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Mercury (Hg2+) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons
121
Brain Research, 632 (1993) 121-126 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19552
Mercury
(Hg 2+) decreases
voltage-gated calcium channel currents in rat D R G and Aplysia neurons M. Pekel, B. Platt, D. Bfisselberg *
Physiologisches Institut 11, Heinrich-Heine Universitiit Diisseldorf, Moorenstrafle 5, D-40225 Diisseldorf, Germany (Accepted 2 August 1993)
Key words: Mercury; Rat dorsal root ganglion (DRG) neuron; Aplysia neuron; Calcium channel current
Inorganic mercury (Hg 2+ ) reduced voltage-gated calcium channel currents irreversibly 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 izM and a Hill coefficient of 1.3. T-currents were blocked with Hg 2÷ in the same concentration range (0.5-2 tzM). With increasing Hg 2+ concentrations a slow membrane current was additionally activated, most obviously at concentrations over 2 / z M Hg 2+. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg 2÷. The current-voltage ( I - V ) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg 2+ to the channel protein a n d / o r 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 Hg 2+ (5 ~M). 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 p.M). No shift of the I - V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg 2+ 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.
INTRODUCTION Calcium is one of the most important messengers for signal transfer in cells. Therefore the intracellular calcium concentration is regulated by a variety of mechanisms including selective voltage-activated channels, which are opened by depolarization. For vertebrates, four different voltage-activated calcium currents have been characterized: a rapidly inactivating low-threshold-activated (T) current, a slowly inactivating high-threshold-activated (L) current, a highthreshold rapidly inactivating (N) current 32 and recently an additional P-type channel has been described 27. Although invertebrate calcium channels do not correspond exactly to any of those in vertebrates 1,1°, the calcium channels of Aplysia neurons have some similarities with the L-channel. Voltage-gated calcium channels are blocked by a variety of divalent and trivalent metal c a t i o n s 6-9,17,19.
* Corresponding author. Fax: (49) (211) 311 4231.
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 0 7 4 - D
Many of these ions are known to be neurotoxic both in inorganic and organic forms 7A4'15. Some studies suggest that methylmercury as well as inorganic mercury have effects on calcium currents in rat forebrain synaptosomes 4'21. Methylmercury produces behavioral changes in rats 11'3°, learning deficits in rats after prenatal exposure 16'28 and visual deficites in monkeys 29. Inorganic mercury, a highly neurotoxic agent z°'33, interacts with GABA-activated chloride channels 3 and increases evoked transmitter release at frog neuromuscular junction 26. In humans Hg 2÷ intoxication results in a loss of coordination, decreased sensation, tremor and abnormal reflexes 2. Hg 2+ persists in the nervous system for a long time 12. It inhibits the synaptosomal N a + / K +ATPase in the brain 25, blocks phosphorylation processes 23, and modulates messenger RNA metabolism 22 possibly via binding to -SH groups 12. Little is known about the effects of inorganic mercury on voltage-activated calcium channels 7 and there
122 TABLE I Composition of the basic external solutions All concentrations are in mM. HEPES, N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid; TEA-Br, tetra-ethylammonium bromide; TEA-CI, tetraethylammonium chloride; 4-AP, 4-aminopyridine; TrisCI, Tris (hydroxymethyl) aminomethane hydrochloride; Tris-base, Tris (hydroxymethyl) a m i n o m e t h a n e base; TTX, tetrodotoxine. Artificial A S W for Tyrodes h Ba- T T X ~ seawater (ASW) " calcium current a NaCI 480 10 KCI 10 CaCt 2 BaCI 31) MgCI 2 5 HEPES TEA-Br TEA-CI 4-AP Tris-Cl Tris-base TTX Glucose
145 2.5 1.5
10 20
45
10 l 10
1.2 10
200 135 1 234 59
130
10
0.002 10
10
Mercury (11) chloride (HgCI 2, Sigma 99,999'~ purity) was made as a 20 m M stock solution and was added to external Ba-TTX solution just before each application. External solutions were applied by a fast bath perfusion system (bath volume about I ml, perfusion volume 5 - 1 0 ml within 15-30 s). All experiments were performed at room temperature (20-22°C). The cells were routinely clamped at - 8 0 mV, which is close to the resting m e m b r a n e potential of DR(} cells. Voltage steps (one per 10 s) from - 80 to 0 mV for 75 ms werc used to evoke L/N-calcium channel currents (carried by barium to avoid intracellular effects through elevated calcium concentration). T-type currents were evoked by voltage steps from - 80 lo - 35 mV. a potential at which L- or N-channels are not appreciably activated. The total blockade of the resulting currents by 50 # M Cd e" indicates an adaequate isolation of calcium currents 2~~t. C o m m a n d pulses were delivered by an EPC-9 unit (HEKA) controlled by an A T A R I ST computer. Raw data were on-line leak-corrected by a P / 4 protocol, as described by Chad and Eckert L~. To determine the time course of the reduction of the calcium channel currents, command pulses were given every 10 s. Maximum current was measured. Data were filtered on-line at 2.3 kHz, stored on hard disk and analysed off-line using a M2-LAB software package. Cells with a run down of more than 10¢~ within 5 rain were excluded. The Hg 2+ dose response relationship tk)r D R G neurons was determined by fitting mean currents to the Langmuir equation:
~ pH was adjusted to 7.57; h pH was adjusted to 7.4 with NaOH; c pH was adjusted to 7.4 with T E A - O H . lc';,z ~ ( H g 2+ ) = lea2+ (control).
is no electrophysiological investigations on this question in vertebrates. We report here the effects of inorganic mercury on rat DRG and Aplysia neurons. MATERIAL
AND METHODS
D R G preparation For the vertebrate preparation we used primary cultures of dorsal-root ganglion ( D R G ) neurons from 2 - 3 day old rat pups as described by Wood and co-workers 34. Patch-clamp recordings in the whole-cell mode were obtained from 2 - 3 day old cultures to avoid the space-clamping problems which arise from the long axons and dendrites characteristic of older cultures. Electrode resistance was between 2 and 5 M / / . The pipette solution had the following composition (in mM): CsCI 135, HEPES 10, MgCI 2 1.2, Na-ATP 4; pH was adjusted to 7.2 using CsOH. The culture medium was replaced by Tyrodes solution (Table I) and a seal was established before this solution was replaced by an external solution for calcium channel current recordings (Ba-TI'X, Table I).
