Action potential-like responses in B 104 cells with low Na channel densities

Action potential generation and Na⁺ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to −55 mV for depolarizing stimuli, and −56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. −75 mV, close to theV_1/2 for Na⁺ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na⁺ current density of 133 ± 0.14 pA/pF (range, 10.2–296.2 pA/pF) and a lowg_K: g_Na ratio (0.0067 ± 0.0023). Exposure to 0.1 mM Cd²⁺ did not block the generation of action potential-like responses in B104 cells, while 1 μM TTX abolished the responses. We conclude that low densities of Na⁺ channels ( < 3/μm² and < 1/μm² in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K⁺ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.     

Morphine and methadone have different effects on calcium channel currents in neuroblastoma cells

Using patch-clamp technique, cellular calcium channel currents were studied on nine mouse neuroblastoma N1E115 cells. Both morphine and methadone decreased the T-type calcium currents in dose-dependent fashion. These effects were partially blocked by naloxane. On L-type calcium currents, morphine showed no effect. However, methadone inhibited the L-calcium currents in dose-dependent fashion. This effect was not antagonised by naloxone. Hence, the action of methadone on L-calcium channel is probably not associated with μ receptors.     

Changes in Na(+) channel expression and nodal persistent Na(+) currents associated with peripheral nerve regeneration in mice

Patients with peripheral neuropathy frequently suffer from positive sensory (pain and paresthesias) and motor (muscle cramping) symptoms even in the recovery phase of the disease. To investigate the pathophysiology of increased axonal excitability in peripheral nerve regeneration, we assessed the temporal and spatial expression of voltage-gated Na(+) channels as well as nodal persistent Na(+) currents in a mouse model of Wallerian degeneration. Crushed sciatic nerves of 8-week-old C57/BL6J male mice underwent complete Wallerian degeneration at 1 week. Two weeks after crush, there was a prominent increase in the number of Na(+) channel clusters per unit area, and binary or broad Na(+) channel clusters were frequently found. Excess Na(+) channel clusters were retained up to 20 weeks post-injury. Excitability testing using latent addition suggested that nodal persistent Na(+) currents markedly increased beginning at week 3, and remained through week 10. These results suggest that axonal regeneration is associated with persistently increased axonal excitability resulting from increases in the number and conductance of Na(+) channels.     

T-type calcium channel expression in cultured human neuroblastoma cells

Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions.Previous studies have revealed L-and N-type calcium channels in SH-SY5Y cells.However,the distribution of the low-voltage activated calcium channel (namely called T-type calcium channel,including Cav3.1,Cav3.2,and Cav3.3) in SH-SY5Y cells remains poorly understood.The present study detected mRNA and protein expres-sion of the T-type calcium...     

T型钙通道在人类神经母细胞瘤细胞上的表达

Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions.Previous studies have revealed L-and N-type calcium channels in SH-SY5Y cells.However,the distribution of the low-voltage activated calcium channel (namely called T-type calcium channel,including Cav3.1,Cav3.2,and Cav3.3) in SH-SY5Y cells remains poorly understood.The present study detected mRNA and protein expres-sion of the T-type calcium channel (Cav3.1,Cav3.2,and Cav3.3) in cultured SH-SY5Y cells using real-time polymerase chain reaction (PCR) and western blot analysis.Results revealed mRNA and protein expression from all three T-type calcium channel subtypes in SH-SY5Y cells.Moreover,Cav3.1 was the predominant T-type calcium channel subtype in SH-SY5Y cells.     

Dependence of axon initial segment formation on Na+ channel expression

Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results.     

Spectrin expression in neuroblastoma cells

Mouse neuroblastoma cells express a spectrin-related molecule containing 240 kDal (kiloDalton) and 235 kDal subunits in a 1:1 ratio. The 240 kDal and 235 kDal subunits are nearly identical to the alpha and beta subunits respectively of brain spectrin by two dimensional chymotryptic peptide mapping analysis. The neuroblastoma cells do not express a measureable quantity of a red blood cell (rbc)-type spectrin molecule. Neuroblastoma spectrin has been localized throughout the cell body, and neurites of these cells by indirect immunofluorescence studies. As neuroblastoma cells are homogeneous, neuron-like, available in large quantity, and synthesize a single variant of spectrin which is closely related to brain spectrin(240/235), it is the best available model system for the study of the synthesis, assembly and turnover of a neuronal spectrin subtype.     

