ESIPT-AIE active Schiff base based on 2-(2'-hydroxyphenyl)benzo-thiazole applied as multi-functional fluorescent chemosensors.

Three AIE (aggregation-induced emission)-ESIPT (excited-state intramolecular proton transfer) active 2-(2-hydroxyphenyl)benzothiazole derivatives, HL1, HL2 and HL3 with one, two and three rotatable phenyl groups, were obtained and characterized. Their AIE properties in THF/HEPES solution were investigated in detail. HL2 shows the best AIE performance with 71-fold fluorescence enhancement, while HL3 only shows a 9-fold enhancement. With the AIE property, HL1 and HL2 could act as fluorescence chemosensors to detect Cu2+ ions via the "turn off" mode in THF/HEPES media. With the ESIPT property, HL1 and HL2 could also detect Zn2+ ions via the "turn on" mode in EtOH/HEPES media. During the detection process, both demonstrate rapid response and high contrast before and after the addition of metal ions. The species formed in the detection system were investigated. The results of X-ray single-crystal diffraction confirm that Zn2+ is coordinated with the oxygen atom and Schiff base nitrogen atom instead of the benzothiazole nitrogen atom in the tetrahedron geometry. Moreover, the chemosensors were successfully constructed into handy fluorescence test papers for Cu2+ and Zn2+ detection.

The glycine hinge of transmembrane segment 2 modulates the subcellular localization and gating properties in TREK channels.

TWIK-Related K+ channels (TREK), including TREK-1 and TREK-2, belong to the TREK/TRAAK subclass of two-pore domain K+ (K2P) family. The important functions of transmembrane segment 4 (M4)-glycine hinge in TREK channel gating have been characterized, but the roles of M2-hinge (the equivalent residue of M4-hinge) remain unclear. Here, by characterizing the macroscopic currents, subcellular localization and gating properties of their M2-hinge mutants (G166A for TREK-1 and G196A for TREK-2), we investigated the functions of M2-hinge. G166A displayed decreased whole-cell currents, whereas no current was produced by G196A. Subcellular analysis indicated that both mutants were aggregated near the perinuclear region, and most of them were retented within the endoplasmic reticulum (ER). Next, to explore the roles of M2-hinge in the gating mechanism, we tested the responses of the related M2-hinge mutants to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH alteration (ΔpHo). TREK-1mut7-G166A displayed reduced sensitivity to 2-APB activation, but similar sensitivity to ΔpHo, when compared with TREK-1mut7. WT-ΔpCt, a TREK-2 tandom dimer, was used to assess the function of M2-hinge in the cis-type gating of TREK-2. The sensitivities of G196A-ΔpCt to both 2-APB and ΔpHo decreased compared with WT-ΔpCt. Taken together, our results reveal that the M2-hinge of TREK channels control their macroscopic current, subcellular localization and gating process.

Coordination-Directed Stacking and Aggregation-Induced Emission Enhancement of the Zn(II) Schiff Base Complex.

2-(Trityliminomethyl)-quinolin-8-ol (HL) and its Zn(II) complex were synthesized and characterized by single-crystal X-ray diffraction. HL is an unsymmetrical molecule and coordinated with Zn(II) ion to form ZnL2 in the antiparallel-mode arrangement via Zn-O (hydroxyl group) and Zn-N (quinoline ring) of HL. A high degree of ZnL2 molecules ordering stacking is formed by the coordination bonds and intermolecular π-π interactions, in which head-to-tail arrangement (J-mode stacking) for L- is found. HL is nonfluorescent and ZnL2 is weakly fluorescent in THF. The fluorescence emission of ZnL2 enhances in THF/H2O as H2O% (volume %) is above 60% and aggregates particles with several hundred nanometers are formed, which is confirmed by DLS data and TEM images. The J-aggregates stacking for L- in ZnL2 results in aggregation-induced emission enhancement (AIEE) for ZnL2 in THF/H2O. Theoretical computations based on B3LYP/6-31G(d, p) and TD-B3LYP/6-31G(d, p) methods were carried out. ESIPT is the supposed mechanism for fluorescent silence of HL, and fluorescence emission of ZnL2 is attributed to the restriction of ESIPT process. The oscillator strength of ZnL2 increases from 0.017 for monomer to 0.032 for trimer. It indicates that a high degree of ZnL2 molecules ordering stacking in THF/H2O is of benefit to fluorescence enhancement. HL is an ESIPT-coupled AIEE chemosensor for Zn(II) with high selectivity and sensitivity in aqueous medium. HL can efficiently detect intracellular Zn(II) ions because of ESIPT-coupled AIEE property of ZnL2 in mixed solvent.

