Acetoxymethyl Research Articles

Oligosaccharide Blocks PAR1 (Proteinase-Activated Receptor 1)-PAR4-Mediated Platelet Activation by Binding to Thrombin Exosite II and Impairs Thrombosis.

Inappropriate activation and aggregation of platelets can lead to arterial thrombosis. Thrombin is the most potent platelet agonist that activates human platelets via two PARs (proteinase-activated receptors), PAR1 and PAR4. The aim is to study the activity and mechanism of an oligosaccharide HS-11 (the undecasaccharide, derived from sea cucumber Holothuria fuscopunctata) in inhibiting thrombin-mediated platelet activation and aggregation and to evaluate its antithrombotic activity. Platelet activation was analyzed by detecting CD62P/P-selectin expression using flow cytometry. The HS-11-thrombin interaction and the binding site were studied by biolayer interferometry. Intracellular Ca2+ mobilization of platelets was measured by FLIPR Tetra System using Fluo-4 AM (Fluo-4 acetoxymethyl). Platelet aggregation, thrombus formation, and bleeding Assay were assessed. An oligosaccharide HS-11, depolymerized from fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata blocks the interaction of thrombin with PAR1 and PAR4 complex by directly binding to thrombin exosite II, and completely inhibits platelet signal transduction, including intracellular Ca2+ mobilization and protein phosphorylation. Furthermore, HS-11 potently inhibits thrombin-PARs-mediated platelet aggregation and reduces thrombus formation in a model of ex vivo thrombosis. The study firstly report that the fucosylated glycosaminoglycan oligosaccharide has antiplatelet activity by binding to thrombin exosite II, and demonstrates that thrombin exosite II plays an important role in the simultaneous activation of PAR1 and PAR4, which may be a potential antithrombotic target for effective treatment of arterial thrombosis.

Fluorescence-Based Assay for TRPV1 Channels.

The transient receptor potential vanilloid 1 ion channel (TRPV1) is a ligand-gated nonselective calcium-permeant cation channel involved in the detection of a wide variety of chemical and physical noxious stimuli, ranging from exogenous and endogenous ligands to noxious heat (>42°C) and low pH (pH<5.2). Due to its central role in pain and hyperalgesia, TRPV1 is considered a relevant therapeutic target for the development of analgesic and anti-inflammatory drugs potentially useful to relieve chronic, neuropathic, and inflammatory pain and to treat disorders such as inflammatory bowel disease. In this view, the availability of in vitro assays for the screening of novel TRPV1 modulators is highly desirable. Since TRPV1 activation leads to an increase in the intracellular calcium (Ca2+) levels, the use of Ca2+ fluorescent indicators represent a valuable and sensitive tool for monitoring such intracellular changes. In this chapter, we describe methods for recording and monitoring Ca2+ signals through the fluorescent indicators Fluo-4 acetoxymethyl (AM) and Fura-2AM in HEK-293 cells transfected with TRPV1 or other thermoTRP channels.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte’s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte's sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Corneal endothelial regeneration in human eyes using endothelium-free grafts

BackgroundTo report on corneal endothelial regeneration, graft clarity, and vision recovery when using endothelium-free grafts.MethodsWe evaluated the donor’s cell viability using trypan blue staining and dual staining with calcein acetoxy methyl ester and ethidium homodimer-1. To preserve eyeball integrity, we performed therapeutic penetrating keratoplasty using cryopreserved donor tissue without endothelium on 195 consecutive patients who suffered from corneal perforation due to progressive primary corneal disease such as herpes simplex keratitis, fungal keratitis, ocular thermal burns, keratoconus, and phlyctenular keratoconjunctivitis. Of these, 18 eyes recovered corneal graft clarity and underwent periodic slit-lamp microscopy, A-scan pachymetry, and in vivo confocal microscopy to observe the clinical manifestations, variations in corneal thickness, and repopulation of the corneal endothelial cells on the donor grafts.ResultsNo viable cells were detected in the cryopreserved corneas. After the therapeutic penetrating keratoplasty, notable corneal graft edema was observed in all 18 eyes for 1–4 months, and no corneal endothelial cells were detected on the grafts during this period. Thereafter, we observed gradual and progressive regression and final resolution of the stromal edema, with complete recovery of corneal graft clarity. Through periodic confocal microscopy, we observed the corneal endothelium’s regenerating process, along with single cells bearing multiple nuclei and cell division-like morphology. The regenerated endothelium on the grafts reached a mean cell density of 991 cells/mm2. Remarkable vision rehabilitation was achieved in all 18 patients.ConclusionsWe obtained conclusive evidence that host-derived endothelial cells can regenerate a new endothelium over the endothelium-free graft, which possesses normal functions for corneal clarity and vision recovery.

