BACKGROUND: Recently, the phannacologic and therapeutic significance of various types of potassium channels are being realized. Thus it was attempted to delineate the role of voltage-gated K+ -channels on the excitation-contraction coupling in skeletal muscle. METHODS: The effects of tetraethylammonium, a well known K+ -channel blocker, on the electrically-evoked twitch response, train-of-four and tetanic stimulation, and the influence of various agents on the these effects were studied in the isolated rat hemi-diaphragm preparation. RESULTS: Tetraethylammonium (1 & 3 mM) increased the electrically-evoked twitch response, but the large dose (10 mM) decreased the twitch response. And tetraethylammonium decreased the TOF- and tetanus-ratio in a dose-related fashion. d-Tubocurarine(1 microM) decreased the twitch response, and tetraethylammonium recovered the d-tubocurarine-induced-depression of twitch response. When the Ca++(6x) and K+ (2x) concentration of the medium were increased, the twitch response caused by tetraethylammonium were slightly inhibited than that observed in the normal solution, but the fade phenomenon was potentiated. The tetraethylammonium (10 mM)-induced depression of twitch response were reduced by reducing the stimulus frequency to 0.01 Hz and choline (400 microM) treatment. And N-ethylmaleimide inhibited the tetraethylammonium-induced increment of twitch response and also potentiated the tetraethylammonium-induced fade phenomenon. However, it is noteworth the 4-aminopy- ridine, another K+ -channel bloker, potentiated the electrically-evoked twitch response but did not affect the TOF-and tetanusratio. CONCLUSION: These result indicate that tetraethylammonium elicited two districtive types of response in the rat phrenic-hemidiaphragm preparation. The potentiating effects of twitch response is mediated by blocking delayed K+ -rectifier channel and decreasing effects of twitch response, TOF-and tetanus-ratio is may be due to decreased the acetylcholine release from presynaptic nerve terminal.
Acetylcholine ; Animals ; Choline ; Depression ; Ethylmaleimide ; Muscle, Skeletal ; Potassium Channels ; Rats* ; Tetraethylammonium*

When reduced glutathione(GSH) was incubated at neutral pH and at 37degrees, its concentration decteased slowly with formation of oxidized glutathione(GSSG). Autooxidation of GSH was accelerated by Cu2+ and Hg2+, but not by other common mono-, di-, and tri-valent cations. Tranthyretin was found to stimulate autooxidation of GSH in the presence or absence of Cu2+ and Hg2+. EDTA inhibited perfectly the autooxidation of GSH regardless of the presence of transthyretin. The stimulating activity of transthyretin was maximal at pH 7.0, declining progressively with increase or decrease of pH from 7.0. Sulfhydryl-blocking agents such as p-hydroxymercuribenzoic acid and N-ethylmaleimide markedly inhibited the stimulating activity of transthyretin. Transthyretin stimulated autooxidation of other sulfhydryl compounds such as dithiothreitol and cysteine. However, it did not show a significant effect on autooxidation of sulfhydryl group of egg albumin and eye lens proteins. And transthyretin did not cause any oxidative change to thyroxine(T4), 3, 5, 3'-tri iodo thyronine(T3) and 3, 3', 5'-triiodothyronine(rT3) bound to it in the presence of GSH and Cu2+. The above results suggest that transthyretin may play a role in regulation of oxidized status of sulfhydryl groups in blood plasma and cerebrospinal fluid.
Cations ; Cerebrospinal Fluid ; Crystallins ; Cysteine ; Dithiothreitol ; Edetic Acid ; Ethylmaleimide ; Glutathione* ; Hydrogen-Ion Concentration ; Ovum ; Plasma ; Prealbumin* ; Sulfhydryl Compounds

