Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a dee...Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a deeper understanding of ion channel behavior,these techniques significantly accelerate the process of drug discovery and development.In recent years,automated patch-clamp technique has undergone substantial advancements,surpassing traditional manual methods with its high throughput,improved data consistency,and automation.However,fully harnessing these advantages requires meticulous optimization of experimental conditions tailored to specific targets.Without such refinement,the high cost of consumables and operational expenses could severely hinder the widespread adoption of this technique.This study focused on the TRPV1 channel,detailing the establishment of an automated patch-clamp detection system for TRPV1 currents,optimization of experimental parameters,and a comparative evaluation of the results against manual patch-clamp techniques.展开更多
Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission ...Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.展开更多
We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. T...We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.展开更多
One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship whe...One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.展开更多
Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion ...Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.展开更多
Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods...Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.展开更多
Many rat taste receptor cells conduct action potentials(APs).APs had a mean threshold of -35 mV(n=95 cells)and a spike height of 52mV above threshold in current clamp(hold= -80mV).Aps could be classified into two sign...Many rat taste receptor cells conduct action potentials(APs).APs had a mean threshold of -35 mV(n=95 cells)and a spike height of 52mV above threshold in current clamp(hold= -80mV).Aps could be classified into two significantly different (P<0.001) groups-fast,with short half-time durations and large outward currents (mean1.3 ms and 2.7nA),and slow,with long duration and small outward currents(mean9.2ms and 0. 29nA).AP upstrokes were conducted by TTX-sensitive sodium currents whereas the downstroke by TEA-blockable outward currents. Voltage dependent analysis of outward current separated transient and sustained components.The transient component was specifically blocked by 4-AP(1mmol/L).A calcium-dependent outward component was also revealed modulating voltage and external calcium concentration.The fast recovery phase of the AP appears related the sustained outward current whereas the after hyperpolarization(AHP) was blocked by 4AP suggesting a significant contribution of the transient component.Forskolin (FSK),which elevates cAMP,reversibly blocked the majority of the sustained current without influencing the transient. FSK greatly exaggerated the AHP without changing the spike height or duration. These data suggest that several components of the outward current contribute specifically to the gustatory AP and that the AP may be modulated by cyclic nucleotides.展开更多
AIM: To study the effects of crebanine on voltage-gated Na+ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na+ current w...AIM: To study the effects of crebanine on voltage-gated Na+ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na+ current was recorded using the whole cell voltage-clamp technique. RESULTS: Crebanine reversibly inhibited Na+ current with an IC50 value of 0.283 mmol?L-1(95% confidence range: 0.248-0.318 mmol?L-1). Crebanine at 0.262 mmol?L-1 caused a negative shift(about 12 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. CONCLUSION: In addition to blocking other voltage-gated ion channels, crebanine blocked Na+ channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na+ channels in cardiac tissues.展开更多
Patch-clamp recording requires direct accessibility of the cell membrane to patch pipettes and allows the investigation of ion channel properties and functions in specific cellular compartments.The cell body and relat...Patch-clamp recording requires direct accessibility of the cell membrane to patch pipettes and allows the investigation of ion channel properties and functions in specific cellular compartments.The cell body and relatively thick dendrites are the most accessible compartments of a neuron,due to their large diameters and therefore great membrane surface areas.However,axons are normally inaccessible to patch pipettes because of their thin structure;thus studies of axon physiology have long been hampered by the lack of axon recording methods.Recently,a new method of patchclamp recording has been developed,enabling direct and tight-seal recording from cortical axons.These recordings are performed at the enlarged structure（axonal bleb） formed at the cut end of an axon after slicing procedures.This method has facilitated studies of the mechanisms underlying the generation and propagation of the main output signal,the action potential,and led to the finding that cortical neurons communicate not only in action potential-mediated digital mode but also in membrane potential-dependent analog mode.展开更多
