Inhibitory Effects of Neferine on Na_v1.5 Channels Expressed in HEK293 Cells

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.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

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.

The Effect of Coriaria Lactone on NMDA Receptor Mediated Currents in Rat Hippocampal CA1 Neurons

Summary: To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neu- rons was investigated by using nystatin perforated whole-cell patch clamp. 10-6-10-4 mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10-6 mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.

Induced neuronal differentiation of neural stem cells by a combination of cytokines One-step versus two-step methods

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.

Rapid effect of stress concentration corticosterone on glutamate receptor and its subtype NMDA receptor activity in cultured hippocampal neurons

Objective: To study the rapid effect of glucocorticoids (GCs) on NMDA receptor activity in hippocampal neurons in stress and to elucidate its underlying probable membrane mechanisms. Methods: Whole-cell patch-clamp recording was used to assess the effect of stress concentration corticosterone (B) on the responses of cultured hippocampal neurons to glutamate and NMDA (N-methy-D-asparatic acid). To make clear the target of B, intracellular dialysis of B(10 μmol/L)through patch pipette and extracellular application of bovine serum albumin-conjugated corticosterone(B-BSA, 10 μmol/L)were carried out to observe their influence on peak amplitude of NMDA-evoked current. Results: B had a rapid, reversible and inhibitory effect on peak amplitude of GLU- or NMDA-evoked current in cultured hippocampal neurons. Furthermore, B-BSA had the inhibitory effect on INMDA as that of B, but intracellularly dialyzed B had no significant effect on I NMDA. Conclusion: These results suggest that under the condition of stress, GCs may rapidly, negatively regulate excitatory synaptic receptors-glutamate receptors (GluRs), especially NMDA receptor (NMDAR) in central nervous system, which is mediated by rapid membrane mechanisms, but not by classical, genomic mechanisms.

Protein Kinase C Enhances the Swelling-Induced Chloride Current in Human Atrial Myocytes

Swelling-activated chloride currents（ICl.swell） are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C（PKC） in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate（PDBu） enhanced ICl.swellin a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Study of transmembrane La^(3+) movement in rat ventricular myocytes by the patch-clamp technique

We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmo1/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currents increase with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.

Cross-talk between NMDA and GABA_A receptors in cultured neurons of the rat inferior colliculus

Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels,a process called cross-talk.The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABAA receptor) are important excitatory and inhibitory receptors in the central nervous system,respectively.Currently,cross-talk between the NMDA receptor and the GABAA receptor,particularly in the central auditory system,is not well understood.In the present study,we investigated functional interactions between the NMDA receptor and the GABAA receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus,which is an important nucleus in the central auditory system.We found that the currents induced by aspartate at 100 μmol L-1 were suppressed by the pre-perfusion of GABA at 100 μmol L-1,indicating cross-inhibition of NMDA receptors by activation of GABAA receptors.Moreover,we found that the currents induced by GABA at 100 μmol L-1 (IGABA) were not suppressed by the pre-perfusion of 100 μmol L-1 aspartate,but those induced by GABA at 3 μmol L-1 were suppressed,indicating concentration-dependent cross-inhibition of GABAA receptors by activation of NMDA receptors.In addition,inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca2+ channels with CdCl2 in a solution that contained Ca2+,however,CdCl2 effectively attenuated the inhibition of IGABA by aspartate when it was perfused in a solution that contained Ba2+ instead of Ca2+ or a solution that contained Ca2+ and 10 mmol L-1 BAPTA,a membrane-permeable Ca2+ chelator,suggesting that this inhibition is mediated by Ca2+ influx through NMDA receptors,rather than voltage-dependent Ca2+ channels.Finally,KN-62,a potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII),reduced the inhibition of IGABA by aspartate,indicating the involvement of CaMKII in this cross-inhibition.Our study demonstrates a functional interaction between NMDA and GABAA receptors in the inferior colliculus of rats.The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.

Effects of La^(3+) on inward K^+ channels at plasma membr in guard cells

The effects of La3+ on inward K+ channels at plasma membrane in vicia guard cells are investigated using the whole-cell patch-clamp recording mode. It is shown that La3+ on both sides of plasma membrane blocks inward K+ currents in a concentration- dependent manner, indicating that La3+ binding sites may exist on both sides of plasma membrane in guard cells in vicia. The dose response is fitted by the Michaelis-Menten relation characterized by an inhibitor constant Ki of 2.56±0.25 μmol·L?1 (outside membrane) and (1.18±0.11)×10?15 mol·L?1 (inside membrane). Intracellular La3+ has much stronger inhibitory effect on inward K+ currents than ex- tracellular La3+ does, suggesting there may exist stronger binding sites inside membrane than outside membrane. Since ion channel activities of guard cells directly affect plant stomatal movement and water status, our results imply that rare earth elements might have potential practical values in regulating plant water status and strengthening plant drought endurance.

Pathological Networks Involving Dysmorphic Neurons in Type ⅡFocal Cortical Dysplasia

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.

