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Cadmium inhibits GABA-activated ion currents by increasing intracellular calcium level in snail neurons
Brain Research 1008 (2004) 205 – 211 www.elsevier.com/locate/brainres
Research report
Cadmium inhibits GABA-activated ion currents by increasing intracellular calcium level in snail neurons Ga´bor Molna´r *, Ja´nos Sala´nki, Tibor Kiss Department of Experimental Zoology, Balaton Limnological Research Institute, HAS, Tihany, Klebelsberg K. u. 3, P.O. Box 35, H-8237, Hungary Accepted 4 February 2004 Available online 1 April 2004
Abstract Blocking of the GABA-activated chloride current by cadmium was investigated in identified nerve cells (RPeD1, RPaD1) of the pond snail (Lymnaea stagnalis L.), using a two-microelectrode voltage-clamp technique. Cd2 +, at 50 AM extracellular concentration, inhibited GABA-activated chloride currents, both in normal and Ca2 +-free solution. Intracellular injection of Ca2 + or the application of caffeine mimicked the inhibitory effect of Cd2 + on GABA-elicited currents. Cd2 +-block was eliminated, or it was substantially decreased, when neurons were intracellularly loaded with EGTA, or when the Ca2 +-release was blocked by ruthenium red. The blocking effect of Cd2 + was also eliminated by applying G-protein inhibitors: pertussis toxin, suramin or GTP-g-S. Finally, intracellularly injected Cd2 + was ineffective at eliciting an inward current on GABA-activated currents, suggesting that the Cd2 +-binding site resides extracellularly. These results suggest that cadmium inhibited GABA-activated chloride currents by increasing the intracellular Ca2 + level, by releasing it from intracellular stores and by interacting with a putative G-protein-coupled cell-surface ‘‘metal-receptor’’. D 2004 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Ligand gated ion channels Keywords: Lymnaea stagnalis L.; Cadmium; Intracellular calcium; G-protein, neurone; GABA
1. Introduction Because of its high environmental impact cadmium is a frequently examined, heavy metal of biological importance. Cadmium has a long biological half-life ( f 30 years in humans), which contributes to its accumulation in living cells [34]. Exposure to this heavy metal causes carcinogenic, mutagenic and teratogenic effects, damaging of several organs, including the lung, kidney, gastrointestinal tract, bone and also the brain [8,13]. In vertebrates, cadmium enters the central nervous system either through the olfactory bulb [32] or through the blood – brain barrier. Penetration of cadmium into the brain can be facilitated by ethanol [25], chronic exposure to cadmium [29] or by forming complexes with other substances [24]. Once * Corresponding author. Tel.: +36-87-448-244x111; fax: +36-87-448006. E-mail address: [email protected] (G. Molna´r). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.035
cadmium is present in the intercellular spaces, it interferes with a number of cellular and physiological processes. Although the cellular actions of cadmium are not completely understood, the complexity of the underlying mechanisms is discussed in a number of reports [16,21]. Cadmium causes lipid peroxidation, damage to DNA, depletion of sulfhydryls, initiation of expression of heat shock proteins and methallothioneins, and induces alteration of calcium homeostasis by competing with calcium at the Ca2 + binding sites [8,15,35,36]. It is frequently used as an effective blocker of the voltage-sensitive L-type Ca2 + channels [6,28]. It was demonstrated, however, that cadmium reduces almost all kinds of ligand-gated and voltage-dependent ion currents (for review, see Ref. [15]). It was already described that extracellularly applied Cd2 + ions increased the cytosolic-free Ca2 + concentration by binding to the presumptive Cd2 + surface receptors in human skin fibroblast [31], endothelial cells [9], bovine chromaffin cells [37] and Xenopus oocytes [10]. In all cases, the increase
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in intracellular Ca2 +-concentration was elicited by generating inositol triphosphate (IP3) [20]. In our previous work, we have described the blocking effect of extracellularly applied cadmium on GABA-activated chloride-currents in Lymnaea neurones, and also the modulatory effect on GABA-evoked firing patterns [23]. Cadmium inhibited the GABA-mediated current starting from 5 AM concentration (KD = 9.2 AM) in a time- and concentration-dependent manner. The aim of the present study was to describe the signaling pathway by which extracellularly applied Cd2 + blocks GABA-elicited currents in neurones.
