Lead exerts a depression of neurotransmitter release through a blockade of voltage dependent calcium channels in chromaffin cells.

The present work, using chromaffin cells of bovine adrenal medullae (BCCs), aims to describe what type of ionic current alterations induced by lead (Pb2+) underlies its effects reported on synaptic transmission. We observed that the acute application of Pb2+ lead to a drastic depression of neurotransmitters release in a concentration-dependent manner when the cells were stimulated with both K+ or acetylcholine, with an IC50 of 119,57 µM and of 5,19 µM, respectively. This effect was fully recovered after washout. Pb2+ also blocked calcium channels of BCCs in a time- and concentration-dependent manner with an IC50 of 6,87 µM. This blockade was partially reversed upon washout. This compound inhibited the calcium current at all test potentials and shows a shift of the I-V curve to more negative values of about 8 mV. The sodium current was not blocked by acute application of high Pb2+ concentrations. Voltage-dependent potassium current was also shortly affected by high Pb2+. Nevertheless, the calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 24,49 µM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to Ca2+-activated K+-channels (BK) instead a direct linking to these channels. Under current-clamp conditions, BCCs exhibit a resting potential of -52.7 mV, firing spontaneous APs (1-2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of K+ channels. In spite of the effect on ionic channels exerted by Pb2+, we found that Pb2+ didn't alter cellular excitability, no modification of the membrane potential, and no effect on action potential firing. Taken together, these results point to a neurotoxic action evoked by Pb2+ that is associated with changes in neurotransmitter release by blocking the ionic currents responsible for the calcium influx.

Non-Excitatory Amino Acids, Melatonin, and Free Radicals: Examining the Role in Stroke and Aging.

The aim of this review is to explore the relationship between melatonin, free radicals, and non-excitatory amino acids, and their role in stroke and aging. Melatonin has garnered significant attention in recent years due to its diverse physiological functions and potential therapeutic benefits by reducing oxidative stress, inflammation, and apoptosis. Melatonin has been found to mitigate ischemic brain damage caused by stroke. By scavenging free radicals and reducing oxidative damage, melatonin may help slow down the aging process and protect against age-related cognitive decline. Additionally, non-excitatory amino acids have been shown to possess neuroprotective properties, including antioxidant and anti-inflammatory in stroke and aging-related conditions. They can attenuate oxidative stress, modulate calcium homeostasis, and inhibit apoptosis, thereby safeguarding neurons against damage induced by stroke and aging processes. The intracellular accumulation of certain non-excitatory amino acids could promote harmful effects during hypoxia-ischemia episodes and thus, the blockade of the amino acid transporters involved in the process could be an alternative therapeutic strategy to reduce ischemic damage. On the other hand, the accumulation of free radicals, specifically mitochondrial reactive oxygen and nitrogen species, accelerates cellular senescence and contributes to age-related decline. Recent research suggests a complex interplay between melatonin, free radicals, and non-excitatory amino acids in stroke and aging. The neuroprotective actions of melatonin and non-excitatory amino acids converge on multiple pathways, including the regulation of calcium homeostasis, modulation of apoptosis, and reduction of inflammation. These mechanisms collectively contribute to the preservation of neuronal integrity and functions, making them promising targets for therapeutic interventions in stroke and age-related disorders.

Identification of Non-excitatory Amino Acids and Transporters Mediating the Irreversible Synaptic Silencing After Hypoxia.

The contribution of excitatory amino acids (AA) to ischemic brain injury has been widely described. In addition, we reported that a mixture of non-excitatory AA at plasmatic concentrations turns irreversible the depression of synaptic transmission caused by hypoxia. Here, we describe that the presence of seven non-excitatory AA (L-alanine, L-glutamine, glycine, L-histidine, L-serine, taurine, and L-threonine) during hypoxia provokes an irreversible neuronal membrane depolarization, after an initial phase of hyperpolarization. The collapse of the membrane potential correlates with a great increase in fiber volley amplitude. Nevertheless, we show that the presence of all seven AA is not necessary to cause the irreversible loss of fEPSP after hypoxia and that the minimal combination of AA able to provoke a solid, replicable effect is the mixture of L-alanine, glycine, L-glutamine, and L-serine. Additionally, L-glutamine seems necessary but insufficient to induce these harmful effects. We also prove that the deleterious effects of the AA mixtures on field potentials during hypoxia depend on both the identity and concentration of the individual AA in the mixture. Furthermore, we find that the accumulation of AA in the whole slice does not determine the outcome caused by the AA mixtures on the synaptic transmission during hypoxia. Finally, results obtained using pharmacological inhibitors and specific substrates of AA transporters suggest that system N and the alanine-serine-cysteine transporter 2 (ASCT2) participate in the non-excitatory AA-mediated deleterious effects during hypoxia. Thus, these AA transporters might represent therapeutical targets for the treatment of brain ischemia.

