Cytosolic Free Calcium Research Articles

Caspase-3 and -9 are activated in human myeloid HL-60 cells by calcium signal

This study is aimed to determine the role of calcium signaling evoked by the calcium-mobilizing agonist uridine-5'-triphosphate (UTP) and by the specific inhibitor of the endoplasmic reticulum calcium reuptake thapsigargin on caspase activation in human leukemia cell line HL-60. We have analyzed cytosolic free calcium concentration ([Ca(2+)](c)) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric calcium indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with 10 microM UTP or 1 microM thapsigargin induced a transient increase in [Ca(2+)](c) due to calcium release from internal stores. The stimulatory effect of UTP and thapsigargin on calcium signal was followed by a mitochondrial membrane depolarization. Our results also indicated that UTP and thapsigargin were able to increase the caspase-3 and -9 activities. The effect of UTP and thapsigargin on caspase activation was time dependent, reaching a maximal caspase activity after 60 min of stimulation. Loading of cells with 10 microM dimethyl BAPTA, an intracellular calcium chelator, for 30 min significantly reduced both UTP- or thapsigargin-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with 10 microM Ru360 for 30 min, a specific blocker of calcium uptake into mitochondria. The findings suggest that UTP- and thapsigargin-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca(2+)](c) in human myeloid HL-60 cells.

Molecular and functional characterization of voltage-gated sodium channels in human sperm

BackgroundWe have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.MethodsFreshly ejaculated semen was collected from thirty normozoospermic human donors, with each donor supplying 2 different samples. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence techniques were used to detect the mRNAs and proteins of interest. Sperm motility was measured by a computer-assisted sperm analysis system (CASA). Cytosolic free calcium was determined by fluorimetry in cells loaded with the fluorescent calcium indicator Fura-2.ResultsThe mRNAs that encode the different Nav alpha subunits (Nav1.1-1.9) were all expressed in capacitated human spermatozoa. The mRNAs of the auxiliary subunits beta1, beta3 and beta4 were also present. Immunofluorescence studies showed that, with the exception of Nav1.1 and Nav1.3, the Nav channel proteins were present in sperm cells and show specific and different sites of localization. Veratridine, a voltage-gated sodium channel activator, caused time- and concentration-dependent increases in progressive sperm motility. In sperm suspensions loaded with Fura-2, veratridine did not modify intracellular free calcium levels.ConclusionThis research shows the presence of voltage-gated sodium channels in human sperm and supports a role for these channels in the regulation of mature sperm function.

Calmodulin antagonist W7 increases inositol phosphates in insulin secreting RINm5F cells.

W7, a calmodulin antagonist, has been reported to increase cytosolic free calcium concentration [Ca2+]i in non stimulated rat insulinoma cells (RINm5F). And this effect was not due to enhanced calcium uptake. In the present study the effect of calmodulin antagonist W7 on the inositol phosphate turnover of RINm5F cells was studied. Inositol phosphates were separated using a new modified technique of anion-exchange high performance liquid chromatography (HPLC). It was observed that W7 significantly increased inositol trisphosphate and inositol bisphosphate within 5 and 15 sec, respectively. No changes of inositol phosphates were detected employing W5, a chlorine-deficient analogue of W7 without calmodulin antagonistic action. Our data are in favour of the view that (I) calmodulin may be involved in inositol phosphate metabolism of RINm5F cells and that (II) the increase of [Ca2+]i in response to W7 as reported previously may be due to elevation of inositol trisphosphate.

Cellular protective action of angiotensin converting enzyme inhibitors.

In several studies organ protective effects of ACE inhibitors independent from their antihypertensive action have been demonstrated. The mechanisms of the protective effects of angiotensin converting enzyme (ACE) inhibitors on vasculature and kidney are largely unknown. In the present study the modulatory action of captopril on the angiotensin II (AngII), arginine vasopressin (AVP), and platelet derived growth factor (PDGF)-induced increase of cytosolic free calcium concentration ([Ca2+]i) was investigated in cultured vascular smooth muscle cells (VSMC) and cultured glomerular mesangial cells (MC) from rats using the fluorescent dye technique. Resting [Ca2+]i in VSMC or MC was not significantly affected by captopril. The preincubation of VSMC with 1 mumol/l captopril significantly reduced the AngII-induced [Ca2+]i increase in VSMC from 90 +/- 10 nmol/l (n = 78; mean +/- SEM) to 51 +/- 16 nmol/l (n = 53; p < 0.05) and in MC from 102 +/- 42 nmol/l (n = 14) to 43 +/- 12 nmol/l (n = 7; p < 0.05). In the absence of extracellular calcium captopril produced no effect on AngII induced changes of [Ca2+]i. Captopril significantly attenuated the AVP-induced [Ca2+]i increase in VSMC and MC. The preincubation of MC with 1 mumol/l captopril for 40 min significantly reduced the PDGF-induced [Ca2+]i increase in MC from 127 +/- 31 nmol/l (n = 11) to 61 +/- 32 nmol/l (n = 5, p < 0.05). The present results may indicate that part of the protective effects of ACE inhibitors on vasculature and kidney may be promoted by inhibition of growth-factor induced changes of calcium homeostasis.