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents(STOCs)in freshly isolated porcine coronary artery smooth muscle cells(ASMCs).STOCs were recorded using the perforated whole-cell patch-clamp configuration.STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca^(2+)-activated-K+(BKCa)currents.Charybdotoxin(ChTX,200 nmol/L),a selective blocker of BKCa channels,completely inhibited STOCs within 10 min.STOCs activity was greatly suppressed when extracellular Ca^(2+)concentration decreased from 1.8 mmol/L to 200 nmol/L,further removal of Ca^(2+)abolished STOCs activity.Ca^(2+)ionophore A23187(10μmol/L)increased STOCs activity significantly.Verapamil(20μmol/L)and CdCl2(200μmol/L),two kinds of organic L-type voltage-dependent Ca^(2+)channels(L-VDCCs)antagonists,had little effect on STOCs.In addition,the ryanodine receptors(RyRs)agonist caffeine(5 mmol/L)significantly activated STOCs.Application of ryanodine(50μmol/L)to block RyRs abolished STOCs,subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity.Inhibition of inositol 1,4,5-trisphosphate receptors(IP_(3)Rs)by 2APB(40μmol/L)greatly suppressed the activity of STOCs,application of caffeine(5 mmol/L)in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs.These results suggest that STOCs in porcine coronary ASMCs are mediated by BKCa channels.Extracellular Ca^(2+)is essential for STOCs activity,while Ca^(2+)entry through L-VDCCs has little effect on STOCs.Intracellular Ca^(2+)release induced by RyRs is responsible for the regulation of STOCs,whereas IP_(3)Rs might also be involved.

Pertussis toxin modulation of sodium channels in the central neurons of cyhalothrin-resistant and cyhalothrinsusceptible cotton bollworm, Helicoverpa armigera

Pertussis toxin （FIX） inhibits the activation of the α-subunit of the inhibitory heterotrimeric G-proteins （Cαi/o） and modulates voltage-gated sodium channels, which may be one of the primary targets of pyrethroids. To investigate the potential mechanisms of agricultural pests resistance to pyrethroid insecticides, we examined the modulations by PTX on sodium channels in the central neurons of the 3rd-4th instar larvae of cyhalothrin-resistant （Cy-R） and cyhaiothrin-susceptible （Cy-S） Helicoverpa armigera by the whole-cell patch-clamp technique. The isolated neurons were cultured for 12-16 h in an improved L15 insect culture medium with or without PTX （400 ng/mL）. The results showed that both the Cy-R and Cy-S sodium channels exhibited fast kinetics and tetrodotoxin （TTX） sensitivity. The Cy-R sodium channels exhibited not only altered gating properties, including a 8.88-mV right shift in voltage-dependent activation （V0.5act） and a 6.54-mV right shift in voltage-dependent inactivation （V0.5inact）, but also a reduced peak in sodium channel density （Ⅰdensity） （55.2% of that in Cy-S neurons）. Cy-R sodium channels also showed low excitability, as evidenced by right shift of activation potential （Ⅴacti） by 5-10 mV and peak potential （Ⅴpcak） by 20 mV. FIX exerted significant effects on Cy-S sodium channels, reducing sodium channel density by 70.04%, right shifting V0.5act by 14.41 mV and V0.5inact by 9. 38 mV. It did not cause any significant changes of the parameters mentioned above in the Cy-R sodium channels. The activation time （Tpeak） from latency to peak at peak voltage and the fast inactivation time constant （τinact） in both Cy-S and Cy-R neurons were not affected. The results suggest that cotton bollworm resistant to pyrethroid insecticides involves not only mutations and allosteric alterations of voltage-gated sodium channels, but also might implicate perturbation of PTX-sensitive Gαi/o-COupled signaling Wansduction pathways.

Spontaneous firing properties of rat medial vestibular nucleus neurons in brain slices by infrared visual patch clamp technique

Domestic application of infrared patch clamp techniques on brain slices is limited.The key of the tech-nique is to prepare high-quality brain slices.The present paper describes the preparation procedure of brainstem slices and the spontaneous firing properties of rat medial vestibular nucleus(MVN)neurons.By infrared differ-ential interference contrast technique,neurons of rat MVN were visualized directly at the depth of 50–100 mm underneath the surface of slices.Firing activities of MVN neurons were recorded by the whole-cell patch clamp technique in artificial cerebrospinal fluid(ACSF)and low Ca^(2+)-high Mg^(2+) fluid.The firing mode was more irregular and depressive in low Ca^(2+)-high Mg2+fluid than in ACSF.According to the averaged waveform of action potentials,cells were classified as the neurons with mono-phasic after-hyperpolarization potential(AHP),and the neurons with biphasic AHP.The resting membrane potential(RMP),input resistance(Rin)and membrane capacitance(Cm)of neurons were recorded and com-pared between groups.With infrared videomicroscopy,patch clamp recordings could be made under direct obser-vation in freshly prepared brainstem slices.The discharge activities of MVN neurons were spontaneous and the fir-ing mode was modulated by extracellular calcium concen-tration.The basic membrane properties of two types of neurons were not significantly different,while the differ-ences in waveform might play a role in the segregation between tonic and kinetic cells.