E. Sher

ω-Conotoxin binding and effects on calcium channel function in human neuroblastoma and rat pheochromocytoma cell lines

Binding of ω‐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. ω‐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, ω‐conotoxin acts mainly on a subgroup of VOCCs that is resistant to dihydropyridines.

Serotonin release and cell proliferation are under the control of α‐bungarotoxin‐sensitive nicotinic receptors in small‐cell lung carcinoma cell lines

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small‐cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose‐dependent manner; this effect is antagonized by α‐bungarotoxin (αBgtx) and α‐conotoxin MI (αCtx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both αBgtx and αCtx. By PCR analysis, we demonstrate the presence in SCLC of both the α7 and the β2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that αBgtx‐sensitive receptors of the α7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

Neuronal‐type nicotinic receptors in human neuroblastoma and small‐cell lung carcinoma cell lines

A β subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat β4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human β4 subunit is expressed in association with the α3 gene in neuroblastoma and small‐cell lung carcinoma cell lines. Patch‐clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.

Pharmacological Characterization of Cholinergic Receptors in a Human Neuroblastoma Cell Line

A human neuroblastoma cell line, IMR32, has been characterized as far as morphology, membrane receptors for neurotransmitters, and uptake and release of [3H]3,4‐dihydroxyphenylethylamine ([3H]dopamine). These cells expressed at their surface both nicotinic and muscarinic cholinergic receptors, revealed by [125I]α‐bungarotoxin and [3H]quinuclidinylbenzilate ([3H]QNB) binding, respectively. [125I]α‐Bungarotoxin binding was efficiently inhibited by α‐bungarotoxin, nicotine, carbachol, and d‐tubocurarine. [3H]QNB binding was competitively inhibited by atropine, pirenzepine, and carbachol. Hexamethonium did not affect the binding of either ligand. In competition experiments with [3H]QNB, pirenzepine recognized only one binding site with “low affinity,” and carbachol recognized two sites with different affinities, β‐adrenergic receptors were present in a very low amount, whereas α‐adrenergic and dopaminergic receptors were not detectable. IMR32 cells had an imipramine‐sensitive [3H]dopamine uptake, but carbachol, high levels of K+, the calcium ionophore A23187, and α‐latrotoxin were not able to induce release of [3H]dopamine that had been taken up. The ultrastructural analysis showed that IMR32 cells contained very few dense‐core vesicles, suggesting a low storage capacity for neurotransmitter. These cells could be an useful in vitro model for studying neurotransmitter receptors of the human CNS.

N-type Ca2+ channels are present in secretory granules and are transiently translocated to the plasma membrane during regulated exocytosis.

An intracellular pool of N-type voltage-operated calcium channels has recently been described in different neuronal cell lines. We have now further characterized the intracellular pool of N-type calcium channels in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. Intracellular N-type calcium channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. 125I-ω-Conotoxin GVIA binding assays on fixed and permeabilized cells revealed that intracellular N-type calcium channels translocate to the plasma membrane in cells exposed to secretagogues (KCl, ionomycin, and phorbol esters). The kinetics, Ca2+ and protein kinase C dependence, and brefeldin A insensitivity of N-type calcium channels translocation were similar to the regulated release of chromogranin B, while no correlation was found with the constitutive secretion of a heparan sulfate proteoglycan. A PC12 subclone deficient in the regulated but not in the constitutive pathway of secretion had a small intracellular pool of N-type calcium channels, and no secretagogueinduced translocation occurred in these cells. Calcium channel translocation was accompanied by a stronger response of Fura-2-loaded cells to depolarizing stimuli, suggesting that the newly inserted channels are functional.

Sensitivity to dihydropyridines, omega-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic beta-cells

High-voltage-activated (HVA) Ba2+ currents of rat insulinoma (RINm5F) and human pancreatic β-cells were tested for their sensitivity to dihydropyridines (DHPs), ω-conotoxin (ω-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent KD<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 μM and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca2+ agonists and antagonists in human β-cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 μM and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca2+ channels in these cells. In RINm5F cells, but not in human β-cells, DHP-resistant currents were sensitive to ω-CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a “residual” current (25–35%) with similar voltage-dependent characteristics and Ca2+/Ba2+ permeability. Noradrenaline (10 μM) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of ω-CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and ω-CgTx-resistant current although intracellularly applied guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 μM). Both actions were prevented by intracellular perfusion with guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic β-cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during β-cell activity.

Presynaptic localization of ω-conotoxin-sensitive calcium channels at the frog neuromuscular junction

Abstract By the use of anti-ω-CTx antibodies in indirect immunofluorescence we demonstrated the presence of ω-CTx binding sites in the presynaptic compartment of frog nerve-muscle preparations. The images we obtained indicate that ω-CTx-sensitive channels are clustered at discrete sites corresponding in distribution to active zones.

ω-Conotoxin-sensitive, voltage-operated Ca2+ channels in insulin-secreting cells

Abstract The properties of voltage-operated Ca2+ channel subtypes were investigated in insulin-secreting RINm5F cells. Two types of channels were identified: a dihydropyridine-sensitive (L-type) channel, and an ω-conotoxin-sensitive (ω-type) channel. 125I-ω-Conotoxin bound with high affinity (Kd 46.7 pM) to a saturable number of binding sites (10.3 fmol/mg of protein). Toxin binding was not antagonized by L-type channels ligands, but was sensitive to Ca2+ and neomycin. 125I-ω-Conotoxin-labeled Ca2+ channels were recognized by autoantibodies of Lambert-Eaton myasthenic patients. These antibodies are known to be specific for the neuronal ω-type channel. High-voltage-activated Ca2+ currents, investigated with the patch-clamp technique, consisted of a major dihydropyridine-sensitive (L-type) component, and a minor fraction irreversibly blocked by ω-conotoxin. Depolarizing secretagogues, such as D-glyceraldehyde and alanine, induced Ca2+-dependent insulin secretion, which was attenuated by ω-conotoxin. Taken together, these results show that voltage-operated Ca2+ channels in insulin-secreting RINm5F cells are heterogeneous and, in particular, that an ω-type channel, pharmacologically, immunologically and electrophysiologically similar to the neuronal ω-type channel, is also expressed in endocrine cells where it might have a role in the control of hormone secretion.

ω-conotoxin and Cd2+ stimulate the recruitment to the plasmamembrane of an intracellular pool of voltage-operated Ca2+ channels

125I-omega-conotoxin binding to neuroblastoma cells at 37 degrees C continuously increased, reaching a plateau after 6-8 hr; this was up to 6 times higher than that observed at lower temperatures. The same effect was induced by short pulses with omega-conotoxin followed by a chase period at 37 degrees C in control medium. Cd2+ also induced up-regulation of surface 125I-omega-conotoxin-binding sites. Fura-2 and patch-clamp experiments showed that the recruited binding sites corresponded to functional voltage-operated Ca2+ channels. Permeabilization experiments revealed a large intracellular pool of 125I-omega-conotoxin-binding sites, whose recruitment to the plasmamembrane was prevented by brefeldin A and nocodazole. These data suggest that specific stimuli might induce voltage-operated Ca2+ channel translocation to plasmamembrane and, in this way, modulate presynaptic events.

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

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 (τr 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 α2-drenergic receptors and selective for ω-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[γS] and prevented by GDP[βS] 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 μM) and developed with mean time constants of 0.56 s (τon) and 3.6 s (τoff) 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.