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1 µM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 µM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 µM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 µM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

Promising Molecular Targets in Pharmacological Therapy for Neuronal Damage in Brain Injury.

The complex etiopathogenesis of brain injury associated with neurodegeneration has sparked a lot of studies in the last century. These clinical situations are incurable, and the currently available therapies merely act on symptoms or slow down the course of the diseases. Effective methods are being sought with an intent to modify the disease, directly acting on the properly studied targets, as well as to contribute to the development of effective therapeutic strategies, opening the possibility of refocusing on drug development for disease management. In this sense, this review discusses the available evidence for mitochondrial dysfunction induced by Ca2+ miscommunication in neurons, as well as how targeting phosphorylation events may be used to modulate protein phosphatase 2A (PP2A) activity in the treatment of neuronal damage. Ca2+ tends to be the catalyst for mitochondrial dysfunction, contributing to the synaptic deficiency seen in brain injury. Additionally, emerging data have shown that PP2A-activating drugs (PADs) suppress inflammatory responses by inhibiting different signaling pathways, indicating that PADs may be beneficial for the management of neuronal damage. In addition, a few bioactive compounds have also triggered the activation of PP2A-targeted drugs for this treatment, and clinical studies will help in the authentication of these compounds. If the safety profiles of PADs are proven to be satisfactory, there is a case to be made for starting clinical studies in the setting of neurological diseases as quickly as possible.

Novel mechanism of hypoxic neuronal injury mediated by non-excitatory amino acids and astroglial swelling.

In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. Bleeding in hemorrhagic stroke and TBI also release plasma AA. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury. Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Four non-excitatory AA (l-alanine, glycine, l-glutamine, l-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQS-induced increase in LT was due to astroglial but not neuronal swelling. LT raise during hypoxia and AGQS manifested astroglial and neuronal swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volume-regulated anion channels (VRAC) blockers. Our findings suggest that astroglial swelling induced by accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury.

The Coronavirus Disease 2019 (COVID-19): Key Emphasis on Melatonin Safety and Therapeutic Efficacy.

Viral infections constitute a tectonic convulsion in the normophysiology of the hosts. The current coronavirus disease 2019 (COVID-19) pandemic is not an exception, and therefore the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, like any other invading microbe, enacts a generalized immune response once the virus contacts the body. Melatonin is a systemic dealer that does not overlook any homeostasis disturbance, which consequently brings into play its cooperative triad, antioxidant, anti-inflammatory, and immune-stimulant backbone, to stop the infective cycle of SARS-CoV-2 or any other endogenous or exogenous threat. In COVID-19, the corporal propagation of SARS-CoV-2 involves an exacerbated oxidative activity and therefore the overproduction of great amounts of reactive oxygen and nitrogen species (RONS). The endorsement of melatonin as a possible protective agent against the current pandemic is indirectly supported by its widely demonstrated beneficial role in preclinical and clinical studies of other respiratory diseases. In addition, focusing the therapeutic action on strengthening the host protection responses in critical phases of the infective cycle makes it likely that multi-tasking melatonin will provide multi-protection, maintaining its efficacy against the virus variants that are already emerging and will emerge as long as SARS-CoV-2 continues to circulate among us.

Hypoxia-induced depression of synaptic transmission becomes irreversible by intracellular accumulation of non-excitatory amino acids.

