María F. Cano-Abad

Galantamine prevents apoptosis induced by β-amyloid and thapsigargin: involvement of nicotinic acetylcholine receptors

Galantamine is currently used to treat Alzheimers disease patients; it behaves as a mild blocker of acetylcholinesterase (AChE) and has an allosteric modulating action on nicotinic acetylcholine receptors (nAChRs). In this study, we observed that galantamine prevented cell death induced by the peptide beta-amyloid(1-40) and thapsigargin in the human neuroblastoma cell line SH-SY5Y, as well as in bovine chromaffin cells. The protective effect of galantamine was concentration-dependent in both cell types; maximum protection was produced at 300 nM. The antiapoptotic effect of galantamine at 300 nM, against beta-amyloid(1-40) or thapsigargin-induced toxicity, was reversed by alpha-bungarotoxin. At neuroprotective concentrations, galantamine caused a mild and sustained elevation of the cytosolic concentration of calcium, [Ca2+]c, measured in single cells loaded with Fura-2. Incubation of the cells for 48 h with 300 nM galantamine doubled the density of alpha7 nicotinic receptors and tripled the expression of the antiapoptotic protein Bcl-2. These results strongly suggest that galantamine can prevent apoptotic cell death by inducing neuroprotection through a mechanism related to that described for nicotine, i.e. activation of nAChRs and upregulation of Bcl-2. These findings might explain the long-term beneficial effects of galantamine in patients suffering of Alzheimers disease.

ERG K+ channel blockade enhances firing and epinephrine secretion in rat chromaffin cells: the missing link to LQT2-related sudden death?

The ether‐a‐go‐go‐related genes (erg) are expressed in tissues other than heart and brain, in which human erg (HERG) K+ channels are known to regulate the repolarization of heart action potentials and neuronal spike‐frequency accommodation. We provide evidence that erg1 transcripts and ERG proteins are present in rat chromaffin cells in which we could isolate a K+ current that was biophysically and pharmacologically similar to the ERG current. Firing frequency and catecholamine release were analyzed at the single‐cell level by means of perforated patch‐clamp and carbon fiber electrochemical detection. It was found that the blocking of ERG, KATP, and KCa channels led to hyperexcitability and an increase in catecholamine release. Combined immunocytochemical experiments with antibodies directed against phenylethanolamine N‐methyltransferase and ERG channels suggested expression of these channels in epinephrine‐ but not in norepinephrine‐containing cells. It is concluded that, in addition to being crucial in regulating the QT period in the heart, ERG channels play a role in modulating epinephrine, a fundamental neurotransmitter shaping cardiac function. This finding suggests that the sudden death phenotype associated with LQT2 syndrome mutations may be the result of an emotionally triggered increase in epinephrine in a long‐QT running heart.

Voltage inactivation of Ca2+ entry and secretion associated with N‐ and P/Q‐type but not L‐type Ca2+ channels of bovine chromaffin cells

1 In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high‐voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2 The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+‐free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+‐free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. 3 This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura‐2‐loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. 4 A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. 5 The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N‐ and P/Q‐type Ca2+ channels. 6 This was strengthened by two additional findings: (i) nifedipine (3 μM), an L‐type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L‐type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7 In voltage‐clamped cells, switching the holding potential from ‐80 to ‐40 mV promoted the loss of 80 % of the whole‐cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. 8 Thus, it seems that the N‐ and P/Q‐subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L‐subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Cell-Permeable Gomesin Peptide Promotes Cell Death by Intracellular Ca2+ Overload

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

Mitochondria sense with different kinetics the calcium entering into HeLa cells through calcium channels CALHM1 and mutated P86L-CALHM1

The novel Ca(2+) channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca(2+) transients ([Ca(2+)](c)) that regulate the production of amyloid beta (Abeta). Its mutated channel P86L-CALHM1 has been associated to Alzheimers disease (AD). Using cytosolic- and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca(2+) entering into Hela cells and the Ca(2+) released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca(2+) entry in the three cell types; however, the [Ca(2+)](c) and mitochondrial Ca(2+) transients [Ca(2+)](m) had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca(2+) released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca(2+) overload, suggesting how these cells may become more vulnerable to apoptotic stimuli.

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.

Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca2

Dotarizine (a novel piperazine derivative with antimigraine properties) and flunarizine (a Ca2+ channel antagonist) were compared concerning: first, their ability to cause chromaffin cell damage in vitro; second, the possible correlation of their octanol/water partition coefficients and those of another 28 compounds (i.e., Ca2+ channel antagonists, blockers of histamine H1 receptors, antimycotics, beta-adrenoceptor antagonists, neuroleptics), with their ability to cause cell damage; third, their capacity to protect the cells against the damaging effects of veratridine; and fourth, their capabilities to enhance the basal cytosolic Ca2+ concentration in fura-2-loaded single chromaffin cells, or to modify the pattern of [Ca2+]i oscillations elicited by veratridine. After 24-h exposure to 1-30 microM dotarizine, the viability of bovine adrenal chromaffin cells (measured under phase contrast or as lactate dehydrogenase, released into the medium) was similar to that of control, untreated cells; at 100 microM, 80% lactate dehydrogenase release was produced. At 1-3 microM flunarizine caused no cell damage; however 10 microM caused 20% lactate dehydrogenase release and 30 and 100 microM over 90% lactate dehydrogenase release. The time course of cell damage was considerably faster for flunarizine, in comparison to dotarizine. Out of 30 molecules tested (at 10 microM), having different octanol/water partition coefficients (log P), dotarizine was among the molecules causing no cell damage; flunarizine caused 20% cell loss, lidoflazine and verapamil over 50% cell loss, and penfluridol, draflazine, astemizole or nifedipine over 80% cell loss. No correlation was found between log P and cytotoxicity. Both dotarizine (10-30 microM) and flunarizine (3-10 microM) provided protection against veratridine-induced cell death; however, at 30 microM dotarizine afforded a pronounced protection while flunarizine enhanced the cytotoxic effects of veratridine. Dotarizine (30 microM) (but not flunarizine) caused a prompt transient elevation of the basal [Ca2+]i. Both compounds abolished the K+-induced increases of [Ca2+]i as well as the oscillations of [Ca2+]i induced by veratridine. The blocking effects of dotarizine were readily reversed after washout, while those of flunarizine were long-lasting. These differences might be relevant to the clinical use of dotarizine as an antimigraine drug.

Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca2+ release from the endoplasmic reticulum

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7‐nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimers disease. However, its effect on Ca2+ signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca2+ signaling and cell viability. We used human SH‐SY5Y neuroblastoma cells overexpressing α7‐nAChRs (α7‐SH) and their control (C‐SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca2+ with Fura‐2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch‐clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7‐SH but not of C‐SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca2+ concentration; (iii) released Ca2+ from the ER by a Ca2+‐induced Ca2+ release mechanism only in α7‐SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7‐nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7‐nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca2+, overloading of intracellular Ca2+, and neuronal cell death.

Synergism between toxin-γ from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin-γ (Tγ) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tγ was ∼200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tγ (1.2 vs. 12 μg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+concentration ([Ca2+]o), when 30 μM veratridine plus 0.45 μM Tγ were used. Tγ (0.45 μM) doubled the basal uptake of45Ca2+, whereas veratridine (100 μM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tγ and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tγ elevated basal [Ca2+]ilevels, causing only small oscillations. When added together, Tγ and veratridine elevated the basal levels of [Ca2+]iwithout causing large oscillations. Tγ shifted the current-voltage ( I-V) curve for Na+ channel current to the left. The combination of Tγ with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.

Effects of the neuroprotectant lubeluzole on the cytotoxic actions of veratridine, barium, ouabain and 6‐hydroxydopamine in chromaffin cells

1 Incubation of bovine adrenal chromaffin cells with veratridine (10–100 μm) during 24 h, caused a concentration‐dependent release of the cytosolic lactate dehydrogenase (LDH) into the bathing medium, an indicator of cell death. Lubeluzole or its R(−) enantiomer, R91154, did not enhance LDH release. Both lubeluzole and R91154 (0.3–10 μm) decreased the veratridine‐induced LDH release. 2 Penfluridol did not increase LDH release at concentrations 0.003–1 μm; 3–10 μm increased LDH release to 50–60%, after 24 h exposure. Penfluridol (0.03–0.3 μm) did not protect against the cytotoxic effects of veratridine; at 1 μm, 15% protection was produced. Higher concentrations (3–10 μm) enhanced the cytotoxic effects of veratridine. 3 Ba2+ ions caused a concentration‐dependent increase of LDH release. This cytotoxic effect was partially prevented by 3 μm lubeluzole and fully counteracted by 1 μm penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mm Ba2+. 4 Ouabain (10 μm during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3–10 μm) and the lower concentrations of penfluridol (0.003–0.3 μm) prevented the ouabain cytotoxic effects. At higher concentrations (3 μm), penfluridol increased drastically the ouabain cytotoxic effects. 5 6‐Hydroxydopamine (6‐OHDA) caused significant cytotoxic effects at 30 and 100 μm. Lubeluzole (3–10 μm) or penfluridol (0.03–0.3 μm) had no cytoprotective effects against 6‐OHDA. 6 Lubeluzole (3 μm), R91154 (3 μm) and penfluridol (1 μm) blocked the current through Na+ channels in voltage‐clamped chromaffin cells (INa) by around 20–30%. Ca2+ current through Ca2+ channels (ICa) was inhibited 57% by lubeluzole and R91154 and 50% by penfluridol. The effects of penfluridol were not washed out, but those of lubeluzole and R91154 were readily reversible. 7 Lubeluzole (3 μm) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura‐2‐loaded cells exposed to 30 or 100 μm veratridine. Penfluridol (1 μm) inhibited those oscillations in an irreversible manner. 8 The results suggest that lubeluzole and its R‐isomer caused cytoprotection against veratridine cell damage, by blocking the veratridine stimulated Na+ and Ca2+ entry, as well as the [Ca2+]i oscillations. The Ba2+ and ouabain cytotoxic effects were prevented more efficiently by penfluridol, likely by blocking the plasmalemmal Na+/Ca2+ exchanger. It remains dubious whether these findings are relevant to the reported neuroprotective action of lubeluzole in stroke; the doubt rests in the stereoselective protecting effects of lubeluzole in in vivo stroke models, as opposed to its lack of stereoselectivity in the in vitro model reported here.