A
A
-t_
-8(I niV
l-
(
K,,,
t"
1+ ~ [~g~+ ] j
where 1Ca2+(Hg 2+ ) is the calcium current measured in the presence of a given concentration of Hg 2+, Ica2+(control) is the calcium current before addition of Hg 2+, K m is the appearent dissotiation constant and n is the Hill coefficient.
Aplysia preparation The abdominal ganglion of the marine mollusc, Aplysia californica (150-200 g; Pacific Biomarine Supply and Marine Specimens Unlimited, CA), was dissected and incubated in artificial seawater (ASW, Table I). Cells were impaled with two independent microelectrodes with resistances between 3 and 8 M / Z The voltage electrode was filled with 3 M potassium acetate; the current electrode with 3 M CsCl for blocking potassium channels. Neurons were identified by soma location, pigmentation and response properties to acetylcholine TM. All experiments were done in the voltage-clamp mode using a Dagan model 8500 voltage clamp amplifier. Cells were clamped at a holding potential of - 4 5 mV which is close to the
G
B 0 mV
I
-35 m V __3 -80 m ~
0 mV ____] -45 m ~
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,
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Fig. 1. The effect of 0.5 and 2 ~ M Hg z÷ on voltage-activated calcium channel currents (L/N-currents, (A), and T-currents, (B)) of rat dorsal root ganglion cells and of 20 ~ M Hg 2+ on calcium currents of Aplysia neurons (C). Note that in C the two superimposed traces are indicating different times of voltage step activation and not different concentrations of Hg" . Leak currents are subtracted. The voltage steps are shown in the upper trace.
123 m V for D R G neurons and - 4 5 m V for Aplysia neurons). Hg 2÷ caused a reduction of calcium channel currents in both preparations. In D R G ceils two effects by Hg 2÷ were seen: with lower concentrations ( > 2 g M ) there was a fast reduction of the calcium channel currents and at higher concentrations, in addition an increase in m e m b r a n e current was evident. The lower current trace in Fig. 1A results from a voltage step from - 8 0 to 0 mV. The current is long lasting and not inactivating during the step command. Superimposed are two traces measured after application of 0.5 and 2 /~M Hg 2+, respectively, demonstrating the dose-dependent reduction of this current by 15% for 0.5/xM and 70% for 2 /.~M Hg 2÷. The effect of Hg 2÷ on T-type current is shown in Fig. lB. 2 p.M Hg 2÷ reduced the current by more than 75%. Calcium channel currents of Aplysia neurons are shown in Fig. 1C. The transient and the long lasting component are both reduced by Hg 2÷ but as indicated in the two superimposed traces, taken 5 and '13 min after application of 20 ~ M Hg 2÷, the current declined over the whole period of application. Fig. 2A shows a representative t i m e - c o u r s e of calcium currents from a D R G neuron during application of increasing Hg 2÷ concentrations. Increasing concentrations of Hg 2+ cause a stepwise decrease of calcium channel currents. With low concentrations of Hg 2÷ (up to 2 /xM) a steady state is achieved within less than 1 min. Fig. 2B illustrates the effect of 50 g M Hg 2+ on the t i m e - c o u r s e of calcium currents in Aplysia neurons. Data points are means of 4 neurons. The onset of the effect of Hg 2÷ is very slow and failed to reach a plateau during the 8 min of application. U p o n wash there was no obvious recovery in both preparations. The concentration-response curve for Hg 2+ action on the L / N - t y p e currents (caused by a voltage step from the holding potential to 0 mV) of D R G neurons
resting m e m b r a n e potential of these invertebrate neurons. Virtual ground monitor currents were filtered at 3 kHz and a 30 kHz cut-off frequency was used for the pre-amplifier. To record calcium currents the A S W was replaced by an sodium-free A S W for calcium currents (Table I). The experiments were performed at 20-22°C. HgC12 (Fisher, 99.999%)-containing Ca-ASW was applied by a rapid perfusion system for a rapid change of the solution (perfusion rate about 2 m l / m i n ) . The leakage current was subtracted using a P / 3 protocol 13. Current steps of 70 ms from - 45 mV to + 20 mV were applied at 20 s intervals. Peak amplitudes were constant for several hours. Calcium currents were recorded on a videotape via a digitizing unit. Using an IBM A T computer and programs written in ASYST, the leak currents were subtracted and peak currents evaluated.
RESULTS Mercury was applied to 35 D R G neurons and 17
Aplysia neurons. L / N channel currents were measured in all D R G neurons, T-type channel currents in 5 D R G neurons. The I - V relation was investigated in 6 neurons. A comparison of the control traces in Fig. 1 A - C illustrates differences between the typical calcium channel currents of D R G neurons and Aplysia neurons. D R G neurons display different calcium channel currents which can be distinguished by the pulse protocol and their inactivation rate: both L- and N-type currents were activated by depolarizing steps from - 8 0 to 0 m V and gave a peak current of 2.3 nA (Fig. 1A). We did not separate these two calcium channel currents. The transient T-type current was activated by a depolarizion pulse from - 8 0 to - 3 5 mV. The current inactivated rapidly and had a smaller amplitude (500 pAl. The calcium channel current of Aplysia neurons had a transient and a long lasting component. Peak current was 850 nA in the larger neurons of Aplysia. The maximum values of the I - V relation (control traces in Fig. 4A,B) was different for calcium channel currents of D R G and Aplysia neurons. This is due to different charge carriers (Ba 2÷ for D R G neurons and Ca 2÷ for Aplysia cells), different concentrations of the charge carrier and different holding potentials ( - 8 0
A
B In Xi 2
511
11
I
I
I
5
Ill
15
Iminl
i
]
2
9
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Iminl
Fig. 2. T i m e - c o u r s e of reduction of the peak calcium current during application of increasing concentrations of Hg 2÷ for a D R G neuron (A) and the t i m e - c o u r s e for the peak calcium current during application of 5 0 / ~ M Hg 2÷ for Aplysia neurons (mean values for n = 4, vertical lines indicate standard deviation, B).
124
!00
B
A 2 pM
C 5 pM
50 pM
wash
c
' !l
"~ 50 .=.
~5 min
I rain
2 min
0.1
1
Fig. 5. Uncorrected contlnous current registration tbr two DRG neurons (A,B) and for an Aplysia neuron (C). The peaks indicate the currents at a voltage step. As the leak currents are not subtracted, they are superimposed on the calcium currents. Low concentrations of Hg 2+ reduce only the calcium currents in DRG neurons (A), while higher concentrations (5/xM) have an additional effect on the leak current (B). In Aplysia neurons even 50 ~zM Hg z+ do not change the leak current (C).