The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from the hippocampus of...The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from the hippocampus of newborn rats were induced to differentiate with neurotrophic growth factor, and the electrophysiological properties of the voltage-gated potassium ion channels were observed. Immunofluorescence staining showed that the rapidly proliferating neural stem cells formed spheres in vitro that expressed high levels of nestin. The differentiated neurons were shown to express neuron-specific enolase. Flow cytometric analysis revealed that the neural stem cells were actively dividing and the percentage of cells in the S ＋ G2/M phase was high. However, the ratio of cells in the S ＋ G2/M phase decreased obviously as differentiation proceeded. Whole-cell patch-clamp re- cordings revealed apparent changes in potassium ion currents as the neurons differentiated. The potassium ion currents consisted of one transient outward potassium ion current and one delayed rectifier potassium ion current, which were blocked by 4-aminopyridine and tetraethylammonium, respectively. The experimental findings indicate that neural stem cells from newborn rat hippo- campus could be cultured and induced to differentiate into functional neurons under defined condi- tions in vitro. The differentiated neurons expressed two types of outward potassium ion cur＇ents similar to those of mature neurons in vivo.展开更多
Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's dis...Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.展开更多
Focal cortical dysplasia(FCD)is one of the most common causes of drug-resistant epilepsy.Dysmorphic neurons are the major histopathological feature of typeⅡFCD,but their role in seizure genesis in FCD is unclear.Here...Focal cortical dysplasia(FCD)is one of the most common causes of drug-resistant epilepsy.Dysmorphic neurons are the major histopathological feature of typeⅡFCD,but their role in seizure genesis in FCD is unclear.Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients.Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in typeⅡFCD,including an enlarged soma,aberrant dendritic arbors,increased current injection for rheobase action potential firing,and reduced action potential firing frequency.Intriguingly,the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na^(+)-K^(+)-Cl^(−)cotransporter.In addition,we found a depolarizing shift of the GABA reversal potential in the CamKⅡ-cre::PTENflox/flox mouse model of drug-resistant epilepsy,suggesting that enhanced GABAergic inputs might depolarize dysmorphic neurons.Thus,imbalance of synaptic excitation and inhibition of dysmorphic neurons may contribute to seizure genesis in typeⅡFCD.展开更多
Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of nefer...Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.展开更多
Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensi...Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.展开更多
Objective To investigate the effect of ferulic acid,a natural compound,on pancreatic beta cell viability,Ca^(2+)channels,and insulin secretion.Methods We studied the effects of ferulic acid on rat insulinoma cell line...Objective To investigate the effect of ferulic acid,a natural compound,on pancreatic beta cell viability,Ca^(2+)channels,and insulin secretion.Methods We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay.The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca^(2+)channels and insulin secretion,respectively.Results Ferulic acid did not affect cell viability during exposures up to 72 h.The electrophysiological study demonstrated that ferulic acid rapidly and concentration-dependently increased L-type Ca^(2+)channel current,shifting its activation curve in the hyperpolarizing direction with a decreased slope factor,while the voltage dependence of inactivation was not affected.On the other hand,ferulic acid have no effect on T-type Ca^(2+)channels.Furthermore,ferulic acid significantly increased insulin secretion,an effect inhibited by nifedipine and Ca^(2+)-free extracellular fluid,confirming that ferulic acid-induced insulin secretion in these cells was mediated by augmenting Ca^(2+)influx through L-type Ca^(2+)channel.Our data also suggest that this may be a direct,nongenomic action.Conclusion This is the first electrophysiological demonstration that acute ferulic acid treatment could increase L-type Ca^(2+)channel current in pancreaticβcells by enhancing its voltage dependence of activation,leading to insulin secretion.展开更多
