Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis. This article has previously been published in issue 4/4, under doi:.     

Isolation of Stable Human Ceruloplasmin and Its Interaction with Salmon Protamine

An interaction was discovered between ceruloplasmin (CP, a ferro-O₂-oxidoreductase, EC 1.16.3.1), a copper-containing protein of human blood plasma, and salmon protamine (PR), a cationic polypeptide of vertebrates that provides a compact structure of spermatozoid DNA. Addition of PR to CP at a molar ratio of 2 : 1 decreases the CP electrophoretic mobility. Two types of CP binding centers for PR were determined: two centers with a high (K _d1 of 5.31 × 10⁻⁷ M) and four centers with a low affinity (K _d2 of 1.56 × 10⁻⁵ M). PR was shown to form complexes with CPs of various animal species. The CP-PR complex dissociates at an increased ionic strength (0.3 M NaCl), at pH decreased below 4.7, and/or in the presence of added polyanions (DNA, lipopolysaccharides, or heparin) or polylysine, which indicates the electrostatic nature of the interaction. The CP-PR interaction increased 1.5-fold the rate of CP-catalyzed oxidation of Fe²⁺. The preliminary treatment of blood plasma with arginine-Sepharose and heparin-Sepharose (to remove the blood coagulation factors) and affinity chromatography on PR-Sepharose allowed us to isolate the practically unproteolyzed monomeric CP in 90% yield; it remained stable for more than two months at 37°C.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 269–279.Original Russian Text Copyright © 2005 by Sokolov, Zakharova, Shavlovskii, Vasil’ev.     

Inhibitory Effect of di- and Tripeptidyl Aldehydes on Calpains and Cathepsins

Abstract

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of α-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B < calpain II ≈ calpain I < cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (K_i) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (K_i, 36 nM) and calpain II (K_i, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (K_i, 0.5nM); acetyl-Leu-Leu-Met-H for cathepsin B (K_i, 100nM).     

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K _i) of 2450 nM and the first-order rate constant (k ₊₂) for the covalent adduct formation was 0.16 min⁻¹. As a reversible inhibitor it was selective towards rat liver MAO-A (K _i = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k ₊₂ = 0.06 min⁻¹). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K _i = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k ₊₂ = 0.29 min⁻¹). The K _i values were significantly decreased in the presence of KCN but there was little effect on the k ₊₂ values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases. Special issue dedicated to Dr. Moussa Youdim.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.     

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [³H]Octarphin was found to bind to macrophages with high affinity (K _d = 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met⁵]enkephalin (K _i > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.     

Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii

In our previous reports, a Kazal family serine protease inhibitor, male reproduction-related peptidase inhibitor Kazal-type (MRPINK) has been identified from the prawn, Macrobrachium rosenbergii, and discovered having an inhibitory effect on the sperm gelatinolytic activity. MRPINK was predicated to inhibit chymotrypsin since it contains leucine and proline at P₁ positions of the two domains, respectively. In this report, recombinant MRPINK was as expected found to specifically inhibit chymotrypsin, but no inhibition was detected against trypsin or thrombin. By the analysis of kinetic tests, the inhibition mechanism of MRPINK was determined to be typical competitive model with K _i of 354 nM. To elucidate the effects of structure on activity of MRPINK, the mutants (domain-1 only, domain-2 only, MRPINK^P88I, MRPINK^L37K, MRPINK^L37A, and MRPINK^L37G) were prepared and their inhibitory activities assayed. The results showed that domain-2 was the key contributor to the inhibition of chymotrypsin (K _i of 416 nM) and P₁ Pro was crucial for the activity. Nevertheless, whether the P₁ amino acid residue was Leu, or even if it was replaced by Lys, Ala, or Gly, domain-1 was ineffective to the activity.     

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q₁₀ and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K_m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V_m versus 1/6PG and 1/NADP plots gave a V_m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K_i of 31.91±1.31 μM. The relatively small K_i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.     