[pM1
Fig. 3. Dose-response relationship for the inhibitory effect of Hg 2+ on rat DRG neurons. Half of the peak calcium current was inhibited at a concentration of 1.1 ~M.
is shown in Fig. 3. The degree of inhibition in 30 neurons is plotted vs. concentration. The data points were fitted by a sigmoid curve. The cumulative ICs0 is 1.1 /~M and the cumultative Hill coefficient 1.3. Calcium channel currents of Aplysia neurons decreased continously over the time of Hg 2+ application without reaching a steady state - therefore it was impossible to record a concentration-response relationship. The I - V relations plotted in Fig. 4 show remarkable differences between calcium channel currents in D R G neurons (Fig. 4A) and Aplysia neurons (Fig. 4B). These curves were obtained by measuring the calcium channel currents generated by a voltage step from the resting m e m b r a n e potential ( - 8 0 mV in D R G cells and - 4 5 mV in Aplysia cells) to varying potentials which are indicated at the x-axis. The currents increase with voltage steps to more depolarized values (left part of the curves). This reflects the increasing activation of calcium channels. With even greater depolarizating voltage steps there is a reduction of the magnitude of the calcium channel current, which reflects the decreased driving force for Ca 2+ ions due to the increas-
ing depolarization. In D R G neurons a maximum current was generated by a step to - 10 mV (or - 5 mV) (Fig. 4A). The voltage at which the maximum current is elicited shifts in the presence of 2 ~ M Hg 2+ from - 5 mV in the control to + 10 mV (Fig. 4A). This indicates a change in the kinetics of channel activation and suggest toxic actions at a level of charge sites on the channel itself. In Aplysia neurons the 'best' voltage step was from the holding potential to + 20 mV (Fig. 4B). The absence of altered I - V relations in Aplysia neurons shows that Hg 2+ reduces the number of functional channels a n d / o r the conductance rather than the voltage dependence of the channels (Fig. 4B). In D R G neurons high concentrations of Hg 2+ (more than 2 g M ) caused a remarkable sustained increase of the holding current. The time-courses of the raw data in Fig. 5 indicate at 2 g M Hg 2+ a clear suppression of calcium channel currents (Fig. 5 A) without changes in m e m b r a n e currents while 5 ~zM Hg 2+ cause an additional increase in holding current. At concentrations lower than 2 ~ M the non-specific membrane current
B
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Fig. 4. Current-voltage relation of voltage-activated calcium currents for a rat DRG neuron (A) and for Aplysia neurons (n = 4, B). Note that the maximum of the control curves for both preparations differs and that a shift of the curve maximum after application of Hg 2+ appears only for DRG neurons (A).
125 was very small and developed slowly. This effect was not obtained in Aplysia neurons (Fig. 5C). DISCUSSION Hg 2+ blocks voltage-activated calcium channel currents in rat D R G cells as well as in Aplysia neurons at micromolar concentrations. The mammalian L / N - t y p e currents are blocked with an cumulative IC50 of 1.1 ~ M , a concentration which also blocks T-type currents. The Hill coefficient of 1.3 suggests the binding of one Hg 2÷ cation for effective blockade. Other investigations dealing with the blockade of voltage-activated calcium channels by Hg 2÷ describe concentrations of 155 /zM nessecary for a 50% reduction of calcium currents 4'21. This may be explained by differences in the methods: the authors did not use cultured cells but isolated forebrain synaptosomes and measured the Ca 2÷ influx via radioactively marked calcium and they depolarized the synaptosomes by changing the external K ÷ concentration. There are only few other metal ions such as Pb 2÷ which block calcium channel currents at concentrations in the low micromolar range 6'8. Hg 2÷ is unlikely to have a charge screening effect in the used concentration range. The fast rate of onset suggests a direct blockade of the calcium channel currents as the most likely mechanism. For effects of Hg 2+ on Aplysia neurons higher concentrations were needed. While ions have a higher concentration in seawater, Ca 2+ could be expected to bind to the same sites as mercury and so competitively prevent an effect at lower concentrations. The fast onset and the irreversibility of the effect in D R G neurons most likely reflects a tight binding of Hg 2+ to a site at the mouth of or in the calcium channel. In low doses ( < 2 ~ M ) Hg 2÷ is clearly a blocker of voltage-activated calcium channels, but the effect on m e m b r a n e currents in D R G cells is relatively slow and develops with higher concentrations of Hg 2÷, therefore mercury may also act from the inside of the cell membrane. Although it was shown that Hg 2÷ does not pass the m e m b r a n e through calcium channels, it is known to pass through the cell m e m b r a n e and bind very strongly to sulphydryl groups 5'26. The increasing leak current at concentrations abouve 2 /zM indicates the opening of other non-specific ion channel(s) as described by Arakawa and co-workers 3, who also found a slow inward current in rat D R G cells, generated at 10 ~ M Hg 2+. With concentrations this high, we cannot exclude a m e m b r a n e damage, or other general toxic effects by ubiquitous mercury complexation. Three main differences concerning the actions of Hg 2+ on voltage-activated calcium channel currents in
rat D R G and Aplysia neurons were found. (1) The blockade of calcium channels in Aplysia neurons never came to a steady state. This may be due to an intracellular action of Hg z+ on the calcium channels which became stronger with accumulation of Hg 2+ in the cell. Hg 2÷ in D R G neurons might act at an extracellular and an intracellular binding site of the channels. (2) In Aplysia neurons the effective concentration was ten times higher than in D R G neurons. (3) The I-V relation exhibited no maximum shift in Aplysia neurons, in contrast to the mammalian L / N - c a l c i u m channel types, the maximum shifted to more depolarizing values after Hg 2÷ application. Thus in Aplysia neurons Hg 2÷ influences the number of channels which can be opened a n d / o r the mean conductance of the single channels. In D R G neurons the gating properties of the L-type calcium channels and their kinetics may be affected by Hg 2+. Our results suggest some differences concerning the mechanisms of Hg 2÷ action on voltage-activated calcium channel currents between mammalians and invertebrates but in both preparations mercury irreversibly reduces these currents in low doses. The toxic effects of mercury might be in part explained by an action at the calcium channels. REFERENCES 1 Akaike, N., Lee., K.S. and Brown, A.M., The calcium current of Helix neuron, J. Gen. Physiol., 71 (1978) 509-531. 2 Albers, J.W., Kallenbach, L.R., Fine, L.J., Langolf, G.D., Wolfe, R.A., Donofrio, P.D., Alessi, A.G., Stolp-Smith, K.A. and Bromberg, M.B., Neurological abnormalities associated with remote occupational element mercury exposure, Ann. Neurol., 5 (1988) 651-659. 3 Arakawa, O., Nakahiro, M. and Narahashi, T., Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons, Brain Res., 551 (1991) 58-63. 4 Atchison, W.D., Joshi, U. and Thornburg, J.E., Irreversible suppression of calcium entry into nerve terminals by methylmercury, J. PharmacoL Exp. Ther. (1986) 618-624. 5 Blazka, M.E. and Shaikh, Z.A., Differences in cadmium and mercury uptakes by hepatocytes: role of calcium channels, Toxicol. AppL PharrnacoL, 110 (1991) 355-363. 6 Biisselberg, D., Evans, M.L., Rahmann, H., and Carpenter, D.O., Lead and zink block a voltage-activated calcium channel of Aplysia neurons, J. Neurophysiol., 65(4) (1991) 786-795. 7 Biisselberg, D., Evans, M.L., Rahmann, H. and Carpenter, D.O., Effects of inorganic and triethyl lead and inorganic mercury on the voltage-activated calcium channels of Aplysia neurons, NeuroToxicology, 12 (1991) 733-744. 8 Biisselberg, D., Evans, M.L., Haas, H.L. and Carpenter, D.O., Blockade of mammalian and invertebrate calcium channels by lead, NeuroToxicology, 14 (1993) 249-258. 9 Biisselberg, D., Michael, D., Evans, M.L., Carpenter, D.O. and Haas, H., Zink (Zn 2÷ ) blocks voltage-gated calcium channels in cultured rat dorsal root ganglion cells, Brain Res., 593 (1992) 77-81. 10 Byerly, S.C., Chase, P.B. and Stimers, J.R., Permeation and interaction of divalent cations in calcium channels of snail neurons, J. Gen. Physiol., 85 (1985) 491-518.
