Biomedical subjects

E Carbone

Publications and source records attributed to E Carbone.

8 recordsLinked to original sources

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↗

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↗

PubMed1979

K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane.

In the range of pH examined (5.2-10), variations of internal pH from high to low values result in a reversible decrease of the conductance of the open K channels, without significantly affecting the kinetics parameters. A linear plot of the conductance versus internal pH suggests the existence of a titratable group that has an apparent pKa of about 6.9, and that is accessible to protons only from the intracellular side of the membrane.

Animals↗

PubMed1978

Action of extracellular pH on Na+ and K+ membrane currents in the giant axon of Loligo vulgaris.

Voltage-clamp currents and resting membrane potential of squid giant axons have been studied at extracellular pH varying between 4 and 10. The membrane currents, analyzed according to the Hodgkin-Huxley equations, showed that sodium permeability, PNa(E), and potassium conductance gK(E), curves were shifted toward positive voltages by different amounts and slightly depressed as the external pH was lowered. Under the same conditions, taum(E) and taun(E) were found to be enhanced and shifted to a larger extent in the same direction. The rate constants alpham and alphan were shifted substantially toward positive voltages, but betam and betan changed hardly at all. The shift of the alpham(E) curve was analyzed in terms of a fixed surface charge model; it indicates that unspecific negative groups with an approximate pKa of 4.5 are located in the vicinity of sodium active sites with an average charge separation of 8 A. A similar figure is obtained for the potassium system from the shift of the alphan(E) curve.

Animals↗

PubMed1976

Orientation and rotational freedom of fluorescent probes in lecithin bilayers.

The fluorescence polarization properties of lecithin bilayers stained with 2,6-MANS and 1,8 ANS under applied potential steps have been studied. The fluorescence signal components of both dyes were found to have different sign and relative amplitude, suggesting that 1,8-ANS and 2,6-MANS behave differently when bound to black lipid membranes. In order to determine the location and the extent of rotational brownian motions of the bound chromophores, the experimental data were analyzed by using a simplified physico-mathematical model. According to it 2,6-MANS appears to have a ratio rho/tau higher than 1.8-ANS (rho being the rotational relaxation of in plane rotations and tau the lifetime of the excited singlet state of the bound molecules), suggesting that the former chromophore is more tightly held inside the bilayers. Furthermore, 2,6-MANS is found to possess the absorption and emission oscillators more closely oriented to the normal of membrane surface, while 1,8-ANS has both oscillators almost near the plane of the bilayers. The results furnish also a fair estimate of the random molecular motion own by the phospholipid molecules at room temperature. The comparison of the present data with those obtained from squid axon membranes confirms the validity of the proposed physical model, yielding a rough estimate of the axon membrane-area covered by integral protein macromolecules. These preliminary results derived from lecithin model membranes suggest that fluorescence polarization techniques can provide valuable informations if applied to study the macromolecular organization of in vitro reconstituted membranes.

Animals↗

PubMed1975

Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates.

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are soley due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6-MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.

Anilino Naphthalenesulfonates↗