The synthetic peptide octraphin TPLVTLFK is a selective agonist of nonopioid β-endorphin receptor

The peptide TPLVTLFK (coined by the authors “octarphin”), corresponding to the amino acid sequence of β-endorphin fragment 12–19, and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL) were synthesized. The peptide octarphin was labeled with tritium (specific activity, 28 Ci/mol) and its binding to the rat brain cortex membranes and mouse peritoneal macrophages was studied. [³H]Octarphin was found to bind to brain membranes and macrophages with high affinity (K _d = 2.6 ±0.2 and 2.3 +0.2 nM, respectively) and specificity. The specific binding of [³H]octarphin with rat brain membranes and mouse macrophages was inhibited by unlabeled β-endorphin (K _i = 2.4 +0.2 and 2.7 +0.2 nM, respectively) and selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.9 +0.2 and 2.4 +0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met⁵]enkephalin (K _i > 10 mM). Inhibiting activity of unlabeled analogs of octarphin was more than 100 times lower than that of the unlabeled octarphin. Octarphin was shown to stimulate activity of mouse immunocompetent cells in vitro: at the concentration of 1 nM it enhanced the capacity of peritoneal macrophages to digest bacteria Salmonella typhimurium virulent strain 415 in vitro. Thus, octarphin is a selective agonist of nonopioid (insensitive to the opioid antagonist naloxone) β-endorphin receptor of rat brain cortex membranes and mouse peritoneal macrophages.     

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4′ positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-l-phenylalanine residues at 4 and 4′ positions. Binding values are: K_\texti^m = 0.51 \textnM K_{\text{i}}^{\mu } = 0.51\,{\text{nM}} and K_\texti^d = 12.8 \textnM K_{\text{i}}^{\delta } = 12.8\,{\text{nM}} for compound 9, K_\texti^m = 0.09 \textnM K_{\text{i}}^{\mu } = 0.09\,{\text{nM}} and K_\texti^d = 0.11 \textnM K_{\text{i}}^{\delta } = 0.11\,{\text{nM}} for analogue 10.     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

Novel N‐propylphthalimide‐ and 4‐vinylbenzyl‐substituted benzimidazole salts: Synthesis,characterization, and determination of their metal chelating effects and inhibition profiles against acetylcholinesterase and carbonic anhydrase enzymes

The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted N‐heterocyclic carbene (NHC) precursors were synthesized by N‐substituted benzimidazolium with aryl halides. The novel N‐propylphthalimide‐substituted and 4‐vinylbenzyl‐substituted NHC precursors have been characterized by using ¹H NMR, ¹³C NMR, FTIR spectroscopy, and elemental analysis techniques. They were tested for the inhibition of AChE and hCA enzymes and demonstrated efficient inhibition profiles with K_i values in the range of 351.0–1269.9 nM against hCA I, 346.6–1193.1 nM against hCA II, and 19.0–76.3 nM against AChE. On the other hand, acetazolamide, a clinically used molecule, utilized as CA inhibitor, obtained a K_i value of 1246.7 nM against hCA I and 1407.6 nM against hCA II. Additionally, tacrine inhibited AChE and obtained a K_i value of 174.6 nM.     

Synthetic ACTH-Like Peptide GKVLKKRR,Corresponding to the Fragment 81–88 of Human Pro-Interleukin-1α, Acts as an Antagonist of ACTH Receptor