The intracellular accumulation of some amino acids (AAs), mainly glutamine, can contribute to brain edema observed during liver failure. We recently demonstrated that individual applications of high concentrations (10 mM) of some non-excitatory AAs increase the electrical resistance of hippocampal slices, indicating cell swelling. Therefore, we pondered whether an AA mixture's application might cause cell swelling at a physiological concentration range. In rat hippocampal slices, we carried out extra- and intracellular electrophysiological recordings and AAs analysis to address this question. We applied a mixture of 19 AAs at their plasmatic concentrations (Plasma solution: Ala, Gly, Gln, His, Ser, Tau, Thr, Arg, Leu, Met, Pro, Val, Asn, Cys, Phe, Ile, Lys, Tyr, and Trp). This solution was afterward divided into two according to the individual AAs at 10 mM concentration inducing synaptic potentiation (Plasma1, containing the first seven AAs of Plasma) or not (Plasma2, with the remaining AAs). Plasma application increased evoked field potentials requiring extracellular chloride. This effect was mimicked by the Plasma1 but not the Plasma2 solution. Plasma1-induced potentiation was independent of changes in release probability, basic electrophysiological membrane properties, and NMDAR activation. AAs in Plasma1 act cooperatively to accumulate intracellularly and to induce synaptic potentiation. In the presence of Plasma1, the reversible synaptic depression caused by a 40-min hypoxia period turned into an irreversible disappearance of synaptic potentials through an NMDAR-dependent mechanism. The presence of a system A transport inhibitor did not block Plasma1-mediated effects. These results indicate that cell swelling, induced by the accumulation of non-excitotoxic AAs through unidentified transporters, might foster deleterious effects produced by hypoxia-ischemia episodes.

Micromolar concentrations of Zn2+ depress cellular excitability through a blockade of calcium current in rat adrenal slices.

The present work, using chromaffin cells in rat adrenal slices (RCCs), aims to describe what type of ionic current alterations induced by zinc underlies their effects reported on synaptic transmission. Thus, Zn2+ blocked calcium channels of RCCs in a time- and concentration-dependent manner with an IC50 of 391 µM. This blockade was partially reversed upon washout and was greater at more depolarizing holding potentials (i.e. 32 ± 5% at -110 mV, and 43 ± 6% at -50 mV, after 5 min perfusion). In ω-toxins-sensitive calcium channels (N-, P- and Q-types), Zn2+caused a lower blockade of ICa, 33.3%, than in ω-toxins-resistant ones (L-type, 55.3%; and R-type, 90%). This compound inhibited calcium current at all test potentials and shows a shift of the I-V curve to more depolarized values of about 10 mV. The sodium current was not blocked by acute application of high Zn2+concentrations. Voltage-dependent potassium current was marginally affected by high Zn2+ concentrations showing no concentration-dependence. Nevertheless, calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 453 µM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, RCCs exhibit a resting potential of -50.7 mV, firing spontaneous APs (1-2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of voltage and Ca2+-activated K+-channels (BK). We found that the blockade of these ionic currents by Zn2+ led to a drastic alteration of cellular excitability with a depolarization of the membrane potential, the slowdown and broadening of the APs and the severe reduction of the after hyperpolarization (AHP) which led to a decrease in the APs firing frequency. Taken together, these results point to a neurotoxic action evoked by zinc that is associated with changes to cellular excitability by blocking the ionic currents responsible for both the neurotransmitter release and the action potentials firing.

Intracellular accumulation of amino acids increases synaptic potentials in rat hippocampal slices.

The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a transport system with a low affinity for taurine. The prototypic taurine transporter TauT (SLC6A6) was discarded by experimental evidence obtained in TauT-KO mice. The purpose of the present study was to determine whether the proton-coupled amino acid transporter 1 (PAT1; SLC36A1) which is a transport system with low affinity and high capacity for a great variety of amino acids including taurine, contributes to the taurine-induced synaptic potentiation. In rat hippocampal slices, the application of several amino acids (L- and D-alanine, L-glutamine, ß-guanidinopropionic acid, glycine, L-histidine, L- and D-serine, sarcosine, L- and D-threonine) imitated the synaptic potentiation induced by taurine. The magnitude of the potentiation caused by some of these amino acids was even greater than that induced by taurine. By contrast, the application of other amino acids (L-arginine, betaine, L-leucine, L-methionine, L- and D-proline, and L-valine) did not induce potentiation. The behaviour of these different amino acids on synaptic potentiation is not compatible with a role of PAT1 in synaptic potentiation. There was a positive correlation between the accumulation of the different amino acids in the slice and the magnitude of synaptic potentiation induced by them. Some of the amino acids inducing synaptic potentiation, like taurine and L-threonine, also increased electrical resistance of the slice, whereas L-leucine did not modify this parameter. Modifications induced by either taurine or L-threonine in synaptic potentiation, slice resistance and amino acid accumulation were dependent on extracellular chloride concentration. These findings support the idea that the accumulation of amino acids throughout the action of transporters causes cell swelling enhancing the electrical resistance of the slice, which by itself could be sufficient to increase field synaptic potentials.

Discovery of the first dual GSK3ß inhibitor/Nrf2 inducer. A new multitarget therapeutic strategy for Alzheimer's disease.

