Functional Identification of a Multi-product Sesquiterpene Synthase from Ganoderma sinense / 中国实验方剂学杂志

Objective: Ganoderma sinense is one of the most famous medicinal fungi in China. Because it is a model medicinal fungus of basidiomycete,the functional identification of its sesquiterpene synthase is of great significance for the biosynthesis and regulation studies of fungal sesquiterpene. Method: A sesquiterpene synthase gene was discovered by mining the genome data of G. sinense. The sesquiterpene's conservative motifs was analyzed through multiple sequence alignment with two identified sesquiterpene synthases of G. sinense and three terpenoid synthases in the Nr database,which have the highest similarity to it. Subsequently,heterologous expression was observed in Escherichia coli,and protein expression was detected by SDS-PAGE. Volatile compounds were collected by solid phage microextraction (SPME) and detected by GC-MS. Result: The enzyme containing sesquiterpene conserved motifs DDXXDE and NSE/DTE were efficiently expressed in E. coli,and 11 sesquiterpenes were synthesized with endogenous FPP as the substrate. The product α-cadinol at 18.6 min was considered to be the main product,and previous studies showed a significant anticancer activity. According to the comparison with chemical standards,three products were identified as γ-muurlene,α-muurolene and δ-cadinene,respectively. Conclusion: The functional identification of multi-product sesquiterpene synthase from G. sinense can promote the study on the mechanism underlying its product diversity,and lay a foundation for the production of rare or novel sesquiterpenes by improving the catalytic activity of enzymes with enzyme engineering technology.

Bioactivity and Chemical Constituents of Different Polar Parts from Blueberry Leaves / 中国药学杂志

OBJECTIVE: To study the bioactivity and chemical constituents of different polar parts from blueberry leaves. METHODS: Blueberry leaves were extracted by ethanol and then the extract was sequentially partitioned into five fractions. Silicagel and Sephadex LH-20 chromatographic methods were applied to isolate and purify compounds. Their structures were elucidated by physiochemical properties and spectral analysis.The DPPH• radical scavenging activity, α-glycosidase and pancreatic lipase inhibition activity of different polar parts and partial compounds were determined. RESULTS: The n-butyl alcohol fraction(BF) showed the highest DPPH• radical scavenging activity and α-glycosidase inhibition activity. The ethyl acetate fraction(EAF) showed the strongest pancreatic lipase inhibition activity. A total of five compounds were isolated from the EAF, and their structures were identified as β-sitosterol(1), quercetin-3-O-α-L-arabinofuranoside(2), quercetin(3), quercetin-3-O-β-D-glucopyranoside(4) and 1-O-caffeoylquinic acid(5). A total of two compounds were isolated from the BF, and their structures were identified as quercetin-3-O-α-L-arabinoside(6) and quercetin-3-O-β-D-glucuronide(7). The results showed that compounds 3 and 5 had very good DPPH• radical scavenging and pancreatic lipase inhibitory activity, and compounds 1 and 3 had good α-glucosidase inhibitory activity. CONCLUSION: The different polar parts and compounds of blueberry leaves show strong DPPH• radical scavenging activity, α-glycosidase and pancreatic lipase inhibition activity. Compounds 1, 2, 5 and 6 are isolated from blueberry leaves for the first time.

Efficacy of real-time PCR for detecting Clostridium difficile infection: comparison with enzyme-linked fluorescent spectroscopy-based approaches / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the diagnostic efficacy of real?time polymerase chain reaction (q?PCR) for Clostridium difficile infection (CDI) in comparison with routine culture and enzyme?linked fluorescent spectroscopy?based aprroaches.</p><p><b>METHODS</b>Stool samples were collected from suspected CDI cases in General Hospital of Guangzhou Military Command of PLA between May and December in 2016. All the samples were examined with 3 methods, namely enzyme?linked fluorescent spectroscopy for detecting Clostridium difficile toxin A/B (CDAB), detection of glutamate dehydrogenase (GDH), and q?PCR for amplification of Clostridium difficile?specific gene tpi and toxin gene (tcdA/tcdB), with the results of fecal culture as the reference for evaluating the diagnostic efficacy of the 3 methods.</p><p><b>RESULTS</b>Of the total of 70 fecal samples, 13 (18.57%) were found to be positive for Clostridium difficile, including toxin?producing strains in 6 (8.57%) samples. The sensitivity, specificity, positive predictive value, negative predictive value and diagnostic coincidence rate of q?PCR for tpi were 92.31%, 91.23%, 70.59%, 98.11% and 91.43%, respectively, which were significantly higher than those of GDH test (84.62%, 84.21%, 55.00%, 96.00%, and 84.29%, respectively; Χ=24.881, P<0.001). The sensitivity of q?PCR for tcdA/cdB was significantly higher than that of enzyme?linked fluorescent spectroscopy for CDAB in detecting CDI (66.67% vs 33.33%; Χ=35.918, P<0.001).</p><p><b>CONCLUSION</b>Both CDAB detection and q?PCR have a high specificity in detecting CDI, but GDH detection has a good sensitivity, and all these 3 methods have a high negative predictive value. Compared with other detection methods, amplification of tpi and tcdA/tcdB using q?PCR allows more rapid, sensitive and specific detection of CDI.</p>

