Voltage-operated calcium channel heterogeneity in pancreatic beta cells: physiopathological implications.

Voltage-operated calcium channels play crucial roles in stimulus-secretion coupling in pancreatic beta cells. A growing body of evidence indicates that these channels in beta cells are heterogeneous. In particular, not all the high-threshold calcium channels expressed belong to the best known L-type. In rat insulinoma cells, for example, L, N, and P/Q-type channels are present, while in human beta cells L-type and P/Q-type dominate. Where present, N-type and P/Q-type channels participate, alongside with the dominant L-type, in the control of sugar- or depolarization-induced hormone release. Distinct biophysical properties and selective modulation of the channel subtypes are likely to play important physiological roles. T-type channels are involved in beta cell apoptosis, while calcium channel autoantibodies recognizing high-threshold channels in beta cells, have been described both in neurological and diabetic patients. Subtype-selective calcium channel drugs have the potential for being beneficial in beta cell pathological states.

Modulation of N-type calcium channels translocation in RINm5F insulinoma cells.

An intracellular pool of N-type voltage-operated calcium channels has recently been described in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. These channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. Upon exocytosis N-type calcium channels were reversibly inserted in the plasma membrane. We have now extended this study to RINm5F rat insulinoma cells, and characterized the parallelism between the 'regulated' secretion of serotonin and the recruitment of surface calcium channels. Exocytosis was stimulated by different means, such as depolarization with high KCl, high Ba(2+)alone or protein kinase C activation; on the other hand exocytosis was inhibited with the non-selective calcium channel antagonist Cd(2+)or with noradrenaline. Stimulated release was always accompanied, with parallel kinetics, by calcium channel recruitment, while inhibition of secretion blocked calcium channel recruitment too. During repetitive depolarizations we revealed a potentiation of [Ca(2+)]()i transients in single Fura-2 loaded RINm5F cells, that was accompanied by an increase in surface VOCCs, suggesting a physiological role for the newly recruited channels. 2000 Academic Press@p$hr

Metabolism and trafficking of N-type voltage-operated calcium channels in neurosecretory cells.

The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity omega-conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. Omega-conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis.

Nerve growth factor abrogates the tumorigenicity of human small cell lung cancer cell lines.

Nerve growth factor (NGF) has antiproliferative and differentiating effects on adenomas of neuroendocrine origin. Cell lines derived from small-cell lung carcinoma (SCLC), a very aggressive neuroendocrine tumor, express NGF receptors. The role of NGF in the control of proliferation and progression of this carcinoma, however, has never been investigated. Chronic exposure of NCI-N-592 and GLC8 SCLC cell lines to NGF remarkably inhibited their proliferation rate both in vitro and in vivo, prevented their anchorage-independent clonal growth in soft agar, impaired their invasive capacity in vitro, and abolished their tumorigenic potential in nude mice. The proliferative response of SCLC cell lines to nicotine was also remarkably impaired by in vitro NGF treatment. Furthermore, NGF treatment activates in SCLC cell lines the expression and secretion of NGF. NGF thus reverts SCLC cell lines to a noninvasive, nontumorigenic phenotype that does not respond to nicotine and produces NGF.

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release.

Electrophysiological measurements of cell capacitance (Cm) and biochemical assays of [3H] serotonin ([3H]5-hydroxytryptamine or [3H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced Cm changes (DeltaCm), indicative of exocytosis, with the same voltage and Ca2+ dependency as the inward Ca2+ currents (ICa). Ba2+ was able to substitute for Ca2+ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca2+-versus Ba2+-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [3H]5-HT to study hormone release from cell populations. [3H]5-HT was released in a depolarization-, Ca2+- and time-dependent manner. Ba2+ also substituted for Ca2+ in depolarization-induced [3H]5-HT release. Thapsigargin, used to deplete Ca2+ stores, had no effects on Ca2+-triggered Cm increases, but Ca2+-triggered [3H]5-HT release was abolished. Ba2+-triggered [3H]5-HT release, however, was only slightly affected by Ca2+ store depletion. Ba2+ was found to act directly as a secretagogue of [3H]5-HT in intact cells, but not in Cm measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action.