Four new taraxastane-type triterpenoic acids from Cirsium setosum.

Four new taraxastane-type triterpenoids acids 3ß,22α-dihydroxy-20-taraxasten-30-oic acid (1), 3ß-hydroxy-22-oxo-20-taraxasten-30-oic acid (2), 3-oxo-22α-hydroxy-20- taraxasten-30-oic acid (3), and 3ß,19ß-dihydroxy-20-taraxasten-30-oic acid (4) were isolated and characterized from Cirsium setosum (Willd.) MB. Their structures were determined by the combination of 1D and 2D NMR experiments ((1)H-(1)HCOSY, HSQC, HMBC and ROESY) and mass spectrometry. Compound 2 exhibited potent selective cytotoxicity against human ovarian cancer cell line A2780 with an IC50 value of 3.9 µM.

Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2.

In response to diverse stimuli, two-pore-domain potassium channel TREK-2 regulates cellular excitability, and hence plays a key role in mediating neuropathic pain, mood disorders and ischemia through. Although more and more input modalities are found to achieve their modulations via acting on the channel, the potential role of subunit interaction in these modulations remains to be explored. In the current study, the deletion (lack of proximal C-terminus, ΔpCt) or point mutation (G312A) was introduced into TREK-2 subunits to limit K(+) conductance and used to report subunit stoichiometry. The constructs were then combined with wild type (WT) subunit to produce concatenated dimers with defined composition, and the gating kinetics of these channels to 2-Aminoethoxydiphenyl borate (2-APB) and extracellular pH (pHo) were characterized. Our results show that combination of WT and ΔpCt/G312A subunits reserves similar gating properties to that of WT dimmers, suggesting that the WT subunit exerts dominant and positive effects on the mutated one, and thus the two subunits controls channel gating via a concerted cooperative manner. Further introduction of ΔpCt into the latter subunit of heterodimeric channel G312A-WT or G312A-G312A attenuated their sensitivity to 2-APB and pHo alkalization, implicating that these signals were transduced by a cis-type mechanism. Together, our findings elucidate the mechanisms for how the two subunits control the pore gating of TREK-2, in which both intersubunit concerted cooperative and cis-type manners modulate the allosteric regulations induced by 2-APB and pHo alkalization.

Allosteric coupling between proximal C-terminus and selectivity filter is facilitated by the movement of transmembrane segment 4 in TREK-2 channel.

TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.

The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2

TREK-2 (TWIK-related K+ channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K+. Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba2+ inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K+ or mutation of S4 K+ binding site, which suggests that the inhibitory mechanism of Ba2+ is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba2+ and alteration of extracellular pH (pHo), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pHo, which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels

The isoforms generated by alternative translation initiation adopt similar conformation in the selectivity filter in TREK-2.

TREK-2 (TWIK-related K(+) channel-2), a member of two-pore domain potassium (K2P) channel family, tunes cellular excitability via conducting leak or background currents. In TREK-2, the isoforms generated by alternative translation initiation (ATI) mechanism exhibit large divergence in unitary conductance, but similar in selectivity to K(+). Up to now, the structural basis for this similarity in ion selectivity is unknown. Here, we report that externally applied Ba(2+) inhibits the currents of TREK-2 in a concentration- and time-dependent manner. The blocking effect is blunted by elevated extracellular K(+) or mutation of S4 K(+) binding site, which suggests that the inhibitory mechanism of Ba(2+) is due to its competitive docking properties within the selectivity filter (SF). Next, we demonstrate that all the ATI isoforms exhibit analogous behaviors upon the application of Ba(2+) and alteration of extracellular pH (pHo), which acts on the outer position of the SF. These results strongly support the notion that all the ATI isoforms of TREK-2 possess resembled SF conformation in S4 site and the position defined by pHo, which implicates that neither the role of N-terminus (Nt) nor the unitary conductance is associated with SF conformation. Our findings might help to understand the detail gating mechanism of TREK-2 and K2P channels.

