Pro-arrhythmogenic effects of Trypanosoma cruzi conditioned medium proteins in a model of bovine chromaffin cells.

Asymptomatic sudden death is the principal cause of mortality in Chagas disease. There is little information about molecular mechanisms involved in the pathophysiology of malignant arrhythmias in Chagasic patients. Previous studies have involved Trypanosoma cruzi secretion proteins in the genesis of arrhythmias ex vivo, but the molecular mechanisms involved are still unresolved. Thus, the aim was to determine the effect of these secreted proteins on the cellular excitability throughout to test its effects on catecholamine secretion, sodium-, calcium-, and potassium-conductance and action potential (AP) firing. Conditioned medium was obtained from the co-culture of T. cruzi and Vero cells (African green monkey kidney cells) and ultra-filtered for concentrating immunogenic high molecular weight parasite proteins. Chromaffin cells were assessed with the parasite and Vero cells control medium. Parasite-secreted proteins induce catecholamine secretion in a dose-dependent manner. Additionally, T. cruzi conditioned medium induced depression of both calcium conductance and calcium and voltage-dependent potassium current. Interestingly, this fact was related to the abolishment of the hyperpolarization phase of the AP produced by the parasite medium. Taken together, these results suggest that T. cruzi proteins may be involved in the genesis of pro-arrhythmic conditions that could influence the appearance of malignant arrhythmias in Chagasic patients.

Methylmercury reduces synaptic transmission and neuronal excitability in rat hippocampal slices.

In a previous study, we pointed out that the neurotoxic action evoked by methylmercury (MeHg), a potent environmental pollutant responsible for fatal food poisoning, is associated with alterations of cellular excitability by irreversible blockade of sodium and calcium currents. Here, we investigated the MeHg effects on synaptic transmission and neuronal plasticity using extracellular field recording in CA1 area of rat hippocampal slices. MeHg caused a fast and drastic depression of evoked field excitatory postsynaptic potentials (fEPSPs) in a concentration-dependent manner with an IC50 of 25.7 µM. This depression was partially caused by the irreversible reduction of axon recruitment deduced from the decrement of the fiber volley (FV) amplitude. Nevertheless, this MeHg-induced synaptic depression represents a true reduction of synaptic efficacy, as judged by input/output curves. In addition, a reduction on presynaptic release of glutamate was detected with the paradigm of paired-pulse facilitation during MeHg application. Moreover, MeHg also reduced population spike (PS) ampxlitude, and this effect was more prominent when the PS was evoked by ortodromic stimulation than by antidromic stimulation. Interestingly, despite these strong effects of MeHg on synaptic transmission and excitability, this compound did not modify the induction of long-term synaptic potentiation (LTP). The effects described here for MeHg were irreversible or very slowly reversible after drug wash-out. In summary, the blockade of sodium and calcium channels by MeHg affects synaptic transmission and cellular excitability but not synaptic plasticity.

Choline induces opposite changes in pyramidal neuron excitability and synaptic transmission through a nicotinic receptor-independent process in hippocampal slices.

Choline is present at cholinergic synapses as a product of acetylcholine degradation. In addition, it is considered a selective agonist for α5 and α7 nicotinic acetylcholine receptors (nAChRs). In this study, we determined how choline affects action potentials and excitatory synaptic transmission using extracellular and intracellular recording techniques in CA1 area of hippocampal slices obtained from both mice and rats. Choline caused a reversible depression of evoked field excitatory postsynaptic potentials (fEPSPs) in a concentration-dependent manner that was not affected by α7 nAChR antagonists. Moreover, this choline-induced effect was not mimicked by either selective agonists or allosteric modulators of α7 nAChRs. Additionally, this choline-mediated effect was not prevented by either selective antagonists of GABA receptors or hemicholinium, a choline uptake inhibitor. The paired pulse facilitation paradigm, which detects whether a substance affects presynaptic release of glutamate, was not modified by choline. On the other hand, choline induced a robust increase of population spike evoked by orthodromic stimulation but did not modify that evoked by antidromic stimulation. We also found that choline impaired recurrent inhibition recorded in the pyramidal cell layer through a mechanism independent of α7 nAChR activation. These choline-mediated effects on fEPSP and population spike observed in rat slices were completely reproduced in slices obtained from α7 nAChR knockout mice, which reinforces our conclusion that choline modulates synaptic transmission and neuronal excitability by a mechanism independent of nicotinic receptor activation.

