Calcium signaling in prostate cancer cells of increasing malignancy.

Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca2+]i) homeostasis. This study investigates [Ca2+]i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca2+]i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca2+ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca2+]i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca2+ dependence of prostate carcinogenesis.

Epigallocatechin-3-gallate mobilizes intracellular Ca2+ in prostate cancer cells through combined Ca2+ entry and Ca2+-induced Ca2+ release.

AIMS: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. MAIN METHODS: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. KEY FINDINGS: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 µM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SIGNIFICANCE: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.

Posidonia oceanica (L.) Delile Ethanolic Extract Modulates Cell Activities with Skin Health Applications.

Seagrasses are high plants sharing adaptive metabolic features with both terrestrial plants and marine algae, resulting in a phytocomplex possibly endowed with interesting biological properties. The aim of this study is to evaluate the in vitro activities on skin cells of an ethanolic extract obtained from the leaves of Posidonia oceanica (L.) Delile, family Potamogetonaceae, herein named Posidonia ethanolic extract (PEE). PEE showed high radical scavenging activity, high phenolic content, and resulted rich in chicoric acid, as determined through HPLC-MS analysis. The use of MTT assay on fibroblasts showed a PEE cytotoxicity threshold (IC05) of 50 µg/mL at 48 h, while a sub-toxic dose of 20 µg/mL induced a significant increase of fibroblast growth rate after 10 days. In addition, an ELISA assay revealed that PEE doses of 5 and 10 µg/mL induced collagen production in fibroblasts. PEE induced dose-dependent mushroom tyrosinase inhibition, up to about 45% inhibition at 1000 µg/mL, while 50% reduction of melanin was observed in melanoma cells exposed to 50 µg/mL PEE. Finally, PEE lipolytic activity was assessed by measuring glycerol release from adipocytes following triglyceride degradation. In conclusion, we have collected new data about the biological activities of the phytocomplex of P. oceanica seagrass on skin cells. Our findings indicate that PEE could be profitably used in the development of products for skin aging, undesired hyperpigmentation, and cellulite.

Resveratrol induces intracellular Ca(2+) rise via T-type Ca(2+) channels in a mesothelioma cell line.

AIMS: Intracellular calcium (Ca(2+)) is known to play an important role in cancer development and growth. Resveratrol (Res) is a stilbene polyphenol occurring in several plant species and known for various possible beneficial effects, including its ability to inhibit proliferation and to induce apoptosis in cancer cells. This study was designed to determine whether Res affects Ca(2+) signaling in cancer cells. MAIN METHODS: We used the REN human mesothelioma cell line, as an in vitro cancer cell model, and the non-malignant human mesothelial MeT5A cell line, as normal cell model. Cytosolic Ca(2+) concentration was measured by the fluorescent indicator Fura-2. Immunofluorescence, Western blot, and siRNA technique were employed to assess the involvement of T-type Ca(2+) channels. Cell viability was determined by the calcein assay. KEY FINDINGS: REN cells transiently exposed to 1-10µM Res showed increasing peaks of Ca(2+) that were absent in Ca(2+)-free medium and were reduced by non-selective (Ni(2+)), and highly selective (NNC 55-0396) T-type Ca(2+) channels antagonist, and by siRNA knockout of Cav3.2T-type Ca(2+) channel gene. Dose-dependent curve of Res-induced Ca(2+) peaks showed a rightward shift in normal MeT-5A mesothelial cells (EC50=4.9µM) with respect to REN cells (EC50=2.7µM). Moreover, incubation with 3 and 10µM Res for 7days resulted in cell growth inhibition for REN, but not for MeT-5A cells. SIGNIFICANCE: Res induces Ca(2+) influx, possibly mediated through T-type Ca(2+) channels, with significant selectivity towards mesothelioma cells, suggesting a possible use as an adjuvant to chemotherapy drugs for mesothelioma clinical treatment.

Oleuropein-Enriched Olive Leaf Extract Affects Calcium Dynamics and Impairs Viability of Malignant Mesothelioma Cells.

