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E Carbone

Publications and source records attributed to E Carbone.

At least 73 records · Page 4Linked to original sources
PubMed1994

Localized L-type calcium channels control exocytosis in cat chromaffin cells.

Depolarizing 1-s pulses to 0 mV from a holding potential of -70 mV, induced whole-cell currents through Ca2+ channels (ICa) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 microM) reduced the peak current by 47% and the late current by 80%. omega-Conotoxin GVIA (CgTx, 1 microM) reduced the peak ICa by 42% and the late ICa by 55%. Pulses (10 s duration) with 70 mM K+/2.5 mM Ca2+ solution (70 K+/2.5 Ca2+), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca2+ concentration ([Ca2+]i) from 0.1 to 2.21 microM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K+/2.5 Ca2+ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca2+]o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca2+ channels in cat chromaffin cells. It seems, however, that though extracellular Ca2+ entry through both channel types leads to similar increments of averaged [Ca2+]i, the control of catecholamine release is dominated only by Ca2+ entering through L-type Ca2+ channels. This supports the idea of a preferential segregation of L-type Ca2+ channels to localized "hot spots" in the plasmalemma of chromaffin cells where exocytosis occurs.

Adrenal Medulla↗
PubMed1994

omega-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.

Binding Sites↗
PubMed1993

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.

Autoantibodies↗
PubMed1993

Antinociceptive activity of salmon calcitonin: electrophysiological correlates in a rat chronic pain model.

Experimental and clinical evidence testifies to an antinociceptive action of salmon calcitonin (sCT), administered in different ways, on the central nervous system. These studies were performed almost exclusively in acute pain models. The purpose of the present study was to investigate the effects of sCT, injected directly into the lateral cerebral ventriculi, on the firing of single nociceptive thalamic neurons, detected by electrophysiological techniques in an experimental model of prolonged or chronic pain, such as rats rendered arthritic by injection of Freund's adjuvant into the left hindfoot. The noxious test stimuli used were either extension or flexion of the ankle or mild lateral pressure on the heel. With increasing doses of sCT (5, 10, 20, 40 micrograms, 5 microliters/i.c.v.) it was possible to observe correspondingly increasing inhibitory and long-lasting effects on the evoked firing, with a significant dose-effect relationship. In agreement with electrophysiological findings, preliminary data, obtained with a patch clamp technique, on depression of calcium fluxes through neuronal membrane, induced by sCT, oriented the attention to a direct action of sCT on CNS.

Analgesics↗
PubMed1993

Reduced expression of major histocompatibility complex class I free heavy chains and enhanced sensitivity to natural killer cells after incubation of human lymphoid lines with beta 2-microglobulin.

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface--for example as "empty" beta 2-microglobulin (beta 2m)-associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with beta 2m. This was found to shift the equilibrium between beta 2m-associated and non-associated--heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I-deficient cell lines was not affected by beta 2m, arguing against a general nonspecific effect of beta 2m on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of beta 2m) rather than with expression of complexes.

Cell Line↗
PubMed1993

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 beta-cells were tested for their sensitivity to dihydropyridines (DHPs), omega-conotoxin (omega-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 microM 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 beta-cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 microM 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 beta-cells, DHP-resistant currents were sensitive to omega-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 microM) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of omega-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 omega-CgTx-resistant current although intracellularly applied guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) prolonged its activation time course.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

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

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 omega-conotoxin-sensitive (omega-type) channel. 125I-omega-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 channel ligands, but was sensitive to Ca2+ and neomycin. 125I-omega-Conotoxin-labeled Ca2+ channels were recognized by autoantibodies of Lambert-Eaton myasthenic patients. These antibodies are known to be specific for the neuronal omega-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 omega-conotoxin. Depolarizing secretagogues, such as D-glyceraldehyde and alanine, induced Ca(2+)-dependent insulin secretion, which was attenuated by omega-conotoxin. Taken together, these results show that voltage-operated Ca2+ channels in insulin-secreting RINm5F cells are heterogeneous and, in particular, that an omega-type channel, pharmacologically, immunologically and electrophysiologically similar to the neuronal omega-type channel, is also expressed in endocrine cells where it might have a role in the control of hormone secretion.

Animals↗
PubMed1992

Voltage-dependent noradrenergic modulation of omega-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 (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.

Calcium Channel Blockers↗
PubMed1991

Recognition of beta 2-microglobulin-negative (beta 2m-) T-cell blasts by natural killer cells from normal but not from beta 2m- mice: nonresponsiveness controlled by beta 2m- bone marrow in chimeric mice.

