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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.

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.

An acylhydrazone-based derivative, 3-hydroxy-N'-(2-hydroxy-1-naphthalene methylene)-2-naphthalene hydrazide (H 3 L), was synthesized and characterized by elemental analyses, IR, 1 H NMR and mass spectrum. H 3 L is non-fluorescent in DMSO. But the fluorescence emission is enhanced obviously via the formation of aggregates in DMSO/H 2 O mixture. The formed aggregates were confirmed by DLS and SEM. H 3 L is an aggregation-induced emission (AIE)-active fluorophore. H 3 L can act as a fluorescence sensor for Al 3+ and Zn 2+ in DMSO/H 2 O (v/v, 9/1) and THF/H 2 O (v/v, 999/1), respectively. And H 3 L is also a naked-eye probe for Fe 3+ in DMSO/H 2 O (v/v, 9/1). The binding stoichiometry of H 3 L to both Al 3+ and Zn 2+ is 1:1, but that to Fe 3+ is 2:1. The metal complex species formed in the detection procedure are deduced by Job plot, spectroscopic titrations and ESI–MS to be [AlL(DMSO) 2 ], [Zn(HL)(H 2 O)] and [Fe(HL)(H 2 L)]. The detection limit is as low as 3.66 μM for Al 3+ , 1.01 μM for Zn 2+ and 0.34 μM for Fe 3+ . H 3 L can also be used to detect intracellular Al 3+ and Zn 2+ . The sensing event might be attributed to a combinational effect of excited state intramolecular proton transfer (ESIPT) and chelation-enhanced fluorescence (CHEF) mechanism for selective detection of Al 3+ and Zn 2+ ions, and ligand-metal charge transfer (LMCT) mechanism for Fe 3+ ion.

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 f...

A new quinazoline derivative 6-(4-diethylamino)phenol-2-yl-(5,6-dihydrobenzimidazo[1,2-c])-quinazoline (HL) and the two metal Schiff-base complexes [CuL1Cl]·CH3OH (1) and [ZnL1(Ac)]·0.5CH3CH(OH)CH3 (2) (HL1 = 4-{[2-(1H-benzoimidazol-2-yl)-phenyl imino]-methyl}-benzene-1-diethylamino-3-ol) were synthesized and characterized by single-crystal X-ray diffraction. HL is highly selective and sensitive to Cu(II) and Zn(II) ions in aqueous medium with a detection limit of 2.13 × 10−6 and 7.19 × 10−7 M for Cu2+ and Zn2+, respectively. HL can act as a “turn-off” sensor for Cu(II) with the excitation wavelength of 356 nm and a “turn-on” sensor for Zn(II) when excited at 416 nm. The Job plots, fluorescence and UV-vis titrations, 1H NMR as well as ESI data show that the binding stoichiometry of Cu(II)/Zn(II) with HL is 1 : 1. The binding sites of HL with Cu(II)/Zn(II) are two nitrogen atoms and one oxygen atom, forming Cu(II)–L1 and Zn(II)–L1 complexes. This agrees with the crystal structures of 1 and 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.

A chemosensor 3-[(2-hydroxybenzylidene)-amino]-1H-pyridin-2-one (HL) for multi-ions was synthesized and characterized by single-crystal X-ray diffraction. HL is a turn-on fluorescence chemosensor for Al3+, F−, and CN− ions in DMSO. F− and CN− anions could be further distinguished by the ratiometric absorption spectrometry method. Job plot and 1H NMR data showed that the binding stoichiometries of HL with Al3+, F−, or CN− all were 1:1. Further observations of 1H NMR titration and ESI-MS showed that the possible fluorescent species of Al3+ with HL was [AlL(OH)(DMSO)2]+ in DMSO. The spectroscopic observations of the Al(III) complex showed that the two oxygen atoms and one imine nitrogen atom of HL were coordinated to the Al(III) ion. While the crystal structure of the Ni(II)-L complex confirmed the tridentate coordination mode of HL in the complexes. The experimental results revealed that HL and F− ion formed hydrogen bond in DMSO, while CN− attacked the carbon atom at –C N of HL to undergo a nucleophilic addition. The theoretical calculations confirmed that HL showed very weak fluorescence because of the excited-state intramolecular proton transfer (ESIPT). The turn-on sensing of HL to Al3+, F−, and CN− ions arose from the inhibition of ESIPT. MTT tests showed that HL was low toxic to cells. Also, HL could probe intracellular Al3+ and F− ions by bioimaging.