Intracellular calcium homeostasis in patients with early stagesof chronic kidney disease: effects of vitamin D3 supplementation

Chronic renal failure has been referred to as a state of cellular calcium toxicity. The aim of this study was to investigate the status of free cytosolic calcium ([Ca(2+)](i)), intracellular calcium reserves and the capacitative calcium entry in peripheral blood mononuclear cells (PBMCs) of early-stage chronic kidney disease (CKD) patients, and to determine the effect of vitamin D(3) supplementation on these parameters. The study involved 44 patients with CKD stages 2-3; 27 of them were treated with cholecalciferol (5000 IU/week) for 12 months. [Ca(2+)](i) was measured using Fluo-3 AM fluorimetry. Intracellular calcium reserves were emptied by the application of thapsigargin (Tg), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. 2-Aminoethyl-diphenyl borate (2APB) was used to examine the capacitative calcium entry. [Ca(2+)](i) of CKD patients was substantially higher in comparison with healthy subjects: 123 (115-127) versus 102 (98-103) nmol/l, P < 0.001. The calcium concentration of Tg-sensitive stores and the capacitative calcium entry were also significantly increased in CKD patients. After the 12-month vitamin D(3) supplementation, there was a marked decrease in [Ca(2+)](i) [105 (103-112) nmol/l, P < 0.001 versus baseline], independently of the increase in 25(OH)D(3) or the decrease in PTH levels. No significant changes in intracellular calcium reserves and the capacitative calcium entry were found. Our results demonstrate that (1) [Ca(2+)](i), intracellular calcium stores and the capacitative calcium entry were significantly increased already in early stages of CKD; (2) long-term vitamin D(3) supplementation normalized [Ca(2+)](i) without any effect on intracellular calcium reserves or the capacitative calcium entry.

Cold-induced cytosolic free calcium ion concentration changes in wheat

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca(2+)](c)) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca(2+)](c) was detected in vivo by luminometry, and [Ca(2+)](c) elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca(2+)](c) during cold exposure. Temperature-induced changes in [Ca(2+)](c) were found to be more dependent on the change in temperature (dT dt(-1)) than on the absolute value of temperature. [Ca(2+)](c) increased only at cooling rates higher than 8 degrees Cmin(-1), indicating that an overall cellular [Ca(2+)](c) increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called 'calcium signature hypothesis'.

Phosphatidylinositol-linked novel D 1 dopamine receptor facilitates long-term depression in rat hippocampal CA1 synapses

Recent work has demonstrated that a phosphatidylinositol (PI)-linked D 1 dopamine receptor selective agonist, SKF83959, mediates phosphatidylinositol hydrolysis via activation of phospholipase C β in brain. Specific contributions of SKF83959 to synaptic plasticity have not been well elucidated. The aim of the current investigation was to characterize the role of SKF83959 on long-term depression (LTD) in the CA1 region of rat hippocampal slices and to explore the molecular events leading to these changes. The results indicated that SKF83959 stimulation significantly depressed field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner and facilitated the induction of LTD by LFS. SKF83959-facilitated LTD required activation of phospholipase C (PLC). NMDA receptors were involved in this response. Calcium chelator, BAPTA-AM prevented SKF83959-facilitated LTD, indicating that cytosolic free calcium concentration ([Ca 2+] i) elevation could account for this response. Furthermore, SKF83959-facilitated LTD was significantly depressed in the presence of calcineurin (PP2B) inhibitors cyclosporin A (CsA) and associated with a persistent increase in the expression of calcineurin A. Taken together, these findings demonstrate a novel role for PI-linked D 1 dopamine receptor in the neuromodulation of hippocampal LTD.