Phenotypes of ATP-activated current associated with their genotypes of P2X1-6 subunits in neurons innervating tooth-pulp.

To explore the association of the phenotype of ATP-activated current with the genotype of P2X1-6 subunits in nociceptors, we developed a method that allows us to label nociceptive neurons innervating tooth-pulp in rat trigeminal ganglion (TG) neurons using a retrograde fluorescence-tracing method, to record ATP-activated current in freshly isolated fluorescence-labeled neurons, and then to conduct single cell immunohistochemical staining for P2X1-6 subunits in the same neuron. We found that fast application of 100 µM ATP to fluorescence-traced TG neurons produced robust inward current in 87% (96/110) of cells tested. The diameter of cells varied from 16 to 56 µm. Three types of ATP-activated current (F, I and S) were recorded with distinct rise times of the current (R10-90, P < 0.05). There was a positive correlation between the cell diameter and the value of R10-90 (P < 0.05): the value of R10-90 increased with increases in the cell diameter. Cells responsive to ATP with the type F current mainly showed positive staining for P2X3 and P2X5, but negative staining for P2X2; cells responsive to ATP with the type I current showed positive staining for P2X1-3 and P2X5, but negative staining for P2X4; and cells responsive to ATP with the type S current showed positive staining for P2X1-5, but negative staining for P2X6. The present findings suggest that in addition to P2X3 subunits, P2X5 subunits are also involved in the generation of the F type of ATP-activated current in small-sized nociceptive neurons. In addition to the P2X2/3 subunit-containing channels, more complex uncharacterized combinations of P2X1-5 subunits exist in native medium-sized nociceptive neurons exhibiting the I and S types of ATP-activated current. In addition, the P2X6 subunit is not a main subunit involved in the nociceptive signal in rat TG neurons innervating tooth-pulp.

Actions of bis(7)-tacrine and tacrine on transient potassium current in rat DRG neurons and potassium current mediated by K(V)4.2 expressed in Xenopus oocyte.

Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] is a dimeric AChE inhibitor derived from tacrine with a potential to treat Alzheimer's disease. Actions of bis(7)-tacrine on ligand-gated ion channels and voltage-gated cation channels have been identified on neurons of both central and peripheral nervous systems. In the present study, the effect of bis(7)-tacrine was investigated on the K(V)4.2 encoded potassium currents expressed in Xenopus oocytes and the transient A-type potassium current (I(K(A))) on rat DRG neurons. Bis(7)-tacrine suppressed recombinant Kv4.2 potassium channels in a concentration-dependent manner, with IC(50) value of 0.53+/-0.13 muM. Tacrine also inhibited Kv4.2 channels, but with a much lower potency (IC(50) 74+/-15 muM).The possible mechanisms underlying the inhibition on potassium currents by bis(7)-tacrine/tacrine could be that inactivation of the transient potassium currents was accelerated and recovery of the native or Kv4.2 expressed potassium currents was suppressed by bis(7)-tacrine/tacrine.

Potentiation by WIN 55,212-2 of GABA-activated currents in rat trigeminal ganglion neurones.

