Neuromodulator-Mediated Phosphorylation of Specific Proteins in a Neurotumor Hybrid Cell Line (NCB-20)

Mouse neuroblastoma X embryonic Chinese hamster brain explant hybrid cell line (NCB-20) forms functional synapses when intracellular cyclic AMP levels are elevated for a prolonged period of time. NCB-20 cells were labeled with [32P]orthophosphate under conditions where 2-chloroadenosine gave maximum increases of 32P incorporation into tyrosine hydroxylase in nerve growth factor dibutyryl cyclic AMP-differentiated PC12 (pheochromocytoma) cells. When NCB-20 cells were exposed to activators [5-hydroxytryptamine (5-HT), prostaglandin E1, or forskolin], resulting in activation of cyclic AMP-dependent protein kinase, increased 32P incorporation into two major proteins [130 kilodaltons (kDa) and 90 kDa] occurred. 5-HT (in the presence of phosphodiesterase inhibitor, isobutylmethylxanthine) gave a three- to fourfold increase, and forskolin a four- to sevenfold increase in 32P incorporation into the 90-kDa protein. [D-Ala2,D-Leu5]-enkephalin, which decreased cyclic AMP levels and reversed the 2-chloroadenosine-stimulated phosphorylation of tyrosine hydroxylase in differentiated PC12 cells, also reversed the stimulation of phosphorylation of the 90-kDa protein in NCB-20 cells. Pretreatment of NCB-20 cells with a calcium ionophore, A23187, gave increased phosphorylation of the 90- and 130-kDa proteins, but phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (tumor promoting agent), cell depolarization with high K+, or pretreatment with dibutyryl cyclic GMP had no effect on phosphorylation of these proteins. In contrast, phosphorylation of an 80-kDa protein was decreased by forskolin, but increased following activation of the calcium/phospholipid-dependent kinase with tumor promoting agent. Neither the 90-kDa nor the 80-kDa protein showed any immunological cross-reactivity with synapsin, a major synaptic protein known to be phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase, but not calcium/phospholipid-dependent protein kinase. This suggests that in NCB-20 cells, several unique proteins can be phosphorylated by cyclic AMP-dependent protein kinase in response to hormonal elevation of cyclic AMP levels. In contrast, an 80-kDa protein is the primary substrate for calcium/phospholipid-dependent protein kinase, and its phosphorylation is inhibited by agents that elevate cyclic AMP levels and thereby activate cyclic AMP-dependent protein kinase.     

Properties of Large-Conductance Cationic Channels in the Neuronal Nuclear Envelope

The genetic apparatus of the nucleus of eukaryotic cells is surrounded by the nuclear envelope containing ion channels with different biophysical properties. The channels responsible for calcium release from the nuclear envelope into the intranuclear space have attracted special attention. As we found, a great number of large-conductance ion channels selective with respect to monovalent cations and impermeable for bivalent cations are present in all membranes where inositol trisphosphate receptors are expressed. We showed that channels of this type are insensitive to blockers of potassium channels and, according to their properties, cannot be attributed to one well-known type of ion channels or another. In addition, since their blockers remain unknown, these channels at present cannot be isolated and cloned. Channels of this type deserve further investigation; we hypothesize that large-conductance cationic channels can be involved in the control of the duration of Ca²⁺ release from the calcium store.     

Specific Receptor-Mediated Inhibition of Cyclic AMP Synthesis by Dopamine in a Neuroblastoma × Brain Hybrid Cell Line NCB-20

Abstract: Dopamine and dopamine receptor agonists were found to inhibit adenylate cyclase activity dose-de-β ndently in a neuroblastoma × Chinese hamster brain explant hybrid cell line NCB-20. Apomorphine (with an IC₅₀ value of 10 n M ) was the most effective inhibitor, followed by 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-naphthaline (ADTN), dopamine, and N -dipropyldopa-mine. The inhibition was potently reversed by sulpiride, butaclamol, and flupenthixol in a stereospecific manner, but was unaffected by yohimbine, except at high concentrations. Clonidine also inhibited adenylate cyclase activity in these cells and this was reversed by the α₂-adrenoreceptor antagonist yohimbine, but not by sulpiride. [ d -Ala², d -Leu⁵]Enkephalin inhibited adenylate cyclase activity in NCB-20 cells at nanomolar concentrations; this was reversed by naloxone. All three inhibitory neurotransmitters were able to reverse the stimulation of cyclic AMP synthesis by serotonin or prostaglandin E₁The dopamine receptor that modulates cyclic AMP synthesis in NCB-20 cells is pharmacologically quite distinct from a high-affinity spiperone binding site identified in these cells, but shows the pharmacologic specificity of the D₂ receptor previously described in mammalian brain.     