Acutely isolated mouse hippocampal CA3 pyramidal neurons were exposed to 3 mT static magnetic field,and the characteristics of transient outward K+ channel were studied using the whole-cell patch-clamp technique.The e...Acutely isolated mouse hippocampal CA3 pyramidal neurons were exposed to 3 mT static magnetic field,and the characteristics of transient outward K+ channel were studied using the whole-cell patch-clamp technique.The experiment revealed that the amplitude of transient outward potassium channel current was reduced.The maximum activated current densities of control group and exposure group were 163.62±20.68 pA/pF and 98.74±16.57 pA/pF(n=12,P<0.01) respectively.The static magnetic field exposure affected the activation and inactivation process of transient outward potassium channel current.Due to the magnetic field exposure,the half-activation voltage of the activation curves changed from 5.59±1.96 mV to 27.87±7.24 mV(n=12,P<0.05) ,and the slope factor changed from 19.43±2.11 mV to 25.87±4.22 mV(n=12,P<0.05) .The half-inactivation voltage of the inactivation curves also changed from-56.09±0.89 mV to-57.16±1.10 mV(n=12,P>0.05) and the slope factor of the inactivation curves from 8.69±0.80 mV to 10.87±1.02 mV(n=12,P<0.05) .The results show that the static magnetic field can change the characteristics of transient outward K+ channel,and affect the physiological functions of neurons.展开更多
Objectives To investigate the effects of simvastatin on membrane ionic currents in left ventricular myocytes of rabbit heart suffering from acute myocardial infarction （ AMI）, so as to explore the ionic mechanism of...Objectives To investigate the effects of simvastatin on membrane ionic currents in left ventricular myocytes of rabbit heart suffering from acute myocardial infarction （ AMI）, so as to explore the ionic mechanism of statin treatment for antiarrhythmia. Methods Forty-five New Zealand rabbits were randomly divided into three groups： AMI group, simvastatin intervention group （ Statin group） and sham-operated control group （CON）. Rabbits were infarcted by ligation of the left anterior descending coronary artery after administration of oral simvastatin 5 mg · kg^-1·d^-1 （Statin group） or placebo （AMI group） for 3 days. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region 72 h later. Whole cell patch clamp technique was used to record membrane ionic currents, including sodium current （INa）, L-type calcium current （Ica-L） and transient outward potassium current （Ito）. Results （1） There was not significant difference in serum cholesterol concentration among three groups. （2） The peak INa current density （at -30 mV） was significantly decreased in AMI group （ -25.26±5.28, n = 13 ）, comparing with CON （ - 42. 78± 5.48, n = 16）, P 〈 0. 05, while it was significantly increased in Statin group （ - 39.83 ±5.65 pA/pF, n = 12） comparing with AMI group, P 〈0. 01 ; The peak Ica-L current density （ at 0 mV） was significantly decreased in AMI group （ -3. 43 ±0. 92 pA/pF, n = 13） comparing with CON （ -4. 56 ±1.01 pA/pF, n = 15）, P 〈0. 05, while it was significantly increased in Statin group （ -4. 18±0. 96 pA/pF, n = 12） comparing with AMI group, P 〈0. 05; The Ito current density （ at ＋ 60 mV） was significantly decreased in AMI group （ 11.41 ± 1.94 pA/pF, n = 13 ） comparing with CON （17.41 ±3.13 pA/pF, n = 15）, P 〈0. 01, while it was significantly increased in Statin group （16. 11 ± 2. 43 pA/pF, n = 14） comparing with AMI group, P 〈 0. 01. Conclusions AMI induces significant down-regulation of INa, Ica-L and Ito. Pretreatment with simvastatin could attenuate this change without lowering the serum cholesterol level, suggesting that simvastatin could reverse this electrical remodeling, thus contributing to the ionic mechanism of statin treatment for antiarrhythmia.展开更多
Sodium intake is important to maintain proper osmolarity and volume of extracellular fluid in vertebrates. The ability to find sources of sodium ions for managing electrolyte homeostasis relies on the activity of the ...Sodium intake is important to maintain proper osmolarity and volume of extracellular fluid in vertebrates. The ability to find sources of sodium ions for managing electrolyte homeostasis relies on the activity of the taste system to sense salt. Several studies have been performed to understand the mechanisms underlying Na+ reception in taste cells, the peripheral detectors for food chemicals. It is now generally accepted that Na+ interacts with specific ion channels in taste cell membrane, called sodium receptors. As ion channels, these proteins mediate transmembrane ion fluxes (that is, electrical currents) during their operation. Thus, a lot of information on the functional properties of sodium receptors has been obtained by using electrophysiological techniques. Here, I review our current knowledge on the biophysical and physiological features of these receptors obtained by applying the patch-clamp recording techniques to single taste cells.展开更多
