α-Methyl-5-HT, a 5-HT2 receptor agonist, stimulates β2-adrenoceptors in guinea pig airway smooth muscle

Alpha-methyl-5-HT is widely used as a high-affinity 5-HT(2) receptors agonist, though some studies have postulated that this drug also activates other serotonergic receptors. In the present work, we found that a wide range of concentrations of alpha-methyl-5-HT induced biphasic responses (contraction followed by relaxation) in guinea pig tracheal rings. The relaxing phase caused by 32microM alpha-methyl-5-HT was blocked by 0.1microM propranolol. Furthermore, during an ongoing histamine-induced contraction, alpha-methyl-5-HT (0.1-100microM) produced a concentration-dependent relaxation starting at 10microM. This relaxation was fully abolished by 0.1microM propranolol or 1microM ICI 118,551 (a selective beta(2)-adrenoceptor antagonist). Additionally, in electrophysiological recordings, 32microM alpha-methyl-5-HT also enhanced the membrane K(+) currents of single tracheal myocytes, an effect reverted by propranolol and ICI 118,551, and mimicked by 0.1microM salbutamol. Thus, we concluded that alpha-methyl-5-HT activates beta(2)-adrenoceptors in guinea pig tracheal smooth muscle at concentrations >or=10microM. This effect must be taken into account when this drug is used in airway smooth muscle and in other tissues expressing beta(2)-adrenoceptors.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Conformational investigation of α,β-dehydropeptides

Conformations of three series of model peptides: homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHCH₃ (Xaa=Phe, Val, Leu. Abu. Ala) as ivell as α,β-dehydro Ac-Pro-ΔXaa-NHCH_s [ΔXaa = (Z)-ΔPhe, ΔVal. (Z)-ΔLeu, (Z)-ΔAbu] were investigated by CD spectroscopy in 2 % dichloromethanecyclohexane, trifluoroethanol. water. and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane-cyclohexane and unordered ones occur in water. The folded conformers are of the inverse γ-turn type for all the peptides but Ac-Pro-L-Phe-NHCH₃ for which the type-I β-turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the typc-II β–turn in 2% dichloromethanecyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2%, dichloromethanecyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the βII-turn conformation as determined for these coinpounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.     

Effects of the novel antidepressant S-adenosyl-methionine on α1- and β-adrenoceptors in rat brain

α₁- and β-adrenoceptors were studied ex vivo in the brains of rats receiving repeated daily treatment with the standard antidepressant imipramine or the atypical antidepressant S-adenosyl-L-methionine (SAM), which has minimal effects on monoamine reuptake or turnover. Consistent with past studies, a decrease in the density of β receptors at three weeks and an increase in the affinity of α₁ receptors for the agonist phenylephrine at one week of treatment was observed with imipramine. By comparison, an increase in the density of β receptors and a decrease in the affinity of α₁ receptors for phenylephrine was observed at one week of treatment with SAM. These changes were no longer apparent at three weeks of treatment. The results suggest that treatment with SAM does lead to changes in adrenergic neurotransmission, but that down regulation of β receptors or increased agonist affinity of α₁ receptors may not be necessary for the production of antidepressant effects.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Comparison of the binding of α-helical and β-sheet peptides to a hydrophobic surface

The induction and stabilisation of secondary structure for a series of amphipathic α-helical and β-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from β-sheet to α-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Qualitative Differences between β-Adrenergic and α-Adrenergic Inotropic Effects in Rat Heart Muscle

Abstract If β- and α-adrenergic inotropic effects are cyclic AMP dependent and cyclic AMP independent, respectively, they may be qualitatively different. The inotropic effects of β-receptor stimulation (isoprenaline) and α-receptor stimulation (phenylephrine combined with propranolol) were characterized in isolated perfused rat hearts, rat atria and rat papillary muscles. The β-effect reached its maximum before the α-effect. The α-effect followed a three-phasic time-course indicating both stimulatory and inhibitory components. The aortic pressure wave (perfused heart) indicated a shorter contraction phase after β-stimulation than after α-stimulation. The time to peak tension (atrium, papillary muscle) was relatively shorter after isoprenaline than after α-stimulation, which tended to prolong it. The contraction-relaxation cycles (atrium, papillary muscle) were examined by recording the isometric tension (T), its first (T′) and second (T″) derivatives, α- and β-stimulation both increased T_max, T′_max (maximal rate of tension rise), T′_min, (maximal rate of tension decline) and T″_min (maximal rate of transition from rise to decline of tension). Isoprenaline increased T_min, (papillary muscle) and T″_min (atrium, papillary muscle) relatively more than did α-stimulation, i.e. the relaxing processes were activated relatively more by β-stimulation. The results indicate different mechanisms for the two adrenergic inotropic effects. The relatively larger activation of relaxation by β-stimulation is assumed to be caused by cyclic AMP.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

Synthesis,crystal structure and molecular conformation of the tBuCO-D,L-Ala-Δz-Phe-NhiPr α,β-unsaturated dipeptide

The crystal structure of the tBuCO-d,l -Ala-Δ^z-Phe-NHiPr dipeptide has been solved by X-ray diffraction. The peptide crystallizes in monoclinic space group P2JC with a = 13.445 (3) Å, b = 35.088 (4) Å, c = 14.755(3) Å, β= 116.73(1)°, Z = 12 and d_c= 1.151 g.cm^?3. The three independent molecules per asymmetric unit accommodate a βII-folded conformation, but only one of them contains the typical i + 3 → i interaction characterizing a β-turn. In the other two molecules, the N…O distance exceeds 3.2 Å, a value generally considered the upper limit for hydrogen bonds in peptides. In solution, the βII-turn conformation is largely predominant.     