Activation of delta-opioid receptors inhibits neuronal-like calcium channels and distal steps of Ca(2+)-dependent secretion in human small-cell lung carcinoma cells.

Human small-cell lung carcinoma (SCLC) cells express neuronal-like voltage-operated calcium channels (VOCCs) and release mitogenic hormones such as serotonin (5-HT). Opioid peptides, on the other hand, have been shown to reduce SCLC cell proliferation by an effective autocrine pathway. Here we show that in GLC8 SCLC cells, only delta-opioid receptor subtype mRNA is expressed. Consistently, the selective delta-opioid agonist [D-Pen2-Pen5]-enkephalin (DPDPE), but not mu and kappa agonists, potently and dose-dependently inhibits high-threshold (HVA) VOCCs in these cells. As in peripheral neurons, this modulation is largely voltage-dependent, mediated by pertussis toxin (PTX)-sensitive G-proteins, cAMP-independent, and mainly affecting N-type VOCCs. With the same potency and selectivity, DPDPE also antagonizes the Ca(2+)-dependent release of [3H]serotonin ([3H]5-HT) from GLC8 cells. However, DPDPE inhibits not only the depolarization-induced release, but also the Ca(2+)-dependent secretion induced by thapsigargin or ionomycin. This suggests that besides inhibiting HVA VOCCs, opioids also exert a direct depressive action on the secretory apparatus in GLC8 cells. This latter effect also is mediated by a PTX-sensitive G-protein but, contrary to VOCC inhibition, it can be reversed by elevations of cAMP levels. These results show for the first time that opioids effectively depress both Ca2+ influx and Ca(2+)-dependent hormone release in SCLC cells by using multiple modulatory pathways. It can be speculated that the two mechanisms may contribute to the opioid antimitogenic action on lung neuroendocrine carcinoma cells.

Noradrenaline inhibition of Ca2+ channels and secretion in single patch-clamped insulinoma cells.

Noradrenaline effects on voltage-operated calcium channels and exocytosis were studied, for the first time, in single patch-clamped RINm5F insulin-secreting cells. Noradrenaline, despite small and variable inhibition of calcium currents, strongly inhibited the increase in membrane capacitance (a measure of exocytosis) stimulated by both step depolarizations and the calcium ionophore, ionomycin. Noradrenaline similarly inhibited KCl- and ionomycin-induced [3H]serotonin release from RINm5F cell populations. Noradrenaline effects were mediated by PTX-sensitive G proteins. Noradrenaline inhibitory effects on secretion are, therefore, mainly exerted downstream from Ca2+ influx.

alpha-Conotoxin imperialis I inhibits nicotine-evoked hormone release and cell proliferation in human neuroendocrine carcinoma cells.

alpha-Conotoxins are small peptides present in the venom of different species of marine snails of the Conus genus that target nicotinic acetylcholine receptors (nAChRs), with a marked specificity for muscle-type nAChRs. alpha-Conotoxin Imperialis I (alpha-Ctx-Iml), from Conus imperialis, has been recently described as a potent antagonist of mammalian neuronal alpha-bungarotoxin (alpha-Bgtx)-sensitive nAChRs. Human small cell lung carcinoma (SCLC) is a very aggressive tumor composed of neuroendocrine secretory cells. We demonstrated that human SCLC cells express neuronal-type alpha-Bgtx-sensitive nAChRs, and that their activation causes secretion of mitogenic hormones and stimulates cell proliferation, alpha-Ctx ImI inhibits both these nicotinic effects, and could therefore be considered a new important tool for investigating human neuronal-type alpha-Bgtx-sensitive nAChRs.

Serotonin release and cell proliferation are under the control of alpha-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 alpha-bungarotoxin (alpha Bgtx) and alpha-conotoxin MI (alpha Ctx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both alpha Bgtx and alpha Ctx. By PCR analysis, we demonstrate the presence in SCLC of both the alpha 7 and the beta 2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that alpha Bgtx-sensitive receptors of the alpha 7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.

Calcium channel subtypes controlling serotonin release from human small cell lung carcinoma cell lines.