The role of major histocompatibility complex (MHC) class I expression in control of the sensitivity of normal cells to natural killer (NK) cells was studied by the use of mutant mice made deficient for expression of beta 2-microglobulin (beta 2m) through homologous recombination in embryonal stem cells. T-cell blasts from beta 2m-deficient (beta 2m -/-) mice were killed by NK cells from normal mice in vitro, while beta 2m +/- blasts were resistant. The beta 2m defect also affected the NK effector cell repertoire: NK cells from beta 2m -/- mice failed to kill beta 2m -/- blasts, while they retained the ability to kill the prototype NK cell target lymphoma YAC-1, although at reduced levels. The inability to recognize beta 2m -/- blasts could be transferred with beta 2m -/- bone marrow to irradiated beta 2m-expressing mice. In contrast, the development of CD8+ T cells (deficient in beta 2m -/- mice) was restored in such chimera. These results indicate that loss of MHC class I/beta 2m expression is sufficient to render normal cells sensitive to NK cells, and that the same defect in the hemopoietic system of a mouse renders its NK cells tolerant to beta 2m-deficient but otherwise normal cells. In the beta 2m -/- mice, NK cells may be selected or educated by other bone marrow cells to tolerate the MHC class I deficiency. Alternatively, the specificity may be controlled directly by the class I molecules on the NK cells themselves.

Animals↗
PubMed1991

Noradrenergic inhibition and voltage-dependent facilitation of omega-conotoxin-sensitive Ca channels in insulin-secreting RINm5F cells.

We found that, besides dihydropyridine-sensitive Ca channels, insulin-secreting RINm5F cells also contain a minority (15-25%) of omega-conotoxin (omega-CgTx)-sensitive channels that show a high-affinity binding to [125I] omega-CgTx (Kd 51 pM). Noradrenaline (NA, 10 microM) slows down Ca-channel activation in these cells and produces a sizeable reduction of Ca currents that is relieved by strong pre-conditioning depolarizations (facilitation). The action of NA is mimicked by intracellular application of GTP-gamma-S and is prevented by pertussis toxin (PTX) or by cell pre-incubation with omega-CgTx. This suggests specific noradrenergic inhibition of omega-CgTx-sensitive Ca channels that is modulated by membrane potentials and PTX-sensitive G-protein activation.

Animals↗
PubMed1991

Voltage-dependent inhibition and facilitation of Ca channel activation by GTP-gamma-S and Ca-agonists in adult rat sensory neurons.

Intracellular application of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S, 100 microM) causes a slow down of high-threshold Ca channel activation in adult rat sensory neurons that is relieved by strong depolarizations or by the Ca agonist Bay K 8644 (5 microM). Recovery from GTP-gamma-S inhibition is usually accompanied by an increase in Ca current amplitude (facilitation) and is insensitive to holding potential (-60 to -90 mV). Inhibition and facilitation of Ca currents are also little affected by nifedipine (5 microM) or by cell incubation with omega-conotoxin (omega-CgTx, 3.2 microM). We conclude that both slowdown of Ca channel activation by GTP-gamma-S and facilitation by strong depolarizations or by Ca agonists derive from a common process in which G-protein activation, membrane voltage and Ca agonist receptors interact to modulate neuronal Ca channel gatings.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Do calcium channel classifications account for neuronal calcium channel diversity?

Calcium (Ca2+) ions are involved in the development and control of a variety of neuronal properties and functions such as channel expression, synaptic transmission and neurosecretion. The main pathway by which Ca2+ enters the intracellular space is through voltage-activated Ca2+ channels that can be classified according to their different biophysical and pharmacological properties. Identification and characterization of these channel types are prerequisites for understanding the mechanisms that underlie Ca2(+)-controlled processes. In this article we summarize the efforts made to identify neuronal Ca2+ channel types, and we attempt to evaluate how useful existing classifications are in assigning specific properties and functions to distinct channel types in neurons.

Animals↗
PubMed1991

NK and LAK susceptibility varies inversely with target cell MHC class I antigen expression in a rat epithelial tumour system.

Several cell clones derived from cell lines obtained from a rat thyroid carcinoma, induced by in vivo injection of the Kirsten murine sarcoma virus into thyroid gland, and from its spontaneous lung metastases were analysed for their major histocompatibility complex (MHC) class I antigen expression. The susceptibility to natural killer (NK) cell lysis of these clones, differing in their levels of MHC class I antigen expression, was determined and found to vary inversely with the target cell MHC level, confirming numerous reports of the literature. We then tried to localize the step of the multistage natural cytotoxic process, in which class I antigens could interfere, and tested first whether lymphokine (IL-2) activation of the killer (LAK) cells could overcome the differences in MHC class I expression of target cells. As this did not appear to be the case, we studied the binding step by either a cold target inhibition assay and a target binding assay and found that target cells expressing class I antigens show a lower competitive capacity for effector cells than targets not expressing such antigens, indicating that this interference may occur, at least in our system, in the binding step of the cytotoxic process.