Identification and Characterization of Two Novel Truncated but Functional Isoforms of the Somatostatin Receptor Subtype 5 Differentially Present in Pituitary Tumors

Somatostatin and its related peptide cortistatin exert multiple actions on normal and tumoral tissue targets through a family of receptors termed somatostatin receptor (sst)1-5. Despite the considerable advances in the knowledge on these receptors and their (patho)physiological roles, there is still evidence that additional receptors for these peptides should exist to fully explain their actions. The growing number of spliced variants found in similar receptor families, often present in tumors, and results from our group obtained on sst5 from other species (pig) led us to explore the existence of new human sst5 isoforms. A rapid amplification of cDNA ends PCR approach on samples from a human pituitary tumor and a cell line enabled identification of two novel alternatively spliced sst5 receptor variants. The sequences obtained encode putative proteins that correspond to truncated isoforms of five and four transmembrane domains (TMDs), accordingly named sst5TMD5 and sst5TMD4, respectively. Both novel receptors show a differential expression pattern in normal tissues and are also present in pituitary tumors of diverse etiology including nonfunctioning adenomas, corticotropinomas, somatotropinomas, and a prolactinoma. In contrast to the predominant plasma membrane localization of full-length sst5, both sst5TMD5 and sst5TMD4 show a preferentially intracellular localization. Despite their truncated nature, both receptors are functional, as shown by their ability to mediate selective, ligand-induced rises in free cytosolic calcium concentration. Specifically, whereas sst5TMD5 is selectivity activated by somatostatin compared with cortistatin, cells transfected with sst5TMD4 almost exclusively respond to cortistatin and not to somatostatin. Our results demonstrate the existence of two previously unidentified sst5 spliced variants with distinct distribution in normal tissues and pituitary tumors, unique ligand-selective signaling properties, and subcellular distribution, which could contribute to somatostatin and cortistatin signaling in normal and tumoral cells.

Flubendazole interferes with a wide spectrum of cell homeostatic mechanisms in Echinococcus granulosus protoscoleces

The problem of chemotherapeutic treatments for human echinococcosis has not been completely solved. The benzimidazole–methylcarbamates (BZD), broad-spectrum antihelminthic agents, such as mebendazole and albendazole are the only drugs licensed for treatment of hydatid cysts. These drugs bind directly to β-tubulin causing the disruption of microtubule-based processes in helminths. However, the molecular bases of their multiple biological activities are poorly understood. Recently, the effect of halogenated derivative flubendazole (FLBZ), against E. granulosus larvae has been conclusively demonstrated . The comparative effectiveness of FLBZ, among other BZDs, was shown by means of vitality tests and time of appearance of morphological damage of larvae. In the present study, we examined biochemical and molecular changes on protoscoleces treated with FLBZ. We show that FLBZ induces: 1) an increase in cytosolic free calcium, 2) a decrease in tubulin transcripts, 3) a reduction of mMDH expression and 4) a significant decrease in glycogen levels. These results are consistent with the existence of multiple targets for FLBZ, such as calcium signaling and energy metabolism, and contribute to the understanding of the pharmaceutical properties of FLBZ.

H017 Time dependent activation of AKT-GSK3beta and calcineurin signaling pathways during the development of TAC-induced left ventricular hypertrophy in B6D2/F1 mice

Left ventricular hypertrophy (LVH) is an adaptive response to chronic biomechanical stress that later progresses to maladaptive hypertrophy and heart failure. To better understand the mechanisms responsible for LVH development, we studied the activation of two major kinases, protein kinase B (Akt/PKB) and glycogen synthase kinase3b (GSK3b) and of a phosphatase, calcineurin (Cn) in an experimental model of thoracic aorta constriction (TAC). 4 week-old B6D2/F1 male, Sham-operated (Sham) and TAC mice were studied 3, 7, 15, 30 and 60 days post-TAC. Gene expression and activation of signalling pathways was studied by PCR and Western Blot. TAC-induced LVH (10 – 51 % at days 3-60, respectively) was associated with: 1) fetal gene reexpression characterised by an increase in mRNA levels of BNP (1.7-3 fold) and a-SK (2-4 fold), an a- to b-MHC switch seen at day 30 only ; 2) a significant increase in SERCA2a (1.7 fold), PLB, and NCX (3.6 fold) protein levels at day 7. 3) a 2 fold increase in P-Akt at day 3 and a decrease at day 15 while Akt was increased at days 7 and 15 ; 4) an increase in P-GSK3b at day 15 (1.5 fold), whereas no change in Akt and GSK3b protein levels were seen at days 30 and 60 ; 5) a significant decrease in CnAb at day 15 (2 fold). LVH develops progressively from day 3 in response to TAC, accompanied by re-expression of the fetal gene program. The alpha to beta-MHC mRNA switch occurs late, associated with the transition to HF, which occurs progressively with time. At days 3 and 15, P-Akt acts as an upstream regulator of GSK3b which may favour the development of LVH. 7 days post-TAC, a SERCA2a and NCX increase might contribute to decrease free cytosolic calcium level. At day 15, a further increase in LVH is associated with an increase in Cn activity and an increase GSK3b, which may counteract the hypertrophic response. Taken together, our data suggest a time-dependent cross-talk between Cn and GSK3b to modulate cardiac hypertrophic response to pressure overload.

