Characterization of a Voltage-dependent Calcium Current in the Human Neuroblastoma Cell Line SH-SY5Y During Differentiation

In the presence of retinoic acid, cultured human neuroblastoma SH-SY5Y cells grow processes indicative of neuronal differentiation. A voltage-gated Ca current is already present in undifferentiated cells. A gradual increase of the Ca current density occurs during cell differentiation. According to kinetic and pharmacological properties, Ca currents in differentiated cells are indistinguishable from those elicitable in undifferentiated cells and resemble features of the high-voltage activated currents present in mammalian neuronal cells. omega-conotoxin strongly depresses high-voltage activated currents, both in undifferentiated and in differentiated SH-SY5Y cells. Interestingly, the Ca agonist Bay K 8644 is effective, albeit with great variability from cell to cell, only in differentiated cells and only when barium is the current carrier through the Ca channels. A diversity of high-voltage activated Ca channels of distinct pharmacology has been recently observed in other kinds of neurons. This requires a redefinition of the role that voltage-dependent Ca channel subtypes can play in mammalian neurons.     

Cholinesterase inhibitor use is associated with increased plasma levels of anti-Abeta 1–42 antibodies in Alzheimer's disease patients

Acetyl-cholinesterase inhibitors (AChEI) are drugs frequently prescribed for the treatment of Alzheimer's disease (AD), exerting an effect on cognition, as well as on behavioural and psychological symptoms of dementia and activities of daily living. The efficacy of AChEI may be ascribed not only to the activation of cholinergic transmission, but also to other mechanisms, among which a putative regulation of the immune response has already been hypothesized. In the present study, we evaluated, in a cross-sectional sample of 66 AD patients and 48 healthy controls, the putative influence of AChEI on anti-Abeta 1–42 antibody plasma levels by ELISA assay. AD patients receiving AChEI therapy showed increased plasma levels of anti-Abeta 1–42 antibodies respect to untreated AD patients and antibodies levels similar to those of healthy controls, both before and after normalization by total IgG values. Our results support a potential role of AChEI in the modulation of the immune response against Abeta. We suggest that a strategy aimed at increasing the endogenous response against this peptide might represent an interesting therapeutic target to be further investigated.     

Calcium channels functional roles in the frog semicircular canal

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by omegaconotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and omega-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.     

Pre- and postsynaptic excitatory action of glutamate agonists on frog vestibular receptors

In order to investigate the localization and the type(s) of excitatory amino acid receptors in the frog vestibular system, the exogenous amino acid agonists Quisqualic acid, Kainic acid and N-methyl-D-aspartic acid were tested on the sensory organ of semicircular canals. Intracellular recordings of the resting discharge from single afferents showed that these agonists exerted a complex excitatory action consisting in a rapid and brief increase in frequency of both EPSPs and spikes, followed by a slower and longer lasting membrane depolarization. The progressive impairment of natural transmitter release achieved by adding Mg2+ or Co2+ in the bath caused a dose-dependent decrease of the agonist-induced afferent discharge, without substantially affecting axonal depolarization. These results suggest that the exogenous amino acid agonists act both pre- and postsynaptically on the vestibular organs. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in inducing excitatory effects, suggesting that the amino acid receptors located on both hair cells and afferent endings are mainly of the non-NMDA type. The present findings, while not excluding that an excitatory amino acid may be the afferent transmitter, highlight its possible function as a presynaptic modulator of the afferent transmission in the frog vestibular system.     

Na+ currents in vestibular type I and type II hair cells of the embryo and adult chicken

In birds, type I and type II hair cells differentiate before birth. Here we describe that chick hair cells, from the semicircular canals, begin expressing a voltage-dependent Na current (INa) from embryonic day 14 (E14) and continue to express the current up to hatching (E21). During this period, INa was present in most (31/43) type I hair cells irrespective of their position in the crista, in most type II hair cells located far from the planum semilunatum (48/63), but only occasionally in type II hair cells close to the planum semilunatum (2/35). INa activated close to -60 mV, showed fast time- and voltage-dependent activation and inactivation, and was completely, and reversibly, blocked by submicromolar concentrations of tetrodotoxin (Kd = 17 nM). One peculiar property of INa concerns its steady-state inactivation, which is complete at -60 mV (half-inactivating voltage = -96 mV). INa was found in type I and type II hair cells from the adult chicken as well, where it had similar, although possibly not identical, properties and regional distribution. Current-clamp experiments showed that INa could contribute to the voltage response provided that the cell membrane was depolarized from holding potentials more negative than -80 mV. When recruited, INa produced a significant acceleration of the cell membrane depolarization, which occasionally elicited a large rapid depolarization followed by a rapid repolarization (action-potential-like response). Possible physiological roles for INa in the embryo and adult chicken are discussed.     

Regional distribution of calcium currents in frog semicircular canal hair cells

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.     

Degradation and configurational changes of thioridazine 2-sulfoxide

Thioridazine (THD) is a phenothiazine neuroleptic drug used for the treatment of psychiatric disorders. After oral administration THD is extensively biotransformed to thioridazine 2-sulfone (THD 2-SO(2)), thioridazine 5-sulfoxide (THD 5-SO) and thioridazine 2-sulfoxide (THD 2-SO). THD 2-SO and THD 5-SO have two chiral centres and therefore exist as two diastereoisomeric pairs. The degradation and epimerization of THD 2-SO in human plasma, buffer and methanolic solutions were studied using an enantioselective HPLC method. The samples were prepared by liquid-liquid extraction with diethyl ether and the chiral resolution of the enantiomers was carried out on a Chiralpak AD column using a mobile phase consisting of hexane:ethanol:2-propanol (90:7:3, v/v/v) containing 0.2% diethylamine. The method was validated and used to study the degradation and epimerization under different conditions of incubation. Our results showed that both enantiomers were stable at varying temperatures, pH and ionic strengths; however, solubility problems were observed, mainly at pH 8.5. The influence of light on stability was studied using methanolic solutions and degradation and epimerization of the THD 2-SO enantiomers were observed under UV light of 366 and 254nm, respectively.