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1.
Int J Mol Sci ; 24(13)2023 Jul 04.
Article in English | MEDLINE | ID: mdl-37446245

ABSTRACT

Considering the important cytoprotective and signaling roles but relatively narrow therapeutic index of hydrogen sulfide (H2S), advanced H2S donors are required to achieve a therapeutic effect. In this study, we proposed glutathione dithiophosphates as new combination donors of H2S and glutathione. The kinetics of H2S formation in dithiophosphate solutions suggested a continuous H2S release by the donors, which was higher for the dithiophosphate of reduced glutathione than oxidized glutathione. The compounds, unlike NaHS, inhibited the proliferation of C2C12 myoblasts at submillimolar concentrations due to an efficient increase in intracellular H2S. The H2S donors more profoundly affected reactive oxygen species and reduced glutathione levels in C2C12 myocytes, in which these parameters were elevated compared to myoblasts. Oxidized glutathione dithiophosphate as well as control donors exerted antioxidant action toward myocytes, whereas the effect of reduced glutathione dithiophosphate at (sub-)micromolar concentrations was rather modulating. This dithiophosphate showed an enhanced negative inotropic effect mediated by H2S upon contraction of the atrial myocardium, furthermore, its activity was prolonged and reluctant for washing. These findings identify glutathione dithiophosphates as redox-modulating H2S donors with long-acting profile, which are of interest for further pharmacological investigation.


Subject(s)
Hydrogen Sulfide , Glutathione Disulfide , Hydrogen Sulfide/pharmacology , Glutathione/metabolism , Antioxidants/pharmacology , Antioxidants/metabolism , Oxidation-Reduction
2.
Front Cell Neurosci ; 11: 375, 2017.
Article in English | MEDLINE | ID: mdl-29225568

ABSTRACT

Hydrogen sulfide (H2S) is an endogenously produced neuroactive gas implicated in many key processes in the peripheral and central nervous system. Whereas the neuroprotective role of H2S has been shown in adult brain, the action of this messenger in newborns remains unclear. One of the known targets of H2S in the nervous system is the N-methyl-D-aspartate (NMDA) glutamate receptor which can be composed of different subunits with distinct functional properties. In the present study, using patch clamp technique, we compared the effects of the H2S donor sodium hydrosulfide (NaHS, 100 µM) on hippocampal NMDA receptor mediated currents in rats of the first and third postnatal weeks. This was supplemented by testing effects of NaHS on recombinant GluN1/2A and GluN1/2B NMDA receptors expressed in HEK293T cells. The main finding is that NaHS action on NMDA currents is age-dependent. Currents were reduced in newborns but increased in older juvenile rats. Consistent with an age-dependent switch in NMDA receptor composition, in HEK239T cells expressing GluN1/2A receptors, NaHS increased NMDA activated currents associated with acceleration of desensitization and decrease of the deactivation rate. In contrast, in GluN1/2B NMDA receptors, which are prevalent in newborns, NaHS decreased currents and reduced receptor deactivation without effect on the desensitization rate. Adenylate cyclase inhibitor MDL-12330A (10 µM) did not prevent the age-dependent effects of NaHS on NMDA evoked currents in pyramidal neurons of hippocampus. The reducing agent dithiothreitol (DTT, 2 mM) applied on HEK293T cells prevented facilitation induced by NaHS on GluN1/2A NMDA receptors, however in GluN1/2B NMDA receptors the inhibitory effect of NaHS was still observed. Our data indicate age-dependent effect of H2S on NMDA receptor mediated currents determined by glutamate receptor subunit composition. While the inhibitory action of H2 on GluN1/2B receptors could limit the excessive activation in early age, the enhanced functionality of GluN1/2A receptor in the presence of this gasotransmitter can enlarge synaptic efficacy and promote synaptic plasticity in adults.