Malignant mesothelioma is a poor prognosis cancer in urgent need of alternative therapies. Oleuropein, the major phenolic of olive tree (Olea europaea L.), is believed to have therapeutic potentials for various diseases, including tumors. We obtained an oleuropein-enriched fraction, consisting of 60% w/w oleuropein, from olive leaves, and assessed its effects on intracellular Ca(2+) and cell viability in mesothelioma cells. Effects of the oleuropein-enriched fraction on Ca(2+) dynamics and cell viability were studied in the REN mesothelioma cell line, using fura-2 microspectrofluorimetry and MTT assay, respectively. Fura-2-loaded cells, transiently exposed to the oleuropein-enriched fraction, showed dose-dependent transient elevations of cytosolic Ca(2+) concentration ([Ca(2+)]i). Application of standard oleuropein and hydroxytyrosol, and of the inhibitor of low-voltage T-type Ca(2+) channels NNC-55-0396, suggested that the effect is mainly due to oleuropein acting through its hydroxytyrosol moiety on T-type Ca(2+) channels. The oleuropein-enriched fraction and standard oleuropein displayed a significant antiproliferative effect, as measured on REN cells by MTT cell viability assay, with IC50 of 22 µg/mL oleuropein. Data suggest that our oleuropein-enriched fraction from olive leaf extract could have pharmacological application in malignant mesothelioma anticancer therapy, possibly by targeting T-type Ca(2+) channels and thereby dysregulating intracellular Ca(2+) dynamics.

Ion channel and lipid scramblase activity associated with expression of TMEM16F/ANO6 isoforms.

TMEM16F is a membrane protein with possible dual function as an ion channel and a phospholipid scramblase. The properties of ion channels associated with TMEM16F and the link between ion channel and scramblase activity are a matter of debate. We studied the properties of four isoforms of TMEM16F generated by alternative splicing. Upregulation of three TMEM16F isoforms or silencing of endogenous TMEM16F increased and decreased, respectively, both scramblase and channel activities. Introduction of an activating mutation in TMEM16F sequence caused a marked increase in phosphatidylserine scrambling and in ion transport indicating direct involvement of the protein in both functions. TMEM16F, also known as ANO6, is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F variants generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular Ca(2+) concentrations and positive membrane potentials. These variants showed different degrees of Ca(2+) sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for Cl(-) over Na(+) . A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent Ca(2+) sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel.

Epigallocatechin-3-gallate elicits Ca2+ spike in MCF-7 breast cancer cells: essential role of Cav3.2 channels.

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca(2+) channels toward a mechanistic study on the effect of EGCG on [Ca(2+)]i. Confocal Ca(2+) imaging showed that EGCG induces a [Ca(2+)]i spike which is due to extracellular Ca(2+) entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca(2+)channel blockers. siRNA knockdown of T-type Ca(2+) channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K(+) currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K(+) channel induced a reduction of the EGCG [Ca(2+)]i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K(+) current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca(2+)]i by EGCG may be relevant in breast cancer treatment.

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

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

Multiple effects of copper on NMDA receptor currents.

Copper (Cu) is an essential metal present in the human brain and released from synaptic vesicles following neuronal depolarization. Cu is known to reduce the NMDA receptor (NR) current with IC50≈20 µM. We have studied the effect of Cu on the NR current in cultured neonatal rat cerebellum granule cells (CGC) and in transiently transfected HEK293 cells (HEK), expressing either GluN1/GLUN2A or GluN1/GluN2B receptors. In CGCs, Cu causes a potentiation of the NR current at concentrations <30 µM (EC50=4.6 µM) and a block at higher concentrations (IC50=24 µM). In Fura2 loaded CGCs, Cu (≤30 µM) caused an increase of NMDA-driven calcium influx. This facilitating effect was prevented by pre-treatment with the reducing agent DTT. Cu also caused an increase of the NR current in GluN1/GluN2A receptors (EC50=2 µM) and a block at higher concentrations (IC50=26 µM). Both facilitation and inhibition were independent of voltage. The effect of Cu was quantitatively similar in GluN1/GluN2B receptors, which were potentiated by 10 µM and inhibited by 100 µM Cu. Potentiation was absent in mutants deleted of their entire amino terminal domain (ATD) of the protein, suggesting an involvement of this region in the interaction. These results indicate that Cu can facilitate the NR current at lower concentrations than those required for blocking it; this effect can have consequences on the activity of the metal at synaptic and extrasynaptic sites.