BACKGROUND： A combination of basic fibroblast growth factor （bFGF）, platelet-derived growth factor （PDGF）, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the ...BACKGROUND： A combination of basic fibroblast growth factor （bFGF）, platelet-derived growth factor （PDGF）, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the differentiation of rat bone marrow stromal cells into myelinating Schwann-like cells. OBJECTIVE： To investigate the inducing effects of a combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin on neural stem cell differentiation by one- and two-step methods. DESIGN, TIME AND SETTING： A cytobiology experiment was performed at the Department of Histology and Embryology, Medical School of Nantong University, and Jiangsu Province Key Laboratory of Neuroregeneration, China, between August 2005 and January 2007. MATERIALS： A total of 30 healthy Sprague Dawley rat embryos at gestational days 14-16 were selected, bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol, retinoic acid, and forskolin were purchased from Sigma, USA. METHODS： Passage 3 rat neural stem cells were cultured by a one-step method in serum-free medium plus 10 ng/m/bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1,35 ng/mL all-trans retinoic acid, and 5 pmol/L forskolin or by a two-step method in serum-free medium plus 35 ng/m/ all-trans retinoic acid for 72 hours, followed by serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-t and 5 μmol/L forskolin. The control condition consisted of 10% fetal bovine serum alone or 20 ng/mL bFGF alone. MAIN OUTCOME MEASURES： Differentiated cells were identified by immunocytochemical staining for microtubule associate protein-2 （MAP2） and St 00 protein. Geometric parameters and sodium ion currents of the differentiated cells were measured by image analysis and whole-cell patch-clamp techniques, respectively. RESULTS： Compared with the two-step culture method, neuronal-like cells exhibited longer processes and a similar appearance to mature neurons using the one-step method. The percentage of MAP2 positive cells induced by the one-step method was significantly greater than the serum-alone group （P 〈 0.05）. Furthermore, the MAP2 positive cells induced by the one-step method had greater surface areas, cell body perimeters, and longer process than cells induced by serum-alone and bFGF-alone （P 〈 0.05）. There were no significant differences in these parameters between the one-step and two-step methods （P 〉 0.05）. In addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method displayed inwardly-evoked currents. CONCLUSION： The combination of bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol retinoic acid and forskolin successfully induced neuronal differentiation from neural stem cells, with the one-step induction being more effective than the two-step method.展开更多
基金The National Natural Science Foundation of China(Grant No.32000674).
文摘Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a deeper understanding of ion channel behavior,these techniques significantly accelerate the process of drug discovery and development.In recent years,automated patch-clamp technique has undergone substantial advancements,surpassing traditional manual methods with its high throughput,improved data consistency,and automation.However,fully harnessing these advantages requires meticulous optimization of experimental conditions tailored to specific targets.Without such refinement,the high cost of consumables and operational expenses could severely hinder the widespread adoption of this technique.This study focused on the TRPV1 channel,detailing the establishment of an automated patch-clamp detection system for TRPV1 currents,optimization of experimental parameters,and a comparative evaluation of the results against manual patch-clamp techniques.
基金supported by the National Natural Science Foundation of China,Nos.32070989(to YMZ),31872766(to YMZ),81790640(to XLY),and 82070993(to SJW)the grant from Sanming Project of Medicine in Shenzhen,No.SZSM202011015(to XLY)。
文摘Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.
基金The authors acknowledge the support of the National Natural Science Foundation of ChinaProvincial Natural Science Foundation of Shanxi.
文摘We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.
文摘One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.
文摘Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.
文摘Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.
文摘Many rat taste receptor cells conduct action potentials(APs).APs had a mean threshold of -35 mV(n=95 cells)and a spike height of 52mV above threshold in current clamp(hold= -80mV).Aps could be classified into two significantly different (P<0.001) groups-fast,with short half-time durations and large outward currents (mean1.3 ms and 2.7nA),and slow,with long duration and small outward currents(mean9.2ms and 0. 29nA).AP upstrokes were conducted by TTX-sensitive sodium currents whereas the downstroke by TEA-blockable outward currents. Voltage dependent analysis of outward current separated transient and sustained components.The transient component was specifically blocked by 4-AP(1mmol/L).A calcium-dependent outward component was also revealed modulating voltage and external calcium concentration.The fast recovery phase of the AP appears related the sustained outward current whereas the after hyperpolarization(AHP) was blocked by 4AP suggesting a significant contribution of the transient component.Forskolin (FSK),which elevates cAMP,reversibly blocked the majority of the sustained current without influencing the transient. FSK greatly exaggerated the AHP without changing the spike height or duration. These data suggest that several components of the outward current contribute specifically to the gustatory AP and that the AP may be modulated by cyclic nucleotides.