As it has been reported that the depolarization-induced norepinephrine (NE) release is modulated by activation of presynaptic A-1-adenosine heteroreceptor and various lines of evidence indicate the involvement of adenylate cyclase system in A-1-adenosine post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the A-1-receptor-mediated control of NE release in this study. Slices from rat hippocampus were equilibrated with (3H)-NE and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 V cm-1, 2 ms, rectangular pulses). The influence of various agents on the evoked tritium-outflow was investigated. N-6-Cyclopentyladenosine (CPA), a specific A-1-adenosine receptor agonist, in concentrations ranging from 0.1 to 10 micrometer decreased the (3H)-NE release in a dose-dependent manner without any change of basal rate of release. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 micrometer), a selective A-1-receptor antagonist, inhibited the CPA effect. The responses to N-ethylmaleimide (3 & 10 micrometer), a SH-alkylating agent of G-protein, were characterized by increments of the evoked NE-release and the CPA effects were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.1 to 30 micrometer increased the evoked and basal rate of NE release in a dose-dependent manner and the CPA effects were inhibited by forskolin pretreatment. Rolipram (1 & 10 micrometer), a phosphodiesterase inhibitor, did not affect the evoked NE release, but reduced the CPA effect. And 8-bromo-cAMP (100 & 300 micrometer), a membrane permeable cAMP analogue, inhibited the CPA effect significantly. These results suggest that the A-1-adenosine heteroreceptor plays an important role in NE-release via nucleotide-binding protein G-i in the rat hippocampus and that the adenylate cyclase system might be participated in this process.
8-Bromo Cyclic Adenosine Monophosphate ; Adenylyl Cyclases ; Animals ; Colforsin* ; Electric Stimulation ; Ethylmaleimide ; GTP-Binding Proteins ; Hippocampus* ; Membranes ; Norepinephrine* ; Rats* ; Rolipram

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 microm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 microM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I h) in DRG neurons of rats.
Animals ; Diagnosis-Related Groups ; Ethylmaleimide ; Fires ; Ganglia, Spinal ; Membranes ; Neuralgia ; Neurons ; Pertussis Toxin ; Rats ; Spinal Nerve Roots

BACKGROUNDQacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a variety of antiseptics and disinfectants, possesses a topology of 14 alpha-helical transmembrane segments (TMS). Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5.

METHODSSite-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration (MIC) and transport assay with or without N-ethylmaleimide (NEM) were performed to analyse the function of these mutants.

RESULTSAll of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetric transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity.

CONCLUSIONSThe study identified that residues W149 and S150 situated at the interface of the aqueous: lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

Bacterial Proteins ; chemistry ; physiology ; Drug Resistance, Bacterial ; Ethylmaleimide ; pharmacology ; Indoles ; metabolism ; Membrane Transport Proteins ; chemistry ; physiology ; Structure-Activity Relationship

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions (O2(.-)) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of O2(.-) and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP (10 microM) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-beta-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.
Animals ; Calcium ; Ethylmaleimide ; Humans ; Membranes ; Neurons* ; Nitric Oxide ; Nitrogen* ; Nitroprusside ; Oxidative Stress ; Rats* ; Reactive Oxygen Species* ; Substantia Gelatinosa* ; Superoxides ; Suramin ; Synaptic Transmission ; Thapsigargin ; Tissue Donors ; Xanthine ; Xanthine Oxidase

PURPOSE: In cases of overactive bladders, especially hyperreflexic neurogenic bladders, that arise in patients with spine disorder above sacral micturition center, current primary treatment modalities include the administration of anticholinergics and the intermittent catheterization. Because anticholinergics have many side effects including dry mouth, the demand for new agents has been rising. This study was designed to investigate the effects of ylang-ylang (YY) essential oil, which is currently used in aromatherapy, on the relaxation of urinary bladder muscle in vitro. MATERIALS AND METHODS: Isometric tension changes of isolated rat bladder muscle strips were recorded in an organ bath using a pressure transducer. Effects of YY oil were assessed on resting tension, electrical field stimulation(EFS)-, bethanechol-, ATP- and KCl-induced contraction. In order to determine the mechanism of YY oil, effects of YY oil on above all stimulations were assessed in the presence of methylene blue, L-NAME(N-nitro-L-arginine methyl ester) and N-ethylmaleimide. RESULTS: The contractility of strips pre-treated with YY oil was significantly decreased on all stimulation-induced contractions. There was no statistically significant difference between treated group only with YY oil and pre-treated group with YY oil and methylene blue. Similar findings were obtained when L-NAME(another NOS inhibitor) was used. When N-ethylmaleimide(c-AMP inhibitor) was employed, there was a statistically significant decrease in the rate of contraction induced by EFS, bethanechol, KCl and ATP applications. CONCLUSION: From the obtained data, the results of this study indicate that YY essential oil has relaxing effect on the bladder, and such mechanism is thought to be brought about by a pathway mediated by c-AMP.
Adenosine Triphosphate ; Animals ; Aromatherapy ; Baths ; Bethanechol ; Cananga* ; Catheterization ; Catheters ; Cholinergic Antagonists ; Ethylmaleimide ; Humans ; Methylene Blue ; Mouth ; Muscles* ; Rats* ; Relaxation* ; Spine ; Transducers, Pressure ; Urinary Bladder* ; Urination

