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1.
Opt Express ; 29(11): 16857-16866, 2021 May 24.
Article in English | MEDLINE | ID: mdl-34154238

ABSTRACT

Passive daytime radiative cooling has recently become an attractive approach to address the global energy demand associated with modern refrigeration technologies. One technique to increase the radiative cooling performance is to engineer the surface of a polar dielectric material to enhance its emittance at wavelengths in the atmospheric infrared transparency window (8-13 µm) by outcoupling surface-phonon polaritons (SPhPs) into free-space. Here we present a theoretical investigation of new surface morphologies based upon self-assembled silica photonic crystals (PCs) using an in-house built rigorous coupled-wave analysis (RCWA) code. Simulations predict that silica micro-sphere PCs can reach up to 73 K below ambient temperature, when solar absorption and conductive/convective losses can be neglected. Micro-shell structures are studied to explore the direct outcoupling of the SPhP, resulting in near-unity emittance between 8 and 10 µm. Additionally, the effect of material composition is explored by simulating soda-lime glass micro-shells, which, in turn, exhibit a temperature reduction of 61 K below ambient temperature. The RCWA code was compared to FTIR measurements of silica micro-spheres, self-assembled on microscope slides.

2.
Sci Rep ; 6: 33647, 2016 09 16.
Article in English | MEDLINE | ID: mdl-27633131

ABSTRACT

Macroautophagy (hereafter autophagy) is an evolutionarily highly conserved cellular process that participates in the maintenance of intracellular homeostasis through the degradation of most long-lived proteins and entire organelles. Autophagy participates in some reproductive events; however, there are not reports regarding the role of autophagy in the regulation of sperm physiology. Hence, the aim of this study was to investigate whether autophagy-related proteins are present and functionally active in human spermatozoa. Proteins related to autophagy/mitophagy process (LC3, Atg5, Atg16, Beclin 1, p62, m-TOR, AMPKα 1/2, and PINK1) were present in human spermatozoa. LC3 colocalized with p62 in the middle piece of the spermatozoa. Autophagy activation induced a significant increase in motility and a decrease in PINK1, TOM20 expression and caspase 3/7 activation. In contrast, autophagy inhibition resulted in decreased motility, viability, ATP and intracellular calcium concentration whereas PINK1, TOM20 expression, AMPK phosphorylation and caspase 3/7 activation were significantly increased. In conclusion our results show that autophagy related proteins and upstream regulators are present and functional in human spermatozoa. Modification of mitochondrial proteins expression after autophagy activation/inhibition may be indicating that a specialized form of autophagy named mitophagy may be regulating sperm function such as motility and viability and may be cooperating with apoptosis.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Movement , Spermatozoa/cytology , Spermatozoa/metabolism , AMP-Activated Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Adult , Autophagy/drug effects , Calcium/metabolism , Caspases/metabolism , Cell Movement/drug effects , Cell Survival/drug effects , Chloroquine/pharmacology , Enzyme Activation/drug effects , Humans , Hydrogen-Ion Concentration , Intracellular Space/metabolism , Macrolides/pharmacology , Male , Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Phosphorylation/drug effects , Protein Kinases/metabolism , Receptors, Cell Surface/metabolism , Semen/metabolism , Sequestosome-1 Protein/metabolism , Sirolimus/pharmacology , Spermatozoa/drug effects , Spermatozoa/ultrastructure
3.
Mol Cell Biochem ; 390(1-2): 281-7, 2014 May.
Article in English | MEDLINE | ID: mdl-24488173

ABSTRACT

The present study determines the role of reactive oxygen species (ROS) production and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) on apoptosis in the human leukemia HL-60 and K562 cell lines. The results show that treatment of both cell lines cells with 10 ng/mL TNFα resulted in a rise in the percentage of apoptotic cells after 6 h of treatment. It was also observed that the administration of 10 ng/mL TNFα increased intracellular ROS production, as well as a time-dependent increase in caspase-8, -3, and -9 activities. The present results also show that the pretreatment with well-known antioxidants such as trolox and N-acetyl cysteine partially reduced the caspase activation caused by the administration of TNFα. The findings suggest that TNFα-induced apoptosis is dependent on alterations in intracellular ROS generation in human leukemia HL-60 and K562 cells.


