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.     

An α-adrenoceptor-mediated mechanism of hypoactivity induced by β-amyrin palmitate

Abstract— Inhibitory effects of β-amyrin palmitate in locomotor activity of mice were studied by combining this compound with α-adrenergic agonists or antagonists and a dopaminergic agonist. β-Amyrin palmitate (2·5, 5·0 and 10·0 mg kg^?1, i.p.) decreased locomotor activity of mice in a dose-dependent manner. It enhanced hypoactivity of mice treated with clonidine (0·025 mg kg^?1, i.p.) and antagonized hyperactivity produced by phenylephrine (40 μg, i.c.v.). The inhibitory action of β-amyrin palmitate was not affected by yohimbine (1·5 mg kg^?1, i.p.), but was potentiated by prazosin (0·75 mg kg^?1, i.p.). When combined with a dopaminergic agonist, apomorphine (2·0 mg kg^?1, i.p.), β-amyrin palmitate (5·0 and 10·0 mg kg^?1, i.p.) did not affect locomotor stimulation produced by apomorphine. These results suggest that β-amyrin palmitate might inhibit α₁-adrenoceptors.     

Eugenolol: An eugenol-derived β-adrenoceptor blocker with partial β2-agonist and calcium mobilization inhibition associated vasorelaxant activities

The β-adrenoceptor blocking, vasorelaxant and hypotensive activities of eugenolol (1-(isopropylamino)-3-[(4- allyl-2-methoxy-)phenoxy]-2-propanol) were investigated under in vivo and in vitro conditions. In pentobarbital anesthetized rats, eugenolol (0.1, 0.5, 1.0 mg/kg, i.v.) produced a dose-dependent hypotensive and bradycardia response. Eugenolol also inhibited the tachycardia effects induced by (–)isoproterenol, but did not block arterial pressor responses induced by phenylephrine. In isolated guinea pig tissues, eugenolol competitively antagonized the (–)isoproterenol-induced positive inotropic and chronotropic effects and tracheal relaxation responses. The apparent pA₂ values for eugenolol on right atria, left atria and trachea were 8.23 ± 0.04, 8.36 ± 0.13 and 8.18 ± 0.12, respectively. On tracheal strips of reserpinized guinea pig, cumulative doses of eugenolol (10^–10–10^–7 M) produced dose-dependent relaxant responses. Preincubating the preparation with ICI 118,551 (10^–8, 10^–7, 10^–6 M) shifted the eugenolol concentration-relaxation curve significantly to a region of higher concentrations. Binding characteristics of eugenolol and propranolol were evaluated in [³H]dihydroalprenolol binding to pig ventricular membranes. The Ki values of eugenolol and propranolol were 18.1 ± 3.2 and 3.8 ± 0.5 nM, respectively. In guinea pig isolated thoracic aorta, eugenolol relaxed more potently the contractions induced by phenylephrine than those by high K⁺. The vasorelaxant effect of eugenolol on phenylephrine- or high K⁺-induced contraction was not attenuated by TEA or Bay K 8644 pretreatment. Furthermore, eugenolol pretreatment had a greater inhibitory effect on phenylephrine induced phasic contraction than on tonic contraction. These results indicate that eugenolol exhibits non-selective β-blocking and vasorelaxant activity by inhibiting Ca²⁺ channels, receptor-mediated Ca²⁺ mobilization and by its partial β₂-agonist activity. Drug Dev. Res. 40:239–250, 1997. © 1997 Wiley-Liss, Inc.     