Synthetic adrenocorticotropic hormone (ACTH)-like octapeptide leukocorticotropin (GKVLKKRR), corresponding to the amino acid sequence 81–88 of pro-interleukin-1α, was labeled with tritium (specific activity of 22 Ci/mmol) and was found to bind to rat adrenal cortex membranes with high affinity and specificity (K _d = 2.2 ± 0.1 nM). Synthetic ¹²⁵I-labeled ACTH fragment 11–24 was also obtained (specific activity of 98 Ci/mmol) and shown to bind to ACTH receptor on rat adrenal cortex with high affinity (K _d = 1.8 ± 0.1 nM). Unlabeled leukocorticotropin was found to actively replace ¹²⁵I-labeled ACTH (11–24) in the receptor-ligand complex (K _i = 2.0 ± 0.1 nM). Leukocorticotropin at concentration range of 1–1000 nМ did not affect the adenylate cyclase activity in adrenocortical membranes. Thus, leukocorticotropin is an antagonist of ACTH receptor. ACTH-like peptide GKVLKKRR is an antagonist of ACTH This work financeed by the Russian Foundation for Basic Research (grant No. 05-04-48060), by the programs Leading Scientific Schools (grant No. 312.2003.4), Molecular and Cellular Biology (chairman V.M. Lipkin), and Naukogrady (grant No. 04-04-97200), and by the International Science and Technology Center (project No. 2615). V.V. Yurovsky is supported by the American Heart Association (grant No. 0555415U).     

Two-stage method for purification of ceruloplasmin based on its interaction with neomycin

A two-stage chromatography that yields highly purified ceruloplasmin (CP) from human plasma and from rat and rabbit serum is described. The isolation procedure is based on the interaction of CP with neomycin, and it provides a high yield of CP. Constants of inhibition by gentamycin, kanamycin, and neomycin of oxidase activity of CP in its reaction with p-phenylenediamine were assayed. The lowest K _i for neomycin (11 μM) corresponded to the highest specific adsorption of CP on neomycin-agarose (10 mg CP/ml of resin). Isolation of CP from 1.4 liters of human plasma using ion-exchange chromatography on UNO-Sphere Q and affinity chromatography on neomycin-agarose yields 348 mg of CP with 412-fold purification degree. Human CP preparation obtained with A ₆₁₀/A ₂₈₀ ∼ 0.052 contained neither immunoreactive prothrombin nor active thrombin. Upon storage at 37°C under sterile conditions, the preparation remained stable for two months. Efficient preparation of highly purified CP from rat and rabbit sera treated according to a similar protocol suggests the suitability of our method for isolation of CP from plasma and serum of other animals. The yield of CP in three separate purifications was no less than 78%.     

Antioxidant activity of hydroxy derivatives of coumarin

The inhibition efficiency (antioxidant activity) of hydroxy derivatives of coumarin, such as esculetin, dicumarol, and fraxetin, was studied in the methemalbumin-H₂O₂-tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in a buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) and the inhibition degree (%). The K _i values for esculetin, dicumarol, and fraxetin were 9.5, 15, and 26 μM, respectively. Esculetin and fraxetin inhibited pseudoperoxidase oxidation of TMB in a noncompetitive manner; dicumarol, in a mixed manner. The inhibiting activity of esculetin in peroxidase-catalyzed TMB oxidation at pH 6.4 is characterized by a K _i value equal to 1.15 μM, and the inhibition process is competitive. Esculetin was found to be the most effective antioxidant of plant origin among all derivatives previously studied in model biochemical systems.     

Study of a ligand complexed with Cdk2/Cdk4 by computer simulation

Cyclin-dependent kinases (Cdks) play important roles in the regulation of the cell cycle. Their inhibitors have entered clinical trials to treat cancer. Very recently, Davis et al. (Nat Struct Biol 9:745–749, 2002) have found a ligand NU6102, which has a high affinity with cyclin-dependent kinase 2 (K _i =6 nM) but a low affinity with cyclin-dependent kinase 4 (K _i =1,600 nM). To understand the selectivity, we use homology modeling, molecular docking, molecular dynamics and free-energy calculations to analyze the interactions. A rational 3D model of the Cdk4–NU6102 complex is built. Asp86 is a key residue that recognizes NU6102 more effectively with Cdk2 rather than Cdk4. Good binding free energies are obtained. Energetic analysis reveals that van der Waals interaction and nonpolar contributions to solvent are favorable in the formation of complexes and the sulfonamide group of the ligand plays a crucial role for binding selectivity between Cdk2 and Cdk4. Figure Two-dimensional representative for the interacting model of NU6102 complexed with the Cdk4 from a predicted structure by LIGPLOT.      