基金supported by the Natural Science Foundation of Yunnan Province(No.2002C0006Z)National Natural Science Foundation of China(No.81060371)
文摘AIM: To study the effects of crebanine on voltage-gated Na+ channels in cardiac tissues. METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltage-dependent Na+ current was recorded using the whole cell voltage-clamp technique. RESULTS: Crebanine reversibly inhibited Na+ current with an IC50 value of 0.283 mmol?L-1(95% confidence range: 0.248-0.318 mmol?L-1). Crebanine at 0.262 mmol?L-1 caused a negative shift(about 12 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. CONCLUSION: In addition to blocking other voltage-gated ion channels, crebanine blocked Na+ channels in guinea-pig ventricular myocytes. Crebanine acted as an inactivation stabilizer of Na+ channels in cardiac tissues.
基金supported by the 973 Program(2011CBA00400)the National Natural Science Foundation of China(31025012)+2 种基金the Shanghai Pujiang Program(07PJ14108)the SA-SIBS Scholarship Program,and by the Hundreds of Talents Program,the Strategic Priority Research Program(XDA01020304)the Knowledge Innovation Project(KSCX2YW-R-102) from the Chinese Academy of Sciences
文摘Patch-clamp recording requires direct accessibility of the cell membrane to patch pipettes and allows the investigation of ion channel properties and functions in specific cellular compartments.The cell body and relatively thick dendrites are the most accessible compartments of a neuron,due to their large diameters and therefore great membrane surface areas.However,axons are normally inaccessible to patch pipettes because of their thin structure;thus studies of axon physiology have long been hampered by the lack of axon recording methods.Recently,a new method of patchclamp recording has been developed,enabling direct and tight-seal recording from cortical axons.These recordings are performed at the enlarged structure（axonal bleb） formed at the cut end of an axon after slicing procedures.This method has facilitated studies of the mechanisms underlying the generation and propagation of the main output signal,the action potential,and led to the finding that cortical neurons communicate not only in action potential-mediated digital mode but also in membrane potential-dependent analog mode.
基金supported by the National Natural Science Foundation of China,No.31000514the Scientific Research Project for Talent with High Education of Xinxiang Medical University,No.2007502002
文摘The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from the hippocampus of newborn rats were induced to differentiate with neurotrophic growth factor, and the electrophysiological properties of the voltage-gated potassium ion channels were observed. Immunofluorescence staining showed that the rapidly proliferating neural stem cells formed spheres in vitro that expressed high levels of nestin. The differentiated neurons were shown to express neuron-specific enolase. Flow cytometric analysis revealed that the neural stem cells were actively dividing and the percentage of cells in the S ＋ G2/M phase was high. However, the ratio of cells in the S ＋ G2/M phase decreased obviously as differentiation proceeded. Whole-cell patch-clamp re- cordings revealed apparent changes in potassium ion currents as the neurons differentiated. The potassium ion currents consisted of one transient outward potassium ion current and one delayed rectifier potassium ion current, which were blocked by 4-aminopyridine and tetraethylammonium, respectively. The experimental findings indicate that neural stem cells from newborn rat hippo- campus could be cultured and induced to differentiate into functional neurons under defined condi- tions in vitro. The differentiated neurons expressed two types of outward potassium ion cur＇ents similar to those of mature neurons in vivo.
基金supported by the National Institute on Aging (NIA)National Institutes of Health (NIH)+3 种基金Nos.K99AG065645,R00AG065645R00AG065645-04S1 (to SK)NIH research grants,NINDS,No.R01 NS115834NINDS/NIA,No.R01 NS115834-02S1 (to LG)。
文摘Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.
基金supported by grants from the Ministry of Science and Technology(2019YFA0110103)the National Natural Science Foundation of China(81870898,82071287,and 81870916)+1 种基金the Fundamental Research Funds for the Central Universities(2019FZA7009 and 2021FZZX001-37)the Zhejiang Provincial Natural Science Foundation(LR18H090002).
文摘Focal cortical dysplasia(FCD)is one of the most common causes of drug-resistant epilepsy.Dysmorphic neurons are the major histopathological feature of typeⅡFCD,but their role in seizure genesis in FCD is unclear.Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients.Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in typeⅡFCD,including an enlarged soma,aberrant dendritic arbors,increased current injection for rheobase action potential firing,and reduced action potential firing frequency.Intriguingly,the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na^(+)-K^(+)-Cl^(−)cotransporter.In addition,we found a depolarizing shift of the GABA reversal potential in the CamKⅡ-cre::PTENflox/flox mouse model of drug-resistant epilepsy,suggesting that enhanced GABAergic inputs might depolarize dysmorphic neurons.Thus,imbalance of synaptic excitation and inhibition of dysmorphic neurons may contribute to seizure genesis in typeⅡFCD.