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and ‘tonic’ expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-009-0647-1) contains supplementary material, which is available to authorized users.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO(3)(-))) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO(3)(-)) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO(3)(-)), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol (in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO(3)(-)) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO(3)(-)) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO(3)(-)) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO(3)(-)) in middle proximal tubule.

Effect of 100 mT homogeneous static magnetic field on [Ca2+]c response to ATP in HL‐60 cells following GSH depletion

Calcium is an important molecule in a number of biological systems. Often these systems are signal transduction cascades involving molecules such as ATP. ATP activates second messengers which can interact with ion channels on the endoplasmic/sarcoplasmic reticulum resulting in the emptying of the intracellular calcium stores and an increase in cytosolic free calcium concentration ([Ca2+]c). Changes in [Ca2+]c can be influenced by external factors such as a static magnetic field (SMF). One hypothesis suggests that a SMF affects the cells through the radical pair mechanism. By reducing the number of antioxidant molecules like glutathione (GSH), the proportion of free radicals in the cells is increased and may lead to a greater probability of a biological response to a SMF. The purpose of this study was to determine if the [Ca2+]c response to ATP was affected by depletion of GSH by diethylmaleate (DEM) and the absence or presence of a 100 mT homogeneous SMF. Undifferentiated HL-60 cells were loaded with fura-2 AM. [Ca2+]c was measured in real time using a ratiometric fluorescence spectroscopy system. Various (DEM) ranging from 1 to 15 mM were added to deplete GSH. Cells were either exposed to sham or magnetic field (100 mT) for 13 min (780 s) and challenged with 1 microM ATP. The data show that [Ca2+]c was elevated following treatment with DEM with greater [Ca2+]c at higher [DEM]. The [Ca2+]c response to ATP was decreased as the DEM concentration increased. However, there was no effect of a 100 mT SMF on the average [Ca2+]c peak following ATP activation or the full width at half maximum (FWHM) of the [Ca2+]c response and recovery after ATP activation.

Reduced levels of intracellular calcium releasing in spermatozoa from asthenozoospermic patients

BackgroundAsthenozoospermia is one of the most common findings present in infertile males characterized by reduced or absent sperm motility, but its aetiology remains unknown in most cases. In addition, calcium is one of the most important ions regulating sperm motility. In this study we have investigated the progesterone-evoked intracellular calcium signal in ejaculated spermatozoa from men with normospermia or asthenozoospermia.MethodsHuman ejaculates were obtained from healthy volunteers and asthenospermic men by masturbation after 4–5 days of abstinence. For determination of cytosolic free calcium concentration, spermatozoa were loaded with the fluorescent ratiometric calcium indicator Fura-2.ResultsTreatment of spermatozoa from normospermic men with 20 micromolar progesterone plus 1 micromolar thapsigargin in a calcium free medium induced a typical transient increase in cytosolic free calcium concentration due to calcium release from internal stores. Similar results were obtained when spermatozoa were stimulated with progesterone alone. Subsequent addition of calcium to the external medium evoked a sustained elevation in cytosolic free calcium concentration indicative of capacitative calcium entry. However, when progesterone plus thapsigargin were administered to spermatozoa from patients with asthenozoospermia, calcium signal and subsequent calcium entry was much smaller compared to normospermic patients. As expected, pretreatment of normospermic spermatozoa with both the anti-progesterone receptor c262 antibody and with progesterone receptor antagonist RU-38486 decreased the calcium release induced by progesterone. Treatment of spermatozoa with cytochalasin D or jasplakinolide decreased the calcium entry evoked by depletion of internal calcium stores in normospermic patients, whereas these treatments proved to be ineffective at modifying the calcium entry in patients with asthenozoospermia.ConclusionOur results suggest that spermatozoa from asthenozoospermic patients present a reduced responsiveness to progesterone.