Proline-containing β-turns

The conformations of the dipeptide t-Boc-Pro-d Ala-OH and the tripeptide tBoc-Pro-d Ala-Ala-OH have been determined in the crystalline state by X-ray diffraction and in solution by CD, n.m.r. and i.r. techniques. The unit cell of the dipeptide crystal contains two independent molecules connected by intermolecular hydrogen bonds. The urethane-proline peptide bond is in the cis orientation in both the molecular forms while the peptide bond between Pro and d Ala is in the trans orientation. The single dipeptide molecule exhibits a “bent” structure which approximates to a partial β-turn. The tripeptide adopts the 4 → 1 hydrogen-bonded type II β-turn with all trans peptide bonds. In solution, the CD and i.r. data on the dipeptide indicate an ordered conformation with an intramolecular hydrogen bond. N.m.r. data indicate a significant proportion of the conformer with a trans orientation at the urethane-proline peptide bond. The temperature coefficient of the amide proton of this conformer in DMSO-d₆ points to a 3 → 1 intramolecular hydrogen bond. Taken together, the data on the dipeptide in solution indicate the presence (in addition to the cis conformer) of a C₇ conformation which is absent in the crystalline state. The spectral data on the tripeptide indicate the presence of the type II β-turn in solution in addition to the nonhydrogen-bonded conformer with the cis peptide bond between the urethane and proline residues. The relevance of these data to studies on the substrate specificity of collagen prolylhydroxylase is pointed out.     

Chronic terbutaline treatment desensitizes β-adrenergic inhibition of lymphocyte activation in healthy volunteers

1 A substantial body of evidence has accumulated that β-adrenoceptor mediated increases in human lymphocyte cyclic AMP can inhibit activation of resting lymphocytes. The aim of this study was to determine whether this effect might desensitize during chronic β-adrenoceptor agonist treatment. We assessed the effects of 2 weeks treatment with the β₂-adrenoceptor agonist terbutaline (3 × 5 mg day^?1 p.o.) on isoprenaline-induced inhibition of concanavalin A-evoked lymphocyte activation in nine healthy male volunteers. Lymphocyte activation was determined by [³H]-thymidine incorporation (as a measure of proliferation), and inositol phosphate formation was assessed in [³H]-myo-inositol prelabelled lymphocytes in the presence of 10 mm LiCl. 2 Terbutaline treatment caused a significant reduction in isoprenaline (1 nm – 10 μm )-induced increases in lymphocyte cyclic AMP content; the maximal increase was 14 ± 3 pmol/10⁶ cells before and 7 ± 2 pmol/10⁶ cells (n= 9, P < 0.05) after terbutaline treatment. 3 The mitogen concanavalin A (Con A, 1–32 μg ml^?1)-induced increase in inositol phosphate formation was significantly enhanced after terbutaline treatment (max. increase before treatment: 255 ± 25% above basal; after treatment 453 ± 16% above basal; n= 9, P < 0.001), while isoprenaline (1 nm – 10 μm )-induced inhibition of Con A (16 μg ml^?1)-evoked increases in inositol phosphate formation was significantly reduced after the terbutaline treatment (max. inhibition before treatment: 22 ± 4%; after treatment 9 ± 1%, n= 9, P < 0.01). 4 Con A (1.25 – 10 μg ml^?1)-induced increases in [³H]-thymidine incorporation into the lymphocytes (as a measure of proliferation) was not affected by the terbutaline treatment. On the other hand, isoprenaline (1 nm – 1 μm )-induced inhibition of Con A (5 μg ml^?1)-evoked lymphocyte proliferation (max. inhibition: 33 ± 7%, n= 9) was almost completely abolished after the terbutaline treatment. 5 We conclude that chronic treatment with terbutaline desensitizes lymphocyte β₂-adrenoceptors and, therefore, the inhibitory effect of cyclic AMP on lymphocyte activation.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide Dimeric β-sheet formation

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH₃, monohydrate, (C₁₇H₃₀N₄O₅·H₂O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are mono-clinic, space group P2₁, a = 9.2585(4), b = 9.3541(5). c = 12.4529(4) Å, β= 96.449(3)°, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (Δω=5.2° and Δω=3.7°, respectively). The backbone torsion angles are: φ¹, = -60°, ψ_1/= 143.3°, ω₁= -174.8°, φ₂= 148.4°, ψ₂= -143.1°, ω₂= -179.7°, φ₃= 151.4°, ψ₃= -151.9°, ω₃= -176.3°. The pyrrolidine ring of the proline residue adopts the C₂— C^γ conformation. The molecular packing gives rise to an antiparallel β-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric β-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient β-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH₃.     