文摘Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.
基金the Science and Technology Development Program of Jilin Province, No.20050407-6
文摘Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.
基金This research project was supported by Mahidol University,Thailand.We also thank the Faculty of Medicine Siriraj Hospital,Mahidol University,for additional financial support to K.R.and W.B.W。
文摘Objective To investigate the effect of ferulic acid,a natural compound,on pancreatic beta cell viability,Ca^(2+)channels,and insulin secretion.Methods We studied the effects of ferulic acid on rat insulinoma cell line viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay.The whole-cell patch-clamp technique and enzyme-linked immunosorbent assay were also used to examine the action of ferulic acid on Ca^(2+)channels and insulin secretion,respectively.Results Ferulic acid did not affect cell viability during exposures up to 72 h.The electrophysiological study demonstrated that ferulic acid rapidly and concentration-dependently increased L-type Ca^(2+)channel current,shifting its activation curve in the hyperpolarizing direction with a decreased slope factor,while the voltage dependence of inactivation was not affected.On the other hand,ferulic acid have no effect on T-type Ca^(2+)channels.Furthermore,ferulic acid significantly increased insulin secretion,an effect inhibited by nifedipine and Ca^(2+)-free extracellular fluid,confirming that ferulic acid-induced insulin secretion in these cells was mediated by augmenting Ca^(2+)influx through L-type Ca^(2+)channel.Our data also suggest that this may be a direct,nongenomic action.Conclusion This is the first electrophysiological demonstration that acute ferulic acid treatment could increase L-type Ca^(2+)channel current in pancreaticβcells by enhancing its voltage dependence of activation,leading to insulin secretion.
基金Supported by National Natural Science Foundation of China(No. 60674111)
文摘Acutely isolated mouse hippocampal CA3 pyramidal neurons were exposed to 3 mT static magnetic field,and the characteristics of transient outward K+ channel were studied using the whole-cell patch-clamp technique.The experiment revealed that the amplitude of transient outward potassium channel current was reduced.The maximum activated current densities of control group and exposure group were 163.62±20.68 pA/pF and 98.74±16.57 pA/pF(n=12,P<0.01) respectively.The static magnetic field exposure affected the activation and inactivation process of transient outward potassium channel current.Due to the magnetic field exposure,the half-activation voltage of the activation curves changed from 5.59±1.96 mV to 27.87±7.24 mV(n=12,P<0.05) ,and the slope factor changed from 19.43±2.11 mV to 25.87±4.22 mV(n=12,P<0.05) .The half-inactivation voltage of the inactivation curves also changed from-56.09±0.89 mV to-57.16±1.10 mV(n=12,P>0.05) and the slope factor of the inactivation curves from 8.69±0.80 mV to 10.87±1.02 mV(n=12,P<0.05) .The results show that the static magnetic field can change the characteristics of transient outward K+ channel,and affect the physiological functions of neurons.
