ABSTRACT
Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as "methanobactins," which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.
Subject(s)
Chelating Agents/chemistry , Coordination Complexes/chemistry , Imidazoles/chemistry , Methylocystaceae/chemistry , Oligopeptides/chemistry , Transition Elements/chemistry , Chelating Agents/radiation effects , Coordination Complexes/radiation effects , Fluorescence , Imidazoles/radiation effects , Light , Metals, Heavy/chemistry , Metals, Heavy/radiation effects , Molecular Structure , Oligopeptides/radiation effects , Spectrometry, Fluorescence , Transition Elements/radiation effectsABSTRACT
The semi-metallic element gallium has repeatedly shown bio-activities in preclinical and clinical studies. Gallium derivatives have already entered clinical trials for treatment of various refractory malignancies. To better monitor or track the status of administered gallium compound, herein a novel fluorescent probe N,N',N'',N'''-Tetrakis(2-hydroxybenzylidene)biphenyl-3,3',4,4'-tetramine (bis-salophen) has been designed and synthesized. The bis-salophen probe was found to recognize gallium ions (Ga3+) with high selectivity and sensitivity over other cations via fluorescence "turn on" strategy. The spectroscopy results exhibited a 1:2 stoichiometry for probe and Ga3+, and the association constant and limit of detection were calculated as 8.85â¯×â¯106â¯M-1 and 13.0â¯nM, respectively. Additionally, base on spectroscopy and theoretical research, the mechanism of Ga3+ sensing action was explored by density functional theory (DFT), which indicated suppression of photoinduced electron transfer (PET) action along with the interruption of π-conjugation between salicylaldehyde and 3,3-diaminobenzidine backbone by Ga3+ ions. Furthermore, the biological applicability of bis-salophen probe were evaluated in various normal and cancer cell lines, results have shown that this probe is highly selective and sensitive for cancer cells. Finally, zebrafish imaging confirmed and indicated that the probe is also capable of examining Ga3+ ions. Collectively, these results suggest that we have successfully developed a novel probe for selective and sensitive detection of Ga3+ ions both in living cells and zebrafish. We expect that our work here will shed light on future development of Ga3+ detecting probes and wider application in the filed of biology and medicine shall be anticipated.
Subject(s)
Chelating Agents/chemistry , Fluorescent Dyes/chemistry , Gallium/analysis , 3,3'-Diaminobenzidine/analogs & derivatives , 3,3'-Diaminobenzidine/chemical synthesis , 3,3'-Diaminobenzidine/radiation effects , Animals , Cell Line, Tumor , Chelating Agents/chemical synthesis , Chelating Agents/radiation effects , Density Functional Theory , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/radiation effects , Humans , Microscopy, Fluorescence/methods , Models, Chemical , Schiff Bases/chemical synthesis , Schiff Bases/chemistry , Schiff Bases/radiation effects , Ultraviolet Rays , ZebrafishABSTRACT
Metal ions are essential for a wide range of physiological processes, but they can also be toxic if not appropriately regulated by a complex network of metal trafficking proteins. Intervention in cellular metal distribution with small-molecule or peptide chelating agents has promising therapeutic potential to harness metals to fight disease. Molecular outcomes associated with forming metal-chelate interactions in situ include altering the concentration and subcellular metal distribution, inhibiting metalloenzymes, enhancing the reactivity of a metal species to elicit a favorable biological response, or passivating the reactivity of a metal species to prevent deleterious reactivity. The systemic administration of metal chelating agents, however, raises safety concerns due to the potential risks of indiscriminate extraction of metals from critical metalloproteins and inhibition of metalloenzymes. One can estimate that chelators capable of complexing metal ions with dissociation constants in the submicromolar range are thermodynamically capable of extracting metal ions from some metalloproteins and disrupting regular function. Such dissociation constants are easily attainable for multidentate chelators interacting with first-row d-block metal cations in relevant +1, + 2, and +3 oxidation states. To overcome this challenge of indiscriminate metal chelation, we have pursued a prodrug strategy for chelating agents in which the resulting "prochelator" has negligible metal binding affinity until a specific stimulus generates a favorable metal binding site. The prochelator strategy enables conditional metal chelation to occur preferentially in locations affected by disease- or therapy-associated stimuli, thereby minimizing off-target metal chelation. Our design of responsive prochelators encompasses three general approaches of activation: the "removal" approach operates by eliminating a masking group that blocks a potential metal chelation site to reveal the complete binding site under the desired conditions; the molecular "switch" approach involves a reversible conformational change between inactive and active forms of a chelator with differential metal binding affinity under specific conditions; and the "addition" approach adds a new ligand donor arm to the prochelator to constitute a complete metal chelation site. Adopting these approaches, we have created four categories of triggerable prochelators that respond to (1) reactive oxygen species, (2) light, (3) specific enzymes, and (4) biological regulatory events. This Account highlights progress from our group on building prochelators that showcase these four categories of responsive metal chelating agents for manipulating cellular metals. The creation and chemical understanding of such stimulus-responsive prochelators enables exciting applications for understanding the cell biology of metals and for developing therapies based on metal-dependent processes in a variety of diseases.
