Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

Membrane properties of area postrema neurons

Intrinsic membrane properties, voltage-dependent sodium and voltage-dependent potassium currents of area postrema neurons in culture have been characterized with respect to their voltage dependence, time dependence and sensitivity to specific blocking agents. The area postrema is a hindbrain circumventricular organ which is known to have an important role in the central regulation of cardiovascular function. This study is the first to describe the biophysical properties of ion channels present in rat area postrema neurons. Recordings in current-clamp mode revealed a mean resting membrane potential of -55.0 ± 1.6 (n = 24) mV and an input resistance of 213.6 ± 23 M Ω. For the 24 neurons tested, the evoked action potential had a mean threshold of 38.8 ± 2 mV and a mean amplitude of 107.3 ± 15 mV. Our results show that the area postrema possesses only one principle sodium current which is completely abolished by 5 μM tetrodotoxin (TTX) (n = 28). This current activated near −50 mV and reached peak amplitude at −30 mV. The area postrema does not possess a TTX insensitive sodium current. The area postrema has at least two types of potassium currents. All area postrema neurons studied with tetraethylamonium (TEA) (n = 40) showed the presence of a slowly activating outward current which was present at voltages greater than −40 mV and was blocked by 10 mM TEA. In addition, 75% of the neurons studied (n = 30/40) also showed a rapidly inactivating, 4-AP sensitive IA type current which activated near −30 mV. Angiotensin II attenuated both the peak and the steady-state potassium currents, suggesting that angiotensin 11 may modulate area postrema activity by inhibiting voltage-gated potassium channels.     

Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat

Effects of Zn²⁺ and other polyvalent cations on glycine-induced currents in the freshly dissociated rat dorsal motor nucleus of the vagus neurons were investigated under voltage-clamp conditions by the use of the nystatin-perforated patch recording configuration. Glycine evoked a Cl⁻ current in a concentration-dependent manner with a half-maximum effective concentration (EC₅₀) of 3.3×10⁻⁵ M. Strychnine inhibited the 3×10⁻⁵ M glycine-induced current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 6.8×10⁻⁷ M. At low concentrations (3×10⁻⁸ M–3×10⁻⁶ M), Zn²⁺ potentiated the current elicited by 3×10⁻⁵ M glycine. On the other hand, at concentrations higher than 10⁻⁵ M, Zn²⁺ inhibited the glycine response. The biphasic action of Zn²⁺ was mimicked by Ni²⁺, but La³⁺ and Co²⁺ had only potentiating effect. Zn²⁺ shifted the concentration–response curve for the glycine-induced current without changing the maximum response, and the EC₅₀ values for the glycine response in the absence and presence of 10⁻⁶ M and 10⁻⁴ M Zn²⁺ were 3.3×10⁻⁵ M, 1.3×10⁻⁵ M and 1.3×10⁻⁴ M, respectively. These results suggest that the biphasic modulation of glycine response by Zn²⁺ results from changes in apparent glycine affinity.     

Changes in Ca channel expression upon differentiation of SN56 cholinergic cells

The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca²⁺ channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the α_1H (CaV3.2) Ca²⁺ channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 μM), 37% by the N-type channel antagonist ω-conotoxin-GVIA (1 μM), and 15% by the P/Q-type channel antagonist ω-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca²⁺ channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below −10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance–voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.     

Mercury (Hg) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons

Inorganic mercury (Hg²⁺) reduced voltage-gated calcium channel currents irreversibility in two different preparations. In cultured rat dorsal root ganglion (DRG) neurons, studied with the whole cell patch clamp technique, a rapid concentration-dependent decrease in the L/N-type currents to a steady state was observed with an IC₅₀ of 1.1 μM and a Hill coefficient of 1.3 T-currents were blocked with Hg²⁺ in the same concentration range (0.5–2 μM). With increasing Hg²⁺ concentrations a slow membrane current was additionally activated most obviously at concentrations over 2 μM Hg²⁺. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg²⁺. The current-voltage (I–V) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg²⁺ to the channel protein and/or modifying its gating properties. In neurons of the abdominal ganglion of Aplysia californica, studied with the two electrode voltage clamp technique, a continous decrease of calcium channel currents was seen even with the lowest used concentration of Hg²⁺ (5 μM). A steady state was not reached and the effect was irreversible without any change on resting membrane currents, even with high concentrations (up to 50 μM). No shift of the I–V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg²⁺ concentrations such low have not been reported before. We conclude that neurotoxic effects of inorganic mercury could be partially due to the irreversible blockade of voltage-activated calcium channels.     