2. Materials and methods 2.1. Preparation Experiments were carried out on identified neurones of the pond snail Lymnaea stagnalis L. Animals were collected from the Lake Kis-Balaton during spring and summer, and were kept in aquarium in slowly running, natural lake water, at a temperature of 15– 20 jC. Over the course of the experiments, care was taken to minimize pain and discomfort to, as well as the unnecessary use of, animals. Measurements were performed on the GABAsensitive neurones (RPeD1 and RPaD1) of the isolated suboesophageal ganglionic ring [27]. Following dissection, the ganglionic ring was placed in the experimental chamber. The ganglia were pinned out in Sylgard-lined Petri dishes and the thick connective tissue sheath was removed mechanically. Following an enzymatic treatment with Protease E (type IV, Sigma) for 3 min, the ganglia were kept in refrigerator for at least 2 h and, thereafter, the cells were freed by opening the thin connective sheath using fine forceps. 2.2. Solutions The standard physiological solution (SPS) used in the experiments contained (in mM): NaCl 40, KCl 1.7, CaCl2 4, MgCl2 1.5, glucose 10, Tris – HCl 10, Tris –Cl 10. In the Ca2 +-free solution, Ca2 + was substituted by Ba2 +: NaCl 40, KCl 1.7, BaCl2 4, MgCl2 1.5, glucose 10, HEPES 10; pH was adjusted to 7.5 using NaOH. The following substances were used in the experiments: g-aminobutyric acid (GABA; 25 – 50 AM), ethylene glycol-bis(h-aminoethyl ether)N,N,NV,NV-tetraacetic acid (EGTA), glucose, Tris[hydroxymethyl]amino-methane (Tris), Tris – HCl, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), suramin hexa sodium salt (suramin, 10 AM), ammoniated ruthenium oxychloride (ruthenium red, 2 AM), 1,3,7-trimethylxanthine (caffeine, 1 mM), pertussis toxin (incubation time was 30 h; 2 Ag/ml) and guanosine 5V-O-(3-thiotriphosphate) tetralithium salt (GTP-g-S, 1 mM). Substances listed above were purchased from Sigma. 5(6)-Carboxyfluorescein was
purchased from Eastman Kodak; CdCl2 and other inorganic salts were from Reanal, Hungary. 2.3. Experimental protocol Experiments were performed at room temperature (21 – 24 jC). In order to measure transmembrane ionic currents, two-microelectrode voltage-clamp technique was applied using a DAGAN 8500 voltage clamp amplifier. Glass micropipettes were filled with 2.5 mM KCl, having resistances between 2 and 5 MV. The preparation was continuously superfused with SPS at a rate of 1.1 ml/min, allowing the complete exchange of the recording chamber volume in 2 min. Solution containing CdCl2 was added by perfusion at a concentration of 50 AM. GABA was applied by microperfusion, using a plastic capillary with an orifice of 310 Am positioned over the neurone under study. Changing the hydrostatic pressure applied to the pipette controlled the solution flow from the pipette. Repeated transmitter applications were carried out in 5 min intervals for 1 –2 s, in order to avoid desensitization. The holding potential was set between 75 and 50 mV in all experiments. 2.4. Intracellular injection Several substances were introduced into the cell by pressure injection. Substances for intracellular injection were: 0.1 M CaCl2, 0.1 M K-EGTA (0.1 M EGTA, 0.3 M KOH adjusted with HCl to pH 7.2), 5 mM CdCl2 and 1 mM GTP-g-S. Borosilicate micropipettes (TW150F-3, WPI) were pulled, under the same conditions, with a vertical puller (PC-10, Narishige), and used for ejection without any other modifications of the tip (breaking, beveling, etc.). Intracellular concentrations of these substances were calculated from the ratio of the injected substance and the cell volumes. The ejected volume was calculated before and after experiments, from the diameter of the spherical droplet formed under oil [11,19]. The injected amount was between 3 and 25 pl. The size of the investigated cell was approximated using the formula of an ellipsoid volume: Vcell = d1d2d3p/6 where Vcell is the volume of the cell, d1 is the width and d2 is the length of the investigated cells. It was supposed that the third parameter is the same as the shorter one (d3 = d1). The calculated average volume of both RPaD1 and RPeD1 (n = 6 –10) was f 1200 F 120 pl. 5(6)-Carboxyfluorescein was used as tracer to visually control the success of the injection under blue illumination. CaCl2 was intracellularly delivered using a third microelectrode. Free [Ca2 +]i concentration was determined using a computer program that calculates the free ion concentration from the known concentrations of metal-ligand complexes at defined pH and temperature values [3]. The following intracellular parameters were used for the calculation: K-EGTA 210 4 M, Ca2 +10 4 M, pH = 7.2, temperature 22 jC.
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The intracellular [Ca2 +] value is unlikely to be higher, the EGTA concentration is not supposed to be lower than this. K-EGTA and 5(6)-carboxyfluorescein and the vehicle (intracellular fluid) were tested to have no significant effect on the measured current. 2.5. Statistical analysis Results are presented as mean F standard deviation (S.D.). Statistical significance was determined using a Student’s t-test (paired). A P-value less than 0.05 was considered to be statistically significant.
3. Results Extracellularly applied GABA elicited a Cl -dependent inward current of 8 – 10 nA at a holding potential of 75 mV. The current activated and slowly decayed in 4 s period (Fig. 1A) and was completely blocked by exposure to 50 AM extracellular cadmium within 1 – 2 min (Fig. 1B). This effect was partially reversible after a 5-min washout (34% of control, not shown). The complete recovery of Cd2 + treat-
Fig. 2. The effect of intracellular Ca2 + on the GABA-activated chloride current. (A) The left trace shows the response to 50 AM GABA. On the right Ca2 + was injected into the cell (0.1 mM, RPaD1), followed by application of GABA. Under these conditions, the GABA response was blocked. (B) Caffeine (1 mM) also decreased the GABA-elicited conductance (RPaD1). (C) The effect of caffeine was enhanced with simultaneous 50 AM Cd2 + application. (D) Columns show normalized amplitudes of GABA-elicited responses with and without increased intracellular Ca2 +. The first two columns are taken from Fig. 1.
Fig. 1. The blocking effect of cadmium on GABA-activated chloride ion current. (A) Applying 25 AM GABA directly to the membrane surface of the neurone (RPaD1) induced an inward current. (B) By adding 50 AM Cd2 + to the SPS, the GABA-activated current was completely abolished. (C) The relative mean current amplitudes of the GABA-elicited current obtained in SPS (control) and in the presence of 50 AM Cd2 + in the extracellular saline solution containing Ca2 + (4 mM) and in Ca2 +-free Ba2 + (4 mM) solution.
ment was not followed, because it can last for hours. These results agree well with those described earlier on identified Lymnaea neurones [23,27]. In the next experiments, the role of extracellular Ca2 + was examined. To do this, we replaced Ca2 + ions in the extracellular solution with Ba2 + ions. Both in the control and Ba2 +-containing saline, Cd2 + blocked the GABAelicited current with the same efficiency (Fig. 1C), suggest-
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was almost non-existent (Fig. 2C), which indicates a common mechanism for both caffeine and Cd2 +. Likewise, treatment of the neurone with 2 AM extracellularly applied ruthenium red prevented Cd2 + from blocking the GABA-response (81 F 32%, n = 6, Fig. 3B). No statistically significant differences were observed between GABA-elicited currents recorded in the control saline or in the presence of the Ca2 +-release blocker, ruthenium red (Fig. 3C).