Subject(s)
Chelating Agents/chemistry , Metals/chemistry , Prodrugs/chemistry , Animals , Cell Line , Chelating Agents/metabolism , Chelating Agents/radiation effects , Humans , Hydroxyquinolines/chemistry , Light , Mice , Peptides/chemistry , Peptides/metabolism , Prodrugs/metabolism , Prodrugs/radiation effects , Rats , Reactive Oxygen Species/chemistryABSTRACT
Rutin, the glycoside of quercetin, could be used in topical preparations because of its antioxidant and radical scavenging properties, but its employ in cosmetic and pharmaceutical products is limited by poor physico-chemical stability. These issues were addressed by preparing, characterizing and testing rutin inclusion complexes with MCM-41 mesoporous silica. The effect of surface functionalization with aminopropyl groups (NH2-MCM-41) on the molecules properties was studied. The organic/inorganic interaction was confirmed by many techniques. In particular, the high inclusion of rutin in the pores of NH2-MCM-41 was assessed by XRD, TGA, gas-volumetric analysis (BET), while FTIR spectroscopy allowed to analyse with great detail the molecular interaction with the inorganic surface. Rutin was stabilized against UV degradation, mostly by its inclusion in NH2-MCM-41. Ex vivo studies showed a greater accumulation in porcine skin in the case of rutin complexed with NH2-MCM-41. Not only antioxidant properties of rutin were maintained after immobilization but, with aminopropyl silica, the metal-chelating activity increased noticeably. The immobilization of rutin in aminopropyl silica resulted in better performance in terms of activity and photostability, suggesting the importance of functionalization in stabilizing organic molecules within silica pores.
Subject(s)
Antioxidants/chemistry , Chelating Agents/chemistry , Rutin/chemistry , Silicon Dioxide/chemistry , Administration, Topical , Animals , Antioxidants/administration & dosage , Antioxidants/radiation effects , Chelating Agents/administration & dosage , Chelating Agents/radiation effects , Ferrozine/chemistry , In Vitro Techniques , Rutin/administration & dosage , Rutin/radiation effects , Silicon Dioxide/administration & dosage , Silicon Dioxide/radiation effects , Skin/metabolism , Skin Absorption , Surface Properties , Swine , Ultraviolet RaysABSTRACT
INTRODUCTION: The purpose of this study was to investigate the efficacy of laser-activated irrigation (LAI) of 1320-nm neodymium-doped:yttrium-aluminum-garnet (Nd:YAG) laser on sealer penetration into dentinal tubules in the presence of 5.25% sodium hypochlorite (NaOCl) or 17% ethylenediaminetetraacetic acid (EDTA). METHODS: The curved root canals (>20°) from 63 extracted human molars (negative control, n = 3) were prepared to size #30.06 with NaOCl irrigation. Teeth were divided into 4 groups (n = 15) as follows: group N, NaOCl irrigation without LAI; group E, EDTA irrigation without LAI; group NL, LAI with NaOCl; group EL, LAI with EDTA. In all groups, the laser fiber was inserted and withdrawn 4 times for 5 seconds each. Teeth were obturated with gutta-percha and fluorescent-labeled sealer. Transverse sections at 2 and 5 mm from root apex were examined with confocal laser scanning microscopy, and the percentage of sealer penetration into dentinal tubules was measured. RESULTS: Groups E, NL, and EL showed higher percentage of sealer penetration than group N (P < .05). With NaOCl as irrigant, LAI (group NL) resulted in significantly higher amount of sealer penetration than nonactivated group (group N) in both levels (P < .05). However, with EDTA, no significant differences in sealer penetration were observed between the laser-activated group (group EL) and its nonactivated counterpart (group E) in both levels (P > .05). CONCLUSIONS: The 1320-nm Nd:YAG laser activation with either NaOCl or EDTA was much better than NaOCl irrigation alone and as effective as EDTA final flush for sealer penetration into dentinal tubules. Additional use of laser with EDTA did not improve the quality of obturation in the curved canals.