The formation of neurofibrillary tangles (NFTs), oxidative stress and neuroinflammation have emerged as key targets for the treatment of Alzheimer's disease (AD), the most prevalent neurodegenerative disorder. These pathological hallmarks are closely related to the over-activity of the enzyme GSK3ß and the downregulation of the defense pathway Nrf2-EpRE observed in AD patients. Herein, we report the synthesis and pharmacological evaluation of a new family of multitarget 2,4-dihydropyrano[2,3-c]pyrazoles as dual GSK3ß inhibitors and Nrf2 inducers. These compounds are able to inhibit GSK3ß and induce the Nrf2 phase II antioxidant and anti-inflammatory pathway at micromolar concentrations, showing interesting structure-activity relationships. The association of both activities has resulted in a remarkable anti-inflammatory ability with an interesting neuroprotective profile on in vitro models of neuronal death induced by oxidative stress and energy depletion and AD. Furthermore, none of the compounds exhibited in vitro neurotoxicity or hepatotoxicity and hence they had improved safety profiles compared to the known electrophilic Nrf2 inducers. In conclusion, the combination of both activities in this family of multitarget compounds confers them a notable interest for the development of lead compounds for the treatment of AD.

Chondroitin Sulfate Induces Depression of Synaptic Transmission and Modulation of Neuronal Plasticity in Rat Hippocampal Slices.

It is currently known that in CNS the extracellular matrix is involved in synaptic stabilization and limitation of synaptic plasticity. However, it has been reported that the treatment with chondroitinase following injury allows the formation of new synapses and increased plasticity and functional recovery. So, we hypothesize that some components of extracellular matrix may modulate synaptic transmission. To test this hypothesis we evaluated the effects of chondroitin sulphate (CS) on excitatory synaptic transmission, cellular excitability, and neuronal plasticity using extracellular recordings in the CA1 area of rat hippocampal slices. CS caused a reversible depression of evoked field excitatory postsynaptic potentials in a concentration-dependent manner. CS also reduced the population spike amplitude evoked after orthodromic stimulation but not when the population spikes were antidromically evoked; in this last case a potentiation was observed. CS also enhanced paired-pulse facilitation and long-term potentiation. Our study provides evidence that CS, a major component of the brain perineuronal net and extracellular matrix, has a function beyond the structural one, namely, the modulation of synaptic transmission and neuronal plasticity in the hippocampus.

Different contributions of calcium channel subtypes to electrical excitability of chromaffin cells in rat adrenal slices.

We characterized the ionic currents underlying the cellular excitability and the Ca(2+) -channel subtypes involved in action potential (AP) firing of rat adrenal chromaffin cells (RCCs) preserved in their natural environment, the adrenal gland slices, through the perforated patch-clamp recording technique. RCCs prepared from adrenal slices exhibit a resting potential of -54 mV, firing spontaneous APs (2-3 spikes/s) generated by the opening of Na(+) and Ca(2+) -channels, and terminated by the activation of voltage and Ca(2+) -activated K(+) -channels (BK). Ca(2+) influx via L-type Ca(2+) -channels is involved in reaching threshold potential for AP firing, and is responsible for activation of BK-channels contributing to AP-repolarization and afterhyperpolarization, whereas P/Q-type Ca(2+) -channels are involved only in the repolarization phase. BK-channels carry total outward current during AP-repolarization. Blockade of L-type Ca(2+) -channels reduces BK-current ~60%, whereas blockade of N- or P/Q-type produces little effect. This study demonstrates that Ca(2+) influx through L-type Ca(2+) -channels plays a key role in modulating the threshold potential from RCCs in situ. This study demonstrates that Ca(2+) influx through L-type Ca(2+) channels plays a key role in modulating the threshold potential for action potential firing and activating BK channels contributing to repolarization and afterhyperpolarization from rat adrenal chromaffin cells in situ.

Chondroitin sulfate, a major component of the perineuronal net, elicits inward currents, cell depolarization, and calcium transients by acting on AMPA and kainate receptors of hippocampal neurons.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are the most abundant PGs of the brain extracellular matrix (ECM). Free CS could be released during ECM degradation and exert physiological functions; thus, we aimed to investigate the effects of CS on voltage- and current-clamped rat embryo hippocampal neurons in primary cultures. We found that CS elicited a whole-cell Na(+)-dependent inward current (ICS) that produced drastic cell depolarization, and a cytosolic calcium transient ([Ca(2+)]c). Those effects were similar to those elicited by α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, were completely blocked by NBQX and CNQX, were partially blocked by GYKI, and were unaffected by MK801 and D-APV. Furthermore, ICS and AMPA currents were similarly potentiated by cyclothiazide, a positive allosteric modulator of AMPA receptors. Because CSPGs have been attributed Ca(2) (+) -dependent roles, such as neural network development, axon pathfinding, plasticity and regeneration after CNS injury, CS action after ECM degradation could be contributing to the mediation of these effects through its interaction with AMPA and kainate receptors.