A fluorescent quinazoline derivative, 6-(2,4-dihydroxyphenyl)-5,6-dihydrobenzoimidazo[1,2-c]quinazoline (H2L), was designed and synthesized. Its fluorescence could be quenched by the addition of Cu2+ in an aqueous media. The binding constant for Cu2+ and H2L was determined to be 9.56 × 103 M−2 in DMSO/H2O (1 : 1, v/v). The crystal structure of the Cu(II) complex, [Cu(HL1)(Ac)] (1) (H2L1 = 4-{[2-(1H-benzimidazol-2-yl)-phenylimino]-methyl}-benzene-1,3-diol, Ac = acetate), revealed that Schiff base complex 1, H2L1, was formed in the response system of H2L to Cu2+ via Cu2+-assisted C–N bond breakage of the quinazoline ring in H2L. Consequently, complex 1 was used as a turn-on fluorescence chemosensor for the direct detection of CN−, and showed a high selectivity to CN− over a number of anions in the aqueous media, where CN− replaces HL1 in 1 to form [Cu(CN)x]2−x with HL1 then released and the fluorescence recovered. The detection limit for CN− was 4.0 × 10−6 M. The cell images showed that 1 could be used to detect intracellular CN−.

The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca2+ channel is still unclear. Considering both sigma-1 receptors and N-type Ca2+ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs) in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) and immunofluorescence staining. N-type Ca2+ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084) were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063). Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca2+ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca2+ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET) assays and co-immunoprecipitation (Co-IP) demonstrated that sigma-1 receptors and N-type Ca2+ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T) cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca2+ channels. The mechanism for the inhibition of sigma-1 receptors on N-type Ca2+ channels probably involved a chaperone-mediated direct interaction and agonist-induced conformational changes in the receptor-channel complexes on the cell surface.

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes and provides a mechanism by which water permeability of the plasma membrane can be regulated. Evidence suggests that AQP4 is associated with glutamate transporter-1 (GLT-1) for glutamate clearance and contributes to morphine dependence. Previous studies show that AQP4 deficiency changed the mu opioid receptor expression and opioid receptors' characteristics as well. In this study, we focused on whether AQP4 could form macromolecular complex with GLT-1 and mu opioid receptor (MOR) and participates in morphine dependence. By using immunofluorescence staining, fluorescence resonance energy transfer, and co-immunoprecipitation, we demonstrated that AQP4 forms protein complexes with GLT-1 and MOR in both brain tissue and primary cultured astrocytes. We then showed that the C-terminus of AQP4 containing the amino acid residues 252 to 323 is the site of interaction with GLT-1. Protein kinase C, activated by morphine, played an important role in regulating the expression of these proteins. These findings may help to reveal the mechanism that AQP4, GLT-1, and MOR form protein complex and participate in morphine dependence, and deeply understand the reason that AQP4 deficiency maintains extracellular glutamate homeostasis and attenuates morphine dependence, moreover emphasizes the function of astrocyte in morphine dependence.

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 β-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 β-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.

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.

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.

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here we report that muscarinic acetylcholine receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI). Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum.

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.

Four new taraxastane-type triterpenoids acids 3β,22α-dihydroxy-20-taraxasten-30-oic acid (1), 3β-hydroxy-22-oxo-20-taraxasten-30-oic acid (2), 3-oxo-22α-hydroxy-20- taraxasten-30-oic acid (3), and 3β,19β-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 (1H-1HCOSY, 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.

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.

ZC88 is a novel non-peptide N-type voltage-sensitive calcium channel blocker synthesized by our institute. In the present study, the oral analgesic activity of ZC88 in animal models of acute and neuropathic pain, and functional interactions between ZC88 and morphine in terms of analgesia, tolerance and dependence were investigated. In mice acetic acid writhing tests, ZC88 (10-80 mg/kg) administered by oral route showed significant antinociceptive effects in a dose-dependent manner. The ED50 values of ZC88 were 14.5 and 14.3 mg/kg in male and female mice, respectively. In sciatic nerve chronic constriction injury rats, mechanical allodynia was ameliorated by oral administration of ZC88 at doses of 14, 28 and 56 mg/kg, suggesting ZC88 relieved allodynic response of neuropathic pain. When concurrently administered with morphine, ZC88 (20-80 mg/kg) dose-dependently potentiated morphine analgesia and attenuated morphine analgesic tolerance in hot-plate tests. ZC88 also prevented chronic exposure to morphine-induced physical dependence and withdrawal, but not morphine-induced psychological dependence in conditioned place preference model. These results suggested that ZC88, a new non-peptide N-type calcium channel blocker, had notable oral analgesia and anti-allodynia for acute and neuropathic pain. ZC88 might be used in pain relief by either application alone or in combination with opioids because it enhanced morphine analgesia while prevented morphine-induced tolerance and physical dependence.