Subject(s)
Dental Pulp Cavity/ultrastructure , Dentin/ultrastructure , Lasers, Solid-State/therapeutic use , Root Canal Filling Materials/therapeutic use , Root Canal Irrigants/therapeutic use , Chelating Agents/radiation effects , Chelating Agents/therapeutic use , Edetic Acid/radiation effects , Edetic Acid/therapeutic use , Epoxy Resins/chemistry , Epoxy Resins/therapeutic use , Fluorescent Dyes , Gutta-Percha/chemistry , Gutta-Percha/therapeutic use , Humans , Humidity , Microscopy, Confocal , Molar , Root Canal Filling Materials/chemistry , Root Canal Irrigants/radiation effects , Root Canal Obturation/methods , Root Canal Preparation/instrumentation , Root Canal Preparation/methods , Sodium Hypochlorite/radiation effects , Sodium Hypochlorite/therapeutic use , Temperature , Therapeutic Irrigation/methods , Time FactorsABSTRACT
Using amino-acid histidine as chelating agent, CdS nanoparticles have been synthesized by sonochemical method. It is found that by varying the ultrasonic irradiation time, we can tune the band gap and particle size of CdS nanoparticles. The imidazole ring of histidine captures the Cd ions from the solution, and prevents the growth of the CdS nanoparticles. The deviation in the linear relation in between cube of radius of nanoparticles and ultrasonic irradiation time confirms the growth of CdS nanoparticles occur via two process; one is the diffusion process of the reactants as well as reaction at the surface of the crystallite. CdS nanoparticles synthesized using histidine as organic chelating agent have band edge emission at approximately 481 nm and have greater photoluminescence intensity with blue-shift to higher energy due to typical quantum confinement effect.
Subject(s)
Cadmium Compounds/chemical synthesis , Cadmium Compounds/radiation effects , Histidine/chemistry , Luminescent Agents/chemical synthesis , Luminescent Agents/radiation effects , Nanoparticles/chemistry , Selenium Compounds/chemical synthesis , Selenium Compounds/radiation effects , Sonication/methods , Chelating Agents/chemistry , Chelating Agents/radiation effects , Crystallization/methods , Histidine/radiation effects , Materials Testing , Nanoparticles/radiation effects , Nanoparticles/ultrastructure , Nanotechnology/methods , Particle Size , Radiation DosageABSTRACT
With a tube etching process, conical-ended optical fibers for middle infrared lasers that have lateral emissions can be produced, a feature of benefit for delivering laser energy onto the root canal walls. This study examined the ability of these improved laser tips when Er:YAG and Er,Cr:YSGG lasers were used in root canals in which thick smear layers had been created intentionally to provide a challenge for the laser system. Smear layer was assessed from scanning electron microscopy images with an objective digital method. Lasing improved the action of ethylene diamine tetraacetic acid with cetavlon (EDTAC) in removing smear layer. Conical fibers performed better than plain fibers, but there was no difference in performance between the 2 laser systems when matched for all other parameters. These results provide a "proof of concept" for lateral emitting fibers for endodontic procedures and illustrate the novel contribution of lasing to the action of EDTAC in dissolving smear layer.