β-Endorphin does not protect alkylation of opiate receptor by N-ethylmaleimide

Rat brain membranes were incubated in N-ethylmaleimide (NEM, 0.5–1.0 mM) in the presence and absence of various concentrations of morphine, Leuenkephalin and human β-endorphin (β-EP). After sufficient washing, the binding of dihydromorphine (DHM), [d -Ala-d -Leu]-enkephalin (DADLE) and tritiated β_h-EP was 10–40% above that of membranes treated with NEM alone. There was no additive effect of morphine and Leu-enkephalin with respect to their effect on recovery of β_h-EP binding. Evaluation of β_h-EP as protecting ligand proved to be difficult since preincubation completely inhibits subsequent DHM and DADLE binding unless a more extensive washing protocol is employed. A protocol for washing β_h-EP preincubated membranes using a Tris-phosphate buffer of pH 6 containing 150 mM NaCl, 20 mM MgCl₂ and 10% glycerol was used to recover enough binding potential to evaluate the effects of β_h-EP preincubation towards NEM treatment. Preincubation with β_h-EP itself at 0.1–1.0μM did not result in any increased recovery of opiate binding, in contrast to the findings with the other two ligands.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

Properties of a Resiniferatoxin-stimulated,Calcium Inhibited but Phosphatidylserine-dependent Kinase,which is Distinct from Protein Kinase C Isotypes α, β1γ, δ, ε and η

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca²⁺ the K_a for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nm . Using a phosphate gradient of 20–500 mm , peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nm phosphate and resiniferatoxin kinase recovered in 500 mm phosphate. At the optimum concentration of 160 nm , the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 μm failed to activate the kinase. A Scatchard analysis of [³H] phorbol dibutyrate binding produced a linear plot (K_d 41·6 nm ; B_max 11·6 fmol unit^?1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabeled resiniferatoxin binding assay was also used to demonstrate specific binding of [³H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes α, β₁, γ δ and ε by means of immunological analysis and from the η isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca²⁺ (K_i 0·1-0·5 nm free Ca²⁺). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70–90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.     

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.     

Effect of Serum, α-1 Acid Glycoprotein,Lipoproteins and Albumin on Human Mononuclear Leucocyte β-Adrenoceptors

Abstract: The effects of serum, α-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on ³H-(-)-dihydroalprenolol (³H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of ³H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (K_d) of approximately 0.7 and 65 nM. Stimulation of the MNL β-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 ～0.2 μM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30–35 min. exposure to serum, AAG and SLP increased the number of β-adrenoceptors with 100–200% and the maximal responsiveness to (-)-IPR with 30–90%. The presence of proteins did not change the K_d or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25–40% without effect on the K_d. HSA had no consistent effect on β-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL β-adrenoceptors in vitro.     

Synthesis and properties of human β-endorphin-(1–9) and its analogs

Four analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–9) (I), [D-Ala²]-β_h-EP-(1–9) (II), [Gln⁸]-β_h-EP-(1–9) (III), and [D-Ala², Gln⁸]-β_h-EP-(1–9) (IV). Measurement in a radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: Met-enkephalin, 100; I, 76; II, 100; III, 200; IV, 200. Two new amino acid derivatives were prepared and used for synthesis of the analogs: N^α-t-butyloxycarbonyl-O-(cyclopentyl) -tyrosine and N^α-t-butyloxycarbonyl-γ-(cyclopentyl)-glutamic acid.     