Role of calcium channels in heavy metal toxicity.

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

Disrupted pro- and antioxidative balance as a mechanism of neurotoxicity induced by perinatal exposure to lead.

The aim of this paper was to examine if pre- and neonatal exposure that results in lead (Pb) concentration below 'safe level' (10 µg/dL) in offspring blood may cause disruption of the pro/antioxidant balance in the developing rat brain. We studied oxidative stress intensity (malondialdehyde (MDA) concentration) as well as mRNA, protein expression and the activity of copper/zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (GPx4), catalase (CAT), glutathione reductase (GSR). We also measured glutathione (GSH) concentrations in selected structures of the rat brain (forebrain cortex, FC, cerebellum, C, and hippocampus, H) and showed cellular localization of GPx4, SOD1 and SOD2 expressions in the hippocampus by immunohistochemical examinations. Despite low Pb level in blood we observed decrease of the activity of some antioxidant enzymes as well as mRNA and protein expression downregulation associated with an increase of MDA level and CAT expression upregulation, especially in the hippocampus region. At the subcellular level, downregulation of SOD2 expression and decreased enzyme activity as well as mitochondrial pool of GSH suggest also that mitochondrial mechanisms might account for Pb neurotoxicity mechanism. For some enzymes, we found differences in the effects of Pb on the level of expression and activity. The activity of CAT decreased despite an increase in mRNA and protein expression; and likewise the activities SOD1, GPx1 GPx4 decreased, despite substantially unchanged level of expression. These effects may be the result of impairment of catalytic function of the enzyme protein caused by Pb interaction or of reduction in the availability of cofactors. We conclude that antioxidant system of the hippocampus of immature rat brain is highly vulnerable to perinatal Pb exposure. Therefore, oxidative stress may be one of the possible mechanisms disturbing cellular metabolism in this structure. Disruption of pro- and antioxidant balance should be considered as a potential mechanism of the observed Pb adverse effects, leading to the impaired learning ability caused by Pb exposure in children.

The influence of calcium ions on nickel modulation of NMDA receptor currents.

N-Methyl-D-aspartate (NMDA) receptors (NRs) are glutamate-gated channels critical for the functioning of the nervous system. They are assembled from two types of subunits, the essential GluN1 and at least one type of GluN2 (A, B, C, D) subunit. Nickel (Ni) modulates the NR current in a way dependent on the GluN2 subunit present. Besides voltage-dependent and voltage-independent inhibition, in GluN2B-containing channels Ni enhances channel activity. We have recently identified several domains of the channel involved in Ni interaction, but many aspects of this modulation remain elusive. The purpose of the present work is to investigate the role of calcium (Ca) in the effect of Ni on the NR current measured by voltage- and patch-clamp in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells expressing GluN1/GluN2B recombinant receptors. In both expression systems, in the presence of a physiological concentration of Ca (1.8 mM), Ni increased the NR current with EC(50) in the µM range, but this potentiation was reduced by decreasing Ca concentration or when Ca was substituted with Ba. In injected oocytes, the effect of Ni in 0.3 mM external Ba was only inhibitory (IC(50) = 65 µM). Increasing the internal calcium buffering by EGTA and BAPTA application, as well as incubation with cytoskeleton perturbing agents, colchicine and cytochalasin-D, did not produce major modifications in the Ni effect. These observations indicate that Ni-mediated potentiation is not dependent on Ca influx and internal Ca concentration, but it is dependent on external Ca, which possibly interacts with the extracellular portion of the protein through a modulatory binding site.

Acquisition of neuron-like electrophysiological properties in neuroblastoma cells by controlled expression of NDM29 ncRNA.