BACKGROUND AND PURPOSE: Although both natural and synthetic cannabinoid compounds have been shown to exert an antinociceptive effect on acute and persistent pain, the anatomical locus of the target of cannabinoid-induced analgesia has not been fully elucidated. Here, we investigated the effects of the cannabinoid agonist WIN 55,212-2 on GABA-activated currents (I(GABA)) in rat primary sensory neurones. EXPERIMENTAL APPROACH: In the present study, experiments were performed on neurones freshly isolated from rat trigeminal ganglion (TG) by using whole-cell patch clamp and repatch techniques. KEY RESULTS: GABA-evoked inward currents were potentiated by pretreatment with WIN 55,212-2 in a concentration-dependent manner (10(-10)-10(-8) M). WIN 55,212-2 shifted the GABA concentration-response curve upwards, with an increase of 30.3 +/- 3.7% in the maximal current response but with no significant change in the EC(50) (agonist concentration producing a half-maximal response) value. WIN 55,212-2 potentiated the responses to GABA in a manner independent of holding potential and in the absence of any change in the reversal potential of the current. This potentiation of I(GABA) induced by WIN 55,212-2 was almost completely blocked by AM 251 (3 x 10(-8) M), a CB(1) receptor antagonist, and, using the repatch technique, was found to be abolished after intracellular dialysis with the protein kinase A (PKA) activator cAMP or the PKA inhibitor H89. CONCLUSIONS AND IMPLICATIONS: The potentiation by WIN 55,212-2 of I(GABA) in primary sensory neurones may help to elucidate the mechanism underlying the modulation of analgesia by cannabinoids in the spinal dorsal horn.

Characteristics of ATP-activated current in nodose ganglion neurons of rats.

The characteristics of ATP-activated currents (I(ATP)) in rat nodose ganglion (NG) neurons have not been fully clarified. Especially, the correlation between I(ATP) phenotype and P2X receptor subunit genotype in rat NG neuron is not clear. By whole-cell patch-clamp and single cell immunocytochemical techniques, we explored the characteristics of the I(ATP) phenotype and its correlation with P2X receptor subunits in acutely isolated NG neuron of rats. The results indicated that I(ATP) of NG neurons can be classified into four types: F type (fast type, 8.1%), I type (intermediate type, 14.8%), S type (slowing type, 37.0%) and vS type (very slowing type, 21.5%). The single immunocytochemical studies have demonstrated that F type cells express P2X1 and P2X3 subunits, I type cells P2X1, P2X3 and P2X4 subunits, S type cells P2X1, P2X2 and P2X3 subunits and vS type cells P2X2, P2X3 and P2X4 subunits. The results reveal that there are four types of I(ATP) in NG neurons, differential expression of distinct P2X subunits may underlie the I(ATP) phenotype.

Mechanism of bis(7)-tacrine inhibition of GABA-activated current in cultured rat hippocampal neurons.

Bis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine that shows promise for the treatment of Alzheimer's disease. We have previously reported that bis(7)-tacrine inhibits GABA(A) receptors. In the present study we investigated the mechanism of bis(7)-tacrine inhibition of GABA(A) receptor function using whole-cell patch-clamp recording in cultured rat hippocampal neurons. Bis(7)-tacrine produced a gradual decline of GABA-activated current to a steady-state, but this was not an indication of use-dependence, as the gradually declining component could be eliminated by exposure to bis(7)-tacrine prior to GABA application. In addition, bis(7)-tacrine inhibition did not require the presence of agonist, and GABA-activated current recovered completely from inhibition by bis(7)-tacrine in the absence of agonist. The slow onset of inhibition by bis(7)-tacrine was not apparently due to an action at an intracellular site, as inclusion of 25 microM bis(7)-tacrine in the recording pipette did not alter inhibition by bis(7)-tacrine applied externally. Bis(7)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner and the pA(2) value estimated from a Schild plot was 5.7. Bis(7)-tacrine increased the time constant of activation of GABA-gated ion channels without affecting the time constants of deactivation or desensitization. These results suggest that bis(7)-tacrine is a competitive GABA(A) receptor antagonist with slow onset and offset kinetics. The competitive inhibition of GABA receptors by bis(7)-tacrine could contribute to its ability to enhance memory.

Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors.

Relatively little information is available about the molecular mechanism of ethanol inhibition of P2X receptors. Here, we investigated the possibility that 10 conserved cysteine residues in the extracellular loop of the rat P2X4 receptor may regulate ethanol inhibition of the receptor using a series of individual cysteine to alanine point mutations. Each of the mutated receptors generated robust inward current in response to ATP and the mutations produced less than a sixfold change in the ATP EC50 value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current activated by an EC40 concentration of ATP. By contrast, for the C261A and C270A mutants, ethanol inhibited ATP-activated current in a competitive manner similar to that for the wild-type receptor. Interestingly, for the C132A, C159A, C217A, and C227A mutants, ethanol inhibited ATP-activated current, but decreased the maximal response to ATP by 70-75% without significantly changing the EC50 value of ATP, thus exhibiting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X4 receptor by ethanol.