Tim-1 Signaling Substitutes for Conventional Signal 1 and Requires Costimulation to Induce T Cell Proliferation

Differentiation and clonal expansion of Ag-activated naive T cells play a pivotal role in the adaptive immune response. T cell Ig mucin (Tim) proteins influence the activation and differentiation of T cells. Tim-3 and Tim-2 clearly regulate Th1 and Th2 responses, respectively, but the precise influence of Tim-1 on T cell activation remains to be determined. We now show that Tim-1 stimulation in vivo and in vitro induces polyclonal activation of T cells despite absence of a conventional TCR-dependent signal 1. In this model, Tim-1-induced proliferation is dependent on strong signal 2 costimulation provided by mature dendritic cells. Ligation of Tim-1 upon CD4(+) T cells with an agonist anti-Tim-1 mAb elicits a rise in free cytosolic calcium, calcineurin-dependent nuclear translocation of NF-AT, and transcription of IL-2. Because Tim-4, the Tim-1 ligand, is expressed by mature dendritic cells, we propose that interaction between Tim-1(+) T cells and Tim-4(+) dendritic cells might ensure optimal stimulation of T cells, when TCR-derived signals originating within an inflamed environment are weak or waning.

Computational study of non-homogeneous distribution of Ca 2+ handling systems in cerebellar granule cells

The spatiotemporal distribution of cytosolic free calcium concentration ([Ca 2+] i ) in cerebellar granule cells (GrCs) is thought to be critical in defining the occurrence and direction of long-term changes in synaptic strength at cerebellar mossy fiber–GrC synapses. Despite this, the mechanisms responsible for shaping Ca 2+ transients in GrCs are not well understood. To investigate the interplay between Ca 2+ entry, extrusion, buffering and dendritic morphology in shaping Ca 2+ elevations in GrCs, we developed a model of Ca 2+ regulation in these cells and examined the requirements for reproducing fluorescence responses to depolarization and synaptic stimulation previously described in the literature. Two conclusions can be drawn from our simulation results. First, a significant progressive decrease in the amplitudes of depolarization-evoked fluorescence transients from the dendritic endings (digits) toward the soma of GrCs, can be reproduced in the model only if the density of Ca 2+ channels is considerably higher or the concentration of endogenous buffers is much lower in the digits than in the parent dendrites. In contrast, heterogeneities in the distribution of Ca 2+ pumps or in cytosolic fractional volume cannot account for the formation of [Ca 2+] i gradients in GrCs. Second, much lower amplitudes of fluorescence transients induced by depolarization and synaptic stimulation than expected from typical measurements of Ca 2+ and NMDA receptor-mediated currents can be reconciled with a pronounced slowing of the decay of fluorescence responses in the digits of GrCs after introducing a high-affinity Ca 2+ indicator if a high-capacity immobile Ca 2+ buffer (presumably plasma membrane-associated) is suggested to be present in the soma and apical part of digits. Mitochondria also are likely to modulate synaptically evoked Ca 2+ responses in GrCs. The alternative hypotheses are thoroughly discussed and research avenues for their testing in future experiments are proposed.

Neuropeptide Substance P induces mRNA expression and secretion of CXCL8 chemokine, and HDC in human umbilical cord blood mast cells

Mast cells play an important role in innate and acquired immunity and are thought to be the cellular origin of most proteases and cytokines. Substance P (SP) and its receptor, NK-1R, play critical roles in immune regulation in human and animal models of inflammation. We used mature human cord blood mast cells (HCBMC) differentiated from cord blood CD34+ precursor activated with SP in culture. Our data indicate that Substance P strongly activates mature HCBMC in releasing CXCL8 expression and secretion ( 1.200 +/- 1.0; SP: 4.10 +/- 0.90; P < 0.01). Moreover, in a RT-PCR, HCBMC expressed CXCL8 mRNA after Substance P activation. Since calcium ionophore A23187 is a pharmacological activator that raises cytosolic free calcium ion concentraion and stimulates mast cells in the production and secretion of proinflammatory compounds, it was used as positive control. In addition, we found that HCBMCs generate the transcription of histidine decarboxylase (HDC), the enzyme responsible for the generation of histamine from histidine, after SP treatment. Since CXCL8 is a member of the CXC chemokine subfamily with potent chemotactic activity and is a primary inflammatory cytokine we conclude that our results, obtained from HCBMC cultures, a good and valid model in vitro, support the concept that the neurogenic system modulates inflammatory events by Substance P-mediated HCBMC chemokine CXCL8 release. The expression, synthesis and release of CXCL8 suggest an increase of inflammatory process in vivo mediated by the recruitment and infiltration of inflammatory cells in inflamed tissues.