Subject(s)
Dental Pulp Cavity/drug effects , Dentin/drug effects , Lasers, Solid-State/therapeutic use , Optical Fibers , Root Canal Irrigants/radiation effects , Smear Layer , Tooth Apex/drug effects , Cetrimonium , Cetrimonium Compounds/radiation effects , Cetrimonium Compounds/therapeutic use , Chelating Agents/radiation effects , Chelating Agents/therapeutic use , Dental Pulp Cavity/ultrastructure , Dentin/ultrastructure , Edetic Acid/analogs & derivatives , Edetic Acid/radiation effects , Edetic Acid/therapeutic use , Equipment Design , Humans , Hydrogen Peroxide/radiation effects , Hydrogen Peroxide/therapeutic use , Materials Testing , Microscopy, Electron, Scanning , Root Canal Irrigants/therapeutic use , Root Canal Preparation/instrumentation , Root Canal Preparation/methods , Sodium Hypochlorite/radiation effects , Sodium Hypochlorite/therapeutic use , Tooth Apex/ultrastructureABSTRACT
Photolysis of amino acids that bear a 2-nitrobenzyl protecting group on the amino nitrogen involves a normal 2-nitrobenzyl-type photocleavage to release the amino acid but also a second mechanistic pathway, that appears to be initiated by single electron transfer from the amino group to the excited state of the nitroaromatic. The end result of this pathway is photodecarboxylation. Quantitative experiments suggest that this latter pathway can contribute between 10 and 80% of the total reaction flux in different compounds. It appears that the aminium radical, the first product of the single electron transfer, can be intercepted by certain amino acids, resulting in a transfer of decarboxylation to this "sacrificial" amino acid. Possible implications for precise details of calcium release from photolabile derivatives of EDTA and EGTA are discussed.
Subject(s)
Calcium/chemistry , Calcium/radiation effects , Chelating Agents/chemistry , Chelating Agents/radiation effects , Chelating Agents/chemical synthesis , Decarboxylation , Electron Transport , Free Radicals/chemical synthesis , Free Radicals/chemistry , Free Radicals/radiation effects , Molecular Structure , Photochemistry , Photolysis , Sensitivity and Specificity , Spectroscopy, Fourier Transform Infrared/methods , Ultraviolet RaysABSTRACT
We have prepared and characterized several lanthanide ion complexes of multidentate ligands or chelates in an effort to develop new upconverting luminescent labels that can be immune to autofluorescence and photobleaching. This study has involved the characterization of various chelates of Nd, Er, and Tm with respect to relative luminescent efficiency and excited-state lifetimes and explored various two-photon stepwise excitation mechanisms. Using peak laser powers near 100 kW, the upconversion emissions of Nd in Nd(EDTA)2(5-) at 386 nm, Er in Er(DPA)3(3-) at 550 nm, and Tm in Tm(DPA)3(3-) at 480 nm, at levels of approximately 10(-12) moles can be detected.
Subject(s)
Fluorescent Dyes , Lanthanoid Series Elements/chemistry , Luminescent Measurements/methods , Microscopy, Fluorescence/methods , Spectrometry, Fluorescence/methods , Water/chemistry , Chelating Agents/chemistry , Chelating Agents/radiation effects , Lanthanoid Series Elements/radiation effects , Light , Phase TransitionABSTRACT
Luminescent lanthanide chelates have unusual spectroscopic characteristics that make them valuable alternative probes to conventional organic fluorophores. However, fundamental parameters such as their quantum yield, and radiative and nonradiative decay rates have been difficult or impossible to measure. We have developed a simple and robust method based on resonance energy transfer to accurately measure these parameters. In addition, the excitation/emission process in lanthanide chelates involves several steps, and we are able to quantify each step. These include excitation of an organic antenna, transfer of energy from the antenna to lanthanide, and then lanthanide emission. Overall, the parameters show that lanthanide chelates can be efficient long-lived emitters, making them sensitive detection reagents and excellent donors in resonance energy transfer. The method is also shown to be applicable to photophysical characterization of red-emitting dyes, which are difficult to characterize by conventional means.
Subject(s)
Energy Transfer/radiation effects , Fluorescent Dyes/chemistry , Lanthanoid Series Elements/chemistry , Luminescent Measurements , Chelating Agents/chemistry , Chelating Agents/radiation effects , Fluorescent Dyes/radiation effects , Kinetics , Organometallic Compounds/chemistry , Organometallic Compounds/radiation effectsABSTRACT
The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.
Subject(s)
Acetates/metabolism , Calcium/metabolism , Ethylenediamines/metabolism , Magnesium/metabolism , Acetates/radiation effects , Biophysical Phenomena , Biophysics , Chelating Agents/metabolism , Chelating Agents/radiation effects , Ethylenediamines/radiation effects , Fluorescent Dyes , Fura-2 , In Vitro Techniques , Kinetics , Models, Biological , Photochemistry , Photolysis , Ultraviolet RaysABSTRACT
Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 microm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 microm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of approximately 4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using DM-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artificial calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that the apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significantly reduce the spread of the fluorescence signal from the photolysis spot.