Overexpression of human ether-à-go-go (eag) related gene (herg) contributes to the progression and metastasis of a variety of tumors of different histogenesis, which implies that the herg gene could provide a promising target on tumor therapy. In the present study, plasmid-mediated expression of shRNA-herg1 and shRNA-herg1/1b was employed to silence the herg gene expression in human neuroblastoma SH-SY5Y cell lines. The inhibition of the target gene expression was confirmed by RT-PCR and Western blot. It was found that shRNA-herg1 or shRNA-herg1/1b depressed the cellular growth rate, inhibited cell viability and reduced colony formation of SH-SY5Y cells. The flow cytometry assay revealed that SH-SY5Y cells were retarded in G0-G1 after herg1 or herg1/1b gene was silenced by shRNA-herg1 or shRNA-herg1/1b. In vivo, intra-tumor injection of shRNA-herg1/1b inhibited the growth of SH-SY5Y tumors inoculated subcutaneously in nude mice. The result suggested that the herg played an important role in regulating the growth and proliferation of SH-SY5Y cells. The block of the HERG channel might be a potential therapeutic strategy for neuroblastoma and some other tumors with overexpression of the herg gene.

The effects of agmatine on morphine-induced locomotion sensitization and morphine-induced changes in extracellular striatal dopamine (DA) and DA metabolites were studied. The locomotor response to morphine challenge (3 mg/kg, s.c.) was enhanced in rats 3 days after repeated morphine administration, indicating development of locomotion sensitization. In vivo microdialysis demonstrated a significant increase in striatal basal levels of the DA metabolites DOPAC and HVA, but not in DA itself, and an increase in DA response to morphine challenge in rats 3 days after withdrawal. Agmatine (1, 10, 80 mg/kg) inhibited morphine-induced locomotion sensitization and the changes in DA noted above. Idazoxan attenuated the effects of agmatine on locomotion, suggesting that the effects are mediated by imidazoline receptors. In addition, repeated morphine also increased the expression of tyrosine hydroxylase mRNA in the VTA after 4 days of morphine pretreatment, while decreasing the expression of dynorphin mRNA at 3 days after withdrawal. Agmatine inhibited morphine-induced changes in dynorphin, but not in tyrosine hydroxylase mRNA expression. These data suggest that agmatine, likely by activating imidazoline receptors, inhibits morphine-induced locomotion sensitization and morphine-induced changes in extracellular DA and in dynorphin expression. Thus, agmatine deserves further study as an anti-opioid medication.

Aim: To investigate the underlying mechanism of acid-sensing ion channel (ASIC) 1a involved in the acidosis-induced cytotoxicity of rat C6 glioma cells. Methods: The stable ASIC1a-silenced C6 cells built with the RNA interference technology were confirmed by RT-PCR and Western blot analysis. Intracellular calcium ([Ca 2+ ] i ) in both the wild-type rat C6 glioma cells and the ASIC1a-silenced C6 cells were analyzed before and after acid application/exposure with the calcium imaging experiment. Results: The rapid extracellular pH drop induced the increase of [Ca 2+ ] i in the wild-type C6 cells, but not in the ASIC1a-silenced C6 cells. During the prolonged acid exposure, [Ca 2+ ] i was lower in the ASIC1a-silenced C6 cells than that in the control cells. Conclusion: The resultant toxicity of [Ca 2+ ] i might contribute to the acidosis-induced cytotoxicity.