Animals↗
PubMed1990

Multiple actions of Bay K 8644 on high-threshold Ca channels in adult rat sensory neurons.

omega-Conotoxin (omega-CgTx, 6.4 microM) failed to fully block the high-threshold Ba currents (HVA; L,N) of adult rat sensory neurons, and showed only a minor inhibitory action on the low-voltage activated current (LVA,T). In most of the CgTx-treated neurons the residual high-threshold Ba current was strongly agonized by the 1,4-dihydropyridine Bay K 8644. 1 microM Bay K 8644 enhanced 3- to 4-fold the size of this current at low membrane potentials and prolonged its deactivation kinetics by one order of magnitude. As in cardiac cells, in some neurons Bay K 8644 sped up about 3-fold the inactivation time course of the omega-CgTx-resistant Ba current, suggesting multiple actions of the dihydropyridine on neuronal high-threshold Ca channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Ca currents in human neuroblastoma IMR32 cells: kinetics, permeability and pharmacology.

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Temperature sensitivity of Ca currents in chick sensory neurones.

We have investigated the effects of temperature on the Ca currents of chick sensory neurones. Raising the temperature from 17 to 37 degrees C, caused low-threshold (LVA, T) and high-threshold (HVA, L and N) Ca currents to show a marked amplitude increase and a drastic acceleration of their activation-inactivation gatings. Compared to HVA channels, the LVA type showed a weaker temperature sensitivity. Its average Q10 values were closer to those of other voltage-operated ion channels: 1.7 (permeability), 1.9 (activation) and 2.2 (inactivation). Alternatively, the activation kinetics and peak permeability of HVA Ca channels showed maximal Q10 values of about 5 and 2.8, respectively. HVA channel deactivation was less sensitive to temperature (Q10 1.8). Inactivation of these channels was slow and monoexponential between 17 and 22 degrees C, but faster and double exponential above 30 degrees C, uncovering a fast temperature-sensitive decaying phase. The size and rate of decay of this component decreased with increasing membrane depolarizations and persisted at holding potentials positive to -80 mV, suggesting the involvement of temperature-sensitive Ca-mediated processes in the mechanism of HVA channel inactivation. Our data are consistent with the view that heating from 17 to 37 degrees C causes both an increased probability of Ca channels to open and a drastic acceleration of their activation-inactivation kinetics.

Animals↗
PubMed1990

Na+ currents through low-voltage-activated Ca2+ channels of chick sensory neurones: block by external Ca2+ and Mg2+.

1. Whole-cell currents through low-voltage-activated (LVA) Ca2+ channels carried by monovalent cations were studied in chick dorsal root ganglion (DRG) cells. 2. With 120 mM [Na+] on both sides of the membrane and [Ca2+]o less than or equal to 100 microM, the currents reversed at 0 mV. Their half-times of activation and inactivation were strictly voltage-dependent and decreased to near-constant values of 0.6-0.85 and 40 ms, respectively, at positive membrane potentials. The longer activation times were observed with [Ca2+]o greater than or equal to 50 microM. 3. The selectivity of the Ca2+ channel for monovalent ions with reference to internal Na+ was evaluated from the reversal potential. The Li+ and Na+ permeabilities were similar. The permeability ratios of K+ and Rb+ were 0.45, and 0.33 for Cs+. 4. Micromolar increases in [Ca2+]o produced small voltage shifts of half-times of activation (less than or equal to +3 mV at 10 microM and +10 mV at 500 microM), but strongly depressed the Na+ current. The Ca2(+)-induced block of Na+ current satisfied a 1:1 stoichiometry with an apparent KD of 1.8 microM at -20 mV. The block was, however, relieved with more positive and negative potentials, with KDs of 55 and 8.5 microM at +90 and -110 mV, respectively. 5. Relaxation time constants of block and unblock of Na+ currents through the LVA Ca2+ channel were measured on step changes to and from membrane potentials at which pronounced Ca2(+)-induced block occurred. 6. At -20 mV, the time constants of block decreased with micromolar increase in [Ca2+]o in line with a blocking rate coefficient of 1.9 x 10(8) M-1 s-1, but settled to values of 0.18 ms at [Ca2+]o beyond 50 microM. The Na+ currents were unblocked with time constant (tau u) of around 0.25 ms at strongly positive and negative membrane potentials at 22 degrees C. 7. Tau u failed to show any obvious dependence on [Ca2+]o up to the millimolar range. This finding contradicts suggestions that removal of the block occurs in a [Ca2+]o-dependent manner as a result of an increased probability of Ca2+ ion mobilization by repulsive forces with increased Ca2+ occupation of the channel. 8. The time course of unblock of Na+ currents was strongly temperature-dependent showing a Q10 of 2.5 for tau u. 9. The voltage dependence of the Na+ current block by Ca2+ ions is best accounted for by a single, centrally located Ca2+ binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