3.
Neuroscience ; 340: 153-165, 2017 01 06.
Article in English | MEDLINE | ID: mdl-27984177

ABSTRACT

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with neuroprotective properties that participates in the regulation of transmitter release and neuronal excitability in various brain structures. The role of H2S in the growth and maturation of neural networks however remains unclear. The aim of the present study is to reveal the effects of H2S on neuronal spontaneous activity relevant to neuronal maturation in hippocampal slices of neonatal rats. Sodium hydrosulfide (NaHS) (100µM), a classical donor of H2S produced a biphasic effect with initial activation and subsequent concentration-dependent suppression of network-driven giant depolarizing potentials (GDPs) and neuronal spiking activity. Likewise, the substrate of H2S synthesis l-cysteine (1mM) induced an initial increase followed by an inhibition of GDPs and spiking activity. Our experiments indicate that the increase in initial discharge activity by NaHS is evoked by neuronal depolarization which is partially mediated by a reduction of outward K+ currents. The subsequent decrease in the neuronal activity by H2S appears to be due to the rightward shift of activation and inactivation of voltage-gated Na+ currents, thus preventing network activity. NaHS also reduced N-methyl-d-aspartate (NMDA)-mediated currents, without essential effect on AMPA/kainate or GABAA-mediated currents. Finally, H2S abolished the interictal-like events induced by bicuculline. In summary, our results suggest that through the inhibitory action on voltage-gated Na+ channels and NMDA receptors, H2S prevents the enhanced neuronal excitability typical to early hippocampal networks.


Subject(s)
Anticonvulsants/pharmacology , Epilepsy/drug therapy , Hippocampus/drug effects , Hydrogen Sulfide/pharmacology , Animals , Animals, Newborn , Cations, Monovalent/metabolism , Epilepsy/physiopathology , Hippocampus/growth & development , Hippocampus/physiopathology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neuroprotective Agents/pharmacology , Patch-Clamp Techniques , Potassium/metabolism , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Rats, Wistar , Receptors, AMPA/metabolism , Receptors, GABA-A/metabolism , Receptors, Kainic Acid/metabolism , Receptors, N-Methyl-D-Aspartate , Sodium/metabolism , Tetrodotoxin/pharmacology , Tissue Culture Techniques
4.
FEBS Lett ; 590(19): 3375-3384, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27586872

ABSTRACT

In this study, we investigated the effects of L-homocysteine (Hcy) on maxi calcium-activated potassium (BK) channels and on exocytosis of secretory granules in GH3 rat pituitary-derived cells. A major finding of our study indicates that short-term application of Hcy increased the open probability of oxidized BK channels in inside-out recordings. Whole-cell recordings show that extracellular Hcy also augmented BK currents during long-term application. Furthermore, Hcy decreased the exocytosis of secretory granules. This decrease was partially prevented by the BK channel inhibitor paxilline and fully prevented by N-acetylcysteine, a reactive oxygen species scavenger. Taken together, our data show that elevation of cellular Hcy level induces oxidative stress, increases BK channel activity, and decreases exocytosis of secretory granules. These findings may provide insight into some of the developmental impairments and neurotoxicity associated with Hyperhomocysteinemia (HHcy), a disease arising due to abnormally elevated levels of Hcy in the plasma.


Subject(s)
Exocytosis/drug effects , Homocysteine/pharmacology , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Secretory Vesicles/drug effects , Acetylcysteine/pharmacology , Action Potentials/drug effects , Animals , Cell Line , Indoles/pharmacology , Potassium Channel Blockers/pharmacology , Rats , Secretory Vesicles/metabolism
5.
Biochem Biophys Res Commun ; 465(4): 825-31, 2015 Oct 02.
Article in English | MEDLINE | ID: mdl-26319431

ABSTRACT

The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 µM (equals effective H2S of 16-19 µM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.


Subject(s)
Hydrogen Sulfide/metabolism , Pituitary Neoplasms/physiopathology , Action Potentials/drug effects , Action Potentials/physiology , Animals , Cell Line, Tumor , Exocytosis/drug effects , Exocytosis/physiology , Hydrogen Sulfide/pharmacology , KATP Channels/drug effects , KATP Channels/metabolism , Large-Conductance Calcium-Activated Potassium Channels/deficiency , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Membrane Potentials/drug effects , Membrane Potentials/physiology , Patch-Clamp Techniques , Rats , Secretory Vesicles/drug effects , Secretory Vesicles/physiology , Sulfides/metabolism , Sulfides/pharmacology
6.
Nano Lett ; 14(6): 2994-3001, 2014 Jun 11.
Article in English | MEDLINE | ID: mdl-24754795