126 11 Cagiano, R., De Salvia, M.A., Renna, G., Tortella, E., Braghiroli, D., Parenti, C., Zanoli, P., Baraldi. M., Annam Z.. and Cuomo, V., Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats, Neurotoxicol. TeratoL, 12 (1990) 573-576. 12 Carry, A.J. and Malone, S.F., The chemistry of mercury in biological system. In J.O. Nriagu (Ed.), The Biogeochemistr), of Mercury in the Em'ironment, Elsevier. New York, 1979, pp. 43348{I. 13 Chad, J.E. and Eckert, R., An enzymatic mechanism for calcium current inactivation in dialysed Helix" neurons, J. Physiol., 378 (1986) 31-51. 14 Clarcson, T.W., Metal toxicology in the central nervous system, Era'iron. Health Persp., 75 (1987) 59-64. 15 Damastra, T., Toxicological properties of lead, Era'iron. Health Persp., 19 (1977) 297-307. 16 Eccles, C.U. and Annau, Z , Prenatal methylmercury exposure. 11. Alterations in learning and psychotropic drug sensitivity in adult offspring, Neurobehac. Toxicol. Teratol. 4 (19821 377-382. 17 Evans, M.L., Biisselberg, D. and Carpenter, D.O., Pb -'+ blocks calcium currents of cultured dorsal root ganglion cells, Neurosci. Left., 129 (19911 103-106. 18 Frazier, W.T., Kandel, E.R., Kupfermann, I., Waziri, R. and Coggeshall, R.E, Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia californica, J. Neurophysio[., 30 (19671 1288-1351. 19 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rec. Neurosci.. 4 (19811 69-125. 211 Hare, M.F., Rezazadeh, S.M., Cooper, G.P, Minnema, D.J. and Michaelson, 1.A., Effects of inorganic mercury on [3H]dopamine release and calcium homeostasis in rat striatal synaptosomes, Toxicol. Appl. Pharmacol., 86 (1990) 316-330. 21 Hewett, S.J. and Atchison, W.D., Effects of charge and lipophilicity on mercurial-induced reduction of 4SCa~+ uptake in isolated nerve terminals of the rat, Toxicol. AppL Pharmaeol., 113 (1992) 267-273. 22 Kuznetov, D.A. and Richter, V., Modulation of messenger RNA metabolism in experimentel methyl mercury neurotoxicity, Int. Z Neurosci., 34 (1987) 1-17.
23 Kuznetov, D.A., Zavijalov, N.V., Govorkov, A.V. and Sibileva, T.M., Methylmercury-induced nonselective blocking of phosphorylation processes as a possible cause of protein synthesis inhibition in vitro and in vivo, ToaicoL Lett., 36 (19871 153-160. 24 Lansman, J.B., Hess, P. and Tsien, R.W., Blockade of current through single calcium channels by Cd 2~, Mg :~ and Ca2": w)ltage and concentration dependence of calcium entry_ into the pore, J.Gen. Physiol., 88 (1986) 321-347. 25 Magour S., Studies on the inhibition of the brain synaptosomal Na +/Ka ~-ATPase by mercury chloride and methyl mercury chloride, Arch. ToxicoL, 9 (1987) 393-396. 26 Manalis, R.S. and Cooper, G., P., Evoked transmitter release increased by inorganic mercury at frog neuromuscular junction, Nature. 257 (1975) 690-691. 27 Mintz, I.M.; Adams, M.E. and Bean B.P., P-type calcium channels in rat central and peripheral neurons. Neuron, 9 (19921 85-95. 28 Musch, H.R., Bornhausen, M., Kriegel, H. and Greim, H., Methylmercury chloride induces learning deficits in prenatally treated rats, Arch. ToxicoL, 40 (1978) 103-108. 29 Rice, D.C., Methodological approaches to primate behavioral toxicology testing, Neurotoxicol. Teratol, 9 (1987) 161-169. 30 Schalock, R.L., Brown, W.J., Kark, R.A. and Menon, N.K., Perinatal methylmercury intoxication: behavioral effects in rats, Dev. t~ychobiol., 14 (1981) 213-219. 31 Swandulla, D. and Armstrong, C.M., Calcium channel block by cadmium in chicken sensory neurons, Proc. Natl. Acad. Sci. USA, 86 (19891 1736-174/I. 32 Tsien, R.W., Lipscombe, D., Madison, D.V., Bley, K.R. and Fox, A.P., Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci., 11 (1988)431-438. 33 Umbach, J.A. and Gundersen, C.B., Mercuric ions are potent noncompetitive antagonists of human brain kainate receptors expressed in Xenopus oocytes, Mol. Pharmacol. (1989) 582-588. 34 Wood, J.N., Winter, J., James, I.F., Rong, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988), 3208-32211.