β1- and β2-Adrenoceptor Affinity and Stimulatory Effects of (S)-Pindolol and Iodinated (S)-Pindolol

Abstract: The β₁- and β₂-adrenoceptor affinity and stimulatory effects of iodinated (S)-pindolol (IPIN) and (S)-pindolol were investigated in vitro using β-adrenoceptor binding technique and isolated right atrium (rate increase, β₁) and uterus (relaxation, β₂) of the rat. IPIN had a higher affinity towards β-adrenoceptors compared to (S)-pindolol, with some β₂-adrenoceptor selectivity. In the rat uterus, IPIN produced only marginal stimulatory effects, while (S)-pindolol caused a concentration-dependent relaxation with a maximal effect that was 55% of that generated by isoprenaline. In the right atrium IPIN caused an increase in the atrial rate similar to that caused by (S)-pindolol. The concentration of IPIN required in the right atrium for a half-maximal response (pD₂ = 7.81) was markedly greater than that required for occupation of half the β-adrenoceptor population (pK_B = 9.81). The β₁-selective blocker metoprolol antagonized the effect of (S)-pindolol and IPIN on the atrial rate but a greater concentration of metoprolol (5 × 10^?6 M compared with 5 × 10^?7 M) was required to antagonize the effect of IPIN significantly. It is concluded that iodination of (S)-pindolol increased its affinity and decreased its efficacy towards β-adrenoceptors.     

β-Endorphin: synthesis and properties of human β-endorphin-(1–28) and its analogs

Three analogs of human β-endorphin (β_h-EP) were synthesized by the solid-phase method: β_h-EP-(1–28) (II), [D-Ala², Gln⁸] - β_h-EP-(1–28) (III). Radioreceptor binding assay with use of tritiated β_h-EP as primary ligand gave relative potencies as follows: β_h-EP, 100; I, 85; II, 380; III, 146. Relative potencies in an analgesic assay were: β_h-EP; 100; I, 18; II, 36; III, 13.     

Functional identification of β3‐adrenoceptors in the guinea‐pig ileum using the non‐selective β‐adrenoceptor antagonist (±)‐bupranolol

1 To clarify whether there is a species difference or a tissue difference in β₃‐adrenoceptors, the β₃‐adrenoceptors mediating relaxations to catecholamines ((–)‐isoprenaline, (–)‐noradrenaline and (–)‐adrenaline), a selective β₃‐adrenoceptor agonist BRL37344 and a non‐conventional partial β₃‐adrenoceptor agonist (±)‐CGP12177A (a potent β₁‐ and β₂‐adrenoceptor antagonist with a partial β₃‐adrenoceptor agonist property) were investigated in the guinea‐pig ileum. 2 Catecholamines and β₃‐adrenoceptor agonists induced concentration‐dependent relaxations of pre‐contracted strips of the guinea‐pig ileum. The rank order for their relaxing potency was (–)‐isoprenaline (pD₂: 7.60) > BRL37344 (7.05) > (–)‐noradrenaline (6.38) > (±)‐CGP12177A (6.25) > (–)‐adrenaline (6.07). 3 In the presence of the non‐selective β₁‐ and β₂‐adrenoceptor antagonist (±)‐propranolol (1 μM ), only small rightward shifts of the concentration–response curves (CRCs) to these agonists were observed and the rank order of potency of agonists was BRL37344 (pD₂: 7.00) > (±)‐CGP12177A (6.17) > (–)‐isoprenaline (6.01) > (–)‐noradrenaline (5.69) > (–)‐adrenaline (5.41). 4 In the presence of (±)‐propranolol (1 μM ), the additional presence of (±)‐bupranolol (3–30 μM ), a non‐selective β₁‐, β₂‐ and β₃‐adrenoceptor antagonist, caused a concentration‐dependent rightward shift of the CRCs to catecholamines and β₃‐adrenoceptor agonists. Schild plot analyses of (±)‐bupranolol against these agonists gave pA₂ values of 6.02 ((–)‐isoprenaline), 6.03 ((–)‐noradrenaline), 6.01 ((–)‐adrenaline), 6.56 (BRL37344) and 5.74 ((±)‐CGP12177A), respectively. All Schild plot slopes were not significantly different from unity. The pA₂ values of (±)‐bupranolol obtained for the guinea‐pig β₃‐adrenoceptors were about one log unit less than the values obtained for the rat β₃‐adrenoceptors and about two log units less than the values obtained for dog β₃‐adrenoceptors. 5 These results confirm that functional β₃‐adrenoceptors are present in the guinea‐pig ileum and that the relaxations of these agonists are mainly mediated via β₃‐adrenoceptors in this tissue. The differential antagonistic potency of (±)‐bupranolol may suggest that there is a species difference between the three species (guinea‐pig, dog and rat) in their β₃‐adrenoceptors.     

