dihydropyridines and Neuroblastoma

Neuroblastoma is the most common and deadliest tumor in infancy. WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC

Topics: Biphenyl Compounds; Cell Line, Tumor; Dihydropyridines; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; N-Myc Proto-Oncogene Protein; Neuroblastoma

Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.

Topics: alpha-Synuclein; Benzodiazepines; Dihydropyridines; Humans; Molecular Docking Simulation; Neuroblastoma; Parkinson Disease

Topics: Alzheimer Disease; Antioxidants; Calcium; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Humans; Melatonin; Neuroblastoma; Nimodipine; Tumor Cells, Cultured

Heat shock proteins (Hsps) have chaperone activity and play a pivotal role in the homeostasis of proteins by preventing misfolding, by clearing aggregated and damaged proteins from cells, and by maintaining proteins in an active state. Alzheimer's disease (AD) is thought to be caused by amyloid-β peptide that triggers tau hyperphosphorylation, which is neurotoxic. Although proteostasis capacity declines with age and facilitates the manifestation of neurodegenerative diseases such as AD, the upregulation of chaperones improves prognosis. Our research goal is to identify potent Hsp co-inducers that enhance protein homeostasis for the treatment of AD, especially 1,4-dihydropyridine derivatives optimized for their ability to modulate cellular stress responses. Based on favorable toxicological data and Hsp co-inducing activity, LA1011 was selected for the in vivo analysis of its neuroprotective effect in the APPxPS1 mouse model of AD. Here, we report that 6 months of LA1011 administration effectively improved the spatial learning and memory functions in wild type mice and eliminated neurodegeneration in double mutant mice. Furthermore, Hsp co-inducer therapy preserves the number of neurons, increases dendritic spine density, and reduces tau pathology and amyloid plaque formation in transgenic AD mice. In conclusion, the Hsp co-inducer LA1011 is neuroprotective and therefore is a potential pharmaceutical candidate for the therapy of neurodegenerative diseases, particularly AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Brain; Cell Line, Tumor; Dendritic Spines; Dihydropyridines; Disease Models, Animal; Gene Expression Regulation; Heat-Shock Proteins; Humans; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neuroblastoma; Neuroprotective Agents; Presenilin-1; tau Proteins

In this work we describe the synthesis and biological evaluation of the tacrine-1,4-dihydropyridine (DHP) hybrids (3-11). These multipotent molecules are the result of the juxtaposition of an acetylcholinesterase inhibitor (AChEI) such as tacrine (1) and a 1,4-DHP such as nimodipine (2). Compounds 3-11 are very selective and potent AChEIs and show an excellent neuroprotective profile and a moderate Ca2+ channel blockade effect. Consequently, these molecules are new potential drugs for the treatment of Alzheimer's disease.

Topics: Acetylcholinesterase; Alzheimer Disease; Animals; Antioxidants; Calcium; Calcium Channels; Cell Proliferation; Cells, Cultured; Dihydropyridines; Eels; Humans; Hydrogen Peroxide; L-Lactate Dehydrogenase; Neuroblastoma; Neuroprotective Agents; Structure-Activity Relationship; Tacrine

Although the anti-sympathetic mechanisms of the antihypertensive drug cilnidipine have been analyzed in neuronal cells derived from rodents, there is little information regarding the effects of cilnidipine in human neuronal cells. We investigated the effects of cilnidipine on N-type Ca2+ channels in IMR-32 human neuroblastoma cells using fura-2-based microfluorimetry. The ratio of the intensities of the emitted fluorescence at an excitation wavelength of 340 nm to that at 380 nm was calibrated to estimate the intracellular concentration of Ca2+. Stimulation of IMR-32 cells by 40 mmol/l KCl immediately increased the intensities ratio. In the presence of 10 micromol/l of nifedipine to block L-type Ca2+ channels, omega-conotoxin GVIA, a selective N-type Ca2+ channel blocker, in a concentration of 1 micromol/l suppressed the elevation of the intensities ratio induced by 40 mmol/l KCl. Similarly, cilnidipine in a concentration of 10 micromo/l suppressed the elevation of the ratio induced by 40 mmol/l KCl, and this suppression was effectively inhibited after the treatment with omega-conotoxin GVIA. These results suggest that cilnidipine potentially inhibits N-type Ca2+ channels in human neuronal cells and might be applied as a prospective therapeutic tool to provide neuronal protection as well as its antihypertensive effects and anti-sympathetic actions.