Aim: To study the role of acid-sensing ion channel (ASIC) 1a in the cell death and apoptosis induced by extracellular acid in C6 glioma cells. Methods: The stable ASIC1a-silenced C6 cell line, built with RNA interference technology, were confirmed by RT-PCR and Western blot analysis. The cell viability following acid exposure was analyzed with lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptotic cells dyed with Annexin-V and propidium iodide were measured with a flow cytometer, while the changes of cell cycle were also assayed. Results: The downregulation of ASIC1a proteins by stable transfection of short hairpin RNA decreased the cell death percentage and increased cell viability following acid exposure with LDH and the MTT assay. The rate of apoptosis was lower in the ASIC1a-silenced cell line than that in the wild-type C6 cell line. The percentage of sub-G0 cells was lower in the ASIC1a-silenced C6 cells than that in the wild-type cells. Conclusion: Extracellular acid induced cell death and apoptosis via ASIC1a mechanisms in the C6 glioma cells.

The corticotropin releasing factor (CRF) plays a central role in regulating the activities of hypothalamic-pituitary-adrenal (HPA) axis in the presence of a variety of stressful stimuli via binding to its type 1 receptors (CRFR1). Despite that many peptidic or non-peptidic antagonists of CRFR1 have been developed to serve as therapeutic tools to CRF-related pathologies, none of them have been utilized clinically. Targeting the extracellular domain 1 (EC1) of CRFR1, the CRF-binding site, represents a new strategy to inhibit the function of the receptor. However, no such agents have been identified up to now. Herein, by using an 87-amino acid fragment corresponding to the EC1 region as the bait, we screened the binding polypeptides from a phage display (Ph.D.-12) peptide library. After 3-round biopanning, positive clones were selected and the polypeptides carried by them were identified. 5 polypeptides were found to bind with the target specifically. Among them, the P7 exhibited the highest affinity. By evaluating the cAMP accumulation in the CRFR1 or CRFR2-expressing HEK293 cells, we demonstrated that P7 blocking the function of CRFR1, but not CRFR2. In addition, we also found that P7 and CRF act on CRFR1 competitively. Taken together, we reveal that P7, a novel polypeptide identified from phage display library, inhibits the function of CRFR1 effectively and specifically by binding at its EC1 domain. The new polypeptide might provide a promising agent for diagnostic or therapeutic utilities in CRF-related disorders.

The present study was designed to examine the effect of agmatine, the decarboxylated product of l -arginine by l -arginine decarboxylase, on convulsion in the mouse maximal electroshock (MES) test and mouse glutamate-induced convulsant test. MES convulsion and glutamate convulsion were respectively induced by an electrical stimulation (110 V, 0.3 s, 8 Hz) and by intracerebroventricular injection of glutamate (0.5 M, pH 7.4, 5μl). The results were expressed as the tonic and clonic time of convulsion in MES or percentage of mice with tonic hind-limb extension in glutamate-induced convulsant assay. Agmatine given intracerebroventricularly (2–16 mg/kg) or subcutaneously (10–160 mg/kg) significantly shortened the tonic and clonic times of convulsion in a dose-dependent manner in the mouse MES test. Glutamate (0.5 M, 5μl icv per mouse) induced an obvious convulsive response indicated by tonic hind-limb extension in mice, and agmatine (2–16 mg/kg icv) decreased the rate of mice with tonic hind-limb extension like NMDA receptor antagonist MK-801. The anticonvulsive effect of agmatine (80 mg/kg sc) on both the tonic and clonic times of convulsion lasted for more than 4 h after administration in the mouse MES test, which was twice that of barbital. Taken together, the results implicate that agmatine has obvious anticonvulsive effects, and its possible mechanism might be related to the antagonism of the function of NMDA receptors.

The present experiment investigates the effect of neostigmine on nicotinic acetylcholine receptors (nAChRs) in the cultured neurons from neonatal rat superior cervical ganglia (SCG). Using whole-cell patch clamp techniques, we found that the amplitudes of the currents induced by 50 microM dimethylphenylpiperazinium (DMPP) were 21.5+/-10.7%, 52.9+/-9.2% and 86.9+/-4.9% depressed at the increased concentrations of neostigmine 100, 200 and 400 microM, respectively. The inhibition of neostigmine decreased gradually with the increased concentration of nicotine from 10 to 160 microM. Lineweaver-Burk's double-reversible plot illustrated that neostigmine blocked neuronal nAChRs in a competitive manner. Hyperpolarization of membrane potential from -40 mV to -100 mV did not significantly influence the blockade of neostigmine. Neostigmine could not accelerate the decay of the DMPP-induced currents, neither evoke any detectable currents in SCG neurons. The results indicate that neostigmine depress neuronal nAChRs in a competitive, concentration-dependent and voltage-independent manner, and can not facilitate desensitization of the receptors. The present data suggest that neostigmine blocks neuronal nAChRs by interacting with the ACh binding sites of the receptors.