Presynaptic muscarinic receptors reduce synaptic depression and facilitate its recovery at hippocampal GABAergic synapses.

Hippocampal gamma oscillation, involved in cognitive processes, can be induced by muscarinic acetylcholine receptors activation and depends in large part on the activation of γ-aminobutyric acidergic (GABAergic) interneurons. The precise role of the modulatory action of muscarinic receptors on GABAergic transmission still remains unclear due to the great heterogeneity of observed effects. We have examined the presynaptic and postsynaptic mechanisms involved. Methacholine induces a down-regulation of evoked inhibitory postsynaptic currents (eIPSCs) not associated with the change of postsynaptic receptors. The significant decrease in the paired-pulse depression strongly suggested a presynaptic mechanism of action. We have used cumulative amplitude profile analysis to show that the impairment of eIPSCs is not related to a decreased size of the readily releasable pool, but rather depends on the reduced release probability by a down-modulation of voltage-gated calcium channels. The decreased neurotransmitter release probability only partially accounts for the dramatic reduction in the rate of synaptic depression evoked by short- and long-lasting tetanic stimuli. This effect is accompanied by a significant enhancement in the rate of recovery from synaptic depression that demonstrates the reinforcement of the synaptic recycling processes. These results show that muscarinic modulation of hippocampal GABAergic synapses confers a greater resistance to sustain periods of intense synaptic activity in the gamma frequency range.

Methylmercury decreases cellular excitability by a direct blockade of sodium and calcium channels in bovine chromaffin cells: an integrative study.

Methylmercury, a potent environmental pollutant responsible for fatal food poisoning, blocked calcium channels of bovine chromaffin cells in a time- and concentration-dependent manner with an IC50 of 0.93 µM. This blockade was not reversed upon wash-out and was greater at more depolarising holding potentials (i.e. 21 % at -110 mV and 60 % at -50 mV, after 3 min perfusion with methylmercury). In ω-toxins-sensitive calcium channels, methylmercury caused a higher blockade of I Ba than in ω-toxins-resistant ones, in which a lower blockade was detected. The sodium current was also blocked by acute application of methylmercury in a time- and concentration-dependent manner with an IC50 of 1.05 µM. The blockade was not reversed upon wash-out of the drug. The drug inhibited sodium current at all test potentials and shows a shift of the I-V curve to the left of about 10 mV. Intracellular dialysis with methylmercury caused no blockade of calcium or sodium channels. Voltage-dependent potassium current was not affected by methylmercury. Calcium- and voltage-dependent potassium current was also drastically depressed. This blockade was related to the prevention of Ca(2+) influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, the blockade of ionic current present during the generation and termination of action potentials led to a drastic alteration of cellular excitability. The application of methylmercury greatly reduced the shape and the number of electrically evoked action potentials. Taken together, these results point out that the neurotoxic action evoked by methylmercury may be associated to alteration of cellular excitability by blocking ionic currents responsible for the generation and termination of action potentials.

Modulation by endogenously released ATP and opioids of chromaffin cell calcium channels in mouse adrenal slices.