Subject(s)
Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Acetates/radiation effects , Aniline Compounds , Animals , Calcium/chemistry , Calcium/metabolism , Cheese/analysis , Chelating Agents/radiation effects , Ethylenediamines/radiation effects , Fluorescent Dyes , Heart Ventricles/anatomy & histology , Heart Ventricles/cytology , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Myocardium/cytology , Myocardium/metabolism , Patch-Clamp Techniques , Photolysis , Photons , Rats , XanthenesABSTRACT
A new technique for the determination of the two-photon uncaging action cross section (deltau) of photolyzable calcium cages is described. This technique is potentially applicable to other caged species that can be chelated by a fluorescent indicator dye, as well as caged fluorescent compounds. The two-photon action cross sections of three calcium cages, DM-nitrophen, NP-EGTA, and azid-1, are studied in the range of excitation wavelengths between 700 and 800 nm. Azid-1 has a maximum deltau of approximately 1.4 GM at 700 nm, DM-nitrophen has a maximum deltau of approximately 0.013 GM at 730 nm, and NP-EGTA has no measurable uncaging yield. The equations necessary to predict the amount of cage photolyzed and the temporal behavior of the liberated calcium distribution under a variety of conditions are derived. These equations predict that by using 700-nm light from a Ti:sapphire laser focused with a 1.3-NA objective, essentially all of the azid-1 within the two-photon focal volume would be photolyzed with a 10-micros pulse train of approximately 7 mW average power. The initially localized distributions of free calcium will dissipate rapidly because of diffusion of free calcium and uptake by buffers. In buffer-free cytoplasm, the elevation of the calcium concentration at the center of the focal volume is expected to last for approximately 165 micros.
Subject(s)
Acetates/radiation effects , Calcium/radiation effects , Chelating Agents/radiation effects , Egtazic Acid/analogs & derivatives , Ethylenediamines/radiation effects , Photolysis , Biophysical Phenomena , Biophysics , Egtazic Acid/radiation effects , Kinetics , Models, Chemical , PhotonsSubject(s)
Calcium/chemistry , Calcium/radiation effects , Photons , Acetates/chemistry , Acetates/radiation effects , Chelating Agents/chemistry , Chelating Agents/radiation effects , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , Egtazic Acid/radiation effects , Ethylenediamines/chemistry , Ethylenediamines/radiation effects , Lasers , Photochemistry/instrumentation , PhotolysisABSTRACT
Nitrophenyl-EGTA and DM-nitrophen are Ca2+ cages that release Ca2+ when cleaved upon illumination with near-ultraviolet light. Laser photolysis of nitrophenyl-EGTA produced transient intermediates that decayed biexponentially with rates of 500,000 s-1 and 100,000 s-1 in the presence of saturating Ca2+ and 290,000 s-1 and 68,000 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. Laser photolysis of nitrophenyl-EGTA in the presence of Ca2+ and the Ca2+ indicator Ca-orange-5N produced a monotonic increase in the indicator fluorescence, which had a rate of 68,000 s-1 at pH 7.2 and 25 degrees C. Irradiation of DM-nitrophen produced similar results with somewhat slower kinetics. The transient intermediates decayed with rates of 80,000 s-1 and 11,000 s-1 in the presence of Ca2+ and 59,000 s-1 and 3,600 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. The rate of increase in Ca(2+)-indicator fluorescence produced upon photolysis of the DM-nitrophen: Ca2+ complex was 38,000 s-1 at pH 7.2 and 25 degrees C. In contrast, pulses in Ca2+ concentration were generated when the chelator concentrations were more than the total Ca2+ concentration. Photoreleased Ca2+ concentration stabilized under these circumstances to a steady state within 1-2 ms.
Subject(s)
Acetates/radiation effects , Calcium/administration & dosage , Chelating Agents/radiation effects , Egtazic Acid/analogs & derivatives , Ethylenediamines/radiation effects , Acetates/chemistry , Biophysical Phenomena , Biophysics , Chelating Agents/chemistry , Egtazic Acid/chemistry , Egtazic Acid/radiation effects , Ethylenediamines/chemistry , Fluorescent Dyes , Hydrogen-Ion Concentration , In Vitro Techniques , Indicators and Reagents , Kinetics , Lasers , Models, Chemical , Organic Chemicals , Photolysis , TemperatureABSTRACT
New procedures are described for producing brief transients and reversible elevations in [Ca] that can be used to quantitatively control the concentration of cytoplasmic calcium. If the photolabile calcium chelator DM-nitrophen, partially bound to calcium, is exposed to steady illumination, [Ca] can be raised from a few nM to up to 10 microM for durations of 100 ms or longer, depending on light intensity and duration. An association rate of calcium with nitrophen of 1.5 x 10(6) M-1s-1 was estimated from measurements of [Ca] using the fluorescent indicator Fluo-3, and calcium was found to speed the photolysis of nitrophen 2.5-times. Partial photolysis of DM-nitrophen partly loaded with calcium elicits a [Ca] spike of over 100 microM lasting about 1 ms, depending on intensity and duration of the light flash. Simulations of the reactions involved predict changes in Fluo-3 fluorescence measured at high time resolution with a laser scanning confocal microscope. These procedures have been applied in physiological experiments to generate cytoplasmic [Ca] spikes and pulses and study the cellular responses to them.
