Search

Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca2+ and Na+ channels were analyzed by electrophysiological techniques (using Ba2+ as charge carrier through Ca2+ channels) and by binding studies with specific ligands. With NGF, Na+ current ( I Na) density increased in parallel with neurite extension. Ba2+ current ( I Ba) density and Ca2+ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I Na density. With forskolin, neurite outgrowth was linked to an apparent increase in I Ba density similar to the one induced by NGF, while no change in I Na was observed. Dihydropyridine-sensitive (L-type) as well as ω-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I Ba, binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca2+ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca2+ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics. [ABSTRACT FROM AUTHOR]

The effects of the potassium channel openers (KCO), cromakalim or pinacidil, were evaluated in an anesthetized porcine model of pacing- and ischemia-induced ventricular fibrillation (VF). Hearts were paced at 180 bpm and the left anterior descending coronary artery was occluded until VF was induced. Reproducible times to VF (in seconds) were obtained allowing at least 20 min recovery following defibrillation. Cromakalim (0.3 mg/kg) or pinacidil (3 mg/kg) produced equivalent drops in mean arterial blood pressure. At these doses, cromakalim reduced monophasic action potential duration measured at 90% repolarization (APD). Although time to VF in the cromakalim group was significantly greater than the vehicle treated group, it was not significantly different from its predrug value. In contrast, pinacidil reduced APD, and significantly increased time to VF from 134±5 to 322±62 s ( p<0.05). Neither cromakalim nor pinacidil affected whole-cell calcium currents recorded in guinea pig myocytes. During ischemia, cromakalim or pinacidil further reduced APD; however, pinacidil had a two-fold greater effect than did cromakalim. The Class III antiarrhythmic agent, dofetilide, prolonged APD, but did not increase time to VF. In conclusion, the increased time to VF observed with pinacidil coincides with its ability to shorten APD, and is consistent with activation of ATP-sensitive K channels (K). It is suggested that indirect reduction of calcium influx through K activation and APD shortening is sufficient to increase time to VF in this model. However, the inability of dofetilide to be effective suggests that this model would not be useful to test for Class III antiarrhythmic agents. [ABSTRACT FROM AUTHOR]

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel. [ABSTRACT FROM AUTHOR]

Introduction: Steady-State Na/K pump current (Ip) in adult guinea pig ventricular myocytes was studied to determine the effect on the Na/K pump of transmembrane Na leak, membrane potential, and pipette Na concentration. Methods and Results: Using conventional whole cell, patch clamp techniques, Ip was identified as either Ko-sensitive or ouabain-sensitive current when most other membrane currents were inhibited. Control experiments showed that there were no Kp-sensitive currents other than Ip under the conditions of our experiments. Ip was found to be similar to that reported by others being voltage dependent between -130 and 0 mV and having a half maximal activation by Nai of 28 mM. Ouabain sensitivity was also measured, and it was found that there were two binding sites with the high affinity site comprising 5% to 10% of the total and having an apparent affinity 1000-fold higher than the low affinity site. Apparent affinity of both sites was shifted about 10-fold (higher affinity) by increasing Na; from 10 to 85 mM. When internally perfused with 0 Na solution, Na leak through the membrane was found to be linearly related to Na/K pump activity. In contrast to prior suggestions, Ip was not correlated with series resistance when there was a large transmembrane Na gradient. Conclusion: These data suggest that, under conditions of high transmembrane Na gradient, Na leak through the membrane plays a significant role in determining Na/K pump activity. [ABSTRACT FROM AUTHOR]

Three synthetic peptides corresponding to parts of S of the first repeat of eel electroplax sodium channel were synthesized. The basic peptide was Cwhich corresponds to amino acids 210-223 (eel channel numbering) and two subfractions: an external fraction, C(amino acid 210-217); and an internal part, C(amino acid 218-221). Peptide Cincludes four of the charged amino acids of this domain; peptide Cincludes three of the charged amino acids and is closer to the external membrane surface (according to channel models) than peptide Cwhich includes the fourth charged amino acid alone. Antibodies generated in rabbits against these peptides were shown to be site specific. Using the whole-cell patch-clamp technique, we found that in rat dorsal root ganglion (DRG) cells, the antibodies against Cbut not against Chad an effect on the gating parameters. They shifted the Na-channel inactivation curve towards hyperpolarization and decreased the slope of the Na-channel activation curve. These results demonstrate that during the conformational changes associated with channel gating, the fourth charged amino acid of S must be accessible to antibodies given to the external solution. Furthermore, they indicate a specific involvement of S in the voltage dependency of the gating processes. This study was supported by a basic research grant of The Israel Academy of Sciences and Humanities (#430.87 to H.M. and G.S.). [ABSTRACT FROM AUTHOR]