Real‐time measurement of cytosolic free calcium concentration in DEM‐treated HL‐60 cells during static magnetic field exposure and activation by ATP

This study investigated whether glutathione depletion affected the sensitivity of HL-60 cells to static magnetic fields. The effect of Diethylmaleate (DEM) on static magnetic field induced changes in cytosolic free calcium concentration ([Ca(2+)](c)) was examined. Cells were loaded with a fluorescent dye and exposed to a uniform static magnetic field at a strength of 0 mT (sham) or 100 mT. [Ca(2+)](c) was monitored during field and sham exposure using a ratiometric fluorescence spectroscopy system. Cells were activated by the addition of ATP. Metrics extracted from the [Ca(2+)](c) time series included: average [Ca(2+)](c) during the Pre-Field and Field Conditions, peak [Ca(2+)](c) following ATP activation and the full width at half maximum (FWHM) of the peak ATP response. Comparison of each calcium metric between the sham and 100 mT experiments revealed the following results: average [Ca(2+)](c) measured during the Field condition was 53 +/- 2 nM and 58 +/- 2 nM for sham and 100 mT groups, respectively. Average FWHM was 51 +/- 3 s and 54 +/- 3 s for sham and 100 mT groups, respectively. An effect of experimental order on the peak [Ca(2+)](c) response to ATP in sham/sham experiments complicated the statistical analysis and did not allow pooling of the first and second order experiments. No statistically significant difference between the sham and 100 mT groups was observed for any of the calcium metrics. These data suggested that manipulation of free radical buffering capacity in HL-60 cells did not affect the sensitivity of the cells to a 100 mT static magnetic field.

The endocannabinoid anandamide inhibits potassium conductance in rat cortical astrocytes

Endocannabinoids are a family of endogenous signaling molecules that modulate neuronal excitability in the central nervous system (CNS) by interacting with cannabinoid (CB) receptors. In spite of the evidence that astroglial cells also possess CB receptors, there is no information on the role of endocannabinoids in regulating CNS function through the modulation of ion channel-mediated homeostatic mechanisms in astroglial cells. We provide electrophysiological evidence that the two brain endocannabinoids anandamide (AEA) and 2-arachidonylglycerol (2-AG) markedly depress outward conductance mediated by delayed outward rectifier potassium current (IK(DR)) in primary cultured rat cortical astrocytes. Pharmacological experiments suggest that the effect of AEA does not result from the activation of known CB receptors. Moreover, neither the production of AEA metabolites nor variations in free cytosolic calcium are involved in the negative modulation of IK(DR). We show that the action of AEA is mediated by its interaction with the extracellular leaflet of the plasma membrane. Similar experiments performed in situ in cortical slices indicate that AEA downregulates IK(DR) in complex and passive astroglial cells. Moreover, IK(DR) is also inhibited by AEA in NG2 glia. Collectively, these results support the notion that endocannabinoids may exert their modulation of CNS function via the regulation of homeostatic function of the astroglial syncytium mediated by ion channel activity.

In Vitro Activity of Acanthamoeba castellanii on Human Platelets and Erythrocytes

The effect of Acanthamoeba on human platelets and erythrocytes has not been fully elucidated. This paper reports that cell-free supernatants prepared from A. castellanii can activate human platelets, causing both a significant increase in the cytosolic free-calcium concentration and platelet aggregation. In addition, we demonstrated that platelet activation depends on the activity of ADP constitutively secreted into the medium by trophozoites. This study also showed that A. castellanii can affect human red blood cells, causing hemolysis, and provided evidence that hemolysis occurs in both contact-dependent and contact-independent ways; there are differences in kinetics, hemolytic activity, and calcium dependency between the contact-dependent and contact-independent mechanisms. Partial characterization of contact-independent hemolysis indicated that ADP does not affect the plasma membrane permeability of erythrocytes and that heat treatment of amoebic cell-free supernatant abolishes its hemolytic activity. These findings suggest that some heat-labile molecules released by A. castellanii trophozoites are involved in this phenomenon. Finally, our data suggest that human platelets and erythrocytes may be potential cell targets during Acanthamoeba infection.