Many studies have provided convincing evidence for hERG as an important diagnostic and prognostic factor in human cancers, as well as a useful target for antineoplastic therapy. Our previous study also revealed that knockdown of herg gene expression by shRNA interference inhibited the growth of neuroblastoma cells in vitro and in vivo. In the experiment, a novel 4-amino piperidine analog, ZC88, was examined for its effect on hERG potassium channels and its antitumor potency was observed in vitro and in vivo. The results showed that ZC88 could block hERG1 and hERG1b channels expressed in Xenopus oocytes in a concentration-dependent manner. ZC88 displayed significant antiproliferative activity in several tumor cell lines and the tumor cells with higher expression of hERG presented higher sensitivity to ZC88. The mitotic progression of tumor cells was markedly suppressed in the presence of ZC88 through arresting cells in G0/G1 phase. ZC88 significantly inhibited the tumor growth in nude mice at a dosage with slight influence on the cardiac QT interval. The antitumor effect of ZC88 was correlated at least partly with its blockage of hERG channels, which implicated a positive role of hERG potassium channel in tumor cell proliferation.

Summary 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.

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.

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.

To investigate the effects of melatonin on voltage-gated delayed rectifier potassium channels.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.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.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.

N-type Ca 2+ channels located on presynaptic nerve terminals regulate neurotransmitter release, including that from the spinal terminations of primary afferent nociceptors. Pharmacological and ion-channel gene knockdown approaches in animals have revealed N-type Ca 2+ channels to be particularly attractive molecular targets for the discovery and development of new analgesic drugs. In recent years, some non-peptide small molecular N-type Ca 2+ channel blockers have been reported. However, low selectivity and some side effects limit their further development. To overcome these disadvantages, some new compounds were designed and synthesized in our institute by optimizing the 4-amino-piperidine template. C101, one of these compounds, was demonstrated to block N-type Ca 2+ channels with higher selectivity. It was found that C101 produced concentration-dependent inhibition on N-type Ca 2+ channels expressed in Xenopus oocytes with an IC 50 is 2.2 ± 0.6 μM. The current–voltage relationship was not altered after 2-min exposure to C101. However, the steady-state inactivation relationship curve was shifted to more negative potentials for channels. Therefore, it seemed that C101 blocks the inactivated channel. C101 did not present any remarkable effects on voltage-gated potassium, sodium channels in cultured rat hippocampal neurons, and L-, P/Q-, R-type calcium channels and HERG channels expressed in Xenopus oocytes. The results suggested that C101 was a high selective blocker targeting N-type Ca 2+ channels, and may have a potential to be developed as a novel analgesic agent.

Imidazoline receptor antisera-selected protein (IRAS) is considered as a candidate for the I1-imidazoline receptor (I1R), but the signaling pathway mediated by IRAS remains unknown. In our study, the signal transduction pathways of IRAS were investigated in CHO cells stably expressing IRAS (CHO-IRAS), and compared to the native I1R signaling pathways. Rilmenidine or moxonidine (10 nM-100 microM), I1R agonists, failed to stimulate [35S]-GTPgammaS binding in CHO-IRAS cell membrane preparations, suggesting that G protein may not be involved in IRAS signaling pathway. However, incubation of CHO-IRAS with rilmenidine or moxonidine for 5 min could induce an upregulation of phosphatidylcholine-selective phospholipase C (PC-PLC) activity, and an increase in the accumulation of diacylglycerol (DAG), the hydrolysate of PC-PLC, in a concentration-dependent manner. The elevated activation of PC-PLC by rilmenidine or moxonidine (100 nM) could be blocked by efaroxan, a selective I1R antagonist. Cells treated with rilmenidine or moxonidine showed an increased level of extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, which could be reversed by efaroxan or D609, a selective PC-PLC inhibitor. These results suggest that the signaling pathway of IRAS in response to I1R agonists coupled with the activation of PC-PLC and its downstream signal transduction molecule, ERK. These findings are similar to those in the signaling pathways of native I1R, providing some new evidence for the relationship between I1R and IRAS.