The effects of toxin II ( AaH II) isolated from the scorpion Androtonus australis Hector on sodium current in neuroblastoma X glioma NG 108-15 hybrid cells were analysed under patch clamp conditions in the whole cell configuration. AaH II (70 n M)_induced a maintained sodium current, as well as increasing both fast and slow inactivation time constants and the amplitude of the peak current. This latter effect occurred via a shift of the activation-voltage curve towards negative voltage values by about 9 mV. Oleic acid (5 μ M), which had no effect on I under control conditions, decreased the AaH II-induced maintained current. It also reversed, or prevented the increase of the peak current induced by AaH II. However, it neither prevented nor modified the AaH II-induced increase in inactivation time constants. The binding of the toxin to its specific site and the number of binding sites for AaH II were not significantly modified by oleic acid. The oleic acid-induced effects could not be related to the activation of protein kinase C since PMA, a potent activator of this enzyme, did not produce oleic acid-like effects. From these results, it is concluded that AaH II has several independent effects on sodium channels, some of which could be modulated by the lipid environment of sodium channels in the membrane. [ABSTRACT FROM AUTHOR]

• The carotid body's glomus cells are the primary sensors of hypoxia in mammals. • Lactate is a potential contributing mechanism for the hypoxia sensitivity of glomus cells from mice. • Lactate did not affect the outward currents, membrane conductance, and membrane potential of Wistar rats' glomus cells. • Lactate applied into carotid body did not activate the chemoreceptive petrosal neurons of in situ preparation of Wistar rats. • Carotid body of Wistar rat is not sensitive to lactate. The carotid body's glomus cells are the primary sensors of hypoxia in mammals. Previous studies suggested that the glomus cells' hypoxia sensitivity is mediated by lactate in mice. This molecule increases the intracellular [Ca2+] and induces exocytosis in glomus cells, activating the carotid sinus nerve (the axons of chemoreceptive petrosal neurons). On the other hand, how lactate affects the activity of carotid body of rats is still unknown. We hypothesized that lactate activates the carotid body of rats. In Wistar rats, we measured the changes in the electrical properties of isolated glomus cells and petrosal chemoreceptive neurons in in situ preparations in response to different concentrations of lactate. Superfusion of both physiological and supraphysiological concentrations of lactate did not affect the membrane conductance and potential of glomus cells. Moreover, lactate injected into the carotid body did not activate the anatomically and physiologically identified chemoreceptive petrosal neurons. We conclude that the carotid body of Wistar rats is not sensitive to lactate. [ABSTRACT FROM AUTHOR]

Long-term consequences of stroke significantly impair the quality of life in a growing population of stroke survivors. Hippocampal adult neurogenesis has been hypothesized to play a role in the pathophysiology of cognitive and neuropsychiatric long-term sequelae of stroke. Reliable animal models of stroke are paramount to understanding their biomechanisms and to advancing therapeutic strategies. We present a detailed protocol of a transient cerebral ischemia model which does not cause direct ischemic damage in the hippocampus, allowing investigations into the pathophysiology of long-term neurocognitive deficits of stroke. Furthermore, we describe a protocol for obtaining acute hippocampal slices for the purpose of electrophysiological and morphological characterization of adult-borne granule cells. Particularities relating to performing electrophysiological recordings from small cells, such as immature adult-borne granule cells, are also discussed. The present protocol may be complemented by multi-modal investigations (behavioral, morpho-structural, biochemical), to hopefully facilitate research and advances into the long-term sequelae of stroke and the discovery of new therapeutic opportunities., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