New LC-MS Method Development and Validation for Quantitative Estimation of 2-Acetoxy Ethyl Acetoxy Methyl Ether in Acyclovir

The objective of the proposed method is to develop and validate a specific, precise and accurate LC-MS method for the determination of 2-acetoxy ethyl acetoxy methyl ether (AEM) in Acyclovir. An isocratic separation was done by using YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column with a mobile phase composition of Buffer (0.1% Ammonia in water) : Methanol(25 :75v/v)at a flow rate of 0.4mL/min, diluent solution of 0.2% of 0.1N NaOH in Methanol were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) Acquisition mode and possitive polarity mode. The Retention time of AEM was found to be 5.344 minutes, LOD and LOQ were observed at 0.03 ppm and 0.11 ppm concentration respectively by signal to noise ratio method, linearity was found in the concentration range of 0.18-0.55 ppm with correlation coefficient of 0.996 and accuracy in the range of 97.7- 101% was obtained by performing percentage recovery studies. The % RSD was observed to be less than 10% for six replicates said to be precise. The proposed method was validated for accuracy, precision, sensitivity, and linearity successfully employed for quantitative determination of AEM in acyclovir.

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter.

Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨm) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨm indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-dependent accumulation. We redirected the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxymethyl (AM) ester. Once within mitochondria, esterases remove the AM ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨm. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage Reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨm induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca2+, plasma membrane potential, and reversible ΔΨm dynamics.

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies. Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging. Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm. The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Comparing the effect of three Descemet membrane endothelial keratoplasty injectors on endothelial damage of grafts.

Purpose:Various injectors are commercially available for Descemet membrane endothelial keratoplasty (DMEK) but not all injectors have been studied for endothelial damage of grafts. The aim of the study was to compare endothelial damage in pre-stripped DMEK tissue from three clinically used injector devices: the modified Jones tube, the STAAR intraocular (IOL) injector, and the Geuder glass cannula in a laboratory setting.Methods:Twenty-four human donor corneas were used for this study, eight for each study arm. Each endothelial graft was pre-stripped, trephined to 8.0 mm diameter, then loaded into either the modified Jones tube, the STAAR IOL injector, or the Geuder glass cannula by an eye bank technician who had no prior experience with any of the injectors. Grafts were then ejected, stained with Calcein acetoxymethyl (AM), and quantitatively analyzed using FIJI image software. The primary outcome was the percent of endothelial damage from injector loading and injection. Donor demographics were analyzed using Fisher’s exact test. The percentage of endothelial cell loss was compared across groups using the Kruskal–Wallis test.Results:The mean percent of endothelial damage from after injection of the graft was 37.8% (±SD 12.2%) for the modified Jones tube, 37.0% (±SD 13.9%) for the STAAR IOL injector, and 23.5% (±SD 5.1%) for the Geuder cannula (P = 0.008).Conclusion:DMEK injectors contribute to intraoperative endothelial damage of transplanted grafts. The Geuder glass cannula may offer increased ease of use and less endothelial damage compared to the modified Jones tube or STAAR IOL injector for the novice user in early cases.

Calcium Imaging in the Zebrafish.

The zebrafish (Danio rerio) has emerged as a widely used model system during the last four decades. The fact that the zebrafish larva is transparent enables sophisticated in vivo imaging, including calcium imaging of intracellular transients in many different tissues. While being a vertebrate, the reduced complexity of its nervous system and small size make it possible to follow large-scale activity in the whole brain. Its genome is sequenced and many genetic and molecular tools have been developed that simplify the study of gene function in health and disease. Since the mid 90's, the development and neuronal function of the embryonic, larval, and later, adult zebrafish have been studied using calcium imaging methods. This updated chapter is reviewing the advances in methods and research findings of zebrafish calcium imaging during the last decade. The choice of calcium indicator depends on the desired number of cells to study and cell accessibility. Synthetic calcium indicators, conjugated to dextrans and acetoxymethyl (AM) esters, are still used to label specific neuronal cell types in the hindbrain and the olfactory system. However, genetically encoded calcium indicators, such as aequorin and the GCaMP family of indicators, expressed in various tissues by the use of cell-specific promoters, are now the choice for most applications, including brain-wide imaging. Calcium imaging in the zebrafish has contributed greatly to our understanding of basic biological principles during development and adulthood, and the function of disease-related genes in a vertebrate system.

Development of a Novel Quinoline Derivative as a P-Glycoprotein Inhibitor to Reverse Multidrug Resistance in Cancer Cells.

