Effect of natsudaidain isolated from Citrus plants on TNF‐α and cyclooxygenase‐2 expression in RBL‐2H3 cells

Objectives Flavonoids inhibit the activity of chemical mediators released from mast cells. Our aim was to investigate the effects of natsudaidain, a polymethoxyflavone isolated from Citrus plants, on mast cells. Methods We investigated the inhibitory effects of natsudaidain, which is a polymethoxy‐flavone isolated from Citrus plants, on histamine release, tumour necrosis factor‐α production and cyclooxygenase‐2 expression in Ca ionophore‐stimulated rat basophilic leukemia cells (A23187‐stimulated RBL‐2H3 cells) by spectrofluorometric, ELISA and immunoblotting methods. Key findings The percent of histamine release from A23187‐stimulated RBL‐2H3 cells pretreated with natsudaidain at 5, 25 and 50 μM was not changed as compared with non‐treated A23187‐stimulated cells. At 100 and 200 μM, natsudaidain pretreatment resulted in slightly reduced histamine release (% histamine release, 89.8 ± 3.5% and 71.5 ± 5.6% at 100 and 200 μM). Thus, natsudaidain hardly affects histamine release from RBL‐2H3 cells, except at high concentrations. On the other hand, natsudaidain dose‐dependently inhibited tumour necrosis factor‐α protein and mRNA levels in A23187‐stimulated RBL‐2H3 cells; a concentration of 6.8 μM was required for a 50% reduction. In addition, all concentrations of this compound that we tested also inhibited cyclooxygenase‐2 protein expression. The mRNA levels of cyclooxygenase‐2 in A23187‐stimulated RBL‐2H3 cells treated with natsudaidain were also markedly decreased. The phosphorylated‐p38 MAPK protein levels in A23187‐stimulated RBL‐2H3 cells treated with natsudaidain were lower than in the non‐treated cells. Conclusions These findings suggest that natsudaidain inhibits tumour necrosis factor‐α and cyclooxygenase‐2 production by suppressing p38 MAPK phosphorylation but not p65 NFKB phosphorylation, and that natsudaidain might alleviate inflammatory diseases.     

Peroxisome proliferator‐activated receptor‐γ coactivator‐1α in muscle links metabolism to inflammation

1. In higher eukaryotes, metabolism and immunity are tightly coupled. However, whereas in evolutionary terms a compromised immune response due to undernourishment has been the predominant problem, the inflammatory response to obesity and other lifestyle‐associated diseases has increased in relevance in Western societies in the past 100 years. 2. Traditionally, fat tissue has been considered as the major source of pro‐inflammatory secreted factors in these pathologies. However, in recent years the contribution of other tissues to disease‐causing chronic inflammation has been increasingly appreciated. 3. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) is one of the key regulatory factors in active skeletal muscle. Aberrant expression of PGC‐1α in inactive muscle fibres could be linked to a sedentary lifestyle, persistent systemic inflammation and a higher risk for many chronic diseases. Accordingly, modulation of PGC‐1α activity in skeletal muscle may have a broad range of therapeutic effects. Here, recent advances in the understanding of the role of muscle PGC‐1α in health and disease are reviewed.     

RNAi Silencing of IL‐1β and TNF‐α in the Treatment of Post‐traumatic Arthritis in Rabbits

Post‐traumatic arthritis is a secondary complication to severe joint trauma. With the disease progression, it may eventually lead to osteoarthritis in patients whose age is considerably younger than patients with traditional bone arthritis. The main objective of this study was to explore the feasibility of using lentiviral‐mediated RNA interference silencing of IL‐1β and TNF‐α to treat post‐traumatic arthritis in rabbits. About 48 New Zealand rabbits underwent bilateral knee joint surgery to stimulate traumatic arthritis. They were then randomly divided into four groups of 12 rabbits each. The histopathology of the cartilage was observed, and the changes were assessed by Mankin scoring. ELISA was used to detect the expression of IL‐1β and TNF‐α in the synovial fluid. (i) Compared with the control group, the transfection and co‐transfected groups displayed reduced cartilage damage and speed of degeneration. The co‐transfected group showed the greatest alleviation of symptoms. The Mankin score was statistically different (p < 0.01). (ii) Compared with the control group, the expression of IL‐1β or TNF‐α was reduced in the respective transfection groups (p < 0.01 in both groups) and IL‐1β and TNF‐α were reduced in the co‐transfected group (p < 0.01). The co‐transfected group showed the lowest expression of the three experimental groups of both IL‐1β and TNF‐α (p < 0.01). Lentivirus‐mediated RNA interference can knock down the expression of IL‐1β and TNF‐α in joint fluids and, in a synergistic effect when two siRNAs are co‐transfected, ease cartilage degeneration.     