The transient outward current was studied, using the whole-cell patch-clamp technique, in isolated ventricular cells from the ferret heart. In the presence of 4-aminopyridine and cadmium chloride which respectively blocked the Ca-insensitive and the Ca-dependent outward currents, a residual transient outward current was observed in about 30% of the cells tested. This current was suppressed in external hypochloride solution, completely inhibited by SITS (3mM) and reversed at the equilibrium potential for chloride ions. This suggests the presence of a chloride permeability which could contribute to the repolarization phase of the cardiac action potential. [ABSTRACT FROM AUTHOR]

Background: High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line.Methods: We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells.Results: The GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways.Conclusions: The GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells. [ABSTRACT FROM AUTHOR]

Objective: Motor neuron differentiation from human pluripotent stem cells (hPSC) is a goal in regenerative medicine for cell therapy in diseases that loss these cells. Most protocols have low efficiency in generate functional motoneurons, therefor small molecules are new idea for dominance this challenge. Materials and Methods: Under this approach we tried to use an efficient, short time and cost effective protocol than reported protocols to generate functional motoneurons from hPSC. Therefor we used small molecules Dorsomorphin, A8301, XAV, Retinoic acid and Purmorphamine in this protocol. Results: Immunofleoroscent staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) showed these structures are 92.68% NESTIN+, 64.40% PAX6+, 82.11%% SOX1+ in a chemically defined adherent culture. After replating of hESC-derived NE, the differentiated cells are Tuj1+, MAP2+, HB9+ and ISL1+.level of gene expression evaluated with real time RTPCR in different stages of differentiation protocol. Electrophysiological properties of differentiated cells recorded by whole cell Patch clamp technique. hESC_drived motor neurons displayed voltage gated delay rectifier K+ and Na+-Ca2+ inward currents. Conclusion: Our findings suggested that, hESC-derived neurons expressed special motor neuron proteins such as HB9 and ISL1 but electrophysiological properties showed that these cells were not mature because the resting membrane potential was more positive than mature motor neuron and Na+-Ca2+ inward currents were not sufficient for firing action potentials. It seems that the cells should be more developed for functional maturation. [ABSTRACT FROM AUTHOR]

Lipid rafts, specialized membrane microdomains in the plasma membrane rich in cholesterol and sphingolipids, are hot spots for a number of important cellular processes. The novel nicotinic acetylcholine receptor (nAChR) mutation αC418W, the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome was shown to be cholesterol-sensitive and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1. The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP), quantitative RT-PCR, and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells to assess the role of cholesterol and caveolin-1 (CAV-1) in the diffusion, expression, and functionality of the nAChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in specialized membrane microdomains that upon cholesterol depletion become disrupted and release the cholesterol-sensitive nAChRs to the pool of activable receptors. In addition, our results in HEK 293 cells show an interdependence between CAV-1 and αC418W that could confer end plates rich in αC418W nAChRs to a susceptibility to changes in cholesterol levels that could cause adverse drug reactions to cholesterol-lowering drugs such as statins. The current work suggests that the interplay between cholesterol and CAV-1 provides the molecular basis for modulating the function and dynamics of the cholesterol-sensitive αC418W nAChR., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

Abstract It is generally accepted that neurons in the ventral spinal grey matter, a substantial proportion of which can be regarded as constituents of the spinal motor apparatus, receive and integrate synaptic inputs arising from various peripheral, spinal and supraspinal sources. Thus, a profound knowledge concerning the integrative properties of interneurons in the spinal ventral grey matter appears to be essential for a fair understanding of operational principles of spinal motor neural assemblies. Using the whole cell patch clamp configuration in a correlative physiological and morphological experimental approach, here we demonstrate that the intrinsic membrane properties of neurons vary widely in laminae V–VII of the ventral grey matter of the neonatal rat lumbar spinal cord. Based on their firing patterns in response to depolarizing current steps, we have classified the recorded neurons into four categories: ‘phasic’, ‘repetitive’, ‘single’ and ‘slow’. Neurons with firing properties characteristic of the ‘phasic’, ‘repetitive’ and ‘single’ cells have previously been reported also in the superficial and deep spinal dorsal horn, but this is the first account in the literature in which ‘slow’ neurons have been recovered and described in the spinal cord. The physiological heterogeneity in conjunction with the morphological correlation and distribution of neurons argues that different components of motor neural assemblies in the spinal ventral grey matter possess different signal processing characteristics. [ABSTRACT FROM AUTHOR]

Aim: To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells., Methods: We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C., Results: About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed., Conclusion: We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.