Brain Research, 632 (1993) 121-126 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 19552
Mercury
(Hg 2+) decreases
voltage-gated calcium channel currents in rat D R G and Aplysia neurons M. Pekel, B. Platt, D. Bfisselberg *
Physiologisches Institut 11, Heinrich-Heine Universitiit Diisseldorf, Moorenstrafle 5, D-40225 Diisseldorf, Germany (Accepted 2 August 1993)
Key words: Mercury; Rat dorsal root ganglion (DRG) neuron; Aplysia neuron; Calcium channel current
Inorganic mercury (Hg 2+ ) reduced voltage-gated calcium channel currents irreversibly 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 izM and a Hill coefficient of 1.3. T-currents were blocked with Hg 2÷ in the same concentration range (0.5-2 tzM). With increasing Hg 2+ concentrations a slow membrane current was additionally activated, most obviously at concentrations over 2 / z M Hg 2+. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg 2÷. The current-voltage ( I - V ) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg 2+ to the channel protein a n d / o r 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 Hg 2+ (5 ~M). 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 p.M). No shift of the I - V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg 2+ 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.
INTRODUCTION Calcium is one of the most important messengers for signal transfer in cells. Therefore the intracellular calcium concentration is regulated by a variety of mechanisms including selective voltage-activated channels, which are opened by depolarization. For vertebrates, four different voltage-activated calcium currents have been characterized: a rapidly inactivating low-threshold-activated (T) current, a slowly inactivating high-threshold-activated (L) current, a highthreshold rapidly inactivating (N) current 32 and recently an additional P-type channel has been described 27. Although invertebrate calcium channels do not correspond exactly to any of those in vertebrates 1,1°, the calcium channels of Aplysia neurons have some similarities with the L-channel. Voltage-gated calcium channels are blocked by a variety of divalent and trivalent metal c a t i o n s 6-9,17,19.
* Corresponding author. Fax: (49) (211) 311 4231.
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 0 7 4 - D
Many of these ions are known to be neurotoxic both in inorganic and organic forms 7A4'15. Some studies suggest that methylmercury as well as inorganic mercury have effects on calcium currents in rat forebrain synaptosomes 4'21. Methylmercury produces behavioral changes in rats 11'3°, learning deficits in rats after prenatal exposure 16'28 and visual deficites in monkeys 29. Inorganic mercury, a highly neurotoxic agent z°'33, interacts with GABA-activated chloride channels 3 and increases evoked transmitter release at frog neuromuscular junction 26. In humans Hg 2÷ intoxication results in a loss of coordination, decreased sensation, tremor and abnormal reflexes 2. Hg 2+ persists in the nervous system for a long time 12. It inhibits the synaptosomal N a + / K +ATPase in the brain 25, blocks phosphorylation processes 23, and modulates messenger RNA metabolism 22 possibly via binding to -SH groups 12. Little is known about the effects of inorganic mercury on voltage-activated calcium channels 7 and there
122 TABLE I Composition of the basic external solutions All concentrations are in mM. HEPES, N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid; TEA-Br, tetra-ethylammonium bromide; TEA-CI, tetraethylammonium chloride; 4-AP, 4-aminopyridine; TrisCI, Tris (hydroxymethyl) aminomethane hydrochloride; Tris-base, Tris (hydroxymethyl) a m i n o m e t h a n e base; TTX, tetrodotoxine. Artificial A S W for Tyrodes h Ba- T T X ~ seawater (ASW) " calcium current a NaCI 480 10 KCI 10 CaCt 2 BaCI 31) MgCI 2 5 HEPES TEA-Br TEA-CI 4-AP Tris-Cl Tris-base TTX Glucose
145 2.5 1.5
10 20
45
10 l 10
1.2 10
200 135 1 234 59
130
10
0.002 10
10
Mercury (11) chloride (HgCI 2, Sigma 99,999'~ purity) was made as a 20 m M stock solution and was added to external Ba-TTX solution just before each application. External solutions were applied by a fast bath perfusion system (bath volume about I ml, perfusion volume 5 - 1 0 ml within 15-30 s). All experiments were performed at room temperature (20-22°C). The cells were routinely clamped at - 8 0 mV, which is close to the resting m e m b r a n e potential of DR(} cells. Voltage steps (one per 10 s) from - 80 to 0 mV for 75 ms werc used to evoke L/N-calcium channel currents (carried by barium to avoid intracellular effects through elevated calcium concentration). T-type currents were evoked by voltage steps from - 80 lo - 35 mV. a potential at which L- or N-channels are not appreciably activated. The total blockade of the resulting currents by 50 # M Cd e" indicates an adaequate isolation of calcium currents 2~~t. C o m m a n d pulses were delivered by an EPC-9 unit (HEKA) controlled by an A T A R I ST computer. Raw data were on-line leak-corrected by a P / 4 protocol, as described by Chad and Eckert L~. To determine the time course of the reduction of the calcium channel currents, command pulses were given every 10 s. Maximum current was measured. Data were filtered on-line at 2.3 kHz, stored on hard disk and analysed off-line using a M2-LAB software package. Cells with a run down of more than 10¢~ within 5 rain were excluded. The Hg 2+ dose response relationship tk)r D R G neurons was determined by fitting mean currents to the Langmuir equation:
~ pH was adjusted to 7.57; h pH was adjusted to 7.4 with NaOH; c pH was adjusted to 7.4 with T E A - O H . lc';,z ~ ( H g 2+ ) = lea2+ (control).
is no electrophysiological investigations on this question in vertebrates. We report here the effects of inorganic mercury on rat DRG and Aplysia neurons. MATERIAL
AND METHODS
D R G preparation For the vertebrate preparation we used primary cultures of dorsal-root ganglion ( D R G ) neurons from 2 - 3 day old rat pups as described by Wood and co-workers 34. Patch-clamp recordings in the whole-cell mode were obtained from 2 - 3 day old cultures to avoid the space-clamping problems which arise from the long axons and dendrites characteristic of older cultures. Electrode resistance was between 2 and 5 M / / . The pipette solution had the following composition (in mM): CsCI 135, HEPES 10, MgCI 2 1.2, Na-ATP 4; pH was adjusted to 7.2 using CsOH. The culture medium was replaced by Tyrodes solution (Table I) and a seal was established before this solution was replaced by an external solution for calcium channel current recordings (Ba-TI'X, Table I).
A
A
-t_
-8(I niV
l-
(
K,,,
t"
1+ ~ [~g~+ ] j
where 1Ca2+(Hg 2+ ) is the calcium current measured in the presence of a given concentration of Hg 2+, Ica2+(control) is the calcium current before addition of Hg 2+, K m is the appearent dissotiation constant and n is the Hill coefficient.