Subject(s)
Apoptosis/genetics , Caspases/biosynthesis , Leukemia/genetics , Tumor Necrosis Factor-alpha/administration & dosage , Apoptosis/drug effects , Caspase Inhibitors/administration & dosage , Chromans/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , HL-60 Cells , Humans , K562 Cells , Leukemia/metabolism , Leukemia/pathology , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolism
4.
Physiol Behav ; 118: 8-13, 2013 Jun 13.
Article in English | MEDLINE | ID: mdl-23643827

ABSTRACT

AIM: This study evaluated the effect of the administration of melatonin, the chief secretory product of the pineal gland, on the body weight in male Wistar rats. MAIN METHODS: The animals were housed for 4months in cages equipped to log horizontal activity within a thermostatically-controlled chamber, under a 12h/12h light/dark photoperiod (lights on at 08:00h). After acclimatization, the animals were divided into two groups: (1) control animals, and (2) melatonin-treated animals. Melatonin was administered in tap water (20µg/ml), and fresh drinking fluid was changed twice weekly. Rats were fed a standard diet ad libitum. KEY FINDINGS: Food and water intake, body weight, the amplitude of the activity/rest rhythm (motor activity), and blood melatonin and glucose concentrations were measured. The administration of melatonin did not influence either food or water intake or glucose levels relative to those found in the control animals. However, melatonin administration reduced body weight gain and increased nocturnal locomotor activity. The peak concentration of melatonin was found at night coinciding with the increase in nocturnal activity. SIGNIFICANCE: The results show that exogenous melatonin reduces body weight gain without having marked effects on metabolism. This may be due in part to the increased nocturnal activity shown by the animals treated with the indoleamine.


Subject(s)
Melatonin/pharmacology , Motor Activity/drug effects , Weight Gain/drug effects , Animals , Blood Glucose/metabolism , Circadian Rhythm/drug effects , Darkness , Drinking/drug effects , Eating/drug effects , Light , Male , Melatonin/metabolism , Photoperiod , Rats , Rats, Wistar
5.
J Anim Physiol Anim Nutr (Berl) ; 97(1): 137-45, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22074327

ABSTRACT

The decline in melatonin secretion with age seems to be one of the major reasons for increased sleep disruption in older animals. Previously, we showed that the administration with melatonin or its precursor, tryptophan, improved activity/rest rhythms in aged individuals. Here, it was evaluated the effect of a 10-day consumption of a Jerte Valley cherry-based nutraceutical product (patent no. ES2342141B1), which contains high levels of tryptophan, serotonin and melatonin, on the activity/rest rhythms of young and old rats (Rattus norvegicus) and ringdoves (Streptopelia risoria) as representatives of animals with nocturnal and diurnal habits, respectively, and its possible relationship with the serum levels of melatonin and glucose. Total diurnal and nocturnal activity pulses were logged at control, during, and up to 3 days after the treatment. Melatonin and glucose were measured with ELISA and testing kits respectively. In both young and old rats, the intake of the cherry nutraceutical decreased diurnal activity, whereas nocturnal activity increased. The opposite effect was observed for ringdoves. The treatment increased the circulating levels of melatonin in both species and restored the amplitude of the activity rhythm in the old animals to that of the non-treated young groups. The consumption of a Jerte Valley cherry-based nutraceutical product may help to counteract the impaired activity/rest rhythm found in aged animals.


Subject(s)
Aging , Behavior, Animal/drug effects , Circadian Rhythm , Columbidae/physiology , Dietary Supplements , Prunus/chemistry , Animals , Diet , Drug Administration Schedule , Female , Male , Rats
6.
Physiol Behav ; 105(3): 683-92, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22019785