Acetylcholine-activated currents in mouse neuroblastoma cells

The nicotinic and muscarinic responses of differentiated mouse neuroblastoma cells from the clonal line N1E 115 to applied cholinergic agents were recorded using single channel and whole cell patch clamp techniques. An inward macroscopic current induced by acetylcholine (ACh) at the resting potential was blocked by curare; cell-attached recordings revealed a single channel conductance of 18 pS and a lifetime of 36 ms at 30°C, with 200 nM ACh. The zero current potential was close to 0 mV. The kinetics of these nicotinic currents were described by multiexponential functions for both the open and closed time distributions. An outward single channel current, present at resting and slightly depolarized potentials, was also observed and has been tentatively described as being dependent on muscarinic receptor activation, as it was usually blocked by atropine. Under our conditions of whole cell clamp, no macroscopic outward current sensitive to ACh was observed.     

Release of endogenous brain epinephrine by the calcium ionophores X537A and A23187

The Ca²⁺ ionophores X537A and A23187 produced dose-dependent release of endogenous epinephrine (Epi) as well as norepinephrine (NE) and dopamine (DA) from chopped rat hypothalamus and brainstem. The X537A-induced release of these catecholamines (CAs) was found to be Ca²⁺-independent, whereas the A23187-induced release was Ca²⁺-dependent. X537A and A23187 were approximately equipotent in causing the release of the hypothalamic Epi, NE and DA, but X537A was much more effective than A23187 in causing the maximal release. X537A, but not A23187, reduced the total endogenous CA content and increased the total 3,4-dihydroxyphenylacetic acid (DOPAC) content in the chopped hypothalamus. Ca²⁺-independent release of Epi, NE and DA in the chopped hypothalamus was also observed with indirectly acting sympathomimetic amines (e.g. tyramine and amphetamine). Tyramine and amphetamine did not affect the total endogenous CA contents nor the total DOPAC content. These results suggest that X537A caused release of endogenous brain Epi, NE and DA by transporting thebiogenic amines across vesicular or intracellular storage sites. However, A23187 caused release of these CAs by exocytosis via transport of Ca²⁺ into the neurons.     

A 23187-stimulated calcium uptake and GABA release by cerebrocortical synaptosomes: effects of high pressure

Summary Guinea pig cerebrocortical synaptosome preparations were used to study the effect of compression to 62 ATA on⁴⁵Ca²⁺ uptake and [³H]GABA release using a calcium ionophore A23187, which bypasses the voltage-sensitive calcium channel. Pressure was found to exert a suppressive effect on the A 23187-induced release of [³H]GABA, while having no significant effect on A 23187-stimulated⁴⁵Ca²⁺ uptake. On the other hand, both depolarization-induced⁴⁵Ca²⁺ uptake and [³H]GABA release were inhibited by pressure exposure. These results suggest that pressure may suppress GABA release by affecting pre-synaptic events subsequent to calcium influx.     

Neurogenic origin of an experimental myopathy induced by the ionophore A23187

An experimental myopathy was induced in frog skeletal muscle incubated in vitro with the calcium ionophore A23187. This myopathy could be prevented if Ca2+ ions in the incubating medium were replaced by either Mg2+ or Co2+ ions. Similarly, preincubation of the preparation with acetylcholine (ACh) receptor blockers such as d-tubocurarine (d-TC) or alpha-bungarotoxin (alpha-BuTX) prevented the development of muscle damage. In muscles that had been previously denervated or treated with botulinum toxin (BTX) the ionophore failed to induce morphological alterations. These results suggest that spontaneous transmitter release, greatly increased by the ionophore A23187, triggers Ca2+ influx into the muscle fibres at the endplate region. Elevated levels of intracellular Ca2+ presumably activate proteolytic systems leading to myofilament disassembly.     

Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve

Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than-50 mV and reached a maximum value at about-10 mV, while no rectifying channel was present. The inward current was carried by Na⁺ ions, because the extrapolated reversal potential of the current agreed with the calculated E_Na, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was-82 mV. The inactivation curve indicated that more than 90% of the Na⁺ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13°C). The electrophysiological characteristics of the Na⁺ current in the cultured frog Schwann cells were compared with those in other tissues. This Na⁺ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course. © 1994 Wiley-Liss, Inc.     

Opioids potentiate transmitter release from SK-N-SH human neuroblastoma cells by modulating N-type calcium channels

Opioids induce dual (inhibitory and excitatory) regulation of depolarization-evoked [³H]dopamine release in SK-N-SH cells through either μ or δ receptors. The potentiation of dopamine release by opioid agonists is mediated by N-type voltage-dependent calcium channels and does not involve G_i/G_o proteins. Removal of the excitatory opioid effect by blockade with ω-conotoxin, an N-channel antagonist, reveals the inhibitiory effect of opioids on release, thus suggesting that both modulatory effects of opioids are exerted in parallel.     

Cholinergic regulation of VIP gene expression in human neuroblastoma cells

The effects of carbamylcholine (CCh) on the gene expression of the neuropeptide vasoactive intestinal polypeptide (VIP) were studied using two human neuroblastoma cell lines, NB-1 and BE(2)M17. CCh caused a fast increase in VIP mRNA level in both cell lines which was followed by an increase in VIP immunoreactivity. The time-course of the induction of both mRNA and peptide differed, however, between the two cell lines. No morphological changes of the cells were observed during 6 days of stimulation. The effect was mediated by the muscarinic class of acetylcholine receptors, since it could be totally abolished by atropine. Since CCh caused an accumulation of inositol-1,4,5-triphosphate, it is likely that muscarinic receptor subtype M1, M3 or M5 is involved. Experiments with the translational inhibitor, cycloheximide, showed that CCh mediated a direct effect on the VIP gene expression. By combining gel permeation chromatography with radioimmunoassays using antisera specific for the various VIP-precursor products, immunoreactive peaks eluting as the synthetic peptides were found in both cell lines. In addition, earlier eluting peaks which could represent partially processed or extended VIP forms were found. After CCh induction the concentration of all preproVIP-derived products increased, and there was a tendency towards a shift to more fully processed VIP. The findings give new evidence for a direct regulation of VIP gene expression in human neuronal cells by cholinergic agents.     

Tetanus toxin: a rapid and selective blockade of the calcium, but not sodium, component of action potentials in cultured neuroblastoma N1E-115 cells

The slow action potential of mouse neuroblastoma N1E-115 cells, which was elicited in Na-free medium and whose amplitude was directly dependent on extracellular Ca concentrations, was blocked by tetanus toxin (TNTX) without change in resting membrane potential and resistance. The average value of action potential overshoot (peak potential level of spikes) in the absence and presence of TNTX was -1 and -11 mV, respectively. The concentration of TNTX required for 50% inhibition of Ca-dependent spikes (IC50) was about 65 ng/ml. The amplitude of spikes was decreased within 10-20 min after adding TNTX. The inhibition of Ca-dependent spikes was mimicked by 10 mM CoCl2 and MnCl2, but not by the boiled or neutralized toxin. The results indicate that TNTX inhibited electrogenesis of Ca, but not Na, components of action potentials of the neuroblastoma cells.     

Restriction of μM-calcium–requiring calpain activation to the plasma membrane in human neuroblastoma cells: Evidence for regionalized influence of a calpain activator protein