Fig. 3. The effect of intracellular Ca2 + concentration on the Cd2 +-block of the GABA-activated ion current. (A) Following a control GABA application (50 AM), intracellular injection of 0.1 mM EGTA and extracelullarly applied 50 AM Cd2 + failed to result in a blocking effect on RPeD1 neurone. (B) Simultaneous application of Cd2 + and 2 AM ruthenium red eliminated the Cd2 + effect. (C) Summarized data did not show any significant alteration in mean amplitude. Bars represent mean F S.D.
ing that extracellular Ca2 +-entry plays no significant role in the Cd2 +-induced blocking mechanisms. To investigate the role of free intracellular Ca2 + in a 2+ Cd -induced block of a GABA-elicited current, Ca2 + was intracellularly injected into RPaD1 neurone. A few seconds thereafter, GABA was applied, in order to elicit an inward current. By injecting Ca2 + into the cell, intracellular Ca2 + levels reached a concentration of about 1.6 10 4 mM, which completely eliminated the GABA-activated inward Cl current (Fig. 2A). The effect was repeatable after 5 min of washout and caused the same result (not shown). Methylxanthine caffeine has been used for inducing Ca2 + release from intracellular stores, especially from the endoplasmic reticulum (for review, see Ref. [38]). Neurones were perfused in 1 mM caffeine, which caused a 45% decrease in GABA-elicited response, compared to the control (Fig. 2B). Cd2 +, in the presence of extracellular caffeine, further reduced the GABA-induced response until it
Fig. 4. The effect of intracellular Cd2 + injection on GABA-activated chloride ion currents. (A) Current elicited by GABA application (50 AM). (B) Cd2 + was injected into the cell (RPeD1); GABA elicited an inward current comparable to that of the control. A mechanical artifact appeared at the moment of pressure injection (arrow). After Cd2 + injection, the GABAactivated current was recorded every 5 min. (C and E) show currents recorded at the 5th, 10th and 30th min. (F) Intracellular cadmium failed to produce any significant changes in the amplitude of the measured currents. Bars represent mean F S.D.
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Extracellular application of cadmium (50 AM), 1 min later of intracellular injection of the Ca2 +-chelator, EGTA (0.1 M), was performed to ascertain whether the blocking effect could be reduced by lowering the intracellular free Ca2 + concentration. After the injection of EGTA, the permanently
Fig. 5. The effect of different G-protein inhibitors on the Cd2 +-block of GABA-induced currents. (A) In RPeD1 neurone pretreated with pertussis toxin (PT), Cd2 + decreased the GABA elicited current, but did not block it. (B) In the presence of suramin, Cd2 + proved to be ineffective at blocking the GABA-current (RPaD1). (C) Intracellular injection of 1mM GTP-g-S effectively eliminated the Cd2 + effect on RPaD1 neurone. (D) A summary of the effects induced by PT, suramin and GTP-g-S on the GABA-elicited current block by Cd2 +. The results represent the mean (S.D.) for three to eight experiments. No statistical differences were found between means, except in the column showing the Cd2 + effect. The first two columns are taken from Fig. 1.
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present Cd2 + failed to significantly decrease the GABAactivated Cl current, compared to the control (85 F 25%, n = 5, Fig. 3A). The intracellular free Ca2 + concentration was estimated to be less than 1.5 10 7 M. EGTA itself had no significant effect on the GABA-elicited current. In order to investigate the site of action, Cd2 + was also applied intracellularly. The concentration of Cd2 + in the pipette was 5 mM, which decreased to approximately 30– 50 AM after diffusion into the cell. Cadmium, when applied extracellularly at this concentration, significantly decreased the GABA-activated ion current, as was shown in Fig. 1. Following the intracellular injection of Cd2 +, GABA was applied by microperfusion, and the amplitude of the GABAinduced Cl current was measured (Fig. 4A). Intracellular application of Cd2 + had no effect on the GABA-induced current (Fig. 4B). Supposing that a possible delayed Cd2 + effect may take place, measurements were done every 5 min for up to 30 min. Fig. 4 shows an example of the GABAactivated chloride current on RPeD1 neurone measured at the 5th (Fig. 4C), 10th (Fig. 4D) and 30th (Fig. 4E) minutes after Cd2 + injection. Mean amplitude of the currents was reduced by 6 F 11% ( F, n = 4). These values, however, did not show significant differences from the control values. Hence, cadmium is essentially ineffective when injected intracellularly, suggesting that the metal ion binding sites are extracellular. We hypothesized a possible second messenger signaling pathway between Cd2 + binding and the increase of [Ca2 +]i via G-proteins. Therefore, the involvement of G-proteins was studied using pertussis toxin (PT), suramin and GTP-gS. Lymnaea ganglia were incubated in 2 Ag/ml pertussis toxin-containing saline for 30 h, and the blocking effect of Cd2 + (50 AM) on GABA-elicited ion currents was investigated. In the presence of 50 AM Cd2 +, the amplitude of GABA-activated currents decreased to 66 F 10% (n = 3) that of the control (Fig. 5A,D). Furthermore, Cd2 + (50 AM) when applied simultaneously with the G-protein inhibitor, suramin (10 AM) did not eliminate the GABA-activated current (102 F 11%, n = 6, Fig. 5B,D). Intracellular injection of a non-hydrolyzable GTP analogue, GTP-g-S (1 mM), also prevented blocking of the GABA-activated currents by extracellular Cd 2 + (112 F 34%, n = 3, Fig. 5C,D). Injection of GTP-g-S, alone, evoked an inward current in some neurones (not shown). All these experiments support our suggestion, that a G-protein is involved in the blocking of a GABA-elicited current by Cd2 + in Lymnaea neurones.