Aplysia preparation The abdominal ganglion of the marine mollusc, Aplysia californica (150-200 g; Pacific Biomarine Supply and Marine Specimens Unlimited, CA), was dissected and incubated in artificial seawater (ASW, Table I). Cells were impaled with two independent microelectrodes with resistances between 3 and 8 M / Z The voltage electrode was filled with 3 M potassium acetate; the current electrode with 3 M CsCl for blocking potassium channels. Neurons were identified by soma location, pigmentation and response properties to acetylcholine TM. All experiments were done in the voltage-clamp mode using a Dagan model 8500 voltage clamp amplifier. Cells were clamped at a holding potential of - 4 5 mV which is close to the
G
B 0 mV
I
-35 m V __3 -80 m ~
0 mV ____] -45 m ~
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,
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Fig. 1. The effect of 0.5 and 2 ~ M Hg z÷ on voltage-activated calcium channel currents (L/N-currents, (A), and T-currents, (B)) of rat dorsal root ganglion cells and of 20 ~ M Hg 2+ on calcium currents of Aplysia neurons (C). Note that in C the two superimposed traces are indicating different times of voltage step activation and not different concentrations of Hg" . Leak currents are subtracted. The voltage steps are shown in the upper trace.
123 m V for D R G neurons and - 4 5 m V for Aplysia neurons). Hg 2÷ caused a reduction of calcium channel currents in both preparations. In D R G ceils two effects by Hg 2÷ were seen: with lower concentrations ( > 2 g M ) there was a fast reduction of the calcium channel currents and at higher concentrations, in addition an increase in m e m b r a n e current was evident. The lower current trace in Fig. 1A results from a voltage step from - 8 0 to 0 mV. The current is long lasting and not inactivating during the step command. Superimposed are two traces measured after application of 0.5 and 2 /~M Hg 2+, respectively, demonstrating the dose-dependent reduction of this current by 15% for 0.5/xM and 70% for 2 /.~M Hg 2÷. The effect of Hg 2÷ on T-type current is shown in Fig. lB. 2 p.M Hg 2÷ reduced the current by more than 75%. Calcium channel currents of Aplysia neurons are shown in Fig. 1C. The transient and the long lasting component are both reduced by Hg 2÷ but as indicated in the two superimposed traces, taken 5 and '13 min after application of 20 ~ M Hg 2÷, the current declined over the whole period of application. Fig. 2A shows a representative t i m e - c o u r s e of calcium currents from a D R G neuron during application of increasing Hg 2÷ concentrations. Increasing concentrations of Hg 2+ cause a stepwise decrease of calcium channel currents. With low concentrations of Hg 2÷ (up to 2 /xM) a steady state is achieved within less than 1 min. Fig. 2B illustrates the effect of 50 g M Hg 2+ on the t i m e - c o u r s e of calcium currents in Aplysia neurons. Data points are means of 4 neurons. The onset of the effect of Hg 2÷ is very slow and failed to reach a plateau during the 8 min of application. U p o n wash there was no obvious recovery in both preparations. The concentration-response curve for Hg 2+ action on the L / N - t y p e currents (caused by a voltage step from the holding potential to 0 mV) of D R G neurons
resting m e m b r a n e potential of these invertebrate neurons. Virtual ground monitor currents were filtered at 3 kHz and a 30 kHz cut-off frequency was used for the pre-amplifier. To record calcium currents the A S W was replaced by an sodium-free A S W for calcium currents (Table I). The experiments were performed at 20-22°C. HgC12 (Fisher, 99.999%)-containing Ca-ASW was applied by a rapid perfusion system for a rapid change of the solution (perfusion rate about 2 m l / m i n ) . The leakage current was subtracted using a P / 3 protocol 13. Current steps of 70 ms from - 45 mV to + 20 mV were applied at 20 s intervals. Peak amplitudes were constant for several hours. Calcium currents were recorded on a videotape via a digitizing unit. Using an IBM A T computer and programs written in ASYST, the leak currents were subtracted and peak currents evaluated.
RESULTS Mercury was applied to 35 D R G neurons and 17
Aplysia neurons. L / N channel currents were measured in all D R G neurons, T-type channel currents in 5 D R G neurons. The I - V relation was investigated in 6 neurons. A comparison of the control traces in Fig. 1 A - C illustrates differences between the typical calcium channel currents of D R G neurons and Aplysia neurons. D R G neurons display different calcium channel currents which can be distinguished by the pulse protocol and their inactivation rate: both L- and N-type currents were activated by depolarizing steps from - 8 0 to 0 m V and gave a peak current of 2.3 nA (Fig. 1A). We did not separate these two calcium channel currents. The transient T-type current was activated by a depolarizion pulse from - 8 0 to - 3 5 mV. The current inactivated rapidly and had a smaller amplitude (500 pAl. The calcium channel current of Aplysia neurons had a transient and a long lasting component. Peak current was 850 nA in the larger neurons of Aplysia. The maximum values of the I - V relation (control traces in Fig. 4A,B) was different for calcium channel currents of D R G and Aplysia neurons. This is due to different charge carriers (Ba 2÷ for D R G neurons and Ca 2÷ for Aplysia cells), different concentrations of the charge carrier and different holding potentials ( - 8 0
A
B In Xi 2
511
11
I
I
I
5
Ill
15
Iminl
i
]
2
9
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Fig. 2. T i m e - c o u r s e of reduction of the peak calcium current during application of increasing concentrations of Hg 2÷ for a D R G neuron (A) and the t i m e - c o u r s e for the peak calcium current during application of 5 0 / ~ M Hg 2÷ for Aplysia neurons (mean values for n = 4, vertical lines indicate standard deviation, B).
124
!00
B
A 2 pM
C 5 pM
50 pM
wash
c
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"~ 50 .=.
~5 min
I rain
2 min
0.1
1
Fig. 5. Uncorrected contlnous current registration tbr two DRG neurons (A,B) and for an Aplysia neuron (C). The peaks indicate the currents at a voltage step. As the leak currents are not subtracted, they are superimposed on the calcium currents. Low concentrations of Hg 2+ reduce only the calcium currents in DRG neurons (A), while higher concentrations (5/xM) have an additional effect on the leak current (B). In Aplysia neurons even 50 ~zM Hg z+ do not change the leak current (C).