Modulation of high-threshold voltage-dependent calcium channels by neurotransmitters has been the subject of numerous studies in cultures of neurons and chromaffin cells. However, no studies on such modulation exist in chromaffin cells in their natural environment, the intact adrenal medullary tissue. Here we performed such a study in voltage-clamped chromaffin cells of freshly prepared mouse adrenal slices under the whole cell configuration of the patch-clamp technique. The subcomponents of the whole cell inward Ca(2+) current (I(Ca)) accounted for 49% for L-, 28% for N-, and 36% for P/Q-type channels. T-type Ca(2+) channels or residual R-type Ca(2+) currents were not seen. However, under the perforated-patch configuration, 20% of I(Ca) accounted for a toxin-resistant R-type Ca(2+) current. Exogenously applied ATP and methionine-enkephalin (Met-enk) inhibited I(Ca) by 33%. Stop-flow and Ca(2+) replacement by Ba(2+), which favored the release of endogenous ATP and opioids, also inhibited I(Ca), with no changes in activation or inactivation kinetics. This inhibition was partially voltage independent and insensitive to prepulse facilitation. Furthermore, in about half of the cells, suramin and naloxone augmented I(Ca) in the absence of exogenous application of ATP/Met-enk. No additional modulation of I(Ca) was obtained after bath application of exogenous ATP and opioids to these already inhibited cells. Augmentation of I(Ca) was also seen upon intracellular dialysis of guanosine 5'-[ß-thio]diphosphate (GDPßS), indicating the existence in the intact slice of a tonic inhibition of I(Ca) in resting conditions. These results suggest that in the intact adrenal tissue a tonic inhibition of I(Ca) exists, mediated by purinergic and opiate receptors.

Presynaptic muscarinic receptor subtypes involved in the enhancement of spontaneous GABAergic postsynaptic currents in hippocampal neurons.

We investigated the effects of muscarinic acetylcholine receptor (mAChR) activation on GABAergic synaptic transmission in rat hippocampal neurons. Current-clamp recordings revealed that methacholine produced membrane depolarization and action potential firing. Methacholine augmented the bicuculline-sensitive and GABA(A) -mediated frequency of spontaneous inhibitory postsynaptic currents (sIPSCs); the action of methacholine had a slow onset and longer duration. The increase in methacholine-evoked sIPSCs was completely inhibited by atropine and was insensitive to glutamatergic receptor blockers. Interestingly, methacholine action was not inhibited by intracellular perfusion with GDP-ß-S, suggesting that muscarinic effects on membrane excitability and sIPSC frequency are mainly presynaptic. McN-A-343 and pirenzepine, selective agonist and antagonist of the m1 mAChR subtype, respectively, neither enhanced sIPSCs nor inhibited the methacholine effect. However, the m3-m5 mAChR antagonist 4-DAMP, and the m2-m4 mAChR antagonist himbacine inhibited the methacholine effect. U73122, an IP(3) production inhibitor, and 2APB, an IP(3) receptor blocker, drastically decreased the methacholine effect. Recording of miniature events revealed that besides the effect exerted by methacholine on membrane firing properties and sIPSC frequency, muscarinic receptors also enhanced the frequency of mIPSCs with no effect on their amplitude, possibly modulating the molecular machinery subserving vesicle docking and fusion and suggesting a tight colocalization at the active zone of the presynaptic terminals. These data strongly suggest that by activating presynaptic m2, m3, m4 and m5 mAChRs, methacholine can increase membrane excitability and enhance efficiency in the GABA release machinery, perhaps through a mechanism involving the release of calcium from the endoplasmic reticulum.

Down-modulation of Ca2+ channels by endogenously released ATP and opioids: from the isolated chromaffin cell to the slice of adrenal medullae.

Modifications in Ca(2+) influx may lead to profound changes in the cell activity associated with Ca(2+)-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca(2+) channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca(2+) concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.

Modulation of calcium channels by taurine acting via a metabotropic-like glycine receptor.

Taurine is one of the most abundant free amino acids in the central nervous system, where it displays several functions. However, its molecular targets remain unknown. It is well known that taurine can activate GABA-A and strychnine-sensitive glycine receptors, which increases a chloride conductance. In this study, we describe that acute application of taurine induces a dose-dependent inhibition of voltage-dependent calcium channels in chromaffin cells from bovine adrenal medullae. This taurine effect was not explained by the activation of either GABA-A, GABA-B or strychnine-sensitive glycine receptors. Interestingly, glycine mimicked the modulatory action exerted by taurine on calcium channels, although the acute application of glycine did not elicit any ionic current in these cells. Additionally, the modulation of calcium channels exerted by both taurine and glycine was prevented by the intracellular dialysis of GDP-ß-S. Thus, the modulation of voltage-dependent calcium channels by taurine seems to be mediated by a metabotropic-like glycinergic receptor coupled to G-protein activation in a membrane delimited pathway.

