omega-conotoxin-(conus-magus) and Neuroblastoma

The voltage-dependent calcium current (ICa) in cultured human neuroblastoma cells (NB-I) was studied by whole-cell recording. The low-threshold current (Il), the high-threshold, fast inactivating current (Ih.f.), and the high-threshold, slow inactivating current (Ih.s.) were identified. Il. was blocked by Ni2+. Ih.f. was blocked by omega-conotoxin GVIA. Ih.s. was blocked by nifedipine, and enhanced by Bay K 8644. These characteristics indicate that Il, Ih.f. and Ih.s. are consistent with the T-, N- and L-type ICa, respectively.

Topics: Calcium; Calcium Channel Blockers; Calcium Channels; Humans; Membrane Potentials; Mollusk Venoms; Neuroblastoma; Nickel; Nifedipine; omega-Conotoxins; Peptides; Tumor Cells, Cultured

Voltage-activated calcium channel currents were recorded from differentiated human neuroblastoma cells. SK-N-SH-SY5Y (SH-SY5Y) line, using patch-clamp techniques. Experimental solutions were designed to suppress sodium and potassium channel currents, and barium ions were used as the charge carrier. Two distinct types of calcium channel currents (N- and L-like) were identified based on their time-dependent inactivation, pharmacology and single-channel conductances. N- and L-like calcium channel currents were evoked by step depolarizing pulses to potentials more positive than -40 mV from a holding potential of -100 mV. The N-like component showed time-dependent inactivation during maintained depolarization with a time constant of tau f approximately 100 ms, whereas the L-like currents showed very slow inactivation with a time constant of tau s approximately 1,000 ms. Steady-state inactivation of currents evoked from a holding potential of -100 mV had two distinct components. One component involved the reduction of the transient current and had a half-maximal current at approximately -66 mV, whereas the other component involved the reduction of the steady-state current in the range of -35 to 0 mV with a half-maximal current at approximately -17 mV. Bay K 8644 (5 microM), had two distinct actions, one was the increase (50%) of the current associated with a depolarizing pulse to +10 mV. The second action was the increase in the peak amplitude of the tail current and the slowing of the deactivation kinetics. Omega-conotoxin at 1 microM irreversibly reduced the N-like current, sparing a component that was still sensitive to 5 microM Bay K 8644. The single-channel currents recorded with the cell-attached configuration of the patch clamp revealed two distinct conductances: a large approximately 28 pS and a small approximately 16 pS, corresponding to the L- and N-like channels, respectively. Bay K 8644 at 5 microM increased the mean open time of L-like single channel currents without changing single-channel conductance.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Calcium Channel Blockers; Calcium Channels; Electrophysiology; Humans; Kinetics; Membrane Potentials; Nervous System Neoplasms; Neuroblastoma; omega-Conotoxins; Peptides, Cyclic; Tumor Cells, Cultured

ATP-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells were studied. Using the fluorescent Ca2+ indicator fura-2, we have shown that the [Ca2+]i increased in response to ATP. ATP at 3 mM caused the greatest increased in [Ca2+]i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5'-thiotriphosphate and 5'-adenylyl-beta, gamma-imidodiphosphate, could not trigger significant [Ca2+]i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, alpha beta-methylene-ATP, beta, gamma-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca2+]i at 3 mM. In the absence of extracellular Ca2+, the effect of ATP was inhibited totally, but could be restored by the addition of Ca2+ to the cells. Upon removal of Mg2+, the maximum increase in [Ca2+]i induced by ATP was enhanced by about 42%. Ca(2+)-channel blockers partially inhibited the ATP-induced [Ca2+]i rise. The ATP-induced [Ca2+]i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca2+]i rise completely. No heterologous desensitization of [Ca2+]i rise was observed between ATP and bradykinin. The magnitude of the [Ca2+]i rise induced by ATP increased between 1.5 and 3.1 times when external Na+ was replaced with Tris, N-methyl-D-glucamine, choline, or Li+. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca2+]i to the basal level in Na(+)-containing or Na(+)-free Tris solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Bradykinin; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Fura-2; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Nifedipine; omega-Conotoxins; Peptides; Terpenes; Thapsigargin; Verapamil