4. Discussion In the mammalian central nervous system, g-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, but, in some cases, its action is excitatory. GABAA receptors gate the transmembrane flow of Cl ions through a Gprotein-independent signaling pathway. This type of receptor is known to be a pentamer with 19 type of subunits in
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mammals [2]. Cloned GABAA receptor subunits in Lymnaea exhibited 36 – 50% identity with vertebrate receptor subunits [7]. The electrophysiological examination of Xenopus laevis oocytes, injected with transcribed molluscan RNA, showed that the receptor gates Cl , and the GABAevoked responses were reversibly blocked by bicucullin and picrotoxin [4,7]. Rubakhin et al. [27] identified giant neurones in Lymnaea (RPeD1, RPaD1, LPaV2), which are members of the respiratory and body withdrawal neuronal networks, possessing a native GABAA receptor subtype. In our previous work we have shown that extracellularly presented Cd2 + ions inhibited GABA-elicited responses in identified Lymnaea neurones. Analysis of the dose – response relationship indicated an EC50 of 9.2 AM and a Hill coefficient of 2.65 F 0.48 [22], suggesting multiple binding sites for Cd2 + ions. The work presented here describes the mechanisms of Cd2 +-block of the GABA elicited Cl inward current. It is suggested that Cd2 +, interacting with a putative Cd2 + binding protein, coupled to G-protein, mediates Ca2 + release from intracellular stores. The increased intracellular Ca2 + concentration consequently caused the decrease in GABA-induced current. The ineffectiveness of the intracellularly injected Cd2 + favours the existence of an extracellular binding site. Furthermore, the binding site could not be on the GABAA receptor/channel complex, because substances acting on Gproteins decreased the Cd2 + effect. We suggest, therefore, that Cd2 + exerted its effect by binding to a special ‘‘metal receptor’’ located at the membrane surface. The idea about the existence of a special metal receptor is not new. It was previously suggested, although not supported by strong experimental evidence in neurones [14]. However, in several non-neuronal cell types, the existence of a specific surface metal-receptor was demonstrated [10,21,31,37]. The strongest argument supporting the existence of metal-receptors was the ineffectiveness of intracellularly injected metal ions [10]. It was also supported by the data of Smith et al. [31], using the cell-permeant heavy metal chelator, TPEN, which has high affinity for Cd2 + and low affinity to Mg2 + and Ca2 +. They found that Cd2 + triggers Ca2 + mobilization from the extracellular side in human skin fibroblasts. After pretreatment of neurones with PT, in the presence of suramin or intracellularly injected GTP-g-S, the Cd2 +block was not present or it was substantially decreased. These results strongly suggest a G-protein involvement in the Cd2 +-signaling pathway. Our results agree with those data of Misra et al. [21], who described that treatment of macrophages with pertussis toxin, prior to Cd2 +, abolished the Cd2 +-induced increase in [Ca2 +]i. Divalent metals, including Cd2 +, appear to trigger Ca2 + release from intracellular stores, by binding to the extracellular metal receptors. Cellular calcium homeostasis is highly susceptible to heavy metals. Ca2 +/GABA-receptor interaction was verified in Lymnaea GABA-sensitive neurones in
the present study, and it was found that elevated intracellular Ca2 + completely abolished the GABA-induced chloride current. Furthermore, simultaneous chelation of intracellular Ca2 + with EGTA protected the cell from extracellular Cd2 + poisoning, similar to EGTA injection in Xenopus oocytes [10]. Also, blocking the Ca2 +-release from intracellular stores with ruthenium red had the same effect. Contrarily, caffeine, due to the release of Ca2 + from intracellular stores, exerted an effect similar to Cd2 +, decreasing the GABAelicited current. We have also confirmed in Ba2 +-substituted experiments, that in the absence of external Ca2 +, Cd2 + blocked GABA currents. These observations exclude the possibility that Cd2 + increases [Ca2 +]i by entering the neurone through voltage-sensitive calcium channels [34]. Our data are in accordance with the results of Yamagami et al. [37], who described that Cd2 + increased [Ca2 +]i in bovine chromaffin cells bathed in lowered external Ca2 + solution. The results also suggest that the inhibitory mechanism of Cd2 +, through an elevation in intracellular Ca2 +, may be a more appropriate explanation than a direct conformational change of the GABA-receptor. Miledi et al. [20] showed that interaction of cadmium, and other metals, with X. laevis oocyte membranes increased the cytosolic levels of inositol 1,4,5-trisphosphate (IP3), and caused the elevation of intracellular Ca2 +. The same was shown in human skin fibroblasts [31], in bovine chromaffin cells [37] and in murine macrophages [21]. It is known that a rise in [Ca2 +]i inactivates NMDA channels in hippocampal neurones [17,26], and ACh-induced conductance in Lymnaea neurones [5]. It was also demonstrated that intracellular Ca2 + has a negative effect on GABAactivated Cl currents by changing the binding affinity or by acting through a diffusible Ca2 +-dependent messenger [1,12,18,33]. This effect was found to be independent of the membrane potential [12]. However, GABAA receptors are differentially regulated by [Ca2 +]i in different brain regions, and the origin of calcium is important in controlling the function of receptors [1]. In summary, based on our data, we suggest the following pathway for the Cd2 +-induced blocking mechanism on GABA-elicited current in Lymnaea neurones: cadmium interacts, at the cell surface, with a putative Cd2 +-binding protein coupled to a G-protein, which presumably mediates IP3 synthesis, which releases stored Ca2 +, and activates Ca2 +-dependent protein kinases and/or phophatases. To reveal the exact toxic pathway of cadmium on GABA receptors, further studies are required. Modification of intracellular Ca2 +-mediated processes is regarded as an important mechanism for modulating the function of GABA receptors and consequently for controlling synaptic plasticity [30].
Acknowledgements Authors wish to thank to Dr. Katalin S.-Ro´zsa and Dr. Attila Szu¨cs for the critical reading of the manuscript, and
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Dr. Ka´roly Elekes for the helpful comments on it. This work was supported by OTKA grants nos. T037505, T032390 and by MEH 2002.
