The Toxic Effects of Ethylene Glycol Tetraacetate Acid, Ferrum Lek and Methanol on the Glutathione System: correction Options.

Objectives: The purpose of this study was to measure the level of lipid peroxidation and investigate the response of the glutathione system to toxic doses of ethylene glycol tetraacetate acid (EGTA), Ferrum Lek, methanol, and Depakine (valproate sodium). Methods: This study focused on analyzing the toxic effects of EGTA, Ferrum Lek and methanol on lipid peroxidation processes and glutathione levels in animals. The study involved 375 outbred adult mice, of both sexes, weighing 28-31 g, and 100 outbred rats, weighing 180-200 g. Results: After 14 days of valproate sodium/ademethionine treatment, the GR (glutathione reductase) activity in experimental animals continued to be higher than in controls. Using EGTA enhanced glutathione reductase and glutathione S transferase activities in the liver and kidney. The activity of glutathione peroxidase, however, increased only in the kidney (2.1-fold, p ≤ 0.001), while in the liver, a 31% drop was observed (p ≤ 0.05). The 15-mg and 30-mg doses of Ferrum Lek caused the liver level of thiobarbituric acid reactive substances to grow 3- and 3.5-fold, respectively (p ≤ 0.001). Conclusion: The results of the study indicate that poisoning affected practically all components of the glutathione system. The oxidative stress was likely to result from an increased generation of reactive oxygen species against the background of inhibited antioxidant protection.

Molecular characterisation and expression of parathyroid hormone-related protein in the caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

Parathyroid hormone-related protein (PTHrP) is a hypercalcemic factor in fish, but the source of circulating PTHrP remains unclear. In this study investigation of the caudal neurosecretory system (CNSS), considered one of major sources of PTHrP in fish, provided valuable insights into this regulatory system. We report pthrpa and pthrpb gene cloning, characterization, expression, and responses to low salinity and hypocalcemia challenge in flounder. The pthrpa and pthrpb precursors, isolated from a European flounder CNSS library, consist of 166 and 192 amino acid residues, respectively, with an overall homology of approximately 59.2%. Both precursors contain a signal peptide and a mature peptide with cleavage and amidation sites. The flounder PTHrPA and PTHrPB peptides share only 41% sequence identity with human PTHrPA. Quantitative PCR analysis demonstrated that the bone and bladder, are respectively major sites of pthrpa and pthrpb expression in flounder. Urophysectomy confirmed the CNSS as a likely contributor to circulating PTHrP peptides. There were no significant differences in CNSS pthrpa and pthrpb mRNA expression or plasma PTHrP levels between seawater (SW) and freshwater (FW)-adapted fish, though plasma total calcium concentrations were higher in FW animals. The intraperitonial administration of EGTA rapidly induced hypocalcemia and concomitant elevation in plasma PTHrP accompanied by increases in both pthrpa and pthrpb expression in the CNSS. Together, these findings support an evolutionary conserved role for PTHrP in the endocrine regulation of calcium.

Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma.

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and in vivo systemic alkalinization with NaHCO3 enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca2+) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca2+ chelator, BAPTA/AM, inhibited cytoplasmic Ca2+ as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high-associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. Mol Cancer Ther; 16(5); 936-47. ©2017 AACR.

Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

Esterase detoxication of acetylcholinesterase inhibitors using human liver samples in vitro.

Organophosphorus (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p-nitrophenyl acetate, a substrate for CaEs. This suggests the importance of CaEs in malaoxon detoxication. Understanding these individual differences in detoxication can inform human variability in pesticide sensitivity.

Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli.