The present study investigated the effects of agmatine action on imidazoline I1 receptor antisera-selected protein (IRAS), a candidate for imidazoline I1 receptor, on prolonged morphine-induced adaptations of calcium signal and long-lasting alterations in gene expression to further elucidate the role of IRAS in opioid dependence. Two cell lines, Chinese hamster ovary cells expressing mu opioid receptor alone (CHO-mu) and expressing mu opioid receptor and IRAS together (CHO-mu/IRAS), were used. After chronic treatment with morphine for 48 h, naloxone induced a significant elevation of intracellular calcium concentration ([Ca2+]i) in CHO-mu and CHO-mu/IRAS cells. Agmatine (0.01-3 microM) concentration-dependently inhibited the naloxone-precipitated [Ca2+]i elevation when co-pretreated with morphine in CHO-mu/IRAS, but not in CHO-mu. Efaroxan, an imidazoline I1 receptor-preferential antagonist, completely reversed the effect of agmatine in CHO-mu/IRAS. Agmatine (1-10 microM) administration after chronic morphine exposure for 48 h partially decreased the [Ca2+]i elevation in CHO-mu/IRAS which was entirely antagonized by efaroxan, but not in CHO-mu. In addition, agmatine (1 microM) co-pretreated with morphine attenuated the naloxone-precipitated increases of cAMP-responsive element binding protein and extracellular signal-regulated kinase 1/2 phosphorylations and c-Fos expression in CHO-mu/IRAS. These effects were blocked by efaroxan as well. Taken together, these results indicate that the agmatine-IRAS action system attenuates the up-regulations of Ca2+ signal and its downstream gene expression in morphine-dependent model in vitro, providing additional evidence to support the contribution of IRAS to opioid dependence.

The patch-clamp technique, a dominant technique in cellular electrophysiology, is always being regarded as the gold standard for ion channel research. Application of the patch-clamp technique can demonstrate the existences of ion channels and provide valuable information for ion channels, including their electrophysiological properties, molecular structures and the mechanism of drug action. Genomics and proteomics research has showed that the development of drugs for ion channel target would be very promising in future. In recent years, numerous improvements have been achieved, which will facilitate the application of patch clamp technique in drug high-throughput screening. These techniques will break through the bottleneck in the development of drugs aiming at ion channels. New advancements in patch-clamp technique and application in drug high-throughput screening are reviewed in the paper.

The 3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (DM-phencynonate hydrochloride, DMCPG) is a demethylated metabolite of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG). (+/-)DMCPG had one chiral center and two enantiomers [R(-) and S(+)DMCPG]. Here we carried out a comparative study of the pharmacological profiles of these optical isomers.Affinity and relative efficacy were tested using a radioligand-binding assay with muscarinic acetylcholine receptors from the rat cerebral cortex. Pharmacological activity was assessed in three individual experiments: (1) potentiating the effect of a sub-threshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol.In the competitive binding assay, R(-)DMCPG (K(i)=763.75 nmol/L) was 4- and 2-fold more potent than (+/-)DMCPG (K(i)=3186 nmol/L) and S(+)DMCPG (K(i)=1699 nmol/L) in inhibiting the binding of [(3)H]QNB. The R(-) and S (+) configurations showed positive cooperation (n(H)1) with the muscarinic receptor, whereas (+/-)DMCPG had a negative cooperation (n(H)1) relationship with the muscarinic receptor in a radio-binding assay. Both the R(-) and S(+) configurations could potentiate the effect of sub-threshold hypnotic dose of sodium pentobarbital in a dose-dependent manner (the ED(50) values were 2.53 and 18.65 mg/kg, respectively), but (+/-)DMCPG did not display significant central depressant effects at doses from 10 to 29.15 mg/kg (P0.05). (+/-)DMCPG and its optical isomers suppressed the guinea pig ileum contractile response to carbachol. The IC(50) values were 7.78 x 10(-9), 1.88 x 10(-7), and 1.038 x 10(-7) nmol/L, respectively. In the anti-salivation study, (+/-)DMCPG and its enantiomers depressed oxotremorine- induced salivation in a dose-dependent manner, and the order of potency was R(-)DMCPG (ED(50)=0.44 mg/kg)(+/-)DMCPG (ED(50)=2.88 mg/kg)S(+)DMCPG (ED(50)=5.05 mg/kg).(+/-)DMCPG and its optical isomers have differences in their pharmacological potencies as anticholinergic agents, and the R(-) configuration is more active than the S(+) configuration.