ABSTRACT

We aimed to investigate the protective effects of melatonin and 2.45 GHz electromagnetic radiation (EMR) on brain and dorsal root ganglion (DRG) neuron antioxidant redox system, Ca(2+) influx, cell viability and electroencephalography (EEG) records in the rat. Thirty two rats were equally divided into four different groups namely group A1: Cage control, group A2: Sham control, group B: 2.45 GHz EMR, group C: 2.45 GHz EMR+melatonin. Groups B and C were exposed to 2.45 GHz EMR during 60 min/day for 30 days. End of the experiments, EEG records and the brain cortex and DRG samples were taken. Lipid peroxidation (LP), cell viability and cytosolic Ca(2+) values in DRG neurons were higher in group B than in groups A1 and A2 although their concentrations were increased by melatonin, 2-aminoethyldiphenyl borinate (2-APB), diltiazem and verapamil supplementation. Spike numbers of EEG records in group C were lower than in group B. Brain cortex vitamin E concentration was higher in group C than in group B. In conclusion, Melatonin supplementation in DRG neurons and brain seems to have protective effects on the 2.45 GHz-induced increase Ca(2+) influx, EEG records and cell viability of the hormone through TRPM2 and voltage gated Ca(2+) channels.


Subject(s)
Brain/cytology , Calcium Channels/metabolism , Ganglia, Spinal/cytology , Melatonin/therapeutic use , Neurons , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , TRPM Cation Channels/metabolism , Animals , Brain/drug effects , Brain/physiology , Brain/radiation effects , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Electroencephalography/methods , Electromagnetic Radiation , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Hydrogen Peroxide/pharmacology , Lipid Peroxidation/drug effects , Lipid Peroxidation/radiation effects , Male , Membrane Potentials/drug effects , Membrane Potentials/radiation effects , Neurons/drug effects , Neurons/metabolism , Neurons/radiation effects , Patch-Clamp Techniques , Rats , Rats, Wistar
7.
J Pineal Res ; 50(2): 132-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20964711

ABSTRACT

Although human seminal fluid contains melatonin and spermatozoa reportedly possess membrane melatonin receptors, there are no experimental studies that have ascertained the relationship between melatonin and male infertility. This study evaluated whether urinary 6-sulfatoxymelatonin and urinary total antioxidant capacity correlate with different seminal parameters including sperm concentration, motility and morphology. Also, the in vitro effects of melatonin on human sperm motility were investigated. Semen samples from 52 men who were counselled for infertility were obtained. Sperm concentration was determined using the haemocytometer method, motility kinematic parameters were assessed using a computer-aided semen analysis system, while morphology and vitality were evaluated after Diff-Quick and Eosin-Nigrosin vital staining, respectively. For the quantification of urinary 6-sulfatoxymelatonin, a commercial ELISA kit was used, and urinary total antioxidant capacity was evaluated by means of a colorimetric assay kit. For the in vitro effects of melatonin, samples were incubated for 30min in the presence or absence of 1mm melatonin. Both urinary 6-sulfatoxymelatonin and total antioxidant capacity levels positively correlated with sperm concentration, motility and morphology, as well as negatively correlated with the number of round cells. Additionally, 30-min exposure of sperm to 1mm melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid cells. Therefore, melatonin improves semen quality, which is important because melatonin supplementation may be potentially used to obtain a successful assisted reproductive technique outcome.


Subject(s)
Melatonin/pharmacology , Semen/drug effects , Sperm Motility/drug effects , Spermatozoa/drug effects , Adult , Humans , In Vitro Techniques , Male , Melatonin/analogs & derivatives , Melatonin/urine , Middle Aged , Semen/metabolism , Semen Analysis , Sperm Count , Spermatozoa/cytology , Young Adult
8.
Mol Cell Biochem ; 333(1-2): 151-7, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19626422

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Caspase 3/metabolism , Caspase 9/metabolism , Calcium/analysis , Caspase 3/analysis , Caspase 9/analysis , Cytosol/chemistry , HL-60 Cells , Humans , Kinetics , Myeloid Cells/metabolism , Thapsigargin/pharmacology , Uridine Triphosphate/pharmacology
9.
J Thromb Haemost ; 7(6): 992-9, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19548908