Neuroblastoma is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumor nodules. It has been previously shown that the over-expression of a specific non-coding RNA, NDM29, reduces neuroblastoma development promoting cell differentiation. We have used neuroblastoma cells expressing NDM29 at its basal level (Mock cells) or at 5.4-fold higher levels (S1 cells) to investigate whether a functional differentiation correlates with morphological and biochemical development induced by NDM29 expression. First, analyzing the expression of specific markers we demonstrated that NDM29 expression is accompanied by a well coordinated differentiation process toward a neuron-like, rather than toward a glial-like, phenotype. Next, we defined the neuron-like traits of S1 in terms of secretion of cytokines involved in axon guidance, synapse formation and neurite outgrowth. Finally, we characterized the ionic channel apparatus of S1 cells by patch-clamp technique and compared with the Mock counterpart. S1 cells showed much higher levels of fast inactivating Na(+) current and were able to generate mature action potentials. Moreover, they developed expression of functional GABA(A) receptors on their membrane. In contrast, the two cell lines shared very similar pools of functional K(+) channels, although slight quantitative differences can be described. Our results suggest that a maturation occurs in neuroblastoma as a consequence of NDM29 expression, inducing the appearance of neuronal-like properties. In this context, S1 cells may represent a novel in vitro tool for electrophysiological and pharmacological studies of human cells of the neural lineage.

South African plants used in traditional medicine to treat epilepsy have an antagonistic effect on NMDA receptor currents.

ETHNOPHARMACOLOGICAL RELEVANCE: Several Searsia species (Anacardiaceae), including Searsia dentata and Searsia pyroides, are used in South Africa traditional medicine to treat epilepsy. Ethanol leaf extracts of these plants have been shown to act as possible antagonists of N-methyl-d-aspartate (NMDA)-type glutamate receptors. MATERIALS AND METHODS: Leaf material of three Searsia species were collected from the Botanical Garden at the University of KwaZulu-Natal, Pietermaritzburg; dried and extracted with ethanol in an ultrasound bath. Filtered and dried extracts were resuspended in DMSO (100mg/ml) and diluted in the recording solution. The effect of Searsia dentata, Searsia pyroides and Searsia glauca extracts was investigated in dissociated cerebellar granule cells (CGCs) from 8-day-old rats and in transiently transfected HEK (human embryonic kidney) 293 cells (HEK), expressing either NR1a/NR2A or NR1a/NR2B receptors. In both systems we measured whole-cell currents elicited by 0.5mM NMDA (CGCs) or 50 µM glutamic acid (HEK) at -60 mV in 0Mg and 30 µM glycine and NMDA driven Ca influx in Fura2-loaded CGC. RESULTS: Searsia dentata and Searsia pyroides ethanol extracts caused a dose-dependent decrease of NR current with ED(50) close to 0.03 mg/ml in CGC and a similar inhibition (80% with 1mg/ml) in HEK cells, while Searsia glauca was much less effective. The inhibition was dependent on time of incubation and slightly favored by opening of the NR channel. It was hardly reversible during the recording time, but was not caused by accelerated run-down or by interaction with the modulatory redox site. Searsia pyroides ethanol extract also depressed the NMDA stimulated increase in intracellular Ca. CONCLUSIONS: The data confirm the specificity of Searsia dentata and Searsia pyroides and justify their use in traditional medicine. These plants may combine one or more γ-aminobutyric acid (GABA)(A) agonists with one or more NMDA antagonists, thus representing an efficient treatment for epilepsy.

Molecular determinants of multiple effects of nickel on NMDA receptor channels.

Nickel (Ni2+) is a toxic metal that affects the function of several neuronal ionic channels. Ni2+ inhibits N-methyl-D: -aspartate receptor (NR) channel in a voltage-dependent manner, but also causes enhancement of NR2B-containing channel activity and voltage-independent inhibition of those containing NR2A. The present work was aimed to find the sites of Ni2+ interaction on the NR2A and NR2B subunits by expressing wild-type and mutated NRs in either HEK293 cells or Xenopus laevis oocytes. The point mutation N616G in the pore region of the NR2B subunit completely removed the voltage-dependent block. In NR2 subunits deleted for their entire amino terminal domain (ATD) and expressed with wild-type NR1 subunit, voltage-independent inhibition of NR2A-containing channels was not modified, but the potentiation effect was abolished in NR2B-containing channels. In the latter channels, potentiation of the current was also removed by H127A, D101A, D104A point mutations and by the double mutation H127AD101A, all located in lobe I of ATD, and reduced by the point mutation T233A in lobe II, suggesting that the interaction site that causes potentiation shares common determinants with the Zn2+ and ifenprodil binding sites. In contrast, in NR2A-containing channels, we postulate the existence of an additional divalent cation binding site in the M3-M4 extracellular loop. In these channels, the point mutation H801A in the NR2A subunit caused an important reduction of the voltage-independent block, with a 7-time increase in IC(50). The block was also partially, but not as prominently, reduced by the double mutation H705AH709A in the same region of NR1. This additional binding site can be responsible of specific heavy metal interaction with NR channels.