Atypical Ca2+ currents in chromaffin cells from SHR and WKY rat strains result from the deficient expression of a splice variant of the α1D Ca2+ channel.

Ca(2+) currents (I(Ca)) recorded from adrenal chromaffin cells (CCs) of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats are similar to one another, but different from those recorded in other rodent species. I(Ca) in WKY/SHR CCs comprises an early, transient (I(Ca(e))) and a late, sustained component (I(Ca(s))). In Wistar CCs, I(Ca(e)) is absent, and I(Ca(s)) is of greater amplitude. Activation and steady-state inactivation of I(Ca(e)) and I(Ca(s)) in WKY/SHR CCs suggest the recruitment of at least two populations of Ca(2+) channels with different voltage dependence and kinetics. In WKY/SHR CCs, I(Ca(e)) is inhibited by nifedipine, enhanced by BAY K 8644, is not blocked by the mibefradil analog NNC 55-0396, and displays Ca(2+)-dependent inactivation and fast deactivation kinetics, suggesting that it results from the opening of L-type rather than T-type Ca(2+) channels. I(Ca(e)) properties suggest that it originates from the opening of Ca(2+) channels formed with the short splice variant (Ca(V)1.3(42A)). RT-PCR showed that expression of Ca(V)1.3(42A) mRNA is similar in both Wistar and WKY/SHR, but that the long variant (Ca(V)1.3(42)) is virtually absent in WKY/SHR. Thus I(Ca(e)) corresponds to the recruitment of Ca(V)1.3(42A) channels, unmasked by the absence of Ca(V)1.3(42) channels. Studies in WKY CCs do not report major functional alterations, despite the unusual expression pattern of Ca(V)1.3 splice variants. It remains to be established if more subtle functional alterations exist, and if the atypical splicing pattern of Ca(V)1.3 could be related to the functional and behavioral alterations reported in WKY/SHR rats, including their susceptibility to develop hypertension.

Functional cGMP-gated channels in cerebellar granule cells.

Cyclic nucleotide-gated channels (CNGCs) are important transducers of external signals in sensory processes. These channels are ubiquitously expressed in a variety of neurons, and are necessary to transduce signals for growth cone guidance and plasticity. Here, we demonstrate that the CNGC subunits (CNGA1 and CNGB1, presumably the 1b isoform) are expressed in rat cerebellar granule cells and that they combine to form functional channels. The expression of the mRNAs that encode these proteins is maximal after 7 days in cell culture, when the channels are expressed at synapses and co-localize with the synaptic marker synapsin I. These ligand-gated channels are functional and can be blocked by Mg(2+) or L-cis-diltiazem. Moreover, channel opening in response to increases in intracellular cGMP results in Ca(2+) entry into the cell. Chronic blockade (96 h) of these channels with L-cis-diltiazem significantly decreases the number of functional boutons, as determined by their capacity to load and unload the styryl dye FM1-43 when stimulated. Moreover, the unloading kinetics is modified from a biphasic to a monophasic profile in a subset of synaptic boutons. These channels are also expressed in early developmental stages, both in the soma and in emerging processes, and CNGA1 can be detected in growth cones. Pharmacological blockade of these channels with L-cis-diltiazem causes an overall change in growth cone morphology, impairing the formation of lamellipodia between filopodia and increasing the number of filopodia. J

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis.

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31 µM. At 10 µM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52 µM). At 10 µM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.