ABSTRACT

BACKGROUND: Apoptosis or programmed cell death involves a number of biochemical events, including the activation of caspases, which lead to specific cell morphology changes and ultimately cell death. Traditionally, two apoptotic pathways have been described: the cell-surface death receptor-dependent extrinsic pathway and the mitochondria-dependent intrinsic pathway. Alternatively, apoptosis has been reported to be induced by endoplasmic reticulum (ER) stress, which is mainly induced by a reduction in intraluminal free Ca(2+) concentration ([Ca(2+)](ER)). OBJECTIVES: The present study aimed to investigate the development of apoptotic events after ER stress induced by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), an ER Ca(2+) chelator, in human platelets. METHODS: Changes in cytosolic free Ca(2+) concentration, caspase activity and phosphatidylserine externalization were determined by fluorimetric techniques. RESULTS: Our results indicate that TPEN reduces the amount of free Ca(2+) releasable by the Ca(2+)-mobilizing agonist thrombin. TPEN induced activation of caspase-3, -8 and -9 and subsequent phosphatidylserine externalization. The ability of TPEN to induce phosphatidylserine externalization was smaller than that of thrombin. In addition, TPEN was able to induce phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2 alpha). TPEN-mediated caspase-3 activation requires functional caspase-8, but is independent of H(2)O(2) generation. Activation of caspase-3 and -8 by TPEN was prevented by salubrinal, an agent that prevents ER stress-induced apoptosis. CONCLUSION: These findings provide experimental evidence for the existence of ER stress-mediated apoptosis in human platelets, a process that might limit platelet life span upon prolonged stimulation with agonists.


Subject(s)
Apoptosis/drug effects , Blood Platelets/drug effects , Caspase 3/metabolism , Caspase 8/metabolism , Endoplasmic Reticulum/enzymology , Ethylenediamines/pharmacology , Blood Platelets/cytology , Blood Platelets/enzymology , Blood Platelets/metabolism , Blotting, Western , Calcium/metabolism , Endocytosis/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Activation , Humans , Phosphatidylserines/metabolism , Phosphorylation , Thrombin/pharmacology
10.
J Thromb Haemost ; 6(10): 1780-8, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18665919

ABSTRACT

BACKGROUND: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. OBJECTIVES: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. METHODS: Changes in the mitochondrial membrane potential were registered using the dye JC-1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. RESULTS: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H(2)O(2) (10 microm) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. CONCLUSION: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.


Subject(s)
BH3 Interacting Domain Death Agonist Protein/metabolism , Blood Platelets/metabolism , Mitochondria/metabolism , Thrombin/pharmacology , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Adenosine Diphosphate/pharmacology , Cells, Cultured , Humans , Membrane Potentials , Mitochondrial Membranes , Protein Transport
11.
J Physiol Pharmacol ; 58(3): 423-40, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17928640

ABSTRACT

In the present study we have employed single cell imaging analysis to monitor the propagation of cholecystokinin-evoked Ca(2+) waves in mouse pancreatic acinar cells. Stimulation of cells with 1 nM CCK-8 led to an initial Ca(2+) release at the luminal cell pole and subsequent spreading of the Ca(2+) signal towards the basolateral membrane in the form of a Ca(2+) wave. Inhibition of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity by 1 microM thapsigargin, preincubation in the presence of 100 microM H(2)O(2) or inhibition of PKC with either 5 microM Ro31-8220 or 3 microM GF-109203-X all led to a faster propagation of CCK-8-induced Ca(2+) signals. The propagation of CCK-8-evoked Ca(2+) signals was slowed down by activation of PKC with 1 microM PMA, and preincubation of cells in the presence of H(2)O(2) counteracted the effect of PKC inhibition. The protonophore FCCP (100 nM) and the inhibitor of the mitochondrial Ca(2+)-uniporter Ru360 (10 microM) led to an increase in the propagation rate of CCK-8-evoked Ca(2+) waves. Finally, depolymerisation of actin cytoskeleton with cytochalasin D (10 microM) led to a faster propagation of CCK-8-evoked Ca(2+) signals. Stabilization of actin cytoskeleton with jasplakinolide (10 microM) did not induce significant changes on CCK-8-evoked Ca(2+) waves. Preincubation of cells in the presence of H(2)O(2) counteracted the effect of cytochalasin D on CCK-8-evoked Ca(2+) wave propagation. Our results suggest that spreading of cytosolic Ca(2+) waves evoked by CCK-8 can be modulated by low levels of oxidants acting on multiple Ca(2+)-handling mechanisms.