[pM1
Fig. 3. Dose-response relationship for the inhibitory effect of Hg 2+ on rat DRG neurons. Half of the peak calcium current was inhibited at a concentration of 1.1 ~M.
is shown in Fig. 3. The degree of inhibition in 30 neurons is plotted vs. concentration. The data points were fitted by a sigmoid curve. The cumulative ICs0 is 1.1 /~M and the cumultative Hill coefficient 1.3. Calcium channel currents of Aplysia neurons decreased continously over the time of Hg 2+ application without reaching a steady state - therefore it was impossible to record a concentration-response relationship. The I - V relations plotted in Fig. 4 show remarkable differences between calcium channel currents in D R G neurons (Fig. 4A) and Aplysia neurons (Fig. 4B). These curves were obtained by measuring the calcium channel currents generated by a voltage step from the resting m e m b r a n e potential ( - 8 0 mV in D R G cells and - 4 5 mV in Aplysia cells) to varying potentials which are indicated at the x-axis. The currents increase with voltage steps to more depolarized values (left part of the curves). This reflects the increasing activation of calcium channels. With even greater depolarizating voltage steps there is a reduction of the magnitude of the calcium channel current, which reflects the decreased driving force for Ca 2+ ions due to the increas-
ing depolarization. In D R G neurons a maximum current was generated by a step to - 10 mV (or - 5 mV) (Fig. 4A). The voltage at which the maximum current is elicited shifts in the presence of 2 ~ M Hg 2+ from - 5 mV in the control to + 10 mV (Fig. 4A). This indicates a change in the kinetics of channel activation and suggest toxic actions at a level of charge sites on the channel itself. In Aplysia neurons the 'best' voltage step was from the holding potential to + 20 mV (Fig. 4B). The absence of altered I - V relations in Aplysia neurons shows that Hg 2+ reduces the number of functional channels a n d / o r the conductance rather than the voltage dependence of the channels (Fig. 4B). In D R G neurons high concentrations of Hg 2+ (more than 2 g M ) caused a remarkable sustained increase of the holding current. The time-courses of the raw data in Fig. 5 indicate at 2 g M Hg 2+ a clear suppression of calcium channel currents (Fig. 5 A) without changes in m e m b r a n e currents while 5 ~zM Hg 2+ cause an additional increase in holding current. At concentrations lower than 2 ~ M the non-specific membrane current
B
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Fig. 4. Current-voltage relation of voltage-activated calcium currents for a rat DRG neuron (A) and for Aplysia neurons (n = 4, B). Note that the maximum of the control curves for both preparations differs and that a shift of the curve maximum after application of Hg 2+ appears only for DRG neurons (A).
125 was very small and developed slowly. This effect was not obtained in Aplysia neurons (Fig. 5C). DISCUSSION Hg 2+ blocks voltage-activated calcium channel currents in rat D R G cells as well as in Aplysia neurons at micromolar concentrations. The mammalian L / N - t y p e currents are blocked with an cumulative IC50 of 1.1 ~ M , a concentration which also blocks T-type currents. The Hill coefficient of 1.3 suggests the binding of one Hg 2÷ cation for effective blockade. Other investigations dealing with the blockade of voltage-activated calcium channels by Hg 2÷ describe concentrations of 155 /zM nessecary for a 50% reduction of calcium currents 4'21. This may be explained by differences in the methods: the authors did not use cultured cells but isolated forebrain synaptosomes and measured the Ca 2÷ influx via radioactively marked calcium and they depolarized the synaptosomes by changing the external K ÷ concentration. There are only few other metal ions such as Pb 2÷ which block calcium channel currents at concentrations in the low micromolar range 6'8. Hg 2÷ is unlikely to have a charge screening effect in the used concentration range. The fast rate of onset suggests a direct blockade of the calcium channel currents as the most likely mechanism. For effects of Hg 2+ on Aplysia neurons higher concentrations were needed. While ions have a higher concentration in seawater, Ca 2+ could be expected to bind to the same sites as mercury and so competitively prevent an effect at lower concentrations. The fast onset and the irreversibility of the effect in D R G neurons most likely reflects a tight binding of Hg 2+ to a site at the mouth of or in the calcium channel. In low doses ( < 2 ~ M ) Hg 2÷ is clearly a blocker of voltage-activated calcium channels, but the effect on m e m b r a n e currents in D R G cells is relatively slow and develops with higher concentrations of Hg 2÷, therefore mercury may also act from the inside of the cell membrane. Although it was shown that Hg 2÷ does not pass the m e m b r a n e through calcium channels, it is known to pass through the cell m e m b r a n e and bind very strongly to sulphydryl groups 5'26. The increasing leak current at concentrations abouve 2 /zM indicates the opening of other non-specific ion channel(s) as described by Arakawa and co-workers 3, who also found a slow inward current in rat D R G cells, generated at 10 ~ M Hg 2+. With concentrations this high, we cannot exclude a m e m b r a n e damage, or other general toxic effects by ubiquitous mercury complexation. Three main differences concerning the actions of Hg 2+ on voltage-activated calcium channel currents in
rat D R G and Aplysia neurons were found. (1) The blockade of calcium channels in Aplysia neurons never came to a steady state. This may be due to an intracellular action of Hg z+ on the calcium channels which became stronger with accumulation of Hg 2+ in the cell. Hg 2÷ in D R G neurons might act at an extracellular and an intracellular binding site of the channels. (2) In Aplysia neurons the effective concentration was ten times higher than in D R G neurons. (3) The I-V relation exhibited no maximum shift in Aplysia neurons, in contrast to the mammalian L / N - c a l c i u m channel types, the maximum shifted to more depolarizing values after Hg 2÷ application. Thus in Aplysia neurons Hg 2÷ influences the number of channels which can be opened a n d / o r the mean conductance of the single channels. In D R G neurons the gating properties of the L-type calcium channels and their kinetics may be affected by Hg 2+. Our results suggest some differences concerning the mechanisms of Hg 2÷ action on voltage-activated calcium channel currents between mammalians and invertebrates but in both preparations mercury irreversibly reduces these currents in low doses. The toxic effects of mercury might be in part explained by an action at the calcium channels. REFERENCES 1 Akaike, N., Lee., K.S. and Brown, A.M., The calcium current of Helix neuron, J. Gen. Physiol., 71 (1978) 509-531. 2 Albers, J.W., Kallenbach, L.R., Fine, L.J., Langolf, G.D., Wolfe, R.A., Donofrio, P.D., Alessi, A.G., Stolp-Smith, K.A. and Bromberg, M.B., Neurological abnormalities associated with remote occupational element mercury exposure, Ann. Neurol., 5 (1988) 651-659. 3 Arakawa, O., Nakahiro, M. and Narahashi, T., Mercury modulation of GABA-activated chloride channels and non-specific cation channels in rat dorsal root ganglion neurons, Brain Res., 551 (1991) 58-63. 4 Atchison, W.D., Joshi, U. and Thornburg, J.E., Irreversible suppression of calcium entry into nerve terminals by methylmercury, J. PharmacoL Exp. Ther. (1986) 618-624. 5 Blazka, M.E. and Shaikh, Z.A., Differences in cadmium and mercury uptakes by hepatocytes: role of calcium channels, Toxicol. AppL PharrnacoL, 110 (1991) 355-363. 6 Biisselberg, D., Evans, M.L., Rahmann, H., and Carpenter, D.O., Lead and zink block a voltage-activated calcium channel of Aplysia neurons, J. Neurophysiol., 65(4) (1991) 786-795. 7 Biisselberg, D., Evans, M.L., Rahmann, H. and Carpenter, D.O., Effects of inorganic and triethyl lead and inorganic mercury on the voltage-activated calcium channels of Aplysia neurons, NeuroToxicology, 12 (1991) 733-744. 8 Biisselberg, D., Evans, M.L., Haas, H.L. and Carpenter, D.O., Blockade of mammalian and invertebrate calcium channels by lead, NeuroToxicology, 14 (1993) 249-258. 9 Biisselberg, D., Michael, D., Evans, M.L., Carpenter, D.O. and Haas, H., Zink (Zn 2÷ ) blocks voltage-gated calcium channels in cultured rat dorsal root ganglion cells, Brain Res., 593 (1992) 77-81. 10 Byerly, S.C., Chase, P.B. and Stimers, J.R., Permeation and interaction of divalent cations in calcium channels of snail neurons, J. Gen. Physiol., 85 (1985) 491-518.