Identification of small molecule estrogen‐related receptor α–specific antagonists and homology modeling to predict the molecular determinants as the basis for selectivity over ERRβ and ERRγ

The estrogen‐related receptor α (ERRα) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor α (ERα). The physiological role of ERRα has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders. A homogeneous time‐resolved fluorescence (HTRF) assay has been developed to screen for ERRα‐specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator‐activated Receptor γ Coactivator‐1α (PGC‐1α) to examine the ability of compounds to antagonize the constitutive interaction between ERRα and the coactivator. A dissociation‐enhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high‐affinity ERRα subtype selective antagonists. Additionally, a homology model of ERRα in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri‐cyclic antagonist, N‐[(2Z)‐3‐(4,5‐dihydro‐1,3‐thiazol‐2‐yl)‐1,3‐thiazolidin‐2‐yl idene]‐5 H dibenzo[a,d][7]annulen‐5‐amine, binds to ERRα with high affinity but does not bind to either ERRβ or ERRγ. Drug Dev Res 69:203–218, 2008. © 2008 Wiley‐Liss, Inc.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

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 ).     

Synthesis of 1,4‐Anthracene‐9,10‐dione Derivatives and Their Regulation of Nitric Oxide,IL‐1β and TNF‐α in Activated RAW264.7 Cells

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono‐ and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL‐1β, TNF‐α and nitric oxide production by LPS/IFN‐γ‐stimulated RAW264.7 cells. The disubstituted 1,4‐anthracene‐9,10‐dione 10 showed significant inhibition of nitric oxide, TNF‐α and IL‐1β production at the concentration of 5 μg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3 , 4 , 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL‐1β production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF‐α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Therapeutic index of methotrexate depends on circadian cycling of tumour necrosis factor‐α in collagen‐induced arthritic rats and mice

Objectives Rheumatoid arthritis is an autoimmune disorder of unknown aetiology. Morning stiffness, a characteristic feature of rheumatoid arthritis, shows a 24‐h rhythm. Noticing this rhythm, we hypothesized the presence of a similar rhythm for a rheumatoid arthritis indicator, in addition to dosing‐time dependency of the anti‐rheumatic effect of methotrexate in arthritis induced by collagen in rats and mice, which reflect the symptomatology of rheumatoid arthritis patients. Methods To measure tumour necrosis factor (TNF)‐α concentration, blood was taken at different times (2, 6, 10, 14, 18 or 22 h after the light was turned on (HALO)) in collagen‐induced arthritic mice. Methotrexate was administered at two different dosing times based on these findings to estimate arthritis. Key findings The arthritis score was significantly lower in the 22 HALO‐treated group than in the control and 10 HALO‐treated groups in collagen‐induced arthritic rats and mice. Plasma TNF‐α concentrations showed obvious 24‐h rhythms, with higher levels at light phase and lower levels at dark phase after rheumatoid arthritis crisis. Arthritis was relieved after administration of methotrexate during the dark phase in synchronization with the 24‐h rhythm. Conclusions Our findings suggest that choosing an optimal dosing time associated with the 24‐h cycling of TNF‐α could lead to effective treatment of rheumatoid arthritis by methotrexate.     