Subject(s)
Calcium Signaling/drug effects , Hydrogen Peroxide/pharmacology , Pancreas, Exocrine/drug effects , Sincalide/pharmacology , Animals , Benzimidazoles/pharmacology , Carbocyanines/pharmacology , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cholecystokinin/pharmacology , Cytochalasin D/pharmacology , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Depsipeptides/pharmacology , Dose-Response Relationship, Drug , Indoles/pharmacology , Intracellular Fluid/drug effects , Intracellular Fluid/physiology , Male , Maleimides/pharmacology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/drug effects , Mitochondria/physiology , Oligomycins/pharmacology , Organotin Compounds/pharmacology , Pancreas, Exocrine/cytology , Pancreas, Exocrine/physiology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Ruthenium Compounds/pharmacology , Salicylates/pharmacology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Thapsigargin/pharmacology
12.
J Thromb Haemost ; 5(6): 1283-91, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17567446

ABSTRACT

BACKGROUND: Thrombin is a major physiological platelet agonist that activates a number of cell functions including aggregation. Platelet stimulation with thrombin has been shown to result in the development of apoptotic events, including activation of caspases-3 and -9, cytochrome c release and phosphatidylserine (PS) exposure; however, the mechanism underlying the activation of apoptosis remains unclear. OBJECTIVES: In the present study, we aim to investigate whether endogenously generated reactive oxygen species upon thrombin stimulation is required for the activation of apoptosis in human platelets. METHODS: Changes in the mitochondrial membrane potential were registered using the dye JC-1; caspase-3 and -9 activity was determined from the cleavage of their respective specific fluorogenic substrates; PS externalization was estimated using annexin V-fluorescein isothicyanate and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. RESULTS: Treatment of platelets with thrombin stimulates mitochondrial membrane potential depolarization and endogenous generation of H(2)O(2) . Platelet exposure to exogenous H(2)O(2) results in cytochrome c release and activation of caspases-9. In addition, H(2)O(2) induces the activation of caspase-3 and PS exposure by a mechanism dependent on cytochrome c release and caspase-9 activation. Finally, thrombin-evoked development of apoptotic events was impaired by treatment with catalase. CONCLUSION: Our results indicate that thrombin-induced apoptosis is likely mediated by endogenous generation of H(2)O(2) in human platelets.


Subject(s)
Apoptosis/drug effects , Apoptosis/physiology , Blood Platelets/drug effects , Blood Platelets/metabolism , Reactive Oxygen Species/metabolism , Thrombin/pharmacology , Blood Platelets/cytology , Caspase 3/blood , Caspase 9/blood , Catalase/pharmacology , Cytochromes c/blood , Enzyme Activation/drug effects , Fluorescent Dyes , Humans , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Membrane Potential, Mitochondrial/drug effects , Phosphatidylserines/blood
13.
Mol Cell Biochem ; 296(1-2): 77-84, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16955226

ABSTRACT

We have studied the effect of exogenous administration of hydrogen peroxide (H(2)O(2)) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H(2)O(2) evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H(2)O(2) resulted in a increase in [Ca(2+)]( i ), indicating the release of calcium from intracellular stores. H(2)O(2) abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H(2)O(2)-induced calcium release. We conclude that H(2)O(2) induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.


Subject(s)
Calcium/metabolism , Hydrogen Peroxide/pharmacology , Neutrophils/drug effects , Oxidants/pharmacology , Phagocytosis/drug effects , Adolescent , Adult , Aged , Chelating Agents/chemistry , Chelating Agents/metabolism , Dose-Response Relationship, Drug , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , Egtazic Acid/metabolism , Female , Humans , Male , Middle Aged , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/cytology , Neutrophils/metabolism , Phagocytosis/physiology , Reactive Oxygen Species/metabolism
14.
J Inorg Biochem ; 100(11): 1861-70, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16963122