Mas-related G protein-coupled receptor D is coupled to endogenous calcium-activated chloride channel in Xenopusoocytes

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopusoocytes by using â-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca2+-activated chloride channels (CaCCs) and intracellular Ca2+ chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the â-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via Gq-PLC-IP3-Ca2+ pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD

Mas-related G protein-coupled receptor D is coupled to endogenous calcium-activated chloride channel in Xenopus oocytes.

Mas-related G protein-coupled receptor D (MrgD) is expressed almost exclusively in nociceptive primary sensory neurons and the neurons located in stratum granulosum of skin. More and more evidence suggest that MrgD plays an important role in pain sensation and/or transduction. Recent studies have demonstrated that the receptor is also involved in itch sensation in both mouse and human. In the present study, we identified a robust inward current in MrgD-expressing Xenopus oocytes by using ß-alanine, a putative ligand of MrgD. The currents were sensitive to inhibitor of Ca(2+)-activated chloride channels (CaCCs) and intracellular Ca(2+) chelator, suggesting they were produced by endogenous CaCCs. Furthermore, it was demonstrated that upon the application of phospholipase C (PLC) inhibitor, or antisense oligonucleotides of inositol trisphosphate receptor (IP3R), the ß-alanine-induced currents were dramatically depressed. However, protein kinase C inhibitor did not display any visible effect on CaCC currents. In summary, our data suggest that the activation of MrgD promotes the open of endogenous CaCCs via G(q)-PLC-IP3-Ca(2+) pathway. The current findings reveal the functional coupling between MrgD and CaCCs in Xenopus oocytes and also provide a facile model to assay the activity of MrgD.

Aquaporin-4 deficiency attenuates opioid dependence through suppressing glutamate transporter-1 down-regulation and maintaining glutamate homeostasis.

BACKGROUND: Glutamate homeostasis plays a critical role in mediating the addiction-related behaviors. Therefore, preventing the disruption or reestablishing of it is a novel strategy for the treatment of addiction. Glutamate transporters are responsible for clearing extracellular glutamate and maintaining glutamate homeostasis. Our previous work demonstrated that aquaporin-4 (AQP4) deficiency attenuated morphine dependence, but the mechanisms are unclear. According to the recent evidence that AQP4 might form a functional complex with glutamate transporter-1 (GLT-1), this study focused on whether AQP4 participates in the modulation of GLT-1 and glutamate homeostasis in morphine-dependent mice. RESULTS: We found that AQP4 knockout prevented the down-regulations of GLT-1 expression and glutamate clearance when mice were repeatedly treated with morphine. Further study revealed that inhibition of GLT-1 by dihydrokainic acid (DHK) initiated morphine dependence in AQP4 knockout mice. In addition, AQP4 knockout abolished both decreases and increases in the extracellular glutamate levels in the prefrontal cortex during repeated morphine treatment and naloxone-precipitated withdrawal. CONCLUSION: AQP4 deficiency suppresses the down-regulation of GLT-1, and the disruption of glutamate homeostasis caused by repeated exposure to morphine, pointing to a strategy for maintaining glutamate homeostasis and thereby treating addiction through the modulation of AQP4 function and expression.

Antidepressant-like effects of 071031B, a novel serotonin and norepinephrine reuptake inhibitor.