Application of comfort care for dental out-patients / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore and apply the nursing methods of comfort care for dental out-patients.</p><p><b>METHODS</b>Control group included 103 dental out-patients who were first treated in General Dentistry Department of West China Hospital of Stomatology from June to August 2008. Experimental group included 105 dental out-patients who were first treated in the same hospital from September to November 2008. Conventional nursing methods were used for control group, and comfort care nursing methods were used for experimental group. The patients' degree of satisfaction with nursing and oral health knowledge rate after first treatment and nursing were investigated.</p><p><b>RESULTS</b>The patients' degree of satisfaction with nursing of the control and experimental groups were 72.816% and 98.095%, and the patients' oral health knowledge rate of the two groups were 57.282% and 93.333%. Both of the investigating results had obvious difference.</p><p><b>CONCLUSION</b>Comfort care for dental out-patients can improve patients' degree of satisfaction with nursing and increase patients' oral health knowledge rate. Simultaneously, comfort care can improve the specialized quality of dental nurses and would advantage to build a harmonious relationship between nurses and patients.</p>

Inhibition of NMDA-gated ion channels by bis(7)-tacrine: whole-cell and single-channel studies.

Bis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine, and has been proposed as a promising agent to treat Alzheimer's disease. We have recently reported that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis by antagonizing NMDA receptors. The purpose of this study was to characterize bis(7)-tacrine inhibition of NMDA-activated current by using patch-clamp recording techniques. In cultured rat hippocampal neurons, bis(7)-tacrine inhibited NMDA-activated whole-cell current in a concentration-dependent manner with an IC(50) of 0.66+/-0.07 microM. Bis(7)-tacrine produced a gradual decline of NMDA-activated current to a steady-state, but this was not an indication of use-dependence. Also, the slow onset of inhibition by bis(7)-tacrine was not apparently due to an action at an intracellular site. Bis(7)-tacrine, 0.5 microM, decreased the maximal response to NMDA by 40% without changing its EC(50). Bis(7)-tacrine inhibition of NMDA-activated current was not voltage-dependent, and was independent of glycine concentration. Results of single-channel experiments obtained from cells expressing NR1 and NR2A subunits revealed that bis(7)-tacrine decreased the open probability and frequency of channel opening, but did not significantly alter the mean open time or introduce rapid closures. These results suggest that bis(7)-tacrine can inhibit NMDA receptor function in a manner that is slow in onset and offset and noncompetitive with respect to both NMDA and glycine. The noncompetitive inhibition of NMDA receptors by bis(7)-tacrine could contribute to its protective effect against glutamate-induced neurotoxicity.

Bis(7)-tacrine prevents glutamate-induced excitotoxicity more potently than memantine by selectively inhibiting NMDA receptors.

We have recently reported that bis(7)-tacrine could prevent glutamate-induced neuronal apoptosis through NMDA receptors. In this study, we demonstrated that in cultured rat cortical neurons, bis(7)-tacrine (IC(50), 0.02 microM) prevented glutamate-induced excitotoxicity more substantially than memantine (IC(50), 0.7 microM). In addition, bis(7)-tacrine was more efficient than memantine in buffering the intracellular Ca(2+) triggered by glutamate. In cultured rat hippocampal neurons, bis(7)-tacrine inhibited 50 microM NMDA-activated current in a concentration-dependent manner with an IC(50) of 0.68+/-0.07 microM, which is five times more potent than that produced by memantine (IC(50), 3.41+/-0.36 microM; p<0.05). By contrast, bis(7)-tacrine, up to 5 microM, did not significantly affect the current activated by 50 microM AMPA or 50 microM kainate. These results suggest that bis(7)-tacrine is more potent than memantine against glutamate-induced neurotoxicity by selectively inhibiting NMDA-activated current.