Small cell lung carcinoma is an aggressive neuroendocrine tumor that secretes several hormones, some of which act as autocrine growth factors. In order to obtain more information on the process of hormone secretion from this tumor, we have studied the role of intracellular free Ca2+ concentrations and voltage-operated calcium channels in the control of [3H]serotonin release from in vitro growing cell lines. We found that the Ca2+ ionophore ionomycin and the Ca(2+)-ATPase antagonist thapsigargin induced a dose-dependent increase of intracellular Ca2+ and a parallel enhancement of [3H]serotonin release. KCl-induced depolarization also stimulated a dose- and Ca(2+)-dependent [3H]serotonin release that in the GLC8 cell line was effectively inhibited by Ca2+ channel antagonists (Cd2+, nitrendipine, verapamil, omega-conotoxin GVIA, and omega-agatoxin IVA) and potentiated by the Ca2+ channel agonist BayK8644. Autoantibodies against Ca2+ channels present in the sera of Lambert-Eaton myasthenic patients antagonized KCl- but not ionomycin-induced [3H]serotonin release. Polymerase chain reaction analysis indicated that GLC8 cells express L-, N-, and P-type neuronal Ca2+ channel alpha 1 subunits, together with two types of Ca2+ channel beta subunits. The presence of three functionally distinct high threshold Ca2+ channels was also revealed by patch clamp experiments; high threshold Ca2+ channels were identified as dihydropyridine-sensitive (L-type), omega-conotoxin GVIA-sensitive (N-type), and omega-agatoxin IVA-sensitive (P-type). Our data demonstrate that [3H]serotonin is released by small cell lung carcinoma cells in a Ca(2+)-dependent manner and that depolarization-induced [3H]serotonin release is mediated by Ca2+ influx through distinct, neuron-like, Ca2+ channel subtypes.

Nicotine stimulates a serotonergic autocrine loop in human small-cell lung carcinoma.

Small-cell lung carcinoma cells express different plasma membrane nicotinic acetylcholine receptor subtypes. We have now found that interacting with these receptors (-)-nicotine induces a dose-dependent and stereoselective release of [3H]serotonin which is dependent on external calcium and blocked by the specific ganglionic nicotinic antagonist mecamylamine. With the same potency (-)-nicotine stimulates tumor cell proliferation, an effect also blocked by mecamylamine. Serotonin itself stimulates cell proliferation in a dose-dependent manner, an effect blocked by the selective serotonergic receptor antagonists methiotepine and metergoline. These data suggest that nicotine might affect proliferation of small-cell lung carcinoma cells by inducing the release of hormones (such as serotonin) with autocrine capabilities and place both the nicotinic and the serotonergic receptors at key positions in the biological and, possibly, pharmacological approach to this human lung cancer.

Amine uptake inhibition by diosmin and diosmetin in human neuronal and neuroendocrine cell lines.

Human neuroblastoma cells of sympathetic origin have been used for studying the effects of diosmin and its metabolite diosmetin (vasotonic agent) on amine reuptake systems. Neuroblastoma cells take up 3H-dopamine in a specific and time-dependent manner. 3H-dopamine uptake was dose-dependently inhibited by the known antagonist desipramine. Diosmin did not affect 3H-dopamine uptake at concentrations as high as 1 mM. On the other hand the aglycone metabolite of diosmin, diosmetin, inhibited 3H-dopamine uptake in a dose-dependent manner (IC50 = 4 microM). Diosmetin inhibited 3H-dopamine uptake in control and differentiated neuroblastoma cells, as well as in small-cell lung carcinoma cells. Furthermore diosmetin also inhibited 3H-serotonin uptake in both cell types. These results demonstrate that some flavonoids act as antagonists of plasma membrane amine transporters at the molecular level and suggest that inhibition of amine reuptake at the level of peripheral sympathetic nerve terminals could be responsible for the increased vascular tone observed in vivo after treatment with these drugs.

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra.

Dendrosomes prepared from substantia nigra are able to take up and release [3H]dopamine in a CA(2+)-dependent manner. The Vmax values of [3H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K(+)-dependency of the [3H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [3H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [3H]dopamine release by 10 mumol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [3H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.