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Research report
Cadmium inhibits GABA-activated ion currents by increasing intracellular calcium level in snail neurons Ga´bor Molna´r *, Ja´nos Sala´nki, Tibor Kiss Department of Experimental Zoology, Balaton Limnological Research Institute, HAS, Tihany, Klebelsberg K. u. 3, P.O. Box 35, H-8237, Hungary Accepted 4 February 2004 Available online 1 April 2004
Abstract Blocking of the GABA-activated chloride current by cadmium was investigated in identified nerve cells (RPeD1, RPaD1) of the pond snail (Lymnaea stagnalis L.), using a two-microelectrode voltage-clamp technique. Cd2 +, at 50 AM extracellular concentration, inhibited GABA-activated chloride currents, both in normal and Ca2 +-free solution. Intracellular injection of Ca2 + or the application of caffeine mimicked the inhibitory effect of Cd2 + on GABA-elicited currents. Cd2 +-block was eliminated, or it was substantially decreased, when neurons were intracellularly loaded with EGTA, or when the Ca2 +-release was blocked by ruthenium red. The blocking effect of Cd2 + was also eliminated by applying G-protein inhibitors: pertussis toxin, suramin or GTP-g-S. Finally, intracellularly injected Cd2 + was ineffective at eliciting an inward current on GABA-activated currents, suggesting that the Cd2 +-binding site resides extracellularly. These results suggest that cadmium inhibited GABA-activated chloride currents by increasing the intracellular Ca2 + level, by releasing it from intracellular stores and by interacting with a putative G-protein-coupled cell-surface ‘‘metal-receptor’’. D 2004 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Ligand gated ion channels Keywords: Lymnaea stagnalis L.; Cadmium; Intracellular calcium; G-protein, neurone; GABA
1. Introduction Because of its high environmental impact cadmium is a frequently examined, heavy metal of biological importance. Cadmium has a long biological half-life ( f 30 years in humans), which contributes to its accumulation in living cells [34]. Exposure to this heavy metal causes carcinogenic, mutagenic and teratogenic effects, damaging of several organs, including the lung, kidney, gastrointestinal tract, bone and also the brain [8,13]. In vertebrates, cadmium enters the central nervous system either through the olfactory bulb [32] or through the blood – brain barrier. Penetration of cadmium into the brain can be facilitated by ethanol [25], chronic exposure to cadmium [29] or by forming complexes with other substances [24]. Once * Corresponding author. Tel.: +36-87-448-244x111; fax: +36-87-448006. E-mail address: [email protected] (G. Molna´r). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.02.035
cadmium is present in the intercellular spaces, it interferes with a number of cellular and physiological processes. Although the cellular actions of cadmium are not completely understood, the complexity of the underlying mechanisms is discussed in a number of reports [16,21]. Cadmium causes lipid peroxidation, damage to DNA, depletion of sulfhydryls, initiation of expression of heat shock proteins and methallothioneins, and induces alteration of calcium homeostasis by competing with calcium at the Ca2 + binding sites [8,15,35,36]. It is frequently used as an effective blocker of the voltage-sensitive L-type Ca2 + channels [6,28]. It was demonstrated, however, that cadmium reduces almost all kinds of ligand-gated and voltage-dependent ion currents (for review, see Ref. [15]). It was already described that extracellularly applied Cd2 + ions increased the cytosolic-free Ca2 + concentration by binding to the presumptive Cd2 + surface receptors in human skin fibroblast [31], endothelial cells [9], bovine chromaffin cells [37] and Xenopus oocytes [10]. In all cases, the increase
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in intracellular Ca2 +-concentration was elicited by generating inositol triphosphate (IP3) [20]. In our previous work, we have described the blocking effect of extracellularly applied cadmium on GABA-activated chloride-currents in Lymnaea neurones, and also the modulatory effect on GABA-evoked firing patterns [23]. Cadmium inhibited the GABA-mediated current starting from 5 AM concentration (KD = 9.2 AM) in a time- and concentration-dependent manner. The aim of the present study was to describe the signaling pathway by which extracellularly applied Cd2 + blocks GABA-elicited currents in neurones.