Azoles are widely applied and largely effective as antifungals; however, the increasing prevalence of clinically resistant isolates has yet to be matched by approaches to improve the efficacy of antimicrobial therapy. In this study, using the model fungus Aspergillus nidulans and one of the most common human pathogen Aspergillus fumigatus as research materials, we present the evidence that calcium signaling is involved in the azole-antifungals-induced stress-response reactions. In normal media, antifungal-itraconazole (ITZ) is able to induce the [Ca(2+)]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca(2+)]c transient. Real-time PCR analysis verified that six-tested Ca(2+)-inducible genes-two calcium channels (cchA/midA), a calmodulin-dependent phosphatase-calcineurin (cnaA), a transcription factor-crzA, and two calcium transporters (pmrA/pmcA)-could be transiently up-regulated by adding ITZ, indicating these components are involved in the azole stress-response reaction. Defect of cnaA or crzA caused more susceptibility to azole antifungals than did single mutants or double deletions of midA and cchA. Notably, EGTA may influence Rh123 accumulation as an azole-mimicking substrate through the process of the drug absorption. In vivo studies of a Galleria mellonella model identified that the calcium chelator works as an adjunct antifungal agent with azoles for invasive aspergillosis. Most importantly, combination of ITZ and EGTA or ITZ with calcium signaling inhibitor-FK506 greatly enhances the ITZ efficacy. Thus, our study provides potential clues that specific inhibitors of calcium signaling could be clinically useful adjuncts to conventional azole antifungals in the Aspergilli.

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells.

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

Results of Membrane-Activated Chelator Stroke Intervention randomized trial of DP-b99 in acute ischemic stroke.

BACKGROUND AND PURPOSE: DP-b99, a lipophilic moderate-affinity chelator of zinc, was postulated to improve recovery after acute ischemic stroke. We evaluated the safety and therapeutic effects of DP-b99 in patients with acute hemispheric ischemic stroke. METHODS: The Membrane-Activated Chelator Stroke Intervention trial was a randomized, double-blind, placebo-controlled, multicenter, parallel-group trial of intravenous DP-b99 administered for 4 consecutive days (NCT00893867). Acute ischemic stroke patients within 9 hours of onset, but untreated by alteplase, with a baseline National Institutes of Health Stroke Scale score of 10 to 16, and evidence of language dysfunction, visual field defect, and neglect were eligible. The primary efficacy analysis compared distributions of functional status measured by modified Rankin score in the intent-to-treat population of patients with any post-treatment outcome, adjusted for initial severity. Functional and neurological recovery were secondary measures. Home time was an exploratory end point. RESULTS: Enrollment terminated at n=446 after the planned interim analysis determined futility; follow-up continued. Final modified Rankin score distributions were equal between DP-b99 and placebo-treated groups (P=0.10; P(adj) adjusted for baseline age and National Institutes of Health Stroke Scale=0.21). Fewer patients recovered to modified Rankin score ≤1 in the DP-b99-treated group (45/218; 20.6%) than after placebo (63/219; 28.8%) (P=0.05; P(adj)=0.10). Similarly, fewer patients attained National Institutes of Health Stroke Scale ≤1 after DP-b99 (42/218; 19.3%) than placebo (56/219; 25.6%; P=0.10; P(adj)=0.26). Mortality was similar between DP-b99 and placebo intent-to-treat groups (36/218; 16.5% vs 33/219; 15.1%; P=0.68). Home time was unchanged by treatment (median 36 vs 36.5 days; P=0.25). CONCLUSIONS: Despite encouraging preclinical and phase II trial data, DP-b99 shows no evidence of efficacy in treating human ischemic stroke.

Quantitative analysis of the effect of irrigant solution sequences on dentin erosion.