To test the antiepileptic effect of phencynonate hydrochloride and investigate its antiepileptic mechanism.Through establishment of different epilepsy models, antiepileptic effects of phencynonate hydrochloride and other drugs were examined. Besides, the effect of phencynonate hydrochloride and other compounds against NMDA-induced lethality in mice, NMDA-induced injury in rat primary hippocampal neuronal cultures and NMDA-induced current were also observed.Phencynonate hydrochloride produced a significant anticonvulsant effect on different epilepsy models. Furthermore, phencynonate hydrochloride also exerted its obvious protection against the lethal effects of NMDA in mice, antagonized the NMDA-induced injury in rat primary hippocampal neuronal cultures and blocked NMDA-induced current in a dose-dependent manner.Phencynonate hydrochloride had a notable anticonvulsant effect on typical epilepsy models, its antiepileptic mechanism might relate to its antagonism against NMDA receptor.

Agmatine, an endogenous ligand for the I1-imidazoline receptor, has previously been shown to prevent morphine dependence in rats and mice. To investigate the role of imidazoline receptor antisera-selected protein (IRAS), a strong candidate for I1R, in morphine dependence, two CHO cell lines were created, in which mu opioid receptor (MOR) was stably expressed alone (CHO-mu) or MOR and IRAS were stably co-expressed (CHO-mu/IRAS). After 48 h administration of morphine (10 microM), naloxone induced a cAMP overshoot in both cell lines, suggesting cellular morphine dependence had been produced. Agmatine (0.1-2.5 microM) concentration-dependently inhibited the naloxone-precipitated cAMP overshoot when co-pretreated with morphine in CHO-mu/IRAS, but not in CHO-mu. Agmatine at 5-100 microM also inhibited the cAMP overshoot in CHO/mu and CHO-mu/IRAS. Efaroxan, an I1R-preferential antagonist, completely blocked the effect of agmatine on the cAMP overshoot at 0.1-2.5 microM in CHO-mu/IRAS, while partially reversing the effects of agmatine at 5-100 microM. L-type calcium channel blocker nifedipine entirely mimicked the effects of agmatine at high concentrations on forskolin-stimulated cAMP formation in CHO-mu and naloxone-precipitated cAMP overshoot in morphine-pretreated CHO-mu. Therefore, IRAS, in the co-transfected CHO-mu/IRAS cell line, appears necessary for low concentrations of agmatine to cause attenuation of cellular morphine dependence. An additional effect of agmatine at higher concentrations seems to relate to both transfected IRAS and some naive elements in CHO cells, and L-type voltage-gated calcium channels are not ruled out. This study suggests that IRAS mediates agmatine's high affinity effects on cellular morphine dependence and may play a role in opioid dependence.

The present research was designed to investigate the interference of Ca 2+ homeostasis by ethanol on the primary cultured superior cervical ganglion (SCG) neurons. (1) Using the whole cell patch clamp recording, the amplitudes of voltage-dependent Ca 2+ channel (VDCC) currents could be reduced by ethanol in a concentration-dependent manner. Ethanol (100 mM) inhibited about 25% of Ca 2+ channel current. However, the activation of Ca 2+ channel was not affected by ethanol at those concentrations. (2) The similar extent inhibitions of 100 mM ethanol on the increments of intracellular Ca 2+ concentration ([Ca 2+ ] i ) induced by 40 mM KCl and 1 μM A23187 were also observed in the fluo-3-AM loaded superior cervical ganglia (SCG) via detecting the change of [Ca 2+ ] i with a laser scanning confocal microscopy. In contrast, the basal [Ca 2+ ] i was significantly increased by ethanol alone in a concentration-dependent manner. These phenomena were also observed even under Ca 2+ free bath solution or the solution added 300 μM cadmium chloride conditions. Together with above results, our data suggest that ethanol increases basal [Ca 2+ ] i , but it also inhibits the extracellular Ca 2+ influx through VDCC and ionophore channel. And the augment of basal [Ca 2+ ] i induced by ethanol might attribute to the Ca 2+ releasing from intracellular Ca 2+ pools.