Subject(s)
Acetates , Calcium/pharmacology , Chelating Agents , Egtazic Acid/analogs & derivatives , Ethylenediamines , Acetates/radiation effects , Aniline Compounds/metabolism , Binding, Competitive , Calcium/administration & dosage , Calibration , Chelating Agents/radiation effects , Egtazic Acid/radiation effects , Ethylenediamines/radiation effects , Hydrogen/metabolism , Hydrogen/pharmacology , Magnesium/metabolism , Magnesium/pharmacology , Neurons/drug effects , Neurons/physiology , Photolysis , Xanthenes/metabolismABSTRACT
1. Intracellular Ca2+ concentration ([Ca2+]i) was raised by photolysis of a caged calcium compound, nitr-5, and its effects on the mechano-electrical transduction (MET) current were studied by a whole-cell patch electrode voltage clamp technique in dissociated hair cells of a chick. Nitr-5 was loaded into the hair cell by incubation with the membrane-permeable form of the compound (nitr-5 AM). 2. Photolysis of nitr-5 by ultraviolet (UV) light irradiation induced outward currents at -50 mV when recorded with a KCl-based intracellular medium without Ca2+ chelating compounds. The average amplitude of the photo-activated outward current was 115 +/- 82 pA (mean +/- S.D., n = 5). 3. The MET current generated at -50 mV showed a decay after step displacement of the hair bundle. This adaptation was accelerated after UV exposure of the cell. The adaptation was further accelerated by hyperpolarization of the membrane and was eliminated in 20-100 microM Ca2+ extracellular media. 4. The displacement-response relationship was shifted towards the positive direction after the UV irradiation. 5. The recovery of the transducer current after step displacement of the hair bundle was accelerated after UV irradiation, for both the inward-going MET current recorded at -50 mV and the outward-going MET current at +54 mV. However, the adaptation was not observed at positive membrane potentials even after the photolysis of nitr-5. 6. The extent of MET current decay was reduced or disappeared in 20-100 microM Ca2+ extracellular media and the offset time course was prolonged at the membrane potential of -50 mV. The current decay was not observed even after the photo-release of intracellular Ca2+ in 50-100 microM Ca2+ extracellular media. 7. These results (paragraphs 3-6) suggest that the MET current adaptation is accelerated by the increase of [Ca2+]i, and that Ca2+ ions entering through MET channels are essential in the development of adaptation. 8. The adaptation of the MET current was reversibly reduced in a dihydrostreptomycin (DHSM, 20-50 microM) medium. The time course of the adaptation changes lagged the changes in the MET current amplitude. 9. The adaptation developed or disappeared with a delay of 10-20 s after the introduction of either the normal-Ca2+ (2.5 mM) or the low-Ca2+ (50-100 microM) extracellular medium, respectively. These delays in the development and the subsidence of adaptation suggest a presence of a Ca2+ buffer site intracellularly between the adaptative site and the MET channel.