Peripheral Metabolism of β-Carboline-Carboxylic Acid Esters

Abstract: Esters of β-carboline-3-carboxylic acid have recently been identified as potent inhibitors of brain benzodiazepine receptors in vitro. Ethyl β-carboline-3-carboxylate (β-CCE), however, is a rather weak inhibitor in vivo of benzodiazepine receptors in mice. The ED50-value was 91 mg/kg intraperitoneally 35 min. after administration (ED50 is that dose which inhibits by 50% the specific binding of ³H-flunitrazepam intravenously). ED50 for β-CCE was 2–20 fold lower in mice pretreated with organophosphorus esterase inhibitors, concomitantly with the observation of strong inhibition of liver and kidney hydrolyzing activity, using ³H-propyl β-carboline-3-carboxylate as substrate. The rat brain contains only approximately 0.1% of the hydrolyzing activity as compared to the liver. It is concluded that some esters of β-carboline-3-carboxylate exhibit only weak effects on benzodiazepine receptors in living animals due to hydrolysis outside the brain.     

β1- and β3-adrenoceptors mediate relaxation in ovine trachealis smooth muscle

1 Isoprenaline (non-selective) and noradrenaline (β₁-selective) concentration-dependently relaxed ovine tracheal strips precontracted with carbachol. The pD₂ values were 7.07 ± 0.08 and 6.13 ± 0.10 for isoprenaline and noradrenaline, respectively. In the same preparation, salbutamol either produced weak relaxation or in some cases, contractile responses indicating the presence of very little or no β₂-adrenoceptors in this preparation. 2 Isoprenaline-and noradrenaline-induced relaxations were antagonized by propranolol and atenolol with pA₂ values in the range reported in the literature for an action on β₁-adrenoceptors. ICI 118551 also antagonized isoprenaline- and noradrenaline-induced relaxation but at concentrations much higher than are required to block β₂-adrenoceptors, confirming that β₂-adrenoceptors do not contribute significantly to these responses. 3 The selective β₃-adrenoceptor agonist, BRL 37344A produced concentration-dependent relaxation of tracheal strips. BRL 37344A was a full agonist producing 100% relaxation of carbachol-induced tone. BRL 37344A-induced relaxation was weakly antagonized by propranolol confirming an action, mainly, on β₃-adrenoceptors. Cyanopindolol antagonized isoprenaline-induced relaxation (in the presence of propranolol, 10^??7 m ) with a pA₂ value of 8.06 ± 0.24. 4 It was therefore concluded that β₁- and β₃-adrenoceptors mediated agonist-induced relaxation in sheep tracheal strips.     

β-LYTIC PROTEASE,A NEUTRAL SORANGIOPEPTIDASE*

The specificity of β-lytic protease of Sorangium is found to be similar to neutral proteases of bacterial origin. The β-enzyme did not hydrolyze any of the common esters of pancreopeptidases; however, an affinity for the peptide bond between R and R¹ in the molecular arrangement X-R-R¹-NH₂ was shown. The substitution occurred at the N-terminal of a hydrophobic amino acid whose carboxyl end was not terminal. 3-(2-furyl-acryloyl)-glycyl-L-leucine amide was the substrate of choice since accurate spectrophotometric rate assays could be accomplished with this substrate. A close relationship existed between the mammalian proteases and Sorangium proteases. The β-enzyme was similar to carboxypeptidase (non-serine protease) in some respects, and the α, δ, enzymes of Sorangium were, to some extent, similar to serine proteases of mammalian origin. In agreement with the nomenclature of other neutral proteases, a more specific name of ‘Neutral Sorangiopeptidase’ has been suggested.