The turnover of voltage-operated calcium channels was studied in two different human neuroblastoma cell lines (IMR32 and SH-SY5Y) using omega-conotoxin. The 125I-omega-conotoxin bound to surface channels was internalized and degraded by the cells in a time- and temperature-dependent manner. The radioactive degradation products released in the medium were all trichloroacetic acid soluble and no longer recognized by anti-omega-conotoxin antibodies. Altering the pH of intracellular organelles with chloroquine and inhibiting lysosomal proteases with leupeptin reduced 125I-omega-conotoxin degradation but had no effect on its internalization. Postlabeling measurements showed that the rates of 125I-omega-conotoxin internalization and degradation were equal to the rate of channel removal from the cell surface after protein synthesis inhibition. The rate of removal of omega-conotoxin binding sites was parallel to the rate of loss of functional channels, as measured by means of the fura-2 technique. Drug-induced differentiation of human neuroblastoma cells slowed down channel internalization and degradation rates, leading to the known increased expression of plasma membrane calcium channels in differentiated cells. On the other hand, both human (from Lambert-Eaton myasthenic patients) and murine (from immunized mice) anti-channel antibodies increased the rates of channel internalization and degradation, leading to channel downregulation. The activity of presynaptic calcium channels is already known to be acutely modulated by a number of different agents (e.g., hormones and neurotransmitters); our studies suggest that a different form of channel modulation (changes in the number of channels due to interference with channel turnover) may be active over a longer time scale in neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies; Calcium Channels; Cell Differentiation; Chloroquine; Electrophysiology; Humans; Leupeptins; Neuroblastoma; omega-Conotoxins; Peptides, Cyclic; Temperature

Cannabinoid receptor ligands irreversibly inhibited peak voltage-activated Ca currents (44%) in NG108-15 cells; this inhibition was Pertussis toxin-sensitive. Inhibition was largely due to a reduction in the omega-conotoxin sensitive portion of high-voltage activated (HVA) current, although there was also a significant decrease in low-voltage activated current (56%) and in the nifedipine-sensitive portion of HVA current (41%).

Topics: Analgesics; Calcium Channel Blockers; Cannabinoids; Cyclohexanols; Dronabinol; GTP-Binding Proteins; Humans; Neuroblastoma; omega-Conotoxins; Peptides, Cyclic; Pertussis Toxin; Receptors, Cannabinoid; Receptors, Drug; Tumor Cells, Cultured; Virulence Factors, Bordetella

High-threshold (HVA) Ca2+ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between -20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%-70% depression of HVA Ba2+ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca2+ channels after facilitation was slow (tau r 36-45 ms) and voltage-independent between -30 mV and -90 mV. The inhibitory action of noradrenaline was dose-dependent (IC50 = 84 nM), mediated by alpha 2-adrenergic receptors and selective for omega-conotoxin-sensitive Ca2+ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[gamma S] and prevented by GDP[beta S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10-50 microM) and developed with mean time constants of 0.56 s (tau on) and 3.6 s (tau off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca2+ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.

Topics: Calcium Channel Blockers; Calcium Channels; Differential Threshold; Dose-Response Relationship, Drug; Electrophysiology; GTP-Binding Proteins; Humans; Neuroblastoma; Norepinephrine; omega-Conotoxins; Peptides, Cyclic; Receptors, Adrenergic, alpha; Time Factors; Tumor Cells, Cultured

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Cadmium; Cadmium Chloride; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Cell Line; Cell Membrane; Chlorides; Evoked Potentials; Fura-2; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Nicotinic Acids; Nifedipine; omega-Conotoxins; Oxadiazoles; Peptides, Cyclic; Potassium; Tretinoin

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.

Topics: Animals; Biological Transport; Bradykinin; Calcium; Calcium Channel Blockers; Cell Line; Cell Membrane; Fluorescent Dyes; Fura-2; Glioma; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Nimodipine; omega-Conotoxins; PC12 Cells; Peptides, Cyclic; Terpenes; Thapsigargin

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