SNRIs (serotonin and norepinephrine reuptake inhibitors) have been proposed to exert increased therapeutic efficacy or be faster acting compared to commonly used antidepressants. In this study, we performed in vitro binding and uptake assays and in vivo behavioral tests to assess the pharmacological properties and antidepressant-like efficacy of the compound 071031B; we also performed cytotoxicity tests using HepG2 cells and SH-SY5Y cells to predict the toxicity of 071031B. In vitro, 071031B had high affinity for both serotonin transporters and norepinephrine transporters prepared from rat cortex tissue (Ki=2.68 and 1.09 nM, respectively) and recombinant cells (Ki=1.57 and 0.36 nM, respectively). Moreover, 071031B also potently inhibited the uptake of serotonin (5-HT) and norepinephrine (NE) into rat cortical synaptosomes (Ki=1.99 and 1.09 nM, respectively) and recombinant cells (Ki=3.23 and 0.79 nM, respectively). In vivo, acute administration of 071031B dose-dependently reduced the immobility time in the tail suspension test in mice and the forced swimming test in mice and rats with higher efficacy than duloxetine and showed no stimulatory effect on the locomotor activity. Chronic 071031B treatment (5 or 10mg/kg) significantly reversed depressive-like behaviors in chronically stressed rats, including reduced sucrose preference, decreased locomotor activity, and prolonged latency to begin eating. Furthermore, 071031B also exhibited lower cytotoxicity in HepG2 cells and SH-SY5Y cells in vitro than duloxetine. These findings suggest that 071031B is a novel, balanced serotonin and norepinephrine reuptake inhibitor, with more potent antidepressant effects and lower hepatotoxicity and neurotoxicity in vitro than duloxetine.

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter.

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Knockdown of ASIC2a subunit aggravates injury of rat C6 glioma cells in acidosis

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Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca2+-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptor-independent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca2+ concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca2+ overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells (AU)

Knockdown of ASIC2a subunit aggravates injury of rat C6 glioma cells in acidosis.

Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca²âº-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptorindependent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca²âº concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca²âº overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells.

New cytotoxic azaphilones from Monascus purpureus-fermented rice (red yeast rice).

Using a cell-based cytotoxicity assay three new cytotoxic azaphilones, including two stereoisomers and designated monapurones A-C (1-3), were isolated from the extract of Monascus purpureus-fermented rice (red yeast rice). Their structures were elucidated by detailed interpretation of spectroscopic and chemical data. The relative configurations were assigned on the basis of analysis of NOE data, and the absolute configurations were determined by direct comparison of their CD spectra with those of known azaphilones and chemical correlations. In the in vitro assays, monapurones A-C (1-3) showed selective cytotoxicity against human cancer cell line A549 with IC50 values of 3.8, 2.8 and 2.4 microM respectively, while exhibiting no significant toxicity to normal MRC-5 and WI-38 cells at the same concentration.

Involvement of tissue transglutaminase in endothelin 1-induced hypertrophy in cultured neonatal rat cardiomyocytes.

A potential link between tissue-type transglutaminase (tTG) and cardiac hypertrophy was suggested recently. However, whether tTG is implicated in hypertrophic agonist-induced cardiac hypertrophy is not yet known. The purpose of this study was to investigate the effects of tTG on cardiomyocyte hypertrophy induced by endothelin (ET) 1. Real-time quantitative RT-PCR and Western blot analysis demonstrated that ET-1 increased the expression of tTG mRNA and protein in cardiomyocytes by activating ET(A) receptors. ET-1 failed to cause increases in cell size and [(3)H]leucine uptake, sarcomere reorganization, and gene induction of the atrial natriuretic factor when cardiomyocytes were treated with monodansylcadaverine, a competitive inhibitor of tTG. Furthermore, the effects of ET-1 on multifunctional activities of tTG were determined by evaluating the incorporation of [(3)H]putrescine into N,N'-dimethylated casein and charcoal absorption, respectively. The results showed that ET-1 did not influence the basal transglutaminase activity of cardiomyocytes but significantly inhibited the 0.1-mmol/L Ca(2+)-stimulated transglutaminase activity. Otherwise, ET-1 elevated the activity of GTPase in a concentration- and time-dependent manner. In vivo, right ventricular hypertrophy induced by 2 weeks of chronic hypoxia was depressed by the tTG inhibitor cystamine (10 to 30 mg/kg, 2 times per day, IP) in a dose-dependent manner. Taken together, our data strongly supported the notion that tTG may act as a positive regulator of the hypertrophic program in response to ET-1. This is probably attributable to the signaling activity of tTG rather than transglutaminase activity.

[Yin-yang relationship between oncogene and antioncogene].

OBJECTIVE: Oncogene and antioncogene play contrary effects on the cell growth and proliferation controlling process, and cancer occurs when the presence of imbalance expression between them. That means there is yin-yang relationship between oncogene (yang) and antioncogene (yin), and also inside both of them. Taking the oncogene myc and antioncogene p53 for example, the yin gene p53 acts, in the yin side, to promote cell apoptosis and inhibit cell growth, while in the yang side, it facilitates for repairing the injured DNA to keep cell survival; the yang gene myc, promoting cell growth and proliferation in the yang side and inducing cell apoptosis in the yin side. To elucidate the yin-yang reactions between oncogene and antioncogene would be of important significance in the all-round and profound research of cancer.

