Carla Marchetti

Pathways of cadmium influx in mammalian neurons.

Abstract: The Influx of the toxic cation Cd2+ was studied in fura 2‐loaded rat cerebellar granule neurons. In cells depolarized with Ca2+‐free, high‐KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 γM Cd2+. This increase was fully reversed by the Cd2+ chelator tetrakis(2‐pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67 ± 5%) by 1 γM nimodipine and enhanced by 1 γM Bay K 8644. Concurrent application of nimodipine and ω‐agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88 ± 5%), whereas ω‐conotoxin MVIIC (2 γM) reduced dR/dt by 24 ± 8%. These results indicate a primary role of voltage‐dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and ω‐agatoxin IVA moderately reduced dR/dt (25 ± 3%). NMDA‐driven Cd2+ entry was almost completely prevented by 1 mM Mg2+, 50 γM memantine, and 10 γM 5,7‐dichlorokynurenic acid, suggesting a major contribution of NMDA‐gated channels in glutamate‐stimulated Cd2+ influx. Moreover, perfusion with α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.

Multiple pathways of Pb2+ permeation in rat cerebellar granule neurones

The pathways of lead (Pb2+) uptake were studied in fura‐2‐loaded cerebellar granule cells from 8‐day‐old rats. In a nominal Ca‐free external bath, Pb2+ (5–50 µm) determined an increase of the fluorescence emission ratio (R = E340/E380) even in the absence of any specific stimulus. This rise was dose‐dependent, was not significantly affected by mm Mg2+ or Ca2+, but it was readily reversed by the membrane‐permeant heavy metal chelator tetrakis(2‐pyridylmethyl) ethylene‐diamine (TPEN, 100 µm), indicating that it was due to Pb2+ influx. The rate of rise, dR/dt, was increased up to a factor of 5 by depolarizing high‐KCl solution, indicating a sizeable permeation through voltage‐dependent channels. This effect was neither antagonized by nimodipine, nor enhanced by BayK8644, but it was slackened by ω‐agatoxin IVA (200 nm), suggesting an involvement of non‐L‐type calcium channels. Pb2+ influx was also stimulated by glutamic acid or NMDA in the presence of 10–30 µm glycine, but only in Mg‐free solution, suggesting that glutamate channels of the NMDA type are an additional pathway of Pb2+ uptake. Pb2+ caused a time‐, dose‐ and stimulus‐dependent saturation of the dye, whose intracellular concentration is ∼ 10 µm, indicating that intracellular Pb2+ can readily reach a concentration in the micromolar range. These results indicate that the particular vulnerability of neurones to Pb2+ poisoning is linked to the presence of specific transport systems, which mediate the rapid uptake of Pb2+ into the neurone.

Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum

Voltage-dependent calcium currents were investigated by the patch-clamp technique in whole-cell recording configuration in cultures from 8-day-old rat cerebella, which contained greater than or equal to 90% granule cells. In solutions designed to minimize the sodium and potassium conductances and in 20 mM barium, an inward current activated around -25 mV, reached a peak amplitude at +20 mV and reversed around +80 mV. In 20 mM calcium, this current was approximately 50% of that in barium. From one to three days in vitro only 16% of the cells tested (n = 20) had a current exceeding 50 pA in maximum amplitude, while after four days in vitro the current reached 100 pA in all neurons tested (n greater than 70). Verapamil (50-100 microM) reversibly depressed this current. The dihydropyridine agonist Bay K 8644 (1 microM) enhanced the maximum conductance by 25 +/- 8% (n = 4), caused a negative shift in the activation of 21 +/- 5 mV and a prolongation of the deactivation time course as the voltage was stepped back from +20 to -80 mV. The GABAB agonist baclofen (50 microM) reversibly depressed the current by 27 +/- 8% in 80% of the cells. The effect was similar to that of GABA (10 microM), when the GABAA response (chloride current) was partially blocked by bicucculine. This current can be classified as a dihydropyridine-sensitive high-voltage-activated calcium current. The modulation by GABAB agonists is likely to be significant for presynaptic inhibition.

NMDA receptors as targets of heavy metal interaction and toxicity

TheN-methyl-D-aspartate (NMDA) receptor (NR) is a ligand-gated channel that carries the slow component of the glutamate-activated postsynaptic current. Divalent metal ions can affect the NR channel activity in a voltage-dependent (Mg2+-like) or voltage-independent (Zn2+-like) manner. We have studied the effect of two toxic metals, lead (Pb2+) and nickel (Ni2+) on recombinant NR1a-NR2A and NR1a-NR2B channels expressed in RNA-injectedXenopus laevis oocytes or in transiently transfected mammalian HEK293 cells. Pb2+ caused a dose-dependent, but voltage-independent reversible inhibition of NMDA-activated channel activity similar for NR2A- and NR2B-containing receptors; it did not modify the single channel conductance, indicating that its binding site is located out of the ionic pathway of permeation. On the contrary, Ni2+ had multiple and complex effects on NR channels. It determined a voltage-dependent, Mg2+-like block by which the single channel amplitude and the mean open time were reduced in both NR2A- and NR2B-containing channels. While high (>100 µM) concentrations caused a dose-dependent reduction of the activity in both channel types, 30 µM determined a voltage-independent decrease in the frequency of NR1a-NR2A channel openings, but an increase in the frequency of NR1a-NR2B channel openings, confirming previous observations of a subunit-dependent effect of this metal. These results were interpreted under the hypothesis that Pb2+mediates a Zn2+-like voltage-independent allosteric modulation that, different from Zn2+, is subunit-independent. In contrast, Ni2+ has different modes of action, which are dependent on the NR2 subunit type present in the receptor and are likely to be related to different interaction sites. The NR2B-dependent facilitation bears close similarities with the polyamine-mediated potentiation.