Molecular determinants of Pb2+ interaction with NMDA receptor channels.

Lead (Pb2+) is a potent neurotoxin that acts as a non-competitive, voltage-independent antagonist of the NMDA receptor (NR) channel. Pb2+ action partially overlaps with that of zinc (Zn2+), but precise coincidence with Zn2+ binding site is debated. We investigated the site of Pb2+ interaction in NR channels expressed in Xenopus laevis oocytes from the clones zeta1, epsilon1 or epsilon2 and mutated epsilon1 or epsilon2 forms. For each epsilon subunit we chose two mutations that have been identified as 'strong mutations' for Zn2+ binding and examined the effect of Pb2+ on channels that contained those mutations. In epsilon1-containing channels, mutations D102A and H128A caused a decrease of Pb2+ inhibition with a 10-fold (D102A) and four-fold (H128A) shift of IC50. In epsilon2-containing channels, the most effective mutation in removing Pb2+ inhibition was H127A, with a five-fold increase of IC50, while D101A was virtually ineffective. Other mutations, D104A, T103A, and T233A, were less effective. The double mutation D101AH127A, while reducing Zn2+ inhibition by nearly nine-fold, caused a minor (less than two-fold) shift in Pb2+ IC50. Competition experiments showed that increasing doses of Zn2+ reduced the apparent affinity for Pb2+ in epsilon1-containing receptors, but not in epsilon2-containing receptors. In addition the effect of Pb2+ on epsilon2-containing channels was additive with that of ifenprodil, with no competition for the site. Although none of the mutations that we have tested abolished the block by Pb2+, our results indicate that the action of this toxic metal on NR channels is more dependent on the receptor composition than previously thought, because Zn2+ is able to displace Pb2+ from its binding site in epsilon1-containing channels, but not in epsilon2-containing channels.

Nickel differentially affects NMDA receptor channels in developing cultured rat neurons.

Nickel (Ni(2+)) is a transition metal that exerts multiple and complex effects on N-methyl-d-aspartate (NMDA) channels. In both HEK293 cells and Xenopus laevis oocytes expressing recombinant NMDA receptors, Ni(2+) (<100 microM) caused a potentiation of NR2B-containing channels but a voltage-independent inhibition in those containing NR2A. We took advantage of this different response to investigate the developmental switch between NR2B and NR2A subunits in neonatal rat cerebellar granule cells up to 16 days in vitro (DIV) and in rat embryo cortical neurons up to 35 DIV. In both cultures, the effect of Ni(2+) on the NMDA current gradually changed from potentiating to inhibitory with progressing DIV, and the decline of potentiation correlated well with the decrease in sensitivity for the NR2B specific antagonist ifenprodil. Dose-dependent experiments confirmed that Ni(2+) has a different effect in younger cultures with respect to older ones, in agreement with an increase of the percentage of NR2A-containing receptors. The developmental switch occurred within the first 5 DIV in cerebellar granule cells and after 20 DIV in cortical neurons. All these data indicate that Ni(2+) is a suitable marker for the identification of NR2A and NR2B native channel subunits and can be used to trace the development of NMDA receptor composition.

NMDA receptors as targets of heavy metal interaction and toxicity.

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

Two-photon analysis of lead accumulation in rat cerebellar granule neurons.

Lead (Pb2+) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb2+ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 microM Pb2+, uptake reached a plateau level (approximately 45% of initial fluorescence) in 4 min and was partially antagonized by 25 microM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb2+ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb2+ sufficient to inactivate the transport system is close to 50 pM.

Susceptibility of insulinoma cells to cadmium and modulation by L-type calcium channels.

Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10-300 microM Cd for 15 min-6 h. While negligible at the end of the treatment, HIT cell death was evident after 18-24 h, and it was time-, dose- and serum-dependent. Short (< or = 60 min) Cd treatments with lower doses (< or = 100 microM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic beta-cell pathology.