OBJECTIVE: The purpose of the study was to examine the level of erosion on root canal wall dentin caused by immersion in different irrigant solutions in alternative sequences. METHODS: Dentin specimens from teeth with one root canal were instrumented and randomly divided into five groups. Each group was subjected to 17% EDTA, 17% ethyleneglycoltetraacetic acid (EGTA), or 10% citric acid (CA) and 5.25% sodium hypochlorite (NaOCl) varying the time of irrigant exposure and the order of the irrigants. The specimens were examined by scanning electron microscopy (SEM), and randomized digital images of the dentin surface were taken. The area of tubule openings was measured by a semiautomatic method. RESULTS: No erosion was detected when demineralizing agents were used as a final rinse after NaOCl. However, the erosion of peritubular and intertubular dentin was detected when EDTA, EGTA, or CA were used first followed by 5.25% NaOCl (P < .05), and an increase over 100% in the area of dentin tubule openings was measured (P < .01). CONCLUSIONS: NaOCl used as a final irrigant solution after demineralization agents causes marked erosion of root canal dentin.

Impaired presynaptic cytosolic and mitochondrial calcium dynamics in aged compared to young adult hippocampal CA1 synapses ameliorated by calcium chelation.

Impaired regulation of presynaptic intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. We studied presynaptic cytosolic and mitochondrial calcium (Ca) dynamics using axonally loaded Calcium Green-AM and Rhod-2 AM fluorescence respectively in young (2-3 months) and aged (23-26 months) CA3 to CA1 Schaffer collateral excitatory synapses in hippocampal brain slices from Fisher 344 rats. After a tetanus (100 Hz, 200 ms), the presynaptic cytosolic Ca peaked at approximately 10 s in the young and approximately 12 s in the aged synapses. Administration of the membrane permeant Ca chelator, bis (O-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA-AM), significantly attenuated the Ca response in the aged slices, but not in the young slices. The presynaptic mitochondrial Ca signal was much slower, peaking at approximately 90 s in both young and aged synapses, returning to baseline by 300 s. BAPTA-AM significantly attenuated the mitochondrial calcium signal only in the young synapses. Uncoupling mitochondrial respiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP) application evoked a massive intracellular cytosolic Ca increase and a significant drop of mitochondrial Ca, especially in aged slices wherein the cytosolic Ca signal disappeared after approximately 150 s of washout and the mitochondrial Ca signal disappeared after 25 s of washout. These signals were preserved in aged slices by BAPTA-AM. Five minutes of oxygen glucose deprivation (OGD) was associated with a significant increase in cytosolic Ca in both young and aged synapses, which was irreversible in the aged synapses. These responses were significantly attenuated by BAPTA-AM in both the young and aged synapses. These results support the hypothesis that increasing intracellular calcium neuronal buffering in aged rats ameliorates age-related impaired presynaptic Ca regulation.

DP-b99, a membrane-activated metal ion chelator, as neuroprotective therapy in ischemic stroke.

BACKGROUND AND PURPOSE: DP-b99 is a chelator of zinc and calcium ions that acts selectively within cell membranes and has neuroprotective properties in animal models of stroke. We present the results of a multicenter, double-blind, placebo-controlled, randomized trial to assess the safety and potential protective effects of DP-b99 in acute ischemic stroke. METHODS: One hundred and fifty stroke patients with signs of cortical involvement and a National Institutes of Health Stroke Scale (NIHSS) score of 7 to 20 received a 4-day course of intravenous 1 mg/kg per day DP-b99 or placebo within 1 to 9 hours of stroke onset. Treatment with recombinant tissue plasminogen activator was not allowed. RESULTS: No major differences in mortality rate, causes of death, adverse events, safety laboratory tests, and ECG parameters were found between the 2 groups. The baseline NIHSS score of the 72 DP-b99- and 75 placebo-treated patients in the intent-to-treat cohort was (mean+/-SD) 12.2+/-4.0 and 12.6+/-3.3, respectively; the time to needle (mean+/-SD) was 6:36+/-1:47 and 6:28+/-1:33 hours, respectively; and the age (mean+/-SD) was 73.3+/-9.9 and 72.0+/-9.6 years, respectively. The 90-day median change from baseline (the primary end point) was -6.0 and -5.0 NIHSS points in the DP-b99 and placebo groups, respectively (nonsignificant). At 90 days, there was a significantly better outcome in the DP-b99 group compared with the placebo group (modified Rankin scale score of 0, 1, or same as prestroke): 30.6% and 16.0%, respectively (P=0.05). The recovery rate was unaffected by the time to needle. Further analyses indicated that the 90-day median change from baseline in patients with an entry NIHSS score of 10 to 16 was 8.0 and 5.0 points in the DP-b99 and placebo groups, respectively (P=0.03). CONCLUSIONS: In this small-scale study, the primary end point of change in NIHSS score from baseline to 90 days was not met. However, secondary end points demonstrated a significantly improved 90-day recovery rate with treatment with DP-b99 when compared with placebo. In addition, in patients with baseline NIHSS scores of 10 to 16, a significant post hoc change in NIHSS score from baseline to day 90 was observed. No major safety problems were identified. These findings need to be confirmed with a larger prospective study of strokes involving the cortex.