Regulation of the μM-calcium–requiring form of calpain (μ calpain) was studied in SH-SY-5Y human neuroblastoma cells. Immunoblot analysis demonstrated that the vast majority of μ calpain is localized within cytosolic pools. Calpain activation was monitored as a function of autolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact neuronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, μ calpain is preferentially activated at the plasma membrane as evidenced by autolytic generation of faster-migrating isoforms. By contrast, similar autolytic profiles for μ calpain in membrane or cytosolic fractions following addition of calcium were observed under cell-free conditions and within cells following death due to extended ionophore-mediated calcium influx. These differential activation profiles for cytosolic μ calpain within living cells and following cellular fractionation/cell death indicate the presence of a regulatory system within neuronal cells. As in previous studies in other systems, we demonstrate the presence of a calpain activator protein. Cycloheximide treatment depleted the autolytic capacity of membrane-associated μ calpain within 4–6 hr without a corresponding decline in total μ calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpain; pulse-chase radiolabeling confirmed the half-life of μ calpain to exceed 24 hr. Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuronal cells, and that, given the tremendous latent μ calpain activity within the cytosol, the interplay of the activator protein and the endogenous inhibitor calpastatin are crucial for maintaining neuronal homeostasis. J. Neurosci. Res. 48:543–550, 1997. © 1997 Wiley-Liss Inc.     

Gene expression profile in beta-amyloid-treated SH-SY5Y neuroblastoma cells

The beta-amyloid peptide, the major component of the senile plaques in Alzheimer's disease (AD), has been probed to be toxic to neurons both in vivo and in vitro. Several mechanisms have been proposed to be involved in the amyloid-induced neurotoxicity; among others it has been suggested that the beta-amyloid peptide exerts its toxic effect mainly by activating the surrounding microglia population, which in turn induces the synthesis and release of preapoptotic and pro-inflammatory factors. In addition, a direct effect of beta-amyloid on neurons has been also described. However, the precise mechanisms involved in the amyloid-induced neurotoxicity have been not yet definitely clarified. To characterize the effects directly induced on neurons, we have analyzed the gene expression profile induced by the 25-35 beta-amyloid fragment in human SH-SY5Y neuroblastoma cells, by using the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Our results confirm that beta-amyloid may directly induce neuronal cell death; activating signals that in vivo have been described as causative of Alzheimer's disease.     

真核表达载体pcDNA3.1-3a的构建及其在神经母细胞瘤SK-N-SH细胞中的表达

目的在pcDNA3.1真核表达载体中构建表达融合myc/his标签的甲基化酶表达载体pcDNA3.1-3a,并在神经母细胞瘤细胞株SK-N-SH细胞中进行验证,以期获得可表达甲基化酶催化结构域的融合基因。方法以甲基化酶的pcDNA4.0-GBD-3a-myc/his质粒为模板,通过PCR的方法扩增获得融合有myc/his标签序列的目的区段--甲基化酶催化结构域3a,将其克隆入真核表达载体pcDNA3.1;以EcoRⅠ、EcoRⅤ双酶切和PCR方法对构建的表达载体进行鉴定,并通过测序证明载体构建正确,同时检测甲基化酶3a在SK-N-SH细胞中的表达。结果通过PCR方法获得了含有甲基化酶催化结构域的真核表达载体pcDNA3.1-3a,并通过免疫印记方法验证了在神经母细胞瘤细胞株SK-N-SH细胞样品中用抗his标签抗体可以特异性识别目的蛋白。结论正确构建了甲基化酶表达载体pcDNA3.1-3a,其可在神经母细胞瘤细胞株SK-N-SH细胞中表达。     

Enhanced oxygen metabolism of peritoneal macrophages in the presence of murine neuroblastoma cells is partly caused by enkephalins

A bioluminescent technique was used to show that murine neuroblastoma (NB) cells or cell-free extracts (H variant) were able to enhance the release of reactive oxygen intermediates (ROI) from peritoneal macrophages in vitro. L-variant NB cells were ineffective. Physiological concentrations of met-enkephalin produced the same effect in vitro but not leu-enkephalin. When both H- and L-variant cells, or their extracts, were incubated together with macrophages, ROI production was not increased. Similar findings were detectable when met- and leu-enkephalin were cultured together with macrophages. In vivo, preliminary studies gave the same results. The concentration rate of met- to leu-enkephalin was higher in H-variant than in L-variant NB cells. We conclude from our results that met-enkephalin can enhance the release of ROI from peritoneal macrophages. The difference in the effects produced by the H and L variants is due to differences in the concentrations of enkephalins released.