Properties and mechanism of the action of the synthetic peptide octarphin

The peptide TPLVTLFK, whose amino acid sequence corresponds to the 12–19 fragment of β-endorphin (the author’s name for the peptide octarphin), and its analogues (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, and TPLVTLFL) have been synthesized. Tritium-labeled octarphin (specific activity of 28 Ci/mol) has been obtained, and its binding to murine peritoneal macrophages has been studied. It was found that [³H]octarphin binds to macrophages with a high affinity (K _d 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin to macrophages was inhibited by the unlabeled β-endorphin and the selective agonist of the nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met5]enkephalin (K _i > 10 μM). The inhibitory activity of the octarphin analogues was more than 100 times lower than that of octarphin. It was shown that octarphin stimulates the activity of mouse immunocompetent cells in vitro and in vivo; at a concentration of 1–10 nM, it increased the adhesion and spreading of peritoneal macrophages and their ability to digest the bacteria of the Salmonella typhimurium virulent strain 415 in vitro. The intraperitoneal injection of the peptide at a dose of 20 μg/animal on day 7, 3, and 1 prior to the isolation of cells led to an increase in the activity of the peritoneal macrophages and the Tand B lymphocytes of the spleen.     

Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5α-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6–8) pH optimum and the type 2 isoform an acidic (5–6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (K_m = 0.5 and 2.9 μM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (K_i (type 2) = 7.3 and 21.7 nM compared to K_i (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (K_i (type 1) = 8.4 nM and 7.2 μg/ml, respectively; K_i (type 2) = 7.4 nM and 4.9 μg/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5α-reductase. Partially purified recombinant 5α-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5α-reductase.     

Effects of Internal K+ and ABA on the Voltage- and Time-dependence of the Outward K+-rectifier in Vicia Guard Cells

One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata to close. K⁺ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K⁺ on the voltage- and time-dependence of the outwardly rectifying K⁺-current (I _K,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative analysis was performed in the presence of ABA at different internal K⁺ concentrations ([K⁺] _i ). Varying [K⁺] _i in the patch pipette from 100 to 270 mm increased the magnitude of I _K,out in a nonlinear manner and caused a negative shift in the midpoint (V _0.5) of its steady-state activation curve. External addition of ABA (10–20 μm) also increased the magnitude of I _K,out at all [K⁺] _i , but caused a shift in V _0.5 of the steady-state activation curve only in those GCP loaded with 150 mm internal K⁺ or less. Indeed, V _0.5 did not shift upon addition of ABA when the [K⁺] _i was above 150 mm and up to 270 mm, i.e., the shift in V _0.5 caused by ABA depended on the [K⁺] _i . Both increase in [K⁺] _i and external addition of ABA, decreased (by ≈ 20%) the activation time constant (τ _n ) of I _K,out. The small decrease in τ _n , in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing cytoplasmic K⁺, and suggest that ABA may increase I _K,out and alter V _0.5 of its steady-state activation curve via an enhancement in cytosolic K⁺. This report describes for the first time the effects of [K⁺] _i on the voltage- and time-dependence of I _K,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics) differences found between intact guard cells and their protoplasts. Received: 1 December 1995/Revised: 8 May 1996     

Binding of synthetic fragments of β‐endorphin to nonopioid β‐endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [³H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (K_d = 2.0 ± 0.1 nM ). The [³H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (K_i = 2.9 ± 0.2 nM ) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met⁵]enkephalin (K_i > 10 µM ). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [³H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (K_i = 3.1 ± 0.3 nM ). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [³H]Octarphin was found to bind to macrophages with high affinity (K_d = 2.3 ± 0.2 nM ). The specific binding of [³H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (K_i = 2.4 ± 0.2 and 2.7 ± 0.2 nM , respectively). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.