Association of β-agonists with corresponding β2- and β1-adrenergic pentapeptide sequences

Synthesized β₁- and β₂-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane α-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the β-adrenoreceptors.     

Peptic proteolysis of esterified β-casein and β-lactoglobulin

Moderate esterification induces slight secondary structure changes in two major milk proteins, β-lactoglobulin and β-casein. Esterification of β-lactoglobulin prompts its tertiary structure‘melting′, opening it to peptic cleavage. Twenty-two new cleavage sites were characterised in β-lactoglobulin and five in β-casein. Some of them are due to esterification-improved peptide bond accessibility, some to the bias of pepsin specificity by glutamate and aspartate esters. The resulting fragmentation yields original and partially amphiphilic peptide populations.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Relaxant β-Receptors in the Trachea,but not Prejunctional α2-Receptors in the Tracheal Cholinergic Neurones,are Subjected to Homologous Desensitization

Abstract: Guinea-pigs were pretreated with either isoprenaline, terbutaline or the α₂ agonist B-HT 920 in order to asses the hypothesis that β- and α₂ receptors in trachea are subjected to homologous desensitization. In these experiments the β receptor activity was investigated on tracheal ring preparations contracted with carbacholine. Both in the isoprenaline and the terbutaline pretreated group the relaxant responses to β agonists were diminished. Pretreatment with B-HT 920 did not affect the sensitivity of the trachea to β stimulation. In order to asses the responsiveness of α₂ receptors the trachea was contracted by electrical field stimulation in the presence of propranolol. During these conditions contractions were mediated by activation of cholinergic neurones and inhibitory effects of alpha stimulation were due to inhibition of the cholinergic neurotransmission by stimulation of prejunctional α₂ receptors. In these tests neither isoprenaline, terbutaline nor B-HT 920 pretreatment affected the responsiveness of α stimulation to inhibit the electrically induced contractions.     

Pharmacological characterization of A-131701, a novel α1-adrenoceptor antagonist selective for α1A- and α1D-compared to α1B-adrenoceptors

New compounds selective for α_1A-adrenoceptors in the prostate may offer enhanced efficacy for benign prostatic hyperplasia (BPH), with fewer side effects than current treatment. A-131701 (3-[2-((3aR,9bR)-cis-6-methoxy-2,3,3a,4,5,9b,hexahydro-[1H]-benz[e]isoindol-2-yl)ethyl]pyrido[3′,4′:4,5]thieno [3,2-d]pyrimidine-2,4(1H,3H)-dione), from a novel class of benz[e]isolindole pyridothienopyrimidines and pyridothienopyrazines, is selective for α_1a- and α_1d-adrenoceptors in radioligand binding studies (0.22 nM at α_1a-, 0.97 nM at α_1d-) compared to α_1b-sites (2.5 nM) and in isolated tissue bioassays (pA₂ values of 8.9–9.0 for α_1A-receptors in rat vas deferens or canine prostate strips, 9.1 at α_1D-sites (rat aorta)), compared to 7.9 at α_1B-sites (rat spleen). A-131701 also potently blocked radioligand binding to α₁-adrenoceptors in canine and human prostatic membranes, but was considerably weaker at α₂-adrenoceptors. In isoflurane-anesthetized dogs, A-131701 antagonized epinephrine-induced increases in intraurethral pressure (IUP) with a pseudo-pA₂ value of 8.17. In spontaneously hypertensive rats, A-131701 caused transient decreases in mean arterial blood pressure (MABP) and transient tachycardia. The area under the curve (AUC₀→₆₀ min) for the hypotensive response was dose-related, with a log index value for A-131701 of 5.33, suggesting a selectivity of >600-fold comparing IUP to MABP effects. In pentobarbital-anesthetized dogs, A-131701 was more potent in blocking phenylephrine (PHE)-induced increases in IUP (pseudo-pA₂ = 8.0) compared to concurrently measured MABP (pseudo-pA₂ = 7.2), or sixfold selective. Doses greater than 1,000 nmol/kg i.v. of A-131701 were required to lower blood pressure by 10 mm Hg in these dogs (pED₁₀ =. 5.57), indicating a uroselectivity ratio of >250, superior to doxazosin, terazosin, or tamsulosin. Thus, A-131701 is selective for α_1A- and α_1D- vs. α_1B-adrenoceptors in vitro, and prostatic function vs. blood pressure effects in vivo, which may provide therapeutic advantages in the treatment of BPH. Drug Dev. Res. 44:140–162, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and receptor binding activity of elephant β-endorphin,a β-endorphin homolog with highly potent analgesic activity#

Elephant β-endorphin and its analog, elephant β-endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant β-endorphin was five to six times more potent than human β-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16 , 33–37), tail flick test for intracerebroventricularly administered β-endor-phin showed that the antinociceptive potency of elephant β-endorphin was seven to eight times higher than that of human β-endorphin in mice. Results from both studies suggest that elephant β-endorphin was a much more potent antinociceptive agent than human β-endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.     

A threonine residue in the M2 region of the β1 subunit is needed for expression of functional α1β1 GABAA receptors

Although there is a high degree of homology in the M2 transmembrane segments of α₁ and β₁ subunits, subunit-specific effects were observed in α₁β₁ GABA_A receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13′ threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13′) was mutated to alanine in the β₁ subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13′) was mutated to alanine in the α₁ subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).