Topics: Antihypertensive Agents; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Cell Culture Techniques; Dihydropyridines; Flow Cytometry; Fura-2; Humans; Neuroblastoma; Neurons

Radiolabeled m-iodobenzylguanidine (MIBG) is a tumor-seeking radioactive drug used in the diagnosis and treatment of pheochromocytomas and neuroblastomas. It is transported into the tumor cells by the neuronal norepinephrine (NE) transporter (NET) which is expressed in almost all neuroblastoma cells. Here, we describe the synthesis and some pharmacological properties of a series of fluorescent compounds structurally related to the NET substrate, MIBG, or to the NET inhibitors, (-)-(2R,3S)-cocaine and nisoxetine. Three of 10 synthesized fluorescent compounds, 1-(1-naphthylmethyl)guanidinium sulfate (1), 1-[2-(dibenz[b, f]azepin-5-yl)ethyl]guanidinium sulfate (2), and (2R, 3S)-2beta-ethoxycarbonyl-3beta-tropanyl 5-(dimethylamino)naphthalene-1-sulfonate (6), exhibited high affinity (IC(50) about 50 nM) for the NET. The nisoxetine derivatives 8 (rac-N-[(3-methylamino-1-phenyl)propyl]-5-(dimethylamino)-1-naphthale nesulfonamide) and 9 (rac-4-[(3-methylamino-1-phenyl)propyl]amino-7-nitro-2,1, 3-benzoxadiazole) and especially the guanidine derivative 4 (1-[4-(4-phenyl-1,3-butadienyl)benzyl]guanidinium sulfate) which are characterized by intermediate affinity for the NET (IC(50) 370-850 nM) caused significant and nisoxetine-sensitive cell fluorescence. At least the guanidine derivative 4 might represent a potentially useful agent for imaging of neuroblastoma cells.

Topics: Carrier Proteins; Cocaine; Dihydropyridines; Fluorescent Dyes; Fluoxetine; Guanidines; Humans; Isoxazoles; Ligands; Neuroblastoma; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Structure-Activity Relationship; Symporters; Tumor Cells, Cultured

Evidence has accumulated that classic L-type Ca2+ channel blockers with a dihydropyridine structure also inhibit T-type Ca2+ channels in certain types of central and peripheral neurons and in smooth muscle cells, albeit with a lower potency. Thus beneficial therapeutic effects of dihydropyridines in cardiovascular and neurological diseases may not only be associated with L-type but also with T-type Ca2+ channel blockade. Little is known about the exact order of potency of dihydropyridine derivatives at T-type Ca2+ channels. Here we investigate the efficacy and potency of four therapeutically used compounds, i.e. nifedipine, nimodipine, nicardipine, niguldipine, in the neuroblastoma-glioma cell line NG108-15. For comparative purposes the Ca2+ channel agonist Bay K 8644 was included. Ca2+ channel currents were measured with the whole-cell voltage clamp technique. Subtype Ca2+ channel currents were separated by clamp protocol and selective blockers. T-type Ca2+ channel currents were inhibited with decreasing potency in the order niguldipine > nicardipine > nimodipine > nifedipine (IC50-values 244 nM, 2.5 microM, 9.8 microM, 39 microM), whereas L-type Ca2+ channel currents were blocked with similar potency (IC50 for nicardipine 75 nM). Bay K 8644 increased T-type Ca2+ channel current at nanomolar concentrations (i.e. 95 +/- 16% increase by 300 nM). T-type Ca2+ channel block was completely reversible with exception of the block by niguldipine. Our results indicate a variability of two orders of magnitude in potency of T-type Ca2+ channel block by the dihydropyridine derivatives investigated. It is speculated that the relation between the L- and T-type Ca2+ channel block may determine the therapeutic profile of a dihydropyridine derivative.