ABSTRACT

The zinc(II) complexes dichloro[2-(3,4-dichlorophenyl)imino-kappaN-(2-thiazolin-kappaN-2-yl)thiazolidine]zinc(II) (1) and dichloro[2-(3,4-dichlorophenyl)imino-kappaN-(4H-5,6-dihydro-1,3-thiazin-kappaN-2-yl)tetrahydrothiazine]zinc(II) (2) have been isolated and characterized in the solid state by X-ray diffraction, elemental analysis and IR spectra. In both complexes, the environment around the zinc(II) ion may be described as a distorted tetrahedral geometry, with the metallic atom coordinated to two chlorine atoms [Zn-Cl(1)=2.218(1)A; Zn-Cl(2)=2.221(1)A], one imino nitrogen [Zn-N(3)=2.042(2)A] and one thiazoline nitrogen [Zn-N(1)=2.022(2)A] in complex 1 and to two chlorine atoms [Zn-Cl(1)=2.216(1)A; Zn-Cl(2)=2.192(1)A], one imino nitrogen [Zn-N(3)=2.045(2)A] and one thiazine nitrogen [Zn-N(1)=2.039(2)A] in complex 2. In addition, we also report in this study the crystal structure of the 2-(3,4-dichlorophenyl)imino-N-(2-thiazolin-2-yl)thiazolidine (TdTn) ligand as well as the synthesis and characterization by X-ray diffraction, (1)H and (13)C NMR spectra, elemental analysis, IR and electronic spectra of the 2-(3,4-dichlorophenyl)imino-N-(4H-5,6-dihydro-1,3-thiazin-2-yl)tetrahydrothiazine (TzTz) ligand. Besides, we study the phagocytic function in humans neutrophils treated with each complex and ligand aforementioned.


Subject(s)
Neutrophils/drug effects , Phagocytosis/drug effects , Thiazines/chemistry , Thiazoles/chemistry , Zinc Compounds/pharmacology , Crystallography, X-Ray , Humans , Ligands , Magnetic Resonance Spectroscopy , Molecular Structure , Neutrophils/cytology , Neutrophils/immunology , Zinc Compounds/chemical synthesis , Zinc Compounds/chemistry
15.
J Physiol Biochem ; 61(4): 507-16, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16669348

ABSTRACT

Intracellular free Ca2+ concentration ([Ca2+]c) is finely regulated by several mechanisms that either increase or reduce [Ca2+]c. Two different Ca2+ pumps have been described so far as the main mechanisms for Ca2+ removal from the cytosol, either by its sequestration into the stores, mediated by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) or by Ca2+ extrusion to the extracellular medium, by the plasma membrane Ca2+-ATPase (PMCA). We have used inhibitors of these pumps to analyze their Ca2+ clearance efficacy in human platelets stimulated by the physiological agonist thrombin. Results demonstrate that, after platelet stimulation with thrombin, activation of SERCA precedes that of PMCA, although the ability of PMCA to remove Ca2+ from the cytosol last longer than that of SERCA. The efficacy of SERCA and PMCA removing Ca2+ from the cytosol is reduced when the concentration of thrombin increases. This phenomenon correlates with the greater increase in [Ca2+]c induced by higher concentrations of thrombin, which further confirms that SERCA and PMCA activities are regulated by [Ca2+]c.


Subject(s)
Blood Platelets/metabolism , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Cytosol/metabolism , Blood Platelets/cytology , Blood Platelets/enzymology , Calcium-Transporting ATPases/antagonists & inhibitors , Cation Transport Proteins/antagonists & inhibitors , Dose-Response Relationship, Drug , Homeostasis , Humans , Plasma Membrane Calcium-Transporting ATPases , Platelet Activation/drug effects , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Thrombin/metabolism , Thrombin/pharmacology , Time Factors
16.
Biochem Pharmacol ; 63(2): 283-92, 2002 Jan 15.
Article in English | MEDLINE | ID: mdl-11841804

ABSTRACT

We studied the role of mitochondria in Ca(2+) signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca(2+) signals under different experimental conditions. Spreading of Ca(2+) waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca(2+) concentration ([Ca(2+)](i)) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca(2+)](i) oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca(2+) concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba(2+) for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca(2+) clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca(2+) waves and in the development of [Ca(2+)](i) oscillations.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Mitochondria/physiology , Pancreas/cytology , Animals , Cytosol/metabolism , In Vitro Techniques , Mice , Pancreas/metabolism
17.
Cell Signal ; 13(6): 451-6, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11384844

ABSTRACT

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca2+-ATPases.