To investigate whether agmatine could selectively block a given type of the voltage-gated calcium channels (VGCC) and whether related receptors are involved in the blocking effect of agmatine on VGCC.The whole-cell patch recording technique was performed to record VGCC currents in the cultured neonatal rat hippocampal neurons.Verapamil (100 micromol/L), a selective blocker of L-type calcium channel, significantly inhibited VGCC current by 80 %+/- 7 %. Agmatine (100 micromol/L) could further depress the remained currents by 25 %+/-6 %. The alpha 2-adrenoceptor antagonist yohimbine (10 micromol/L) and the I2 imidazoline receptor antagonist idazoxon (10 and 40 micromol/L) had no significant effect on VGCC currents when used respectively. When the mixture of yohimbine and agmatine was applied, VGCC currents were still depressed remarkably. However, the blocking effect of agmatine was decreased by 29 %+/- 8 % in the presence of idazoxon (10 micromol/L). The effect of idazoxon did not increase at a higher concentration (40 micromol/L).Agmatine could block the L- and other types of VGCC currents in the cultured rat hippocampal neurons. Blocking effect of agmatine on VGCC was partially related to I2 imidazoline receptor and had no relationship with alpha 2-adrenoceptors.

To investigate the mechanism of agmatine by observing the effect of agmatine on the voltage-gated channels in rat hippocampal neurons.The whole-cell patch recording technique was performed to record the voltage-gated potassium, sodium, and calcium currents in cultured rat hippocampus. Agmatine was applied directly to the single neuron using a pressure injector with microtubules.Agmatine (500 micromol/L) had no significant effect on the voltage-gated potassium and sodium channels. Agmatine reversibly blocked the voltage-gated calcium channel and the blockade was enhanced with the increasing concentration of agmatine. The inhibitory rates were 21%+/-4%, 35%+/-6%, 49%+/-6%, 67%+/-4%, 69%+/-6%, 86%+/-8%, and 87%+/- 9%, at the concentration of 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100 micromol/L, respectively. IC50 was (1.2+/-0.4) micromol/L. Two-way ANOVA revealed that change of membrane potential displayed a significant interaction with the blockade by agmatine.Agmatine reversibly blocked the voltage-gated calcium channel in rat hippocampal neurons in a concentration- and voltage-dependent way. Agmatine might perform its physiological and pharmacological effects partially by blocking the calcium channel.

To study the effect of microwave irradiation on hippocampus cell.Changes of ATPase activity and voltage dependent ion channel of hippocampus cell membrane were observed in mice exposed to 2 450 MHz microwave irradiation of 10 mW/cm2 from a physical therapy machine. Histochemical method and patch clamp method were used to determine the activity of Na+, K(+)-ATPase, Ca2+, Mg(2+)-ATPase and voltage dependent Na+, K+, Ca2+ channels respectively.1) Na+, K(+)-ATPase activity of microwave irradiated mice showed no significant change as compared with the control, but the activity of Ca2+, Mg(2+)-ATPase decreased significantly (P0.05); 2) In microwave irradiated mice, Na+, K+, Ca2+, current inducement rate in hippocampus neuron decreased significantly, the membrane voltage of Na+ current peak shifted to depolarization, and the attenuation rate of Na+ current and current A inducement rate decreased significantly as compared with control mice.Irradiation of 2 450 MHz microwave at a doze of 10 mW/cm2 was not fatal to mice hippocampus cell. But Ca2+, Mg(2+)-ATPase activity of hippocampal cell membrane and voltage dependent Na+, K+, Ca2+ ion channel of hippocampal nervous were affected which would affect study and memory.

To compare the difference in action sites between mecamylamine (MEC) and hexamethonium (HEX) on nicotinic receptors of sympathetic neurons, we investigated the effects of MEC and HEX on the nicotine-induced currents in cultured superior cervical ganglion neurons by whole-cell patch clamp technique. The IC(50) of MEC and HEX for antagonizing the effect of 0.08 mmol/L nicotine was 0.0012 and 0.0095 mmol/L, respectively. Both MEC and HEX accelerated the desensitization of nicotinic receptors. Furthermore, by comparing their effects at holding potentials 30, 70 and 110 mV, it was indicated that their suppressing effect on the nicotine-induced currents was voltage-dependent. However, different from that of HEX, the inhibitory effect of MEC increased with administering the mixture of MEC and nicotine at intervals of 3 min, indicating a use-dependent effect of MEC. It is concluded that the action site of MEC on nicotinic receptors of sympathetic neurons is different from that of HEX.