Topics: Animals; Brain Neoplasms; Calcium Channel Blockers; Cell Differentiation; Dihydropyridines; Glioma; Hybrid Cells; Membrane Potentials; Mice; Neuroblastoma; Rats; Tumor Cells, Cultured

The effects of nifedipine, niguldipine, nimodipine and nitrendipine on the high K+-induced intracellular Ca2+ ([Ca2+]i) transient in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells were studied by using the fluorescent Ca2+ indicator fura-2. It was observed that nifedipine at the concentration of 50 microM inhibited the high K+-induced [Ca2+]i transient by about 60%; niguldipine at the concentration of 10 microM caused a reduction of about 65% in the high K+-induced calcium signal and a further increase in the concentration up to 50 microM did not result in a significant further reduction in the high K+-induced calcium signal. However, on the other hand, nimodipine and nitrendipine at 50 microM inhibited almost completely the high K+-induced [Ca2+]i transient. Consequently, it was demonstrated in the present study that nimodipine and nitrendipine inhibit both L- and N-type calcium channels and thus seem to be unique among the dihydropyridines examined in their effects on calcium channels in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells, whereas nifedipine and niguldipine appear to block mainly L-type calcium channels.

Topics: Animals; Bucladesine; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Dihydropyridines; Evoked Potentials; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Nimodipine; Nitrendipine; Potassium; Rats

A P2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma x glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K+-current. We now report that UTP also inhibits Ca2+-currents in differentiated NG108-15 cells, but probably through a different nucleotide receptor. UTP (100 microM) inhibited the peak of the high-threshold current by 28.4 +/- 3.1% (n = 28) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM omega-conotoxin (CgTx) and one inhibited by 2 microM nifedipine and enhanced by 1 microM BAY K8644. UTP inhibited the former by 31.0 +/- 3.1%, with an IC50 of 2. 8 +/- 1.1 microM, and the latter 34.2 +/- 6.1% with an IC50 of 1.7 +/- 1.3 microM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or cAMP (1mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP4A and ATPgammaS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca2+-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals.. nucleotides, UTP, ATP, calcium currents, neuroblastoma cells

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine; Adenylyl Cyclases; Animals; Calcium; Calcium Channel Blockers; Dihydropyridines; Hybrid Cells; Mice; Mollusk Venoms; Neuroblastoma; Neurons; Nifedipine; omega-Conotoxin GVIA; Peptides; Pertussis Toxin; Rats; Receptors, Cell Surface; Virulence Factors, Bordetella

delta-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i) in ND8-47 cells by activating dihydropyridine-sensitive Ca2+ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [D-Ser2,Leu5]enkephalin-Thr (DSLET)-induced increase in [Ca2+]i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 microM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca2+]i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 microM DSLET for 24 h demonstrate a nearly 90% decrease in [3H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits alpha i2, alpha i3, alpha s, and alpha q were detected in ND8-47 cell membranes by western blot; alpha o and alpha i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the alpha-subunits. These results suggest that the DSLET-induced increase in [Ca2+]i mediated through pertussis toxin-sensitive G proteins (probably Gi2 or Gi3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.