Clinical effects of Fu Fang Ya Tong Ding on gingivitis and pericoronitis / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical effects of Fu Fang Ya Tong Ding on treatment of gingivitis and pericoronitis.</p><p><b>METHODS</b>120 clinical patients with gingivitis or pericoronitis were randomly divided into 3 groups (40 patients in each group). After routine rinse treatment for all patients, patients in the test group were treated with Fu Fang Ya Tong Ding, patients in the positive group were treated with iodine glycerol, while that time patients in the negative group received no treatment anymore. Ten minutes after treatment, visual analogue scale (VAS) was used to record the severity of pain for each patient. 3 days and 7 days later, pain and inflammation degree were also recorded by pain three-degree scoring method and index of gingivitis. The total treatment effects were evaluated under a comprehensive clinical treatment standard.</p><p><b>RESULTS</b>10 minutes after treatment, 40.0% of patients in the test group had almost no pain, while no obvious reduction of pain was found in the control group. 3 days, 7 days after the treatment, 92.5%, 95.0% of patients in the test group had no pain, and 55.0%, 90.0% of patients in the positive group had no pain. In the negative group, there were 47.5% of patients which pain was still remained in 7 days. 7 days after treatment, gingival index in the test group reduced by 25.0% and 42.8% compared with the positive and negative groups (P<0.05). 3 days after treatment, 62.5%, 45.0% and 30.0% patients separately in the test, positive and negative groups manifested good effects under the comprehensive clinical treatment standard; after 7 days, 97.5%, 92.5% and 77.5% patients in the 3 groups manifested good effect. The group using Fu Fang Ya Tong Ding had better effects than groups using iodine glycerol or only applying routine rinsing treatment group (P<0.05).</p><p><b>CONCLUSION</b>Fu Fang Ya Tong Ding can treat gingivitis and pericoronitis through significantly reducing inflammation and pain.</p>

Effect of sFRP2 on the biological behavior of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of sFRP2 on the biological behavior of human hepatoma carcinoma HepG2 cells.</p><p><b>METHODS</b>HepG2 cells were infected with recombinant adenovirus containing mouse sFRP2 gene, and then the proliferation, cell cycle distribution, expression of tumor metastasis related factors (CD44, CD82/KAI1, EMMPRIN) and beta-catenin protein, and migration ability of the cells were detected by MTT, FCM, immunohistochemistry, Western blot and Transwell inserts, respectively.</p><p><b>RESULTS</b>sFRP2 protein inhibited the proliferation of HepG2 cells, and increased the percentage of G0/G1 period cells. Expression of CD44 and CD82/KAI1 proteins, which could inhibit invasion and metastasis of tumor cells, was upregulated. However, EMMPRIN protein, which could promote the above properties of tumor cells decreased in HepG2 cells infected with the recombinant adenovirus containing mouse sFRP-2 gene. Western blot demonstrated that beta-catenin was expressed in HepG2 cells and there was no significant difference between the treated and the control groups. Transwell insert test showed sFRP2 protein decreased the migration ability of HepG2 cells.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing mouse sFRP-2 gene could infect HepG2 cells. sFRP2 protein could significantly reduce the capability of proliferation, invasion and metastasis of HepG2 cells.</p>

5-Hydroxytryptamine directly inhibits neuronal nicotinic acetylcholine receptors in rat trigeminal ganglion neurons.

In the present study, whole-cell patch clamp recording technique was used to investigate the action of 5-hydroxytryptamine (5-HT) on the function of native neuronal nicotinic acetylcholine receptors expressed in the rat trigeminal ganglion neurons. Inward currents (I(nic)) caused by externally-applied nicotine were observed in majority of the examined neurons, which were mediated by alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors. We found that 5-HT could reversibly inhibit I(nic) in a concentration-dependent manner, and the inhibition did not involve 5-HT receptors. Other serotonergic agents, such as 2-methyl-5-HT, alpha-methyl-5-HT, sumatriptan and ICS-205,930, also had similar inhibitory effects on I(nic). 5-HT inhibited nicotinic acetylcholine receptors in a non-competitive manner, as 5-HT decreased the maximal current response to nicotine but had no effect on the threshold and EC(50). The inhibition of I(nic) by 5-HT was voltage-dependent and became stronger at hyperpolarized potentials. These results indicated that 5-HT directly inhibited nicotinic acetylcholine receptors in the trigeminal ganglion neurons. As a local modulator of the nicotinic acetylcholine receptor, 5-HT might play a role in the modulation of sensory information.

Novel dimeric bis(7)-tacrine proton-dependently inhibits NMDA-activated currents.