Allosteric modulation of alpha 7 nicotinic receptors selectively depolarizes hippocampal interneurons, enhancing spontaneous GABAergic transmission.

The role of postsynaptic nicotinic receptors for acetylcholine (nAChRs) in mediating fast neurotransmission processes in the CNS is controversial. Here we have studied the modulation of synaptic transmission by an agonist (choline) and an allosteric modulator (5-OH-indole) of alpha7 nAChRs in rat hippocampal neuronal cultures. Choline evoked a fast inactivating inward current, causing neuron depolarization and action potential discharge, thereby enhancing the spontaneous postsynaptic current activity (sPSCs). This effect was markedly enhanced when both choline and 5-OH-indole were applied together and was blocked by the selective alpha7 nAChR antagonist methyllycaconitine. This choline action was suppressed by the GABA(A) receptor antagonist bicuculline, while the glutamatergic receptor antagonist kynurenic acid had no effect. Frequency, but not amplitude or area, of both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) were drastically reduced when Ca(2+) influx was blocked by Cd(2+). Additionally, nAChR activation did not modify the mIPSCs. These data suggest that Ca(2+) influx through the highly Ca(2+)-permeablealpha7 nAChRs was insufficient to directly activate neurotransmitter release, suggesting that a tight colocalization of this receptor with secretory hot spots is unlikely. In a few cases, the activation of alpha7 AChRs led to a suppression of spontaneous synaptic transmission. This effect may be related to the potentiation of GABAergic interneurons that inhibit the spontaneous activity of neurons making synapses with the cell under study. We suggest that GABA release is modulated by alpha7 nAChRs. Thus, selective allosteric modulators of alpha7 nAChRs could have potential therapeutic applications in brain disorders such as epilepsy and schizophrenia and in alterations of cognition and sensory processing.

L-type calcium channels in adrenal chromaffin cells: role in pace-making and secretion.

Voltage-gated L-type (Cav1.2 and Cav1.3) channels are widely expressed in cardiovascular tissues and represent the critical drug-target for the treatment of several cardiovascular diseases. The two isoforms are also abundantly expressed in neuronal and neuroendocrine tissues. In the brain, Cav1.2 and Cav1.3 channels control synaptic plasticity, somatic activity, neuronal differentiation and brain aging. In neuroendocrine cells, they are involved in the genesis of action potential generation, bursting activity and hormone secretion. Recent studies have shown that Cav1.2 and Cav1.3 are also expressed in chromaffin cells but their functional role has not yet been identified despite that L-type channels possess interesting characteristics, which confer them an important role in the control of catecholamine secretion during action potentials stimulation. In intact rat adrenal glands L-type channels are responsible for adrenaline and noradrenaline release following splanchnic nerve stimulation or nicotinic receptor activation. L-type channels can be either up- or down-modulated by membrane autoreceptors following distinct second messenger pathways. L-type channels are tightly coupled to BK channels and activate at relatively low-voltages. In this way they contribute to the action potential hyperpolarization and to the pace-maker current controlling action potential firings. L-type channels are shown also to regulate the fast secretion of the immediate readily releasable pool of vesicles with the same Ca(2+)-efficiency of other voltage-gated Ca(2+) channels. In mouse adrenal slices, repeated action potential-like stimulations drive L-type channels to a state of enhanced stimulus-secretion efficiency regulated by beta-adrenergic receptors. Here we will review all these novel findings and discuss the possible implication for a specific role of L-type channels in the control of chromaffin cells activity.

A comparison between acetylcholine-like action potentials and square depolarizing pulses in triggering calcium entry and exocytosis in bovine chromaffin cells.