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica).

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.

Does BAPTA leave outer hair cell transduction channels closed?

The calcium chelator BAPTA was iontophoresed into the scala media of the second turn of the guinea pig cochlea. This produced a reduction in low frequency cochlear microphonic (CM) measured in scala media and an elevation of the cochlear action potential (CAP) threshold that lasted for the duration of the experiment. Using two pipettes, one filled with KCl and the other KCl and BAPTA (50, 20 and 5 mM) it was possible to observe the effect of passing current through one electrode while measuring the endolymphatic potential (EP) with the other. The results demonstrated that current passed via the BAPTA pipette caused a sustained increase in EP of 8.2, 12.9 and 7.8 mV in the three animals used. This increase coincided with the decrease in low frequency CM that indicated a causal connection between the two. In a second series of experiments, pipettes with larger tips were inserted into scala media in the first cochlear turn and BAPTA was allowed to diffuse from the pipette. The results confirmed the relationship between EP increase and the fall of scala media CM. One interpretation of these results is that lowering the Ca2+ concentration of endolymph with BAPTA inhibits mechano-electrical transduction in outer hair cells (OHCs) and leaves the hair cell transduction channels in a closed state, thus increasing the resistance across OHCs and increasing the EP. These findings are consistent with a model of hair cell transduction in which tension on stereo cilia opens the transduction channels.

Increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage in the cultured neurons of Lymnaea stagnalis.

PURPOSE: To determine whether the increase in intracellular Ca2+ concentration induced by lidocaine produces neurotoxicity, we compared morphological changes and Ca2+ concentrations, using fura-2 imaging, in the cultured neurons of Lymnaea stagnalis. METHODS: We used BAPTA-AM, a Ca2+ chelator, to prevent the increase in the intracellular Ca2+ concentration, and Calcimycin A23187, a Ca2+ ionophore, to identify the relationship between increased intracellular Ca(2+) concentrations and neuronal damage without lidocaine. Morphological changes were confirmed using trypan blue to stain the cells. RESULTS: Increasing the dose of lidocaine increased the intracellular Ca2+ concentration; however, there was no morphological damage to the cells in lidocaine at 3 x 10(-3) M. Lidocaine at 3 x 10(-2) M increased the intracellular Ca2+ concentration in both saline (from 238 +/- 63 to 1038 +/- 156 nM) and Ca2+-free medium (from 211 +/- 97 to 1046 +/- 169 nM) and produced morphological damage and shrinkage, with the formation of a rugged surface. With the addition of BAPTA-AM, lidocaine at 3 x 10(-2) M moderately increased the intracellular Ca2+ concentration (from 150 +/- 97 to 428 +/- 246 nM) and produced morphological damage. These morphologically changed cells were stained dark blue with trypan blue dye. The Ca2+ ionophore increased the intracellular Ca2+ concentration (from 277 +/- 191 to 1323 +/- 67 nM) and decreased it to 186 +/- 109 nM at 60 min. Morphological damage was not observed during the 60 min, but became apparent a few hours later. CONCLUSION: These results indicated that the increase in intracellular Ca2+ concentration is not the only cause of lidocaine-induced cell damage.