126 11 Cagiano, R., De Salvia, M.A., Renna, G., Tortella, E., Braghiroli, D., Parenti, C., Zanoli, P., Baraldi. M., Annam Z.. and Cuomo, V., Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats, Neurotoxicol. TeratoL, 12 (1990) 573-576. 12 Carry, A.J. and Malone, S.F., The chemistry of mercury in biological system. In J.O. Nriagu (Ed.), The Biogeochemistr), of Mercury in the Em'ironment, Elsevier. New York, 1979, pp. 43348{I. 13 Chad, J.E. and Eckert, R., An enzymatic mechanism for calcium current inactivation in dialysed Helix" neurons, J. Physiol., 378 (1986) 31-51. 14 Clarcson, T.W., Metal toxicology in the central nervous system, Era'iron. Health Persp., 75 (1987) 59-64. 15 Damastra, T., Toxicological properties of lead, Era'iron. Health Persp., 19 (1977) 297-307. 16 Eccles, C.U. and Annau, Z , Prenatal methylmercury exposure. 11. Alterations in learning and psychotropic drug sensitivity in adult offspring, Neurobehac. Toxicol. Teratol. 4 (19821 377-382. 17 Evans, M.L., Biisselberg, D. and Carpenter, D.O., Pb -'+ blocks calcium currents of cultured dorsal root ganglion cells, Neurosci. Left., 129 (19911 103-106. 18 Frazier, W.T., Kandel, E.R., Kupfermann, I., Waziri, R. and Coggeshall, R.E, Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia californica, J. Neurophysio[., 30 (19671 1288-1351. 19 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rec. Neurosci.. 4 (19811 69-125. 211 Hare, M.F., Rezazadeh, S.M., Cooper, G.P, Minnema, D.J. and Michaelson, 1.A., Effects of inorganic mercury on [3H]dopamine release and calcium homeostasis in rat striatal synaptosomes, Toxicol. Appl. Pharmacol., 86 (1990) 316-330. 21 Hewett, S.J. and Atchison, W.D., Effects of charge and lipophilicity on mercurial-induced reduction of 4SCa~+ uptake in isolated nerve terminals of the rat, Toxicol. AppL Pharmaeol., 113 (1992) 267-273. 22 Kuznetov, D.A. and Richter, V., Modulation of messenger RNA metabolism in experimentel methyl mercury neurotoxicity, Int. Z Neurosci., 34 (1987) 1-17.
23 Kuznetov, D.A., Zavijalov, N.V., Govorkov, A.V. and Sibileva, T.M., Methylmercury-induced nonselective blocking of phosphorylation processes as a possible cause of protein synthesis inhibition in vitro and in vivo, ToaicoL Lett., 36 (19871 153-160. 24 Lansman, J.B., Hess, P. and Tsien, R.W., Blockade of current through single calcium channels by Cd 2~, Mg :~ and Ca2": w)ltage and concentration dependence of calcium entry_ into the pore, J.Gen. Physiol., 88 (1986) 321-347. 25 Magour S., Studies on the inhibition of the brain synaptosomal Na +/Ka ~-ATPase by mercury chloride and methyl mercury chloride, Arch. ToxicoL, 9 (1987) 393-396. 26 Manalis, R.S. and Cooper, G., P., Evoked transmitter release increased by inorganic mercury at frog neuromuscular junction, Nature. 257 (1975) 690-691. 27 Mintz, I.M.; Adams, M.E. and Bean B.P., P-type calcium channels in rat central and peripheral neurons. Neuron, 9 (19921 85-95. 28 Musch, H.R., Bornhausen, M., Kriegel, H. and Greim, H., Methylmercury chloride induces learning deficits in prenatally treated rats, Arch. ToxicoL, 40 (1978) 103-108. 29 Rice, D.C., Methodological approaches to primate behavioral toxicology testing, Neurotoxicol. Teratol, 9 (1987) 161-169. 30 Schalock, R.L., Brown, W.J., Kark, R.A. and Menon, N.K., Perinatal methylmercury intoxication: behavioral effects in rats, Dev. t~ychobiol., 14 (1981) 213-219. 31 Swandulla, D. and Armstrong, C.M., Calcium channel block by cadmium in chicken sensory neurons, Proc. Natl. Acad. Sci. USA, 86 (19891 1736-174/I. 32 Tsien, R.W., Lipscombe, D., Madison, D.V., Bley, K.R. and Fox, A.P., Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci., 11 (1988)431-438. 33 Umbach, J.A. and Gundersen, C.B., Mercuric ions are potent noncompetitive antagonists of human brain kainate receptors expressed in Xenopus oocytes, Mol. Pharmacol. (1989) 582-588. 34 Wood, J.N., Winter, J., James, I.F., Rong, H.P., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci., 8 (1988), 3208-32211.