Topics: Calcium; Calcium Channels; Cholera Toxin; Dihydropyridines; Enkephalin, Leucine; Ganglia, Spinal; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Intracellular Membranes; Narcotics; Neuroblastoma; Osmolar Concentration; Pertussis Toxin; Receptors, Opioid; Receptors, Opioid, delta; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cholera Toxin; Dihydropyridines; Egtazic Acid; Fura-2; G(M1) Ganglioside; G(M3) Ganglioside; Manganese; Mice; Microscopy, Fluorescence; Neuroblastoma; Nicardipine; Nickel; Potassium; Tumor Cells, Cultured

Ca2+ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba2+ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 microM Cd2+. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca2+ channels. Currents were also inhibited by the marine snail peptide omega-conotoxin GVIA (omega-CgTx). At a concentration of 10 nM inhibition by omega-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1 microM, supramaximal concentrations reduced the inhibitory actions of omega-CgTx in a concentration-dependent manner. Ca2+ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and omega-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca2+ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by omega-CgTx.

Topics: Brain Neoplasms; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Dihydropyridines; Humans; Membrane Potentials; Neuroblastoma; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Tumor Cells, Cultured

We evaluated the multidrug resistance (MDR)-modulating effects of progesterone (PRG) and an orally active, structurally related compound, megestrol acetate (MA), in several MDR human cell lines. At 100 microM, both steroids inhibited the binding of a Vinca alkaloid photoaffinity analog to P-glycoprotein (P-gp) in MDR human neuroblastic SH-SY5Y/VCR cells [which show greater than 1500-fold resistance to vincristine (VCR) in the tetrazolium dye (MTT) assay]. However, 100 microM MA markedly enhanced the binding of [3H]-azidopine to P-gp in both SH-SY5Y/VCR cells and the MDR human epidermoid KB-GSV2 cell line (which displays 250-fold resistance to VCR in the MTT assay). PRG had little effect on the binding of [3H]-azidopine to P-gp. MA at low doses was more effective than PRG in sensitizing cells to VCR and enhancing their accumulation of [3H]-VCR. The highly resistant SH-SY5Y/VCR subline exhibited significant collateral sensitivity to both steroids. These data suggest that MA may be a clinically useful modulator of MDR.

Topics: Affinity Labels; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Cells, Cultured; Chemical Phenomena; Chemistry, Physical; Cricetinae; Cricetulus; Dihydropyridines; Drug Interactions; Drug Resistance; Epithelial Cells; Epithelium; Humans; Iodine Radioisotopes; Lung; Megestrol; Megestrol Acetate; Membrane Glycoproteins; Neuroblastoma; Progesterone; Tritium; Tumor Cells, Cultured; Vincristine; Vindesine

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The low-threshold (LVA, T) Ca current activated positive to -50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 microM omega-conotoxin (omega-CgTx) or 40 microM Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to -30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of 125I-omega-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 microM omega-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual omega-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 microM) had a clear agonistic action on omega-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the omega-CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T;L,N) may be widely misleading if not supported by pharmacological assays.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amiloride; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Cell Membrane Permeability; Dihydropyridines; Electric Conductivity; Humans; Kinetics; Mollusk Venoms; Neuroblastoma; Neurons; Nickel; omega-Conotoxin GVIA; Tumor Cells, Cultured

Voltage-gated Ca2+ channels were identified in LA-N-5 human neuroblastoma cells using the Ca2+ sensitive fluorescent probe, fura-2. Using a variety of "classical" Ca2+ channel blockers, we have demonstrated the presence of both dihydropyridine (DHP)-sensitive and -insensitive channel types that can be activated by depolarization of the cells with either high K+ or gramicidin in the bathing solution. Brief exposure of LA-N-5 cells to menthol blunted the depolarization-induced Ca2+ influx though both DHP-sensitive and DHP-insensitive channels. This effect is concentration dependent (50% maximal blocking effect with 0.25 mM menthol), rapid in onset, and readily reversible. The specificity of the Ca2(+)-channel blocking effect of menthol was demonstrated in parallel studies using compounds with similar structures: menthone blocked Ca2+ channels with about half the potency of menthol, while cyclohexanol was without effect. Addition of either menthol or menthone to LA-N-5 cultures induced neurite outgrowth, cellular clustering, and reduction of cell growth in a dose-dependent fashion that correlated with the ability of these compounds to inhibit the DHP-insensitive Ca2+ influx. Cyclohexanol had no biologic activity. Taken together, the parallel potency for blockade of DHP-insensitive Ca2+ influx with the biologic activity of menthol suggests a role for certain types of Ca2+ channels in triggering growth and morphologic changes in LA-N-5 cells.