Lead inhibition of NMDA channels in native and recombinant receptors

NMDA channels are key targets for lead (Pb2+) neurotoxicity and Pb2+-induced inhibition of NMDA current is age- and subunit-dependent. In rat cerebellar granule cells maintained in high KCl, glycine affinity as well as sensitivity to ifenprodil change significantly with the days in vitro, indicating a reduction of NR2B subunit expression. Pb2+ blocked NMDA current with IC50 ∼4 μM and this effect decreased significantly during the second week in vitro. In Xenopus laevis oocytes expressing recombinant NR1-NR2A, NR1-NR2B or NR1-NR2C receptors, Pb2+ inhibited glutamate-activated currents with IC50 of 3.3, 2.5 and 4.7 μM respectively. These data indicate that Pb2+ action is dependent on subunit composition and suggest that down-regulation of the NR2B subunit is correlated to a diminished sensitivity to Pb2+ inhibition.

Role of Calcium Channels in Heavy Metal Toxicity

The role of voltage-dependent Ca channels (VDCC) in the membrane permeation of two toxic metals, lead (Pb) and cadmium (Cd), was studied in mammalian cells. Both metals interact with Ca-binding sites, but, while Cd influx appears to occur mainly through the same pathways as Ca, Pb is also rapidly taken up by different passive transport systems. Furthermore, I compared the effect of Cd in two Chinese hamster ovary (CHO) cell lines, a wild-type and a modified cell line, which were permanently transfected with an L-type VDCC. When cultures were subjected to a brief (30–60 min) exposure to 50–100 μM Cd, apoptotic features, metal accumulation, and death were comparable in both cell lines although, in transfected cells, the effect of Cd treatment was partially prevented by nimodipine (VDCC antagonist) and enhanced by BayK8644 (VDCC agonist). Thus, expression of L-type Ca channels is not sufficient to modify Cd accumulation and sensitivity to a toxicological significant extent and while both Cd and Pb can take advantage of VDCC to permeate the membrane, these transport proteins are not the only, and frequently not the most important, pathways of permeation.

Subunit-dependent effects of nickel on NMDA receptor channels.

Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.

Interaction of metal ions with neurotransmitter receptors and potential role in neurodiseases

There is increasing evidence that toxic metals play a role in diseases of unknown etiology. Their action is often mediated by membrane proteins, and in particular neurotransmitter receptors. This brief review will describe recent findings on the direct interaction of metal ions with ionotropic γ-aminobutyric acid (GABAA) and glutamate receptors, the main inhibitory and excitatory neurotransmitter receptors in the mammalian central nervous system, respectively. Both hyper and hypo function of these receptors are involved in neurological and psychotic syndromes and modulation by metal ions is an important pharmacological issue. The focus will be on three xenobiotic metals, lead (Pb), cadmium (Cd) and nickel (Ni) that have no biological function and whose presence in living organisms is only detrimental, and two trace metals, zinc (Zn) and copper (Cu), which are essential for several enzymatic functions, but can mediate toxic actions if deregulated. Despite limited access to the brain and tight control by metalloproteins, exogenous metals interfere with receptor performances by mimicking physiological ions and occupying one or more modulatory sites on the protein. These interactions will be discussed as a potential cause of neuronal dysfunction.

Properties of ionic transport through phospholipid-glycolipid artificial bilayers

The ionic transport properties of dioleoylphosphatidylcholine (DOPC) membranes containing various sphingolipids were studied. Particular attention was paid to membranes formed from beta-D-glucosylceramide (GlcCer) and DOPC. They showed a marked increase in ionic permeability (up to a factor 30 with respect to pure DOPC membranes), slight cation selectivity and almost linear behaviour of the current-voltage characteristic. Bilayers containing GlcCer showed a typical conductance decrease upon increasing the temperature. We suggest the formation of clusters containing GlcCer molecules in a solid-crystalline phase. The conductance increase might be due to ionic pathways through disordered boundary regions. An increase in the mechanical breakdown potential was observed in all membranes which contain sphingolipids.

A Transient Voltage-Dependent Outward Current in Cultured Cerebellar Granules

Granule cells were dissociated from rat cerebella with a procedure that yields a 98% pure cell population. Potassium currents in these cells were studied using the patch-clamp technique. Depolarizing pulses of 10 mV step and 100 ms duration from a holding potential of −80 mV elicited two different potassium outward currents: a transient, low-voltage activated component and a long lasting, high-voltage activated component. At +30 mV, the total current reached an amplitude of 2 nA (mean value of 15 experiments). The reversal potential of the transient current, estimated by measuring tail currents, was −77 mV, close to that predicted by the Nernst equation. The transient current was half inactivated with a holding potential of −78 mV and completely inactivated with −50 mV or more positive holding potentials. Finally, the current decay could be fitted by the sum of two exponentials with time constants of about 20 and 250 ms.