2. Materials and methods 2.1. Preparation Experiments were carried out on identified neurones of the pond snail Lymnaea stagnalis L. Animals were collected from the Lake Kis-Balaton during spring and summer, and were kept in aquarium in slowly running, natural lake water, at a temperature of 15– 20 jC. Over the course of the experiments, care was taken to minimize pain and discomfort to, as well as the unnecessary use of, animals. Measurements were performed on the GABAsensitive neurones (RPeD1 and RPaD1) of the isolated suboesophageal ganglionic ring [27]. Following dissection, the ganglionic ring was placed in the experimental chamber. The ganglia were pinned out in Sylgard-lined Petri dishes and the thick connective tissue sheath was removed mechanically. Following an enzymatic treatment with Protease E (type IV, Sigma) for 3 min, the ganglia were kept in refrigerator for at least 2 h and, thereafter, the cells were freed by opening the thin connective sheath using fine forceps. 2.2. Solutions The standard physiological solution (SPS) used in the experiments contained (in mM): NaCl 40, KCl 1.7, CaCl2 4, MgCl2 1.5, glucose 10, Tris – HCl 10, Tris –Cl 10. In the Ca2 +-free solution, Ca2 + was substituted by Ba2 +: NaCl 40, KCl 1.7, BaCl2 4, MgCl2 1.5, glucose 10, HEPES 10; pH was adjusted to 7.5 using NaOH. The following substances were used in the experiments: g-aminobutyric acid (GABA; 25 – 50 AM), ethylene glycol-bis(h-aminoethyl ether)N,N,NV,NV-tetraacetic acid (EGTA), glucose, Tris[hydroxymethyl]amino-methane (Tris), Tris – HCl, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), suramin hexa sodium salt (suramin, 10 AM), ammoniated ruthenium oxychloride (ruthenium red, 2 AM), 1,3,7-trimethylxanthine (caffeine, 1 mM), pertussis toxin (incubation time was 30 h; 2 Ag/ml) and guanosine 5V-O-(3-thiotriphosphate) tetralithium salt (GTP-g-S, 1 mM). Substances listed above were purchased from Sigma. 5(6)-Carboxyfluorescein was
purchased from Eastman Kodak; CdCl2 and other inorganic salts were from Reanal, Hungary. 2.3. Experimental protocol Experiments were performed at room temperature (21 – 24 jC). In order to measure transmembrane ionic currents, two-microelectrode voltage-clamp technique was applied using a DAGAN 8500 voltage clamp amplifier. Glass micropipettes were filled with 2.5 mM KCl, having resistances between 2 and 5 MV. The preparation was continuously superfused with SPS at a rate of 1.1 ml/min, allowing the complete exchange of the recording chamber volume in 2 min. Solution containing CdCl2 was added by perfusion at a concentration of 50 AM. GABA was applied by microperfusion, using a plastic capillary with an orifice of 310 Am positioned over the neurone under study. Changing the hydrostatic pressure applied to the pipette controlled the solution flow from the pipette. Repeated transmitter applications were carried out in 5 min intervals for 1 –2 s, in order to avoid desensitization. The holding potential was set between 75 and 50 mV in all experiments. 2.4. Intracellular injection Several substances were introduced into the cell by pressure injection. Substances for intracellular injection were: 0.1 M CaCl2, 0.1 M K-EGTA (0.1 M EGTA, 0.3 M KOH adjusted with HCl to pH 7.2), 5 mM CdCl2 and 1 mM GTP-g-S. Borosilicate micropipettes (TW150F-3, WPI) were pulled, under the same conditions, with a vertical puller (PC-10, Narishige), and used for ejection without any other modifications of the tip (breaking, beveling, etc.). Intracellular concentrations of these substances were calculated from the ratio of the injected substance and the cell volumes. The ejected volume was calculated before and after experiments, from the diameter of the spherical droplet formed under oil [11,19]. The injected amount was between 3 and 25 pl. The size of the investigated cell was approximated using the formula of an ellipsoid volume: Vcell = d1d2d3p/6 where Vcell is the volume of the cell, d1 is the width and d2 is the length of the investigated cells. It was supposed that the third parameter is the same as the shorter one (d3 = d1). The calculated average volume of both RPaD1 and RPeD1 (n = 6 –10) was f 1200 F 120 pl. 5(6)-Carboxyfluorescein was used as tracer to visually control the success of the injection under blue illumination. CaCl2 was intracellularly delivered using a third microelectrode. Free [Ca2 +]i concentration was determined using a computer program that calculates the free ion concentration from the known concentrations of metal-ligand complexes at defined pH and temperature values [3]. The following intracellular parameters were used for the calculation: K-EGTA 210 4 M, Ca2 +10 4 M, pH = 7.2, temperature 22 jC.
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The intracellular [Ca2 +] value is unlikely to be higher, the EGTA concentration is not supposed to be lower than this. K-EGTA and 5(6)-carboxyfluorescein and the vehicle (intracellular fluid) were tested to have no significant effect on the measured current. 2.5. Statistical analysis Results are presented as mean F standard deviation (S.D.). Statistical significance was determined using a Student’s t-test (paired). A P-value less than 0.05 was considered to be statistically significant.
3. Results Extracellularly applied GABA elicited a Cl -dependent inward current of 8 – 10 nA at a holding potential of 75 mV. The current activated and slowly decayed in 4 s period (Fig. 1A) and was completely blocked by exposure to 50 AM extracellular cadmium within 1 – 2 min (Fig. 1B). This effect was partially reversible after a 5-min washout (34% of control, not shown). The complete recovery of Cd2 + treat-
Fig. 2. The effect of intracellular Ca2 + on the GABA-activated chloride current. (A) The left trace shows the response to 50 AM GABA. On the right Ca2 + was injected into the cell (0.1 mM, RPaD1), followed by application of GABA. Under these conditions, the GABA response was blocked. (B) Caffeine (1 mM) also decreased the GABA-elicited conductance (RPaD1). (C) The effect of caffeine was enhanced with simultaneous 50 AM Cd2 + application. (D) Columns show normalized amplitudes of GABA-elicited responses with and without increased intracellular Ca2 +. The first two columns are taken from Fig. 1.
Fig. 1. The blocking effect of cadmium on GABA-activated chloride ion current. (A) Applying 25 AM GABA directly to the membrane surface of the neurone (RPaD1) induced an inward current. (B) By adding 50 AM Cd2 + to the SPS, the GABA-activated current was completely abolished. (C) The relative mean current amplitudes of the GABA-elicited current obtained in SPS (control) and in the presence of 50 AM Cd2 + in the extracellular saline solution containing Ca2 + (4 mM) and in Ca2 +-free Ba2 + (4 mM) solution.
ment was not followed, because it can last for hours. These results agree well with those described earlier on identified Lymnaea neurones [23,27]. In the next experiments, the role of extracellular Ca2 + was examined. To do this, we replaced Ca2 + ions in the extracellular solution with Ba2 + ions. Both in the control and Ba2 +-containing saline, Cd2 + blocked the GABAelicited current with the same efficiency (Fig. 1C), suggest-
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was almost non-existent (Fig. 2C), which indicates a common mechanism for both caffeine and Cd2 +. Likewise, treatment of the neurone with 2 AM extracellularly applied ruthenium red prevented Cd2 + from blocking the GABA-response (81 F 32%, n = 6, Fig. 3B). No statistically significant differences were observed between GABA-elicited currents recorded in the control saline or in the presence of the Ca2 +-release blocker, ruthenium red (Fig. 3C).