[Effects of melatonin on the voltage-gated delayed rectifier potassium channels of cultured hippocampal neurons of new-born rats].

OBJECTIVE: To investigate the effects of melatonin on voltage-gated delayed rectifier potassium channels. METHODS: Hippocampus neurons were obtained from newborn Wistar rat and cultured. Primary cultured for 7 to 12 days of new-born Wistar rat were selected as objectives. Patch clamp whole-cell recording technique was used on the hippocampus neurons cultured for 7 to 12 day. to record the delayed rectifier potassium current to analyze the basic electrophysiological characteristics. The effects of melatonin of the concentrations of 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 mol/L, 10 mol/L, 100 mol/L, and 1 mmol/L on the amplitudes and kinetics of delayed rectifier potassium currents were investigated. RESULTS: With different voltage protocols and specific blockers of potassium channel (4-AP and TEA) a delayed rectifier potassium current that activated and inactivated slowly and had the outward rectifying characteristics (Ik) from the outward potassium currents in cultured new-born hippocampus neurons was separated. The effect of melatonin on the delayed rectifier channel was rapid, reversible and voltage-dependent Melatonin had no effect on the kinetic characteristics of the I -V curve. Melatonin increased the potassium current concentration-dependently. 1 - 100 nmol/L melatonin increased the amplitude of potassium current gradually; the effects of 1 - 100 micromol/L melatonin on the potassium current increased concentration-dependently, while the action of 1 mmol/L melatonin decreased. CONCLUSION: Melatonin reversibly increases the rectifier delayed potassium currents of the cultured hippocampus neurons of new-born rat. This may be involved in some aspects of physiological and pathological significance of potassium currents.

Pharmacological characterization of 3-azabicyclo[3,2,1] octane-1-yl-l-leucyl-d-tryptophanyl-d-4-Cl-phenylalanine: A novel ET(A) receptor-selective antagonist.

BACKGROUND AND OBJECTIVES: Pulmonary hypertension is a kind of disease associated with a very high rate of mortality. There are not many effective drugs for the treatment of pulmonary hypertension. Treatment with ET-1 receptor antagonists was proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin A receptor (ET(A)) antagonist for treatment of pulmonary hypertension, 242 peptide compounds were synthesized by structural optimization of a selective ET(A) receptor antagonist BQ-123. Among these, -azabicyclo[3,2,1]octane-1-yl-l-Leucyl-d-tryptophanyl-d-4-Cl-phenylalanine, named ETP-508, was selected for further harmacological characterization. METHODS: Radioligand binding assay was performed to study the binding affinity of ETP-508 for ET(A) and ET(B) receptors. The biological activity of ETP-508 was evaluated in isolated rat aortic ring experiment and in systemic arterial pressure experiment. In addition, hypotensive effect of ETP-508 was investigated on hypoxia-induced pulmonary hypertension. RESULTS: ETP-508 binds to endothelin ET(A) receptor with >10,000-fold higher affinity than to endothelin B receptor in rat lung tissue preparation. ETP-508 inhibited endothelin-1 (ET-1)-induced contraction of isolated rat aortic ring and shifted the cumulative concentration-contraction response curve to ET-1 to right with no change in the maximal response. In vivo, ETP-508 inhibited the increased effect of ET-1 on mean systemic arterial pressure. Pre-treatment with ETP-508 by intravenous infusion significantly inhibited chronic hypoxia-induced pulmonary hypertension and right ventricular hypertrophy. ETP-508 also significantly inhibited the increase in lung ET-1 expression level, hemoglobin, red-cell count and red-cell hematocrit as induced by hypoxia. Furthermore, ETP-508 partially reversed pre-established pulmonary hypertension and right ventricle hypertrophy by chronic hypoxia. CONCLUSION: These results indicated that ETP-508 is a novel highly selective ET(A) receptor antagonist and may have a great potential to be developed as a drug of anti-pulmonary hypertension.