Subject(s)
Calcium/metabolism , Pancreas/metabolism , Vanadates/pharmacology , Animals , Biological Transport , Cell Membrane/metabolism , Dithiothreitol/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/pharmacology , Fura-2/pharmacology , Microscopy, Fluorescence , Organic Chemicals , Pancreas/cytology , Rats , Rats, Wistar , Sincalide/pharmacology , Spectrometry, Fluorescence , Time Factors
18.
J Membr Biol ; 179(1): 27-35, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11155207

ABSTRACT

In the present study we have studied how [Ca2+](i) is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+](i) increase, reaching a stable plateau after 10-15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 microm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 microm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca2+](i). In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+](i) induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.


Subject(s)
Calcium Signaling/drug effects , Hydrogen Peroxide/pharmacology , Pancreas/drug effects , Pancreas/physiology , Animals , Calcium-Transporting ATPases/antagonists & inhibitors , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Separation , Collagenases , Dithiothreitol/pharmacology , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Intracellular Fluid/drug effects , Intracellular Fluid/physiology , Mice , Mitochondria/drug effects , Mitochondria/physiology , Pancreas/cytology , Perfusion , Sincalide/pharmacology , Thapsigargin/pharmacology
19.
Biochem Pharmacol ; 62(12): 1621-7, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11755115

ABSTRACT

In the present study we have employed fura-2 loaded isolated mouse pancreatic acinar cells to monitor the effect that xanthine oxidase (XOD)-catalyzed reactive oxygen species generation presents on Ca(2+) mobilization by the secretagogue cholecystokinin octapeptide (CCK-8). Our results show that perfusion of pancreatic acinar cells with CCK-8 at a physiological concentration (20 pM) induced low frequency oscillations in intracellular free calcium concentration ([Ca(2+)](i)) at a rate of 1 per minute; this oscillatory pattern was completely inhibited by the introduction in the perifusion medium of 20 mU/mL XOD to generate reactive oxygen species. In addition, perfusion of pancreatic acinar cells with 20 mU/mL XOD in the absence of extracellular calcium led to a transient increase in [Ca(2+)](i,) that blocked the initiation of the Ca(2+) signals in response to 20 pM CCK-8. Similarly, XOD was also able to block acetylcholine evoked Ca(2+) spikes. However, reactive oxygen species had no effect either on Ca(2+) extrusion or on re-uptake into intracellular stores, but CCK-8-evoked Ca(2+) entry was reduced by XOD. In conclusion, our results show that XOD-evoked reactive oxygen species generation leads to a reduction either of Ca(2+) mobilization, following stimulation of pancreatic acinar cells with the Ca(2+)-mobilizing agonists CCK-8 and acetylcholine, and Ca(2+) influx evoked by CCK-8 depletion of intracellular stores. The possible XOD inhibitory mechanism on Ca(2+) mobilization by agonists is discussed.


Subject(s)
Calcium/metabolism , Pancreas/drug effects , Reactive Oxygen Species/pharmacology , Sincalide/pharmacology , Xanthine Oxidase/pharmacology , Animals , Biological Transport/drug effects , Catalysis , Drug Interactions , Male , Mice , Pancreas/metabolism , Xanthine Oxidase/metabolism
20.
Biochem Biophys Res Commun ; 271(2): 311-7, 2000 May 10.
Article in English | MEDLINE | ID: mdl-10799293

ABSTRACT

Our objective was to evaluate the role of vacuolar H(+)-ATPase and proton gradients in the refilling of Ca(2+) stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca(2+) stores were replenished with a Ca(2+)-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca(2+) pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H(+)-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca(2+)](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca(2+) stores which are differently operated by vacuolar proton ATPase.


Subject(s)
Calcium/metabolism , Macrolides , Pancreas/enzymology , Proton-Translocating ATPases/physiology , Thapsigargin/metabolism , Vacuoles/enzymology , Animals , Anti-Bacterial Agents/pharmacology , Biological Transport , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fura-2/metabolism , Hydrogen-Ion Concentration , Ionophores/pharmacology , Mice , Microscopy, Confocal , Nigericin/pharmacology , Protons , Time Factors
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