Depending on experimental conditions, cell model, and pattern and type of depolarizing stimuli, the relationship between calcium entry ([Ca2+]c) and the release of neurotransmitters and hormones varies from exponential (power of 3-4) to near linear (power of 1.5) or linear function. Here, we present a study using the more physiological stimulation pattern based on acetylcholine (ACh)-like action potentials, in voltage-clamped bovine chromaffin cells, with the perforated-patch configuration of the patch-clamp technique and 2 mM extracellular calcium. Trains of ACh-like action potentials or square depolarizing pulses of increasing length were applied, and calcium currents (ICa), total calcium entry (QCa), and exocytosis (DeltaCm) measured.

[Anticholinesterases in the treatment of Alzheimer's disease]. / Anticolinesterásicos en el tratamiento de la enfermedad de Alzheimer.

INTRODUCTION: Among the numerous pathophysiological theories that attempt to explain the development of Alzheimer's disease (AD) there are two facts that stand out above the rest: on the one hand, the formation of neurofibrillary tangles inside cells and, on the other, the extra-cellular deposition of beta-amyloid protein. These two mechanisms lead to neurodegeneration and the death of cells by means of a process called 'apoptosis' or 'programmed cell death'. In the early stages of this neurodegenerative process it is more pronounced in cholinergic-type brain centres. This led to the formulation of the so-called cholinergic theory of Alzheimer, which provides the rationale behind the use of the drugs that are currently available to treat this disease, namely, acetylcholine esterase (AChE) inhibitors (rivastigmine, donepezil and galanthamine). DEVELOPMENT AND CONCLUSIONS: We review the possible pharmacological approaches that could help to prevent or delay cell death, and which act on the mechanisms involved in the production of neurofibrillary tangles or the deposition of beta-amyloid protein. We also review the main characteristics of cholinergic neurotransmission, which will help us to understand the therapeutic approaches that have been applied in an attempt to enhance deficient cholinergic neurotransmission. One of the most notable of these is the amount of attention recently being paid to the enzyme AChE, which increases the bioavailability of the neurotransmitter in the cholinergic synapses by preventing the hydrolysis of acetylcholine; these are the only drugs currently available for the symptomatic treatment of this disease.

Opposite action of beta1- and beta2-adrenergic receptors on Ca(V)1 L-channel current in rat adrenal chromaffin cells.

Voltage-gated Ca(2+) channels of chromaffin cells are modulated by locally released neurotransmitters through autoreceptor-activated G-proteins. Clear evidence exists in favor of a Ca(2+) channel gating inhibition mediated by purinergic, opioidergic, and alpha-adrenergic autoreceptors. Few and contradictory data suggest also a role of beta-adrenergic autoreceptors (beta-ARs), the action of which, however, remains obscure. Here, using patch-perforated recordings, we show that rat chromaffin cells respond to the beta-AR agonist isoprenaline (ISO) by either upmodulating or downmodulating the amplitude of Ca(2+) currents through two distinct modulatory pathways. ISO (1 microm) could cause either fast inhibition (approximately 25%) or slow potentiation (approximately 25%), or a combination of the two actions. Both effects were completely prevented by propranolol. Slow potentiation was more evident in cells pretreated with pertussis toxin (PTX) or when beta(1)-ARs were selectively stimulated with ISO + ICI118,551. Potentiation was absent when the beta(2)-AR-selective agonist zinterol (1 microm), the protein kinase A (PKA) inhibitor H89, or nifedipine was applied, suggesting that potentiation is associated with a PKA-mediated phosphorylation of L-channels (approximately 40% L-current increase) through beta(1)-ARs. The ISO-induced inhibition was fast and reversible, preserved in cell treated with H89, and mimicked by zinterol. The action of zinterol was mostly on L-channels (38% inhibition). Zinterol action preserved the channel activation kinetics, the voltage-dependence of the I-V characteristic, and was removed by PTX, suggesting that beta(2)AR-mediated channel inhibition was mainly voltage independent and coupled to G(i)/G(o)-proteins. Sequential application of zinterol and ISO mimicked the dual action (inhibition/potentiation) of ISO alone. The two kinetically and pharmacologically distinct beta-ARs signaling uncover alternative pathways, which may serve the autocrine control of Ca(2+)-dependent exocytosis and other related functions of rat chromaffin cells.