Fig. 3. The effect of intracellular Ca2 + concentration on the Cd2 +-block of the GABA-activated ion current. (A) Following a control GABA application (50 AM), intracellular injection of 0.1 mM EGTA and extracelullarly applied 50 AM Cd2 + failed to result in a blocking effect on RPeD1 neurone. (B) Simultaneous application of Cd2 + and 2 AM ruthenium red eliminated the Cd2 + effect. (C) Summarized data did not show any significant alteration in mean amplitude. Bars represent mean F S.D.
ing that extracellular Ca2 +-entry plays no significant role in the Cd2 +-induced blocking mechanisms. To investigate the role of free intracellular Ca2 + in a 2+ Cd -induced block of a GABA-elicited current, Ca2 + was intracellularly injected into RPaD1 neurone. A few seconds thereafter, GABA was applied, in order to elicit an inward current. By injecting Ca2 + into the cell, intracellular Ca2 + levels reached a concentration of about 1.6 10 4 mM, which completely eliminated the GABA-activated inward Cl current (Fig. 2A). The effect was repeatable after 5 min of washout and caused the same result (not shown). Methylxanthine caffeine has been used for inducing Ca2 + release from intracellular stores, especially from the endoplasmic reticulum (for review, see Ref. [38]). Neurones were perfused in 1 mM caffeine, which caused a 45% decrease in GABA-elicited response, compared to the control (Fig. 2B). Cd2 +, in the presence of extracellular caffeine, further reduced the GABA-induced response until it
Fig. 4. The effect of intracellular Cd2 + injection on GABA-activated chloride ion currents. (A) Current elicited by GABA application (50 AM). (B) Cd2 + was injected into the cell (RPeD1); GABA elicited an inward current comparable to that of the control. A mechanical artifact appeared at the moment of pressure injection (arrow). After Cd2 + injection, the GABAactivated current was recorded every 5 min. (C and E) show currents recorded at the 5th, 10th and 30th min. (F) Intracellular cadmium failed to produce any significant changes in the amplitude of the measured currents. Bars represent mean F S.D.
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Extracellular application of cadmium (50 AM), 1 min later of intracellular injection of the Ca2 +-chelator, EGTA (0.1 M), was performed to ascertain whether the blocking effect could be reduced by lowering the intracellular free Ca2 + concentration. After the injection of EGTA, the permanently
Fig. 5. The effect of different G-protein inhibitors on the Cd2 +-block of GABA-induced currents. (A) In RPeD1 neurone pretreated with pertussis toxin (PT), Cd2 + decreased the GABA elicited current, but did not block it. (B) In the presence of suramin, Cd2 + proved to be ineffective at blocking the GABA-current (RPaD1). (C) Intracellular injection of 1mM GTP-g-S effectively eliminated the Cd2 + effect on RPaD1 neurone. (D) A summary of the effects induced by PT, suramin and GTP-g-S on the GABA-elicited current block by Cd2 +. The results represent the mean (S.D.) for three to eight experiments. No statistical differences were found between means, except in the column showing the Cd2 + effect. The first two columns are taken from Fig. 1.
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present Cd2 + failed to significantly decrease the GABAactivated Cl current, compared to the control (85 F 25%, n = 5, Fig. 3A). The intracellular free Ca2 + concentration was estimated to be less than 1.5 10 7 M. EGTA itself had no significant effect on the GABA-elicited current. In order to investigate the site of action, Cd2 + was also applied intracellularly. The concentration of Cd2 + in the pipette was 5 mM, which decreased to approximately 30– 50 AM after diffusion into the cell. Cadmium, when applied extracellularly at this concentration, significantly decreased the GABA-activated ion current, as was shown in Fig. 1. Following the intracellular injection of Cd2 +, GABA was applied by microperfusion, and the amplitude of the GABAinduced Cl current was measured (Fig. 4A). Intracellular application of Cd2 + had no effect on the GABA-induced current (Fig. 4B). Supposing that a possible delayed Cd2 + effect may take place, measurements were done every 5 min for up to 30 min. Fig. 4 shows an example of the GABAactivated chloride current on RPeD1 neurone measured at the 5th (Fig. 4C), 10th (Fig. 4D) and 30th (Fig. 4E) minutes after Cd2 + injection. Mean amplitude of the currents was reduced by 6 F 11% ( F, n = 4). These values, however, did not show significant differences from the control values. Hence, cadmium is essentially ineffective when injected intracellularly, suggesting that the metal ion binding sites are extracellular. We hypothesized a possible second messenger signaling pathway between Cd2 + binding and the increase of [Ca2 +]i via G-proteins. Therefore, the involvement of G-proteins was studied using pertussis toxin (PT), suramin and GTP-gS. Lymnaea ganglia were incubated in 2 Ag/ml pertussis toxin-containing saline for 30 h, and the blocking effect of Cd2 + (50 AM) on GABA-elicited ion currents was investigated. In the presence of 50 AM Cd2 +, the amplitude of GABA-activated currents decreased to 66 F 10% (n = 3) that of the control (Fig. 5A,D). Furthermore, Cd2 + (50 AM) when applied simultaneously with the G-protein inhibitor, suramin (10 AM) did not eliminate the GABA-activated current (102 F 11%, n = 6, Fig. 5B,D). Intracellular injection of a non-hydrolyzable GTP analogue, GTP-g-S (1 mM), also prevented blocking of the GABA-activated currents by extracellular Cd 2 + (112 F 34%, n = 3, Fig. 5C,D). Injection of GTP-g-S, alone, evoked an inward current in some neurones (not shown). All these experiments support our suggestion, that a G-protein is involved in the blocking of a GABA-elicited current by Cd2 + in Lymnaea neurones.