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (I(Ca(V))). I(Ca(V)) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba2+ as charge carrier. In large diameter neurones (>35 micorm), a concentration of 50 microM was needed to reduce I(Ca(V)) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (< or =30 microm), the IC50 was 0.36 microM. This effect was concentration dependent with a threshold below 0.025 microM and maximal blockade (>80%) at 5 microM. The current-voltage relation was shifted to the hyperpolarized direction with an increase of the current between -40 and -10 mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 microM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I(Ca(V)) at negative and decreases I(Ca(V)) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.     

Ca channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca²⁺ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O₂). Currents activated at potentials of −40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca²⁺ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 μM) always partially inhibited currents and Bay K 8644 (2–5 μM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker ω-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca²⁺ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca²⁺ channels in type I cells.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

Activation of a non-specific cation conductance by intracellular injection of inositol 1,3,4,5-tetrakisphosphate into identified neurons of Aplysia

The ionic mechanism of the effect of intracellulary injected inositol 1,3,4,5-tetrakisphosphate (IP₄) on the membrane of identified neurons (R9–R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure injection, and ion-substitution techniques. Intracellular injection of IP₄ into a neuron voltage-clamped at −45 mV reproducibly induced a slow inward current (20–60 s in duration, 3–5 nA in amplitude) associated with a conductance increase. The current was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential was −21 mV. The IP₄-induced inward current was sensitive to changes in the external Na⁺, Ca²⁺ and K⁺ concentration but not to changes in Cl⁻ concentration, and was resistant to tetrodotoxin (50 μM). When the cell was perfused with tetraethylammonium (5 mM) but not with 4-aminopyridine (5 mM), the IP₄-induced inward current recorded at −45 mV slightly increased. The IP₄-induced inward current was partially reduced by calcium channel blockers (Co²⁺ and Mn²⁺). These results suggest that intracellularly injected IP₄ can activate a non-specific cation conductance.     

The effects of Zn on long-term potentiation of C fiber-evoked potentials in the rat spinal dorsal horn

Tetanic stimuli of peripheral C fibers produces long-term potentiation (LTP) in the spinal cord, which may contribute to sensitization of spinal pain-sensitive neurons. Zn²⁺ is widely distributed in the central nervous system and has blocked (LTP) in the hippocampus. The present study examined the effects of Zn²⁺ on the induction and maintenance of C fiber-evoked LTP in the deep dorsal horn of spinalized rats in vivo. The sciatic nerve was stimulated by tetanic stimuli for inducing LTP. (1) Topical administration of Zinc chloride (15 μM) to the spinal cord 15 min before tetanic stimulation completely blocked the induction of LTP, but not the baseline C responses. When Zn²⁺ was given 2 h after induction of LTP, no significant effect occurred. (2) Chelation of Zn²⁺ by disodium calcium ethylene diaminetelraacetate (CaEDTA) (500 μM) resulted in no effect on LTP. (3) Coadministration of Zn²⁺ (15 μM) and N-methyl-D-aspartic acid (NMDA) (5 μM) significantly attenuated C fiber-evoked potentials, which was prevented by the NMDA receptor antagonist AP-5 (100 μM). The present results showed that Zn²⁺ may contribute to the modulation of the formation, but not the maintenance, of spinal LTP. NMDA receptors may be involved in Zn²⁺-induced modulation.     

L-type Ca channel-mediated Zn toxicity and modulation by ZnT-1 in PC12 cells

In view of evidence that Zn²⁺ neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn²⁺ neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 μM ZnCl₂ for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca²⁺ channel antagonist, nimodipine, and increased by the L-type Ca²⁺ channel opener, S(−)-Bay K 8644. At lower insult levels (200 μM Zn²⁺+Bay K 8644), Zn²⁺-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH₂F (Z-VAD); at higher insult levels (1000 μM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn²⁺ neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative forms of rat ZnT-1 (rZnT-1). Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn²⁺ efflux and reduced vulnerability to Zn²⁺-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.     

Ion channels activated by quinolinic acid in cultured rat hippocampal neurons

The membrane responses to quinolinic acid, an excitotoxic brain metabolite, were studied in cultured rat hippocampal neurons with the patch-clamp technique. In the whole-cell recording mode, pressure applications of quinolinic acid elicited inwardly directed membrane currents over a membrane potential range of −60 to −5 mV. The current response reversed at about 0 mV. The current-voltage (I–V) relation of the response had a negative slope conductance at membrane potentials more negative than −40 mV. On removal of Mg²⁺ from the extracellular solution, the current response showed no region of negative slope conductance at potentials more positive than −60 mV. In Mg²⁺-free solution applications of quinolinic acid elicited discrete pulse-like current flows through the outside-out membrane patch. The single channel conductance was 40–46 pS over a membrane potential range of −40 to −80 mV, and 50–55 pS at membrane potentials more positive than +30 mV, showing an outward rectification. These values of the single channel conductance were similar to those of the main conducting state of the channels activated by (NMDA). The responses to quinolinic acid were completely suppressed by the NMDA receptor antagonist (±)-2-amino-5-phosphonovaleric acid. The results indicate that quinolinic acid selectively activates NMDA receptors in the cultured rat hippocampal neurons.     

Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (I_Ca). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. I_Ca was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to –30 mV from a holding voltage of –80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 μm nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 μm Bay K 8644 or when the external Ca²⁺ was substituted by equimolar Ba²⁺. Omega-conotoxin (10 μm ω-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type I_Ca, while the sustained component is a high-voltage activated L-type I_Ca. T-type I_Ca was recorded in all cells tested, while L-type I_Ca was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type I_Ca is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type I_Ca that had been previously described, bipolar cells of the mammalian retina also contain L-type I_Ca similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.     

Chronic exposure to inorganic lead increases high-threshold voltage-gated calcium currents in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma (PC12) cells were exposed to lead acetate (0, 10, 25 and 50 μM) in their growth media for up to 12 weeks. High-threshold voltage-gated calcium currents were recorded each week from nerve growth factor-differentiated PC12 cells using the whole-cell patch-clamp technique. Chronic exposure for 1 month did not modify peak or sustained calcium current amplitudes in lead-treated cells when compared to sister control cultures. Two month exposure to 25 and 50 μM significantly increased peak and sustained calcium current amplitudes, while 10 μM had little effect. During the third month of exposure, peak and sustained calcium current amplitudes remained increased in the cells exposed to 25 and 50 μM lead acetate. By the end of the second month of exposure to 25 and 50 μM lead acetate, the voltage at which maximal current amplitude was attained shifted from +10 mV to 0 mV. The observed effects of toxicologically relevant lead concentrations on high-threshold calcium currents in chronically exposed mammalian cells provide further support for the notion that at least one cellular target of the heavy metal's neurotoxic action may be the voltage-gated calcium channel.     

Inhibition of NMDA-induced protein kinase C translocation by a Zn chelator: Implication of intracellular Zn

The role of intracellular Zn²⁺ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [³H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-d-aspartate (NMDA, 100 μM) and calcium ionophore A23187 (0.3–30 μM) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [³H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn²⁺ but not by that with Ca²⁺. The addition of 500 μM Zn²⁺ to the lysate of synaptoneurosomes induced an increase of [³H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 μM Ca²⁺. Low concentrations of Zn²⁺ (10 μM), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10 μM Ca²⁺ in the lysate. Under those conditions TPEN inhibited the Zn²⁺-potentiated Ca²⁺-dependent changes in the binding. These results suggest that intracellular Zn²⁺ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes.     

SnCl(2) reduces voltage-activated calcium channel currents of dorsal root ganglion neurons of rats

Stannous dichloride (SnCl₂) occurs in the environment where it has been especially enriched in aquatic ecosystems. Furthermore, it is used in food manufacturing (e.g. for stabilizing soft drinks or as an anti-corrosive substance) and in nuclear medicine where it is employed as a reducing agent for technecium-99m (99mTc) and therefore is applied intravenously to human beings.SnCl₂ is known to have toxic effects on the nervous system which can be related to alterations of intracellular calcium homeostasis ([Ca²⁺]_i). In this study the whole cell patch clamp technique is used on dorsal root ganglion neurons of 3-week-old “Wistar” rats to evaluate the effects of SnCl₂ on voltage-activated calcium channel currents (I_Ca(V)).I_Ca(V) were reduced concentration-dependently by SnCl₂ (1–50 μM). 1 μM SnCl₂ reduced I_Ca(V) by 8.1 ± 4.5% (peak current) and 19.2 ± 8.9% (sustained current), whereas 50 μM inhibited I_Ca(V) by 50.6 ± 4.3% (peak current) and 55.6 ± 11.3% (sustained current). Sustained currents were slightly but not significantly more reduced than peak currents. The effect appeared not to be reversible. The threshold concentration was below 1 μM.The current–voltage relation did not shift which is an indication that different calcium channel subtypes were equally affected. There was a slight but not significant shift of the activation/inactivation curves towards the depolarizing direction.We conclude that voltage-gated calcium channels are affected by Sn²⁺ similarly to other divalent metal cations (e.g. Pb²⁺ or Zn²⁺).The reduction of I_Ca(V) could be related to the neurotoxic effects of SnCl₂.