Topics: Acetylcholinesterase; Axons; Benzofurans; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Division; Cyclohexanols; Dihydropyridines; Fluorescent Dyes; Fura-2; Humans; Menthol; Neuroblastoma; Terpenes; Tumor Cells, Cultured

Neuroblastoma cells (N1E-115) were used as models of transient (T) and long-lasting (L) Ca++ channels. The whole cell version of the patch clamp technique was used to measure inward Ca++ currents, and the fluorescent indicator, Fura-2, was used to measure changes in intracellular Ca++. Cells were cultured and selected during recording so that predominantly T or L channel currents were measured. T channel currents did not respond to dihydropyridine or parathyroid hormone, whereas L channel currents did. BAY-K-8644 increased and nifedipine decreased L channel currents. After a 15 mM KCl challenge, cells with predominantly T channels responded with a transient change in intracellular Ca++, while cells with predominantly L channels showed a sustained response. PTH inhibited the increase in intracellular Ca++ in cells with L channels, but not in those with T channels. PTH may be an example of an endogenous calcium channel blocker, at least in neuroblastoma cells.

Topics: Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Humans; Neuroblastoma; Nifedipine; Parathyroid Hormone; Tumor Cells, Cultured

Topics: Acetylcholine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cadmium; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Electric Conductivity; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Gadolinium; GTP-Binding Proteins; Neuroblastoma; Neurons; Norepinephrine; Tumor Cells, Cultured

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aminoquinolines; Animals; Bucladesine; Calcium; Cricetinae; Dihydropyridines; Fluorescent Dyes; Glioma; Humans; Hybrid Cells; Ion Channels; Membrane Potentials; Neuroblastoma; Potassium Chloride; Sodium; Tetrodotoxin; Veratridine

Binding of omega-conotoxin, a peptide toxin specific for some subtypes of voltage-operated calcium channels (VOCCs), was investigated in IMR32 neuroblastoma and PC12 pheochromocytoma cell lines. In both cell types, binding was specific, saturable and of high affinity. Association was rapid and dissociation almost non-existent. Dihydropyridines and verapamil failed to affect toxin binding, while high concentrations of CaCl2 completely antagonized it. Depolarization with high K+ induced a [Ca2+]i rise (revealed by the fura-2 fluorimetric technique) that consisted of an initial (0.5-1 min) peak followed by a prolonged (several minutes) plateau phase. omega-Conotoxin blocked mainly the first phase, while the dihydropyridine Ca2+ channel blocker, nitrendipine, primarily affected the plateau. This result suggests that in the two cell lines investigated, omega-conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Calcium Channel Blockers; Calcium Chloride; Dihydropyridines; Humans; Ion Channels; Mollusk Venoms; Neuroblastoma; Nitrendipine; omega-Conotoxins; Pheochromocytoma; Potassium; Rats; Tumor Cells, Cultured

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110 +/- 9 nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K+ produced an immediate increase in [Ca2+]i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca2+]i under resting or depolarising conditions. In addition, K+ stimulated 45Ca2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na+ free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202-791 showed opposite effects in modulating K+ stimulated 45Ca2+ uptake. (-)-R-202-791 inhibited K+ induced 45Ca2+ uptake with an IC50 of 100 nM and (+)-S-202-791 enhanced K+ stimulated uptake with an EC50 of 80 nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aminoquinolines; Animals; Brain Chemistry; Calcium; Calcium Channel Blockers; Cell Line; Dihydropyridines; Electric Stimulation; Glioma; In Vitro Techniques; Ion Channels; Neuroblastoma; Neurons; Nifedipine; Potassium; Pyridines; Rats; Synaptosomes; Verapamil