Bis(7)-tacrine has been shown to prevent glutamate-induced neuronal apoptosis by blocking NMDA receptors. However, the characteristics of the inhibition have not been fully elucidated. In this study, we further characterize the features of bis(7)-tacrine inhibition of NMDA-activated current in cultured rat hippocampal neurons. The results show that with the increase of extracellular pH, the inhibitory effect decreases dramatically. At pH 8.0, the concentration-response curve of bis(7)-tacrine is shifted rightwards with the IC(50) value increased from 0.19+/-0.03 microM to 0.41+/-0.04 microM. In addition, bis(7)-tacrine shifts the proton inhibition curve rightwards. Furthermore, the inhibitory effect of bis(7)-tacrine is not altered by the presence of the NMDA receptor proton sensor shield spermidine. These results indicate that bis(7)-tacrine inhibits NMDA-activated current in a pH-dependent manner by sensitizing NMDA receptors to proton inhibition, rendering it potentially beneficial therapeutic effects under acidic conditions associated with stroke and ischemia.

The coupling of acetylcholine-induced BK channel and calcium channel in guinea pig saccular type II vestibular hair cells.

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the alpha9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs(+) completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd(2+) and Ni(2+), respectively. The inhibition potency of these four calcium channel antagonists was nimodipine>nifedipine>cadmium>nickel. The L-type Ca(2+) channels agonist, (-)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.

Inhibition by bis(7)-tacrine of native delayed rectifier and KV1.2 encoded potassium channels.

Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] acts as an AChE inhibitor and also exerts modulatory effects on many ligand-gated ion channels and voltage-gated Ca(2+) and K(+) channels. It has been reported previously that tacrine and some other AChE inhibitors suppressed I(K(A)) in central and peripheral neurons. The present study aimed to explore whether bis(7)-tacrine could modulate the function of native delayed rectifier potassium channels in DRG neurons and K(V)1.2 encoded potassium channels expressed in oocytes. We found that both delayed rectifier potassium currents (I(K(DR))) in rat DRG neurons and the currents recorded from oocytes expressing K(V)1.2 (I(K(K(V)1.2))) were suppressed by bis(7)-tacrine, the potency of which was two orders greater than that of tacrine. The IC(50) values for bis(7)-tacrine and tacrine inhibition of I(K(KD)) in DRG neurons were 0.72+/-0.05 and 58.3+/-3.7 microM, respectively; while the two agents inhibited I(K(K(V)1.2)) in oocytes with an IC(50) of 0.24+/-0.06 and 102.1+/-21.5 microM, respectively. The possible mechanism for bis(7)-tacrine inhibition of I(K(A)) and I(K(K(V)1.2)) was identified as the suppression of their activation, inactivation.

[Electrophysiological characteristic of ATP-activated currents of trigeminal ganglion neurons with different diameter in rat].

AIM: To explore the characteristic of ATP-activated current in trigeminal ganglion (TG) neurons of rat. METHODS: Whole-cell patch-clamp was performed. RESULTS: (1) The majority (92.1%) of TG neurons responded to ATP applied externally with inward currents. We recorded three distinct ATP-activated currents: fast, slow and intermediate, which were concentration-dependent. (2) In general, the fast ATP-activated currents were distributed mainly in small-diameter TG neurons, the slow ATP-activated currents were distributed mainly in large-diameter TG neurons, and the intermediate ATP-activated currents were distributed mainly in intermediate-diameter TG neurons. (3) The time course of rising phase from 10% to 90% of the three distinct ATP-activated currents were as follows: fast: (33.6 +/- 4.5) ms; intermediate: (62.2 +/- 9.9) ms; slow: (302.1 +/- 62.0) ms, and that of desensitizing phase were (399.4 +/- 58.2) ms (fast), and > 500 ms (slow) respectively. (4) From the current-voltage relationship curves, it can be seen that the reversal potential values of the three distinct ATP-activated currents were the same, all being 0-5mV. And they all were characterized by inward rectification. (5) The dose-response curve for fast ATP-activated current shifted downwards as compared with the intermediate ATP-activated current, and that for the slow ATP-activated current shifted upwards. CONCLUSION: The EC50s of the three curves tended to be identical. The results suggested that three kinds of distinct ATP-activated currents could be mediated by various subtypes of P2X receptors assembled by different subunits, and the subtypes existed in TG neurons of different diameters and transmit different information.