Up‐regulation of Gadd45α after exposure to metal nanoparticles: The role of hypoxia inducible factor 1α

The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as cobalt and titanium dioxide (Nano‐Co and Nano‐TiO₂) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage, and apoptosis. The growth arrest and DNA damage‐inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non‐toxic doses of Nano‐Co and Nano‐TiO₂ were selected to treat cells. Our results showed that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression, but Nano‐TiO₂ did not. To investigate the potential pathways involved in Nano‐Co‐induced Gadd45α up‐regulation, we measured the expression of hypoxia inducible factor 1α (HIF‐1α) in PW cells exposed to Nano‐Co and Nano‐TiO₂. Our results showed that exposure to Nano‐Co caused HIF‐1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock‐out cells [HIF‐1α (?/?)] and its wild‐type cells [HIF‐1α (+/+)] were used. Our results demonstrated that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression in wild‐type HIF‐1α (+/+) cells, but only a slight increase in HIF‐1α (?/?) cells. Pre‐treatment of PW cells with heat shock protein 90 inhibitor, 17‐(Allylamino)?17‐demethoxygeldanamycin (17‐AAG), prior to exposure to Nano‐Co significantly abolished Nano‐Co‐induced Gadd45α expression. These results suggest that HIF‐1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano‐Co. These findings may have important implications for understanding the potential health effects of metal nanoparticle exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 490–499, 2015.     

Suppression of tumour necrosis factor‐α by Schizonepeta tenuifolia water extract via inhibition of IκBα degradation and Jun N‐terminal kinase/stress‐activated protein kinase activation

Objectives The anti‐inflammatory effects of an aqueous extract of Schizonepeta tenuifolia on lipopolysaccharide (LPS)‐induced tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) in vivo and in vitro have been investigated. Methods C57BL/6 mice were orally administered phosphate‐buffered saline (control) or S. tenuifolia water extract (50, 200, 500 or 1000 mg/kg) for 10 days before intraperitoneal administration of LPS (1.3 mg/kg). Blood samples were obtained 1 h after LPS challenge, followed by determination of TNF‐α and IL‐6 levels. Peritoneal macrophages from thioglycollate‐injected mice were obtained and stimulated with LPS and S. tenuifolia water extract for viability assay, cytokine analysis, real‐time RT PCR and Western blotting. Key findings Oral administration of S. tenuifolia water extract to mice significantly reduced LPS‐induced serum levels of TNF‐α, but not IL‐6. When peritoneal macrophages were treated in vitro with S. tenuifolia water extract, the inhibition of LPS‐induced TNF‐α was more pronounced than that of IL‐6 at the level of secreted protein and mRNA. S. tenuifolia water extract reduced the degradation of IκBα and the nuclear relocation of p65 NF‐κB, but the phosphorylation of IκBα was not affected. Inhibition of c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) by S. tenuifolia water extract led secondarily to the inhibition of phospho‐c‐Jun and phospho‐ATF‐2. Conclusions These results indicated that the downregulation of TNF‐α by S. tenuifolia water extract may have involved the inhibition of both IκBα degradation and activation of c‐Jun and ATF‐2 involving suppression of JNK/SAPK.     

Conformational specificity of mini‐αA‐crystallin as a molecular chaperone

Abstract: The chaperone activity and biophysical properties of the 19 amino acid peptide DFVIFLDVKHFSPEDLTVK, identified as the functional element in αA‐crystallin and here referred to as mini‐αA‐crystallin, were studied using light scattering and spectroscopic methods after altering its sequence and enantiomerism. The all‐d and all‐l conformers of the peptide do not show marked differences in their chaperone‐like activity against heat‐induced aggregation of alcohol dehydrogenase at 48°C and dithiothreitol‐induced aggregation of insulin. The retro peptide does not show any secondary structure and is also unable to act like a chaperone. Both all‐l and all‐d peptides lose their β‐sheet conformations, hydrophobicity and chaperone‐like activity at temperatures > 50°C. However, upon cooling, a significant portion of those properties was regained, suggesting temperature‐dependent, reversible structural alterations in the peptides under investigation. We propose that both the hydrophobicity and β‐sheet conformation of the functional element of αA‐crystallin are essential for chaperone‐like activity.     

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.     

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.     

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.     

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.     

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.