Transfection of N-Methyl-d-Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 m M ) of the neurotransmitter l -glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N -methyl- d -aspartate (NMDA) receptors and the consequent Ca²⁺ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca²⁺ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca²⁺ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca²⁺ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.     

A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

An Indel in Transmembrane Helix 2 Helps to Trace the Molecular Evolution of Class A G-Protein-Coupled Receptors

Class A G-protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors. Helical distortions play a major role in the overall fold of these receptors. Most are related to conserved proline residues. However, in transmembrane helix 2, the proline pattern is not conserved, and when present, proline may be located at position 2.58, 2.59, or 2.60. Sequence analysis, three-dimensional data mining, and molecular modeling were undertaken to investigate the origin of this unusual pattern. Taken together, the data strongly support the assumption that an indel led to two structural motifs for helix 2: a bulged structure in P2.59 and P2.60 receptors and a “typical” proline kink in P2.58 receptors. The proline pattern of helix 2 can be used as an evolutionary marker and helps to trace the molecular evolution of class A GPCRs. Two indel events yielding functional receptors occurred independently. One indel arose very early in GPCR evolution, in a bilaterian ancestor, before the protostome-deuterostome divergence. This indel led to the split between the P2.58 somatostatin/opioid receptors and other peptide receptors with the P2.59 pattern. A second indel also occurred in insect opsins and corresponds to a deletion. Subfamilies with proline at position 2.59 or no proline expanded earlier, whereas P2.60 receptors remained marginal throughout evolution. P2.58 receptors underwent rapid expansion in vertebrates with the development of the chemokine and purinergic receptor subfamilies from somatostatin/opioid-related ancestors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DIDS (4,4-Diisothiocyanatostilbenedisulphonic Acid) Induces Apoptotic Cell Death in a Hippocampal Neuronal Cell Line and Is Not Neuroprotective against Ischemic Stress

DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF ± DIDS (40 or 400 µM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N-terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.     

The Neuroplastin Adhesion Molecules Are Accessory Proteins That Chaperone the Monocarboxylate Transporter MCT2 to the Neuronal Cell Surface

Background

The neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABA_A receptor subtypes in hippocampal neurones. The brain is dependent not only on glucose but on monocarboxylates as sources of energy. The. monocarboxylate transporters (MCTs) 1–4 are responsible for the rapid proton-linked translocation of monocarboxylates including pyruvate and lactate across the plasma membrane and require association with either embigin or basigin, proteins closely related to neuroplastin, for plasma membrane expression and activity. MCT2 plays a key role in providing lactate as an energy source to neurons.

Methodology/Findings

Here we use co-transfection of neuroplastins and monocarboxylate transporters into COS-7 cells to demonstrate that neuroplastins can act as ancillary proteins for MCT2. We also show that Xenopus laevis oocytes contain endogenous neuroplastin and its knockdown with antisense RNA reduces the surface expression of MCT2 and associated lactate transport. Immunocytochemical studies show that MCT2 and the neuroplastins are co-localised in rat cerebellum. Strikingly neuroplastin and MCT2 are enriched in the same parasagittal zebrin II-negative stripes.

Conclusions

These data strongly suggest that neuroplastins act as key ancillary proteins for MCT2 cell surface localisation and activity in some neuronal populations, thus playing an important role in facilitating the uptake of lactate for use as a respiratory fuel.     

Nerve Growth Factor Amplifies Cyclic AMP Production in the HT4 Neuronal Cell Line

Abstract: There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml 15 min) amplified cAMP accumulation (≈75%) in response to activation of adenosine A₂ receptors (5 min) with 5′-N-ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down-regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF-induced amplification of cAMP accumulation. Although NGF-induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF-mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.     

Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

Purpose

RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.

Methods

661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.

Results

Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.

Conclusions

661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.     

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT_2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.     

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.     

PcExl1 a Novel Acid Expansin-Like Protein from the Plant Pathogen Pectobacterium carotovorum,Binds Cell Walls Differently to BsEXLX1

Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Injury-Related Factors and Conditions Down-Regulate the Thrombin Receptor (PAR-1) in a Human Neuronal Cell Line

Abstract: Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K_D = 3.13 and 5.25 nM, B_max = 190.1 and 67.8 fmol/mg of protein for ¹²⁵I-ATAP2 and ¹²⁵I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.