Calcium-dependent inhibition of L, N, and P/Q Ca2+ channels in chromaffin cells: role of mitochondria.

The hypothesis that the buffering of Ca(2+) by mitochondria could affect the Ca(2+)-dependent inhibition of voltage-activated Ca(2+) channels, (I(Ca)), was tested in voltage-clamped bovine adrenal chromaffin cells. The protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), the blocker of the Ca(2+) uniporter ruthenium red (RR), and a combination of oligomycin plus rotenone were used to interfere with mitochondrial Ca(2+) buffering. In cells dialyzed with an EGTA-free solution, peak I(Ca) generated by 20 msec pulses to 0 or +10 mV, applied at 15 sec intervals, from a holding potential of -80 mV, decayed rapidly after superfusion of cells with 2 microm CCCP (tau = 16.7 +/- 3 sec; n = 8). In cells dialyzed with 14 mm EGTA, CCCP did not provoke I(Ca) loss. Cell dialysis with 4 microm ruthenium red or cell superfusion with oligomycin (3 microm) plus rotenone (4 microm) also accelerated the decay of I(Ca). After treatment with CCCP, decay of N- and P/Q-type Ca(2+) channel currents occurred faster than that of L-type Ca(2+) channel currents. These data are compatible with the idea that the elevation of the bulk cytosolic Ca(2+) concentration, [Ca(2+)](c), causes the inhibition of L- and N- as well as P/Q-type Ca(2+) channels expressed by bovine chromaffin cells. This [Ca(2+)](c) signal appears to be tightly regulated by rapid Ca(2+) uptake into mitochondria. Thus, it is plausible that mitochondria might efficiently regulate the activity of L, N, and P/Q Ca(2+) channels under physiological stimulation conditions of the cell.

Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes.

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (-) enantiomer R91154 on whole-cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of -80 mV, at 10 s intervals had an average magnitude of 1 nA. 2. Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration-dependent manner; their IC50s were 1.94 microM for lubeluzole and 2.54 microM for R91154. In a current-voltage protocol, lubeluzole (3 microM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I-V curve were detected. 3. After 10 min exposure to 3 microM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered. 4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at -110 mV and 87% at -50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at -110 mV, decreased at -80 mV and did not occur at -50 mV. 5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 microM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 microM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa. 6. The effects of lubeluzole (3 microM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At -80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole). 7. In mouse chromaffin cells approximately half of the whole-cell IBa was sensitive to 3 microM nifedipine (L-type Ca2+ channels) and the other half to 3 microM omega-conotoxin MVIIC (non-L-type Ca2+ channels). In omega-conotoxin MVIIC-treated cells, 3 microM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound. 8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca2+ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca2+ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca2+ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca2+ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.

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 microM) 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 microM) decreased the veratridine-induced LDH release. 2. Penfluridol did not increase LDH release at concentrations 0.003-1 microM; 3-10 microM increased LDH release to 50-60%, after 24 h exposure. Penfluridol (0.03-0.3 microM) did not protect against the cytotoxic effects of veratridine; at 1 microM, 15% protection was produced. Higher concentrations (3-10 microM) 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 microM lubeluzole and fully counteracted by 1 microM penfluridol. R91154 was less potent than lubeluzole and only protected against the lesion induced by 0.5 mM Ba2+. 4. Ouabain (10 microM during 24 h) increased LDH release to about 30%. Both lubeluzole (0.3-10 microM) and the lower concentrations of penfluridol (0.003-0.3 microM) prevented the ouabain cytotoxic effects. At higher concentrations (3 microM), penfluridol increased drastically the ouabain cytotoxic effects. 5. 6-Hydroxydopamine (6-OHDA) caused significant cytotoxic effects at 30 and 100 microM. Lubeluzole (3-10 microM) or penfluridol (0.03-0.3 microM) had no cytoprotective effects against 6-OHDA. 6. Lubeluzole (3 microM), R91154 (3 microM) and penfluridol (1 microM) blocked the current through Na+ channels in voltage-clamped chromaffin cells (I(Na)) by around 20-30%. Ca2+ current through Ca2+ channels (I(Ca)) 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 microM) induced reversible blockade of the oscillations of the cytosolic Ca2+, [Ca2+]i, in fura-2-loaded cells exposed to 30 or 100 microM veratridine. Penfluridol (1 microM) 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.