Subject(s)
Calcium/physiology , Hair Cells, Auditory/physiology , Ion Channel Gating/physiology , Adaptation, Physiological/drug effects , Animals , Chelating Agents/radiation effects , Chickens , Dihydrostreptomycin Sulfate/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/radiation effects , In Vitro Techniques , Membrane Potentials/physiology , Time Factors , Ultraviolet RaysABSTRACT
Single twitch fibres from lumbrical muscles of Xenopus have been loaded with the photolysable calcium-chelator diazo-2 by incubation in Ringer solution containing the membrane permeable acetoxymethyl ester (AM) form of diazo-2. Incubation caused a progressive slowing of tetanus rise and relaxation which is ascribed to calcium-buffering by unphotolyzed diazo-2 (Kd = 2.2 microM). After incubation, exposure to a brief UV flash caused a three to four fold increase in the rate of tension fall. A flash given 16-18 ms after the last tetanic stimulus (at 22-24 degrees C) resulted in 10% increase in relaxation rate compared with the control before incubation. A much bigger effect was observed when a flash was given half-way into the slow phase, where an 1.8-1.9-fold increase in relaxation rate, above the preincubation slope, was observed. It is concluded that rapid lowering of [Ca]i, and hence more rapid removal of Ca2+ from troponin, speeds up relaxation, indicating that calcium translocation is the major determinant of the rate of tension fall during the isometric phase of relaxation.
Subject(s)
Calcium/metabolism , Chelating Agents/pharmacology , Muscle Relaxation/drug effects , Photolysis , Animals , Chelating Agents/radiation effects , Diazonium Compounds , Dimethyl Sulfoxide/pharmacology , Isometric Contraction , Phenoxyacetates , Xenopus laevisABSTRACT
1. Transmitter release at the squid giant synapse was stimulated by photolytic release of Ca2+ from the 'caged' Ca2+ compound DM-nitrophen (Kaplan & Ellis-Davies, 1988) inserted into presynaptic terminals. 2. Competing binding reactions cause the amount of Ca2+ released by DM-nitrophen photolysis to depend on the concentrations of DM-nitrophen, total Ca2+, Mg+, ATP and native cytoplasmic Ca2+ buffer. Measurements of presynaptic [Ca2+] changes by co-injection of the fluorescent indicator dye Fura-2 show that DM-nitrophen photolysis causes a transient rise in Ca2+ followed by decay within about 150 ms to an increased steady-state level. 3. Rapid photolysis of Ca2(+)-loaded nitrophen within the presynaptic terminal was followed in less than a millisecond by depolarization of the postsynaptic membrane. As with action potential-evoked excitatory postsynaptic potentials (EPSPs), the light-evoked response was partially and reversibly blocked by 1-3 mM-kainic acid which desensitizes postsynaptic glutamate receptors. 4. Release was similar in magnitude and rate to normal action potential-mediated EPSPs. 5. The release of transmitter by photolysis of Ca2(+)-loaded DM-nitrophen was not affected by removal of Ca2+ from the saline or addition of tetrodotoxin. Photolysis of DM-nitrophen injected into presynaptic terminals without added Ca2+ did not stimulate release of transmitter nor did it interfere with normal action potential-mediated release. 6. Stimulation of presynaptic action potentials in Ca2(+)-free saline during the light-evoked response did not elicit increased release of transmitter if the ganglion was bathed in Ca2(+)-free saline, i.e. in the absence of Ca2+ influx. Increasing the intensity of the light or stimulating presynaptic action potentials in Ca2(+)-containing saline increased the release of transmitter. Therefore the failure of presynaptic voltage change to increase transmitter release resulting from release of caged Ca2+ was not due to saturation or inhibition of the release mechanism by light-released Ca2+. 7. Decreasing the temperature of the preparation increased the delay to onset of the light-evoked response and reduced its amplitude and rate of rise to an extent similar to that observed for action potential-evoked EPSPs.
Subject(s)
Calcium/pharmacology , Neurotransmitter Agents/metabolism , Synapses/metabolism , Acetates/radiation effects , Action Potentials/drug effects , Animals , Binding, Competitive , Chelating Agents/radiation effects , Decapodiformes , Ethylenediamines/radiation effects , Female , Hot Temperature , In Vitro Techniques , Light , Magnesium/pharmacology , Membrane Potentials/drug effects , Photolysis , Stimulation, Chemical , Synapses/drug effectsABSTRACT
A photoregulated chelating agent has been synthesized. It is a photochromic azobenzene compound containing two iminodiacetic acid groups and can exists as cis and trans stereoisomers. The planar trans isomer does not bind zinc ions. On exposure to light of 320 nanometers, the trans isomer is converted to a nonplanar cis isomer, which, because of cooperativity between the two iminodiacetic acid groups, binds zinc ions with the value of the binding constant estimated to be 1.1 x 10(5) +/- 9.2 x 10(5) liters per mole at a ratio of one molecule of chelating agent to one zinc ion. The interconversion of the cis and trans isomers is reversible, suggesting possible application of this class of compounds as photoresponsive ion pumps.