Cellular redox state is known to be perturbed during ischemia and that Ca2+ and K+ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive K+ (KATP) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5, 5'-dithio-bis- (2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the KATP channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate KATP channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the KATP channel in rabbit ventricular myocytes.
Adenosine Triphosphate ; Collagenases ; Cytoplasm ; Dithionitrobenzoic Acid ; Dithiothreitol ; Ethylmaleimide ; Hand ; Ischemia ; KATP Channels* ; Membranes ; Muscle Cells* ; Oxidants ; Oxidation-Reduction* ; Patch-Clamp Techniques ; Perfusion

PURPOSE: Vesicle-associated membrane protein 8 (VAMP8) is a soluble N-ethylmaleimide-sensitive factor receptor protein that participates in autophagy by directly regulating autophagosome membrane fusion and has been reported to be involved in tumor progression. Nevertheless, the expression and prognostic value of VAMP8 in breast cancer (BC) remain unknown. This study aimed to evaluate the clinical significance and biological function of VAMP8 in BC. METHODS: A total of 112 BC samples and 30 normal mammary gland samples were collected. The expression of VAMP8 was assessed in both BC tissues and normal mammary gland tissues via a two-step immunohistochemical detection method. RESULTS: The expression of VAMP8 in BC tissues was significantly higher than that in normal breast tissues. Furthermore, increased VAMP8 expression was significantly correlated with tumor size (p=0.007), lymph node metastasis (p=0.024) and recurrence (p=0.001). Patients with high VAMP8 expression had significantly lower cumulative recurrence-free survival and overall survival (p < 0.001 for both) than patients with low VAMP8 expression. In multivariate logistic regression and Cox regression analyses, lymph node metastasis and VAMP8 expression were independent prognostic factors for BC. CONCLUSION: VAMP8 is significantly upregulated in human BC tissues and can thus be a practical and potentially effective surrogate marker for survival in BC patients.
Autophagy ; Biomarkers ; Breast Neoplasms* ; Breast* ; Humans ; Logistic Models ; Lymph Nodes ; Mammary Glands, Human ; Membrane Fusion ; Methods ; N-Ethylmaleimide-Sensitive Proteins ; Neoplasm Metastasis ; Prognosis ; R-SNARE Proteins* ; Recurrence

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins, composed of two presynaptic membrane proteins [synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin] and a presynaptic vesicular protein [vesicle-associated membrane protein (VAMP) ], serve as a core of exocytotic fusion machinery, which can be affected by ischemia. Synaptic protein in core region, striatum and cortex has been shown to alter after focal ischemia, however, little is known in hippocampus. Hippocampus is remote from ischemic core, but it is one of the most vulnerable regions. Using immunohistochemistry, the present study was undertaken to investigate the alteration of expression of SNAP-25, syntaxin, and VAMP in the hippocampus of rats which were subjected to middle cerebral artery occlusion (MCAO) for 2h and allowed to reperfuse. At 2 weeks of reperfusion, the SNAP-25 and syntaxin immunoreactivity was increased in the stratum oriens of the CA1 and the stratum lucidum of the CA3 in the ipsilateral hippocampus. However, VAMP immunoreactivity didn't show significant change. These results demonstrate that the level of the presynatpic plasma membrane proteins (SNAP-25 and syntaxin) in the rat hippocampus is more sensitively affected by focal ischemia than that of the synaptic vesicle protein (VAMP).
Animals ; Cell Membrane ; Hippocampus* ; Immunohistochemistry ; Infarction, Middle Cerebral Artery* ; Ischemia ; Membrane Proteins ; Middle Cerebral Artery* ; Qa-SNARE Proteins ; Rats* ; Reperfusion ; SNARE Proteins* ; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins ; Synaptic Vesicles