ABSTRACT

Small-molecule chemical calcium (Ca(2+)) indicators are invaluable tools for studying intracellular signaling pathways but have severe shortcomings for detecting local Ca(2+) entry. Nanobiosensors incorporating functionalized quantum dots (QDs) have emerged as promising alternatives but their intracellular use remains a major challenge. We designed cell-penetrating FRET-based Ca(2+) nanobiosensors for the detection of local Ca(2+) concentration transients, using commercially available CANdot565QD as a donor and CaRuby, a custom red-emitting Ca(2+) indicator, as an acceptor. With Ca(2+)-binding affinities covering the range of 3-20 µM, our CaRubies allow building sensors with a scalable affinity for detecting intracellular Ca(2+) transients at various concentrations. To facilitate their cytoplasmic delivery, QDs were further functionalized with a small cell-penetrating peptide (CPP) derived from hadrucalcin (HadUF1-11: H11), a ryanodine receptor-directed scorpion toxin identified within the venom of Hadrurus gertschi. Efficient internalization of QDs doubly functionalized with PEG5-CaRuby and H11 (in a molar ratio of 1:10:10, respectively) is demonstrated. In BHK cells expressing a N-methyl-d-aspartate receptor (NMDAR) construct, these nanobiosensors report rapid intracellular near-membrane Ca(2+) transients following agonist application when imaged by TIRF microscopy. Our work presents the elaboration of cell-penetrating FRET-based nanobiosensors and validates their function for detection of intracellular Ca(2+) transients.


Subject(s)
Biosensing Techniques/methods , Calcium Signaling/physiology , Calcium/metabolism , Cell-Penetrating Peptides/chemistry , Fluorescence Resonance Energy Transfer , Quantum Dots/chemistry , Animals , Cricetinae , HEK293 Cells , Humans , Scorpion Venoms/chemistry
7.
J Am Chem Soc ; 134(36): 14923-31, 2012 Sep 12.
Article in English | MEDLINE | ID: mdl-22816677

ABSTRACT

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.


Subject(s)
Calcium/analysis , Egtazic Acid/analogs & derivatives , Fluorescent Dyes/chemistry , Indicators and Reagents/chemistry , Photons , Animals , Astrocytes/chemistry , Astrocytes/drug effects , Egtazic Acid/chemical synthesis , Egtazic Acid/chemistry , Egtazic Acid/pharmacokinetics , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacokinetics , Indicators and Reagents/chemical synthesis , Indicators and Reagents/pharmacokinetics , Mice , Mice, Inbred Strains , Molecular Structure
8.
ACS Nano ; 4(9): 5487-97, 2010 Sep 28.
Article in English | MEDLINE | ID: mdl-20822159

ABSTRACT

The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD's surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis-Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide-alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor-acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.


Subject(s)
Biosensing Techniques/methods , Calcium/analysis , Caspase 3/metabolism , Peptides/chemistry , Quantum Dots , Amino Acid Sequence , Engineering , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Humans , Molecular Sequence Data
9.
Langmuir ; 25(5): 3232-9, 2009 Mar 03.
Article in English | MEDLINE | ID: mdl-19437725

ABSTRACT

Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Nanoparticles/chemistry , Nanotechnology/methods , Cyclohexanes/chemistry , Diffusion , Emulsions , Ethanol/chemistry , Hydrolysis , Kinetics , Magnetic Resonance Spectroscopy , Micelles , Microscopy, Electron, Transmission/methods , Models, Statistical , Oils , Temperature
10.
Cell Calcium ; 45(3): 275-83, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19167753

ABSTRACT

The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.


Subject(s)
Calcium/metabolism , Chromaffin Cells/metabolism , Cytoplasm/metabolism , Dextrans/metabolism , Green Fluorescent Proteins/metabolism , Mitochondria/metabolism , Rhodamines/metabolism , Adrenal Medulla/cytology , Animals , Calcium Signaling , Cattle , Dextrans/chemistry , Imaging, Three-Dimensional , Membrane Microdomains/metabolism , Rhodamines/chemistry , Spectrometry, Fluorescence , Staining and Labeling
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