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Analysis of neurotransmitters and their metabolites is useful for the diagnosis of central nervous system diseases. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation was developed to monitor levels of adrenaline (AD), noradrenaline (NA), glutamic acid (Glu), γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in rat brain tissue. Isoprenaline was used as an internal standard (IS). Neurotransmitters and metabolites were eluted with a reverse phase column under gradient conditions through a mobile phase consisting of 0.2% formic acid water solution/acetonitrile. The compounds were detected and quantified by LC-MS/MS with positive or negative electrospray ionization, which operates in multiple-reaction monitoring mode. The method was linear or polynomial (R(2)>0.99) for AD, NA, Glu, GABA, DA, 5-HT, 5-HIAA, and MHPG in the range of 0.25-200, 0.5-200, 250-20,000, 250-20,000, 0.25-200, 10-3000, 1-50, and 1-50ng/mL, respectively. The validation assays for accuracy and precision, matrix effect, extraction recovery, stability and carry-over of the samples for neurotransmitters and metabolites were consistent with the requirements of regulatory agencies. The method enables rapid quantification of neurotransmitters and their metabolites and has been applied in the nuclear factor (erythroid 2-derived)-like 2 (Nrf2) knockout mouse model of depression., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A simple, reproducible and fast (4 min chromatogram) method of liquid chromatography in tandem with mass spectrometry (LC/MS-MS) was developed to determine simultaneously the plasma levels of albendazole (ABZ) and its metabolite albendazole sulfoxide (ABZOX) for pharmacokinetic and clinical analysis. Each plasma sample was extracted by solid phase extraction (SPE) using phenacetin as internal standard (IS). The extracted sample was eluted with a Zorbax XDB-CN column using an isocratic method. The mobile phase consisting of water with 1% acetic acid (40%, A) and MeOH (60%, B), was used at a flow rate of 1 mL/min. ABZ and ABZOX were detected and identified by mass spectrometry with electrospray ionization (ESI) in the positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 5-1000 ng/mL for ABZ and 10-1500 ng/mL (full validation) or 10-5000 ng/mL (partial validation) for ABZOX, with 5 and 10 ng/mL lower limit of quantification (LLOQ) for ABZ and ABZOX, respectively. The tests of accuracy and precision, matrix effect, extraction recovery and stability of the samples for both ABZ and ABZOX did not deviate more than 20% for the LLOQ and no more than 15% for other quality controls (QCs), according to regulatory agencies., (© 2013.)

The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 Alzheimer's 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., (Copyright 2009 Elsevier Inc. All rights reserved.)

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.

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.