文摘Objectives To investigate the effects of simvastatin on membrane ionic currents in left ventricular myocytes of rabbit heart suffering from acute myocardial infarction （ AMI）, so as to explore the ionic mechanism of statin treatment for antiarrhythmia. Methods Forty-five New Zealand rabbits were randomly divided into three groups： AMI group, simvastatin intervention group （ Statin group） and sham-operated control group （CON）. Rabbits were infarcted by ligation of the left anterior descending coronary artery after administration of oral simvastatin 5 mg · kg^-1·d^-1 （Statin group） or placebo （AMI group） for 3 days. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region 72 h later. Whole cell patch clamp technique was used to record membrane ionic currents, including sodium current （INa）, L-type calcium current （Ica-L） and transient outward potassium current （Ito）. Results （1） There was not significant difference in serum cholesterol concentration among three groups. （2） The peak INa current density （at -30 mV） was significantly decreased in AMI group （ -25.26±5.28, n = 13 ）, comparing with CON （ - 42. 78± 5.48, n = 16）, P 〈 0. 05, while it was significantly increased in Statin group （ - 39.83 ±5.65 pA/pF, n = 12） comparing with AMI group, P 〈0. 01 ; The peak Ica-L current density （ at 0 mV） was significantly decreased in AMI group （ -3. 43 ±0. 92 pA/pF, n = 13） comparing with CON （ -4. 56 ±1.01 pA/pF, n = 15）, P 〈0. 05, while it was significantly increased in Statin group （ -4. 18±0. 96 pA/pF, n = 12） comparing with AMI group, P 〈0. 05; The Ito current density （ at ＋ 60 mV） was significantly decreased in AMI group （ 11.41 ± 1.94 pA/pF, n = 13 ） comparing with CON （17.41 ±3.13 pA/pF, n = 15）, P 〈0. 01, while it was significantly increased in Statin group （16. 11 ± 2. 43 pA/pF, n = 14） comparing with AMI group, P 〈 0. 01. Conclusions AMI induces significant down-regulation of INa, Ica-L and Ito. Pretreatment with simvastatin could attenuate this change without lowering the serum cholesterol level, suggesting that simvastatin could reverse this electrical remodeling, thus contributing to the ionic mechanism of statin treatment for antiarrhythmia.
文摘Sodium intake is important to maintain proper osmolarity and volume of extracellular fluid in vertebrates. The ability to find sources of sodium ions for managing electrolyte homeostasis relies on the activity of the taste system to sense salt. Several studies have been performed to understand the mechanisms underlying Na+ reception in taste cells, the peripheral detectors for food chemicals. It is now generally accepted that Na+ interacts with specific ion channels in taste cell membrane, called sodium receptors. As ion channels, these proteins mediate transmembrane ion fluxes (that is, electrical currents) during their operation. Thus, a lot of information on the functional properties of sodium receptors has been obtained by using electrophysiological techniques. Here, I review our current knowledge on the biophysical and physiological features of these receptors obtained by applying the patch-clamp recording techniques to single taste cells.
基金the National Natural Science Foundation of China,No.30870643Natural Science Foundation of Jiangsu Province,No. BK2002036
文摘BACKGROUND： A combination of basic fibroblast growth factor （bFGF）, platelet-derived growth factor （PDGF）, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the differentiation of rat bone marrow stromal cells into myelinating Schwann-like cells. OBJECTIVE： To investigate the inducing effects of a combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin on neural stem cell differentiation by one- and two-step methods. DESIGN, TIME AND SETTING： A cytobiology experiment was performed at the Department of Histology and Embryology, Medical School of Nantong University, and Jiangsu Province Key Laboratory of Neuroregeneration, China, between August 2005 and January 2007. MATERIALS： A total of 30 healthy Sprague Dawley rat embryos at gestational days 14-16 were selected, bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol, retinoic acid, and forskolin were purchased from Sigma, USA. METHODS： Passage 3 rat neural stem cells were cultured by a one-step method in serum-free medium plus 10 ng/m/bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1,35 ng/mL all-trans retinoic acid, and 5 pmol/L forskolin or by a two-step method in serum-free medium plus 35 ng/m/ all-trans retinoic acid for 72 hours, followed by serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-t and 5 μmol/L forskolin. The control condition consisted of 10% fetal bovine serum alone or 20 ng/mL bFGF alone. MAIN OUTCOME MEASURES： Differentiated cells were identified by immunocytochemical staining for microtubule associate protein-2 （MAP2） and St 00 protein. Geometric parameters and sodium ion currents of the differentiated cells were measured by image analysis and whole-cell patch-clamp techniques, respectively. RESULTS： Compared with the two-step culture method, neuronal-like cells exhibited longer processes and a similar appearance to mature neurons using the one-step method. The percentage of MAP2 positive cells induced by the one-step method was significantly greater than the serum-alone group （P 〈 0.05）. Furthermore, the MAP2 positive cells induced by the one-step method had greater surface areas, cell body perimeters, and longer process than cells induced by serum-alone and bFGF-alone （P 〈 0.05）. There were no significant differences in these parameters between the one-step and two-step methods （P 〉 0.05）. In addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method displayed inwardly-evoked currents. CONCLUSION： The combination of bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol retinoic acid and forskolin successfully induced neuronal differentiation from neural stem cells, with the one-step induction being more effective than the two-step method.