4. Discussion In the mammalian central nervous system, g-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, but, in some cases, its action is excitatory. GABAA receptors gate the transmembrane flow of Cl ions through a Gprotein-independent signaling pathway. This type of receptor is known to be a pentamer with 19 type of subunits in
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mammals [2]. Cloned GABAA receptor subunits in Lymnaea exhibited 36 – 50% identity with vertebrate receptor subunits [7]. The electrophysiological examination of Xenopus laevis oocytes, injected with transcribed molluscan RNA, showed that the receptor gates Cl , and the GABAevoked responses were reversibly blocked by bicucullin and picrotoxin [4,7]. Rubakhin et al. [27] identified giant neurones in Lymnaea (RPeD1, RPaD1, LPaV2), which are members of the respiratory and body withdrawal neuronal networks, possessing a native GABAA receptor subtype. In our previous work we have shown that extracellularly presented Cd2 + ions inhibited GABA-elicited responses in identified Lymnaea neurones. Analysis of the dose – response relationship indicated an EC50 of 9.2 AM and a Hill coefficient of 2.65 F 0.48 [22], suggesting multiple binding sites for Cd2 + ions. The work presented here describes the mechanisms of Cd2 +-block of the GABA elicited Cl inward current. It is suggested that Cd2 +, interacting with a putative Cd2 + binding protein, coupled to G-protein, mediates Ca2 + release from intracellular stores. The increased intracellular Ca2 + concentration consequently caused the decrease in GABA-induced current. The ineffectiveness of the intracellularly injected Cd2 + favours the existence of an extracellular binding site. Furthermore, the binding site could not be on the GABAA receptor/channel complex, because substances acting on Gproteins decreased the Cd2 + effect. We suggest, therefore, that Cd2 + exerted its effect by binding to a special ‘‘metal receptor’’ located at the membrane surface. The idea about the existence of a special metal receptor is not new. It was previously suggested, although not supported by strong experimental evidence in neurones [14]. However, in several non-neuronal cell types, the existence of a specific surface metal-receptor was demonstrated [10,21,31,37]. The strongest argument supporting the existence of metal-receptors was the ineffectiveness of intracellularly injected metal ions [10]. It was also supported by the data of Smith et al. [31], using the cell-permeant heavy metal chelator, TPEN, which has high affinity for Cd2 + and low affinity to Mg2 + and Ca2 +. They found that Cd2 + triggers Ca2 + mobilization from the extracellular side in human skin fibroblasts. After pretreatment of neurones with PT, in the presence of suramin or intracellularly injected GTP-g-S, the Cd2 +block was not present or it was substantially decreased. These results strongly suggest a G-protein involvement in the Cd2 +-signaling pathway. Our results agree with those data of Misra et al. [21], who described that treatment of macrophages with pertussis toxin, prior to Cd2 +, abolished the Cd2 +-induced increase in [Ca2 +]i. Divalent metals, including Cd2 +, appear to trigger Ca2 + release from intracellular stores, by binding to the extracellular metal receptors. Cellular calcium homeostasis is highly susceptible to heavy metals. Ca2 +/GABA-receptor interaction was verified in Lymnaea GABA-sensitive neurones in
the present study, and it was found that elevated intracellular Ca2 + completely abolished the GABA-induced chloride current. Furthermore, simultaneous chelation of intracellular Ca2 + with EGTA protected the cell from extracellular Cd2 + poisoning, similar to EGTA injection in Xenopus oocytes [10]. Also, blocking the Ca2 +-release from intracellular stores with ruthenium red had the same effect. Contrarily, caffeine, due to the release of Ca2 + from intracellular stores, exerted an effect similar to Cd2 +, decreasing the GABAelicited current. We have also confirmed in Ba2 +-substituted experiments, that in the absence of external Ca2 +, Cd2 + blocked GABA currents. These observations exclude the possibility that Cd2 + increases [Ca2 +]i by entering the neurone through voltage-sensitive calcium channels [34]. Our data are in accordance with the results of Yamagami et al. [37], who described that Cd2 + increased [Ca2 +]i in bovine chromaffin cells bathed in lowered external Ca2 + solution. The results also suggest that the inhibitory mechanism of Cd2 +, through an elevation in intracellular Ca2 +, may be a more appropriate explanation than a direct conformational change of the GABA-receptor. Miledi et al. [20] showed that interaction of cadmium, and other metals, with X. laevis oocyte membranes increased the cytosolic levels of inositol 1,4,5-trisphosphate (IP3), and caused the elevation of intracellular Ca2 +. The same was shown in human skin fibroblasts [31], in bovine chromaffin cells [37] and in murine macrophages [21]. It is known that a rise in [Ca2 +]i inactivates NMDA channels in hippocampal neurones [17,26], and ACh-induced conductance in Lymnaea neurones [5]. It was also demonstrated that intracellular Ca2 + has a negative effect on GABAactivated Cl currents by changing the binding affinity or by acting through a diffusible Ca2 +-dependent messenger [1,12,18,33]. This effect was found to be independent of the membrane potential [12]. However, GABAA receptors are differentially regulated by [Ca2 +]i in different brain regions, and the origin of calcium is important in controlling the function of receptors [1]. In summary, based on our data, we suggest the following pathway for the Cd2 +-induced blocking mechanism on GABA-elicited current in Lymnaea neurones: cadmium interacts, at the cell surface, with a putative Cd2 +-binding protein coupled to a G-protein, which presumably mediates IP3 synthesis, which releases stored Ca2 +, and activates Ca2 +-dependent protein kinases and/or phophatases. To reveal the exact toxic pathway of cadmium on GABA receptors, further studies are required. Modification of intracellular Ca2 +-mediated processes is regarded as an important mechanism for modulating the function of GABA receptors and consequently for controlling synaptic plasticity [30].
Acknowledgements Authors wish to thank to Dr. Katalin S.-Ro´zsa and Dr. Attila Szu¨cs for the critical reading of the manuscript, and
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Dr. Ka´roly Elekes for the helpful comments on it. This work was supported by OTKA grants nos. T037505, T032390 and by MEH 2002.
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