Blockade by agmatine of catecholamine release from chromaffin cells is unrelated to imidazoline receptors.

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC(50)=366 microM); the K(+)-evoked release of catecholamines was unaffected. Clonidine (100 microM) and moxonidine (100 microM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at -80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents (I(ACh)) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast (tau(on) = 31 ms); the recovery of the current after washout of agmatine also occurred very rapidly (tau(off = 39 ms). Efaroxan (10 microM) did not affect the inhibition of I(ACh) elicited by 1 mM agmatine. I(ACh) was blocked 90% by 100 microM clonidine and 50% by 100 microM moxonidine. The concentration-response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 microM agmatine. The blockade of I(ACh) caused by agmatine (100 microM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block I(ACh). At 1 mM, agmatine blocked I(Na) by 23%, I(Ba) by 14%, I(K(Ca)) by 16%, and I(K(VD)) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.

Differential variations in Ca2+ entry, cytosolic Ca2+ and membrane capacitance upon steady or action potential depolarizing stimulation of bovine chromaffin cells.

AIMS: This study looks into the physiology of the exocytosis of catecholamines released by adrenal medullary chromaffin cells. We have comparatively explored the exocytotic responses elicited by two different patterns of depolarizing stimulation: the widely employed square depolarizing pulses (DPs) and trains of acetylcholine-like action potentials (APs), likely the physiological mode of stimulation in the intact innervated adrenal medulla. APs were applied at 30 Hz, a frequency similar to that produced in a stressful situation. METHODS: Patch-clamp, cell membrane capacitance, single cell amperometry and fluorescence were the techniques used. The variations of calcium entry measured as the integral of the calcium current, cytosolic calcium (measured with the calcium-sensitive fluorescent probe fluo-4) and exo-endocytosis (membrane capacitance variations) were the parameters measured. RESULTS: Trains of AP depolarizations produced distinct responses compared to those of square depolarizations: (1) Calcium current amplitude decreased to a lesser extent along the AP train; (2) calcium entry and capacitance increments raised linearly with stimulation time whereas they deviated from linearity when square depolarizations were used; (3) slower activation and faster delayed decay phase of cytosolic calcium transients; (4) capacitance increments varied linearly with calcium entry with APs and deviated from linearity with longer depolarizations; (5) little endocytosis after stimulation with longer trains of APs and pronounced endocytosis with longer square depolarizations. CONCLUSIONS: Stimulation of chromaffin cells with trains of APs produced patterns of cytosolic calcium transients, exocytotic and endocytotic responses quite different from those elicited by the widely employed DPs. Our study is relevant from the methodological and physiological points of view.

[Is good old minocycline a new neuroprotective drug?]. / Es la vieja minociclina un nuevo fármaco neuroprotector?

INTRODUCTION: During the last decade, the neuroprotective effects of minocycline have been a matter of an intense debate. A broad amount of contradictory studies can be found in the scientific literature, going from neuroprotection to the exacerbation of toxicity in diverse experimental models. Such differences could be the result of minocycline acting on multiple pharmacological targets. DEVELOPMENT: In the present review we will go over these pharmacological targets and the effects derived from their modulation by minocycline. Among others, its antioxidant activity derived from its chemical structure or its modulator effect on several enzymes such as nitric oxide synthase will be reviewed. Furthermore, the effects of minocycline on the intracellular pathways implicated in neurodegenerative processes including apoptosis stages, activation decision and execution will be addressed. CONCLUSIONS: All the mechanisms described herein have not escaped to a scientific community needed of new therapeutic drugs for the treatment of neurodegenerative conditions. However, the sparse clinical trials carried out so far are mainly aimed at assessing its tolerability and safety or are still in progress. We believe that more studies, both clinical and pre-clinical, should be carried out in order to ascertain the therapeutic window and the neurodegenerative disorders in which minocycline could be useful.