Comparison of Ca2+ currents of chromaffin cells from normotensive Wistar Kyoto and spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) are widely used as model to investigate the pathophysiological mechanisms of essential hypertension. Catecholamine plasma levels are elevated in SHR, suggesting alterations of the sympathoadrenal axis. The residual hypertension in sympathectomized SHR is reduced after demedullation, suggesting a dysfunction of the adrenal medulla. Intact adrenal glands exposed to acetylcholine or high K+ release more catecholamine in SHR than in normotensive Wistar Kyoto (WKY) rats, and adrenal chromaffin cells (CCs) from SHR secrete more catecholamines than CCs from WKY rats. Since Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers exocytosis, alterations in the functional properties of these channels might underlie the enhanced catecholamine release in SHR. This study compares the electrophysiological properties of VGCC from CCs in acute adrenal slices from WKY rats and SHR at an early stage of hypertension. No significant differences were found in the macroscopic Ca2+ currents (current density, I­V curve, voltage dependence of activation and inactivation, kinetics) between CCs of SHR and WKY rats, suggesting that Ca2+ entry through VGCC is not significantly different between these strains, at least at early stages of hypertension. Ca2+ buffering, sequestration and extrusion mechanisms, as well as Ca2+ release from intracellular stores, must now be evaluated to determine if alterations in their function can explain the enhanced catecholamine secretion reported in CCs from SHR.

Blockade by nanomolar resveratrol of quantal catecholamine release in chromaffin cells.

The cardiovascular protecting effects of resveratrol, an antioxidant polyphenol present in grapes and wine, have been attributed to its vasorelaxing effects and to its anti-inflammatory, antioxidant, and antiplatelet actions. Inhibition of adrenal catecholamine release has also been recently implicated in its cardioprotecting effects. Here, we have studied the effects of nanomolar concentrations of resveratrol on quantal single-vesicle catecholamine release in isolated bovine adrenal chromaffin cells. We have found that 30 to 300 nM concentrations of resveratrol blocked the acetylcholine (ACh) and high K(+)-evoked quantal catecholamine release, amperometrically measured with a carbon fiber microelectrode. At these concentrations, resveratrol did not affect the whole-cell inward currents through nicotinic receptors or voltage-dependent sodium and calcium channels, neither the ACh- or K(+)-elicited transients of cytosolic Ca(2+). Blockade by nanomolar resveratrol of secretion in ionomycin- or digitonin-treated cells suggests an intracellular site of action beyond Ca(2+)-dependent exocytotic steps. The fact that nanomolar resveratrol augmented cGMP is consistent with the view that resveratrol could be blocking the quantal secretion of catecholamine through a nitric oxide-linked mechanism. Because this effect occurs at nanomolar concentrations, our data are relevant in the context of the low circulating levels of resveratrol found in moderate consumers of red wines, which could afford cardioprotection by mitigating the catecholamine surge occurring during stress.

Coste-eficacia del tratamiento de la hepatitis C crónica en España / Cost-effectiveness of chronic hepatitis C treatment in Spain

En esta revisión se evalúa el coste farmacológico del tratamiento de la hepatitis C y se consideran las recomendaciones más eficientes en cuanto a la duración del tratamiento y a la respuesta virológica sostenida (RVS). Para esto, se han analizado las publicaciones más significativas de los últimos 10 años en esta área considerando los genotipos más frecuentes en España. Con este análisis, se ha calculado un coste global para el tratamiento de la hepatitis C de 1.636.524,58 a 1.761.365,73 € al utilizar el pegIFN-α (pegylated interferon alpha ‘interferón pegilado alfa’) 2a y de 1.794.586,39 a 1.917.013,73 € al utilizar el pegIFN-α-2b. La RVS de ambos tratamientos fue del 59,18 y del 64,58%, respectivamente, sin mostrarse una diferencia significativa entre ambas formulaciones. Adicionalmente, se justifica el coste económico de la introducción de altas dosis de pegIFN-α-2a y de ribavirina para los pacientes con genotipo 1 difíciles de tratar (con ácido ribonucleico del virus de la hepatitis C basal superior a 800.000U/ml, que no logran una respuesta virológica precoz en la semana 12 y con un peso superior a 85kg) (AU)

In this study we evaluated the pharmacologic costs of hepatitis C treatment, considering recommendations on both the duration of therapy and sustained virological response. With this aim, we analyzed relevant scientific articles published in the previous 10 years, considering the most common genotypes present in Spain. In this analysis, we estimated overall costs to be 1,636,524.58–1,761,365.73 € and 1,794.586.39–1,917,013.73 € with the use of pegylated interferon (PegIFN)-alpha-2a and PegIFN-alpha-2b, respectively. Sustained virological response was 59.18% and 64.58% respectively, with no significant difference between the two formulations. Finally, we assess the economic costs of the use of high-dose PegIFN-alpha-2a and ribavirin in patients with genotype 1 and treatment resistance (baseline HCV-RNA values >800.000UI/ml, without early viral response at 12 weeks and weight >85kg) (AU)