Differential involvement of intracellular Ca2+ in 1-methyl-4-phenylpyridinium- or 6-hydroxydopamine-induced cell viability loss in PC12 cells.

1-Methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP(+)-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP(+ )or 6-OHDA evoked the elevation of intracellular Ca(2+) levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca(2+) levels due to both toxins. The results show that the MPP(+)-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca(2+) levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca(2+)-independent manner.

The role of Ca2+ in (-)-menthol-induced human promyelocytic leukemia HL-60 cell death.

A human promyelocytic leukemia HL-60 cell line was selected to examine the effect of (-)-Menthol on cell death. Based on the results from morphological changes and the percentage of viable cells in HL-60 cells after treatment with various concentrations of (-)-Menthol, it was shown that (-)-Menthol induced cell death through necrosis, not apoptosis. No cell cycle arrest was found in HL-60 cells examined by flow cytometry analysis. Also, the DNA gel electrophoresis method showed that (-)-Menthol did not induce apoptosis in HL-60 cells. However, it was found that (-)-Menthol induced the production of Ca2+ in these examined cells, dose-dependently. When HL-60 cells were pretreated with the chelator (BAPTA) of Ca2+ for 3 hours before addition of (-)-Menthol to the culture, a decrease of Ca2+ production was observed. Under the same conditions, the percentage of viable HL-60 cells was increased. Apparently Ca2+ production is associated with the induction of (-)-Menthol-induced cell death.

The effect of BAPTA and 4AP in scala media on transduction and cochlear gain.

We have injected by iontophoresis 4-amino-pyridine, a K+ channel blocker and BAPTA, (a Ca++ chelator), into scala media of the first three turns of the guinea pig cochlea. We measured the reduction in outer hair cell (OHC) receptor current, as indicated by cochlear microphonic measured in scala media evoked by a 207 Hz tone, and compared this with the elevation of the cochlear action potential (CAP) threshold. We found that in the basal turn, for frequencies between 12 and 21 kHz, CAP threshold was elevated by about 30 dB, while in the second turn, at the 3 kHz place, the maximum elevation was 15 dB. In the third turn, iontophoresis of 4AP and BAPTA reduced CM by similar amounts to that in the basal and second turn, but caused negligible elevation of CAP threshold. We conclude that the gain of the cochlear amplifier is maximal for basal turn frequencies, is halved at 3 kHz, and is reduced to close to one for frequencies below 1 kHz (no active gain). The effect of 4AP and BAPTA on neural threshold and the receptor current represented by CM may be explained by their action on OHC transduction without the involvement of IHCs.

The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human beta-amyloid precursor protein transgenic mice.

Metals such as zinc, copper and iron contribute to aggregation of amyloid-beta (Abeta) protein and deposition of amyloid plaques in Alzheimer's disease (AD). We examined whether the lipophilic metal chelator DP-109 inhibited these events in aged female hAbetaPP-transgenic Tg2576 mice. Daily gavage administration of DP-109 for 3 months markedly reduced the burden of amyloid plaques and the degree of cerebral amyloid angiopathy in brains, compared to animals receiving vehicle treatment. Moreover, DP-109 treatment appeared to facilitate the transition of Abeta from insoluble to soluble forms in the cerebrum. These results further support the hypothesis that endogenous metals are involved in the deposition of aggregated Abeta in brains of AD patients, and that metal chelators may be useful therapeutic agents in the treatment of AD.

Effects of long-term high-altitude hypoxia and troponin I phosphorylation on cardiac myofilament calcium responses in fetal and nonpregnant sheep.

OBJECTIVE: We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep. METHODS: Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing no PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state. RESULTS: In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significantly less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left ventricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small. CONCLUSION: In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphosphatase activity observed previously. The larger decrease in tension after PKA phosphorylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.