneous isomerization between its inactive state (favored in the absence of agonist) and its active state (induced or stabilized by the agonist) (6). Movement of TMD helices through translational or rotational displacement is believed to be essential to achieve the active state (7, 8). It has been proposed that TMD3, TMD5, TMD6, and TMD7 may participate in the activation process of the AT₁ receptor by providing a network of interactions through the AngII-binding pocket (9). The dynamics of this network are thought to be modified following agonist binding, thereby forcing the receptor to form new interactions between the TMDs.Based on homology with the high resolution structure of rhodopsin, the archetypal GPCR (10), it was expected that the binding site of the AT₁ receptor would involve the seven mostly hydrophobic TMDs and would be accessible to charged water-soluble ligands, like AngII. For this receptor, the binding site would thus be contained within a water-accessible crevice, the binding pocket, extending from the extracellular surface of the receptor to the transmembrane portion. Using a photoaffinity labeling approach, we directly identified ligand-contact points within the second extracellular loop and the seventh TMD of the AT₁ receptor (11–13). Interestingly, numerous mutagenesis studies have provided the basis for a model in which an interaction between Asn-111 in TMD3 and Tyr-292 in TMD7 maintains the AT₁ receptor in the inactive conformation. The agonist AngII would disrupt this interaction and promote the active conformational state (14). In support of this model, it was further shown that substitution of Asn-111 for a residue of smaller size (Ala or Gly) confers constitutive activity on the AT₁ receptor (15–17).The substituted cysteine accessibility method (SCAM) (18–20) is an ingenious approach for systematically identifying the residues in a TMD that contribute to the binding site pocket of a GPCR. Consecutive residues within TMDs are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substituted mutant is unchanged compared with wild-type receptor, it is assumed that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and therefore that the substituted cysteine lies in an orientation similar to that of the wild-type residue. In TMDs, the sulfhydryl of a cysteine oriented toward the binding site pocket should react faster with a positively charged sulfhydryl reagent like methanethiosulfonate-ethylammonium (MTSEA) than sulfhydryls facing the interior of the protein or the lipid bilayer. Two criteria are used for identifying engineered cysteines on the surface of the binding site pocket: (i) the reaction with MTSEA alters binding irreversibly, and (ii) the reaction is retarded by the presence of ligand. We previously used this approach to identify the residues in TMD3, TMD6, and TMD7 that form the surface of the binding site pocket in the wild-type AT₁ receptor and in the constitutively active N111G-AT₁ receptor (21–23). Here we report the application of SCAM to probe TMD2 in the wild-type and constitutively active receptors.     

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.     

Characterization of AT2 Receptor Expression in NIH 3T3 Fibroblasts

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts.2. Characterization with selective ligands, dithiothreitol, and GTPS, indicated that only the AT₂ subtype was expressed.3. AT₂ receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT₂ receptors being expressed earlier than ACE. In contrast, high expression of AT₂ receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras.4. Our results imply a possible relation of AT₂ receptors to cell growth and cell–cell contact.     

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by the angiotensin II analogue Leu⁸ angiotensin II, with a pA₂ value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA₂ value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu⁸ angiotensin II (10^-8 mol·l^-1) and DuP 753 (10^-6 mol·l^-1) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn¹-Val⁵] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu⁸ angiotensin II. The hydrosmotic response to [Asn¹-Val⁵] angiotensin II (10^-7 mol·l^-1) was significantly inhibited by DuP 753 (10^-6 and 5×10^-6 mol·l^-1), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10^-6 mol·l^-1) also inhibited the hydrosmotic response to angiotensin III (10^-7 mol·l^-1). These results suggest that receptors for angiotensin II present in isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.Abbreviations AT ₁ angiotensin receptor subtype 1 - AT ₂ angiotensin receptor subtype 2 - AT II angiotensin II - AT III angiotensin III - CDRC cumulative doseresponse curve(s) - NE norepinephrine - SCC short-circuit current     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Analysis of T cell receptor Vβ gene usage during the course of disease in patients with chronic hepatitis B

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virusinfected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR V gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR V₇ gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR V₁ and V₂ were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific V gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.     

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.     

Muscarinic acetylcholine receptor in chick limb bud during morphogenesis

Summary In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate ((³H)QNB), a muscarinic antagonist, to homogenates of chick limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of (³H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 M). Specific binding of (³H)QNB reflected a muscarinic receptor. The K_d in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol (³H)QNB/mg protein and 12.0 fmol (³H)QNB/mg protein, respectively. Data on displacement of specific bound (³H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the (³H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis.     

Molecular structural requirements for binding and activation of L-alanine taste receptors

Summary L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[³H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC₅₀s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of the-carboxylic acid and the-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[³H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.     

Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine β-casein

Summary The action of the cell-envelope proteinase (P_III-type) from Lactococcus lactis ssp. cremoris AM₁ on bovine -casein was studied. The results were compared with those obtained earlier with (P_I-type) proteinases from the cell envelope of other L. lactis strains. From a 4-h digest (pH 6.2; 15°C) of -casein made with the P_III-type proteinase, 24 peptides were isolated and purified by selective precipitation followed by semi-preparative reversed-phase HPLC. Altogether, these peptides accounted for the preferential splitting of 16 peptide bonds in -casein by the P_III-type proteinase. In nine cases the primary cleavage site (P₁-P₁) was a Glx-X or X-Glx peptide bond. In ten cases at least one large hydrophobic residue (Met, Leu, Tyr, Phe) formed part of the cleavable bond. The P₂-P₃ and/or P₂-P₃ regions of the substrate consisted of hydrophobic and/or negatively charged side chains or of side chains potentially involved in hydrogen bonds. Nine of the peptide bonds split were reported previously to be also susceptible to cleavage by P_I-type proteinases, although the kinetics may be different. The P_III-type proteinase shows a broader specificity in its initial cleavage of -casein than does the P_I-type. Offprint requests to: S. Visser     

Atypical SCH23390 Binding Sites Are Present on Bovine Adrenal Medullary Membranes

D1-selective dopamine receptor agonists inhibit secretagogue-stimulated catecholamine secretion from bovine adrenal chromaffin cells. The purpose of the studies reported here was to use the radiolabeled D1-selective dopamine receptor antagonist, SCH23390, to characterize putative D1-like dopamine receptors responsible for this effect. Characterization of SCH23390 binding sites demonstrated an unusual pharmacological profile inconsistent with classical D1-like receptors. [¹²⁵I]SCH23390 bound to adrenal medullary membranes was competed for by non-radioactive iodo-SCH23390 (Kd = 490 ± 50 nM), but not by (+)butaclamol. Other classical D1 antagonists had little, if any, effect. Competition with dopamine receptor agonists demonstrated a relative rank order of potency profile characteristic of D1-like dopamine receptors, however, K_is were higher than those found in other tissues. The K_is for competition of [¹²⁵I]SCH23390 binding by C1-APB and SKF38393 (16 and 118 M, respectively) are nearly identical to the IC₅₀s previously observed for inhibition of secretion (9 and 100 M, respectively). Combined these data suggest that adrenal medullary membranes contain a novel SCH23390 binding site involved in the inhibition of secretion by D1-selective agonists.     

AT1 Receptor Induced Alterations in Histone H2A Reveal Novel Insights into GPCR Control of Chromatin Remodeling

Chronic activation of angiotensin II (AngII) type 1 receptor (AT₁R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT₁R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT₁R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT₁R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, {"type":"entrez-protein","attrs":{"text":"Q96QV6","term_id":"74752099","term_text":"Q96QV6"}}Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT₁R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT₁R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Use of scanning cytometry in studying bradykinin binding in MRC-5 cells

Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a larger cell population, calcium flux was measured in MRC-5 cell line expressing the B₂ receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin. This study confirmed that only the B₂ receptor is present on the plasma membrane of MRC-5 cells. Bradykinin binding to the B₂ receptor was not modified by a B₁ agonist (Des-Arg⁹-bradykinin) or by a B₁ antagonist (Des-Arg⁹-[Leu⁸]-bradykinin) but was inhibited by a B₂ agonist ([Hyp³]-bradykinin) and a B₂ antagonist (HOE 140). The source of free calcium was also studied in comparison with ionomycin. The intensity of the calcium peak after binding of bradykinin is independent of the concentration of extracellular calcium. Preincubation with diltiazem or TMB-8 did not modify calcium flux indicating that transduction of the signal after bradykinin binding in this cell line is independent of voltage-dependent channels and does not require mobilization of intracellular calcium blocked by TMB-8. In conclusion, scanning cytometry can be used to study ligand-receptor binding and to obtain results rapidly from multiple cells. Recording of individual cell variations and kinetics enables identification of active agonists or antagonists and consequently the selection of new compounds.Abbreviations 9AA 9 amino acids - CCD charged-coupled device - DMEM Dulbecco's Modified Eagle's Medium - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol-bis (-amino-ethyl ether)N,N,N,N-tetraacetic acid - FCS Fetal Calf Serum - GTP guanosine triphosphate - HBSS Hank's Buffer Salt Solution - IP3 inositol triphosphate     

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied ³H-glycine and ³H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a K_D of 2.5 and 2.0 M, respectively. Specific binding of glycine was displaced by -alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.