Multidrug resistance (MDR) is one of conventional cancer chemotherapy’s limitations. Our group previously synthesized a series of quinoline-based compounds in an attempt to identify novel anticancer agents. With a molecular docking analysis, the novel compound 160a was predicted to target p-glycoprotein, an MDR candidate. The purpose of this study is to evaluate 160a’s MDR reversal effect and investigate the underlying mechanism at the molecular level. To investigate 160a’s inhibitory effect, we used a series of parental cancer cell lines (A549, LCC6, KYSE150, and MCF-7), the corresponding doxorubicin-resistant cell lines, an MTS cytotoxicity assay, an intracellular doxorubicin accumulation test, and multidrug resistance assays. The Compusyn program confirmed, with a combination index (CI) value greater than 1, that 160a combined with doxorubicin exerts a synergistic effect. Intracellular doxorubicin accumulation and transported calcein acetoxymethyl (AM) (a substrate for p-glycoprotein) were both increased when cancer cells with MDR were treated with compound 160a. We also showed that compound 160a’s MDR reversal effect can persist for at least 1 h. Taken together, these results suggest that the quinoline compound 160a possesses high potential to reverse MDR by inhibiting p-glycoprotein-mediated drug efflux in cancer cells with MDR.

Kinetics-Based Measurement of Hypoxia in Living Cells and Animals Using an Acetoxymethyl Ester Chemiluminescent Probe

Oxygenation and tissue hypoxia play critical roles in mammalian biology and contribute to aggressive phenotypes in cancerous tumors, driving research to develop accurate and easy-to-implement methods for monitoring hypoxia in living cells and animal models. This study reports the chemiluminescent probe HyCL-4-AM, which contains a nitroaromatic sensing moiety and, importantly, an acetoxymethyl (AM) ester that dramatically improves operation in cells and animals. HyCL-4-AM provides a selective 60 000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM). For cellular operation, the chemiluminescence response kinetics is sharply dependent on oxygen levels, enabling highly significant and reproducible measurement of hypoxia in living cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.

Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.

Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides adramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca2+) as shown by comparison of transport assays in Ca2+-rich and Ca2+-free buffers and upon treatment with inhibitors of plasma membrane Ca2+-permeable channels Al3+ and ruthenium red, both abolishing the effect of AgNO3. Confocal microscopy of Ca2+-sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca2+-permeable channels at the plasma membrane.

Acetoxymethyl Concept for Intracellular Administration of Carbon Monoxide with Mn(CO)3 -Based PhotoCORMs.

Targeted administration of carbon monoxide with CO releasing molecules (CORMs) inside of cells proved to be very challenging. Consequently, there are only very few reports on intracellular uptake of CORMs requiring high extracellular CORM loading because an equilibrium between extra- and intracellular concentrations can be assumed. Here we present a strategy for a targeted intracellular administration of manganese(I)-based CORMs that are altered inside of cells to trap these complexes. Thereafter, carbon monoxide can be liberated by irradiation (photoCORMs). To achieve this innovative task, acetoxymethyl (AM) groups are attached at the periphery of the hydrophobic manganese(I) carbonyl complexes to not influence the CO release behavior. Inside of cells these AM substituents are cleaved by esterases yielding hydrophilic manganese(I) carbonyl compounds which are captured inside of cells. This objective is realized by using the bidentate bases 4-(acetoxymethoxycarbonyl)phenyl-bis(3,5-dimethylpyrazolyl)methane (1) and 4-(acetoxymethoxy)phenyl-bis(3,5-dimethylpyrazolyl)methane (4) at facial (OC)3 MnBr fragments yielding CORM-AM1 (2) and CORM-AM2 (5), respectively. Besides synthesis, crystal structures and spectroscopic properties we present targeted administration and intracellular accumulation of these AM-containing CORMs.

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions.

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.

Rapid Bacterial Detection during Endodontic Treatment

Bacteria present in the root canal (RC) space following an RC treatment (RCT) can lead to persistent infections, resulting in treatment failure and the need for reintervention or extraction. Currently, there are no standardized methods in use to clinically detect bacterial presence within RC spaces. The use of paper point sampling and fluorescence staining was shown to be a rapid method, able to detect residual bacteria following treatment. The study demonstrated that Calcein acetoxymethyl (AM) proved to be a suitable dye for detecting vital bacteria within mature endodontic biofilms, with an improved sensitivity over colony-forming unit counting in a stressed biofilm model. Furthermore, in a clinical trial with primary RCTs, 53 infected teeth were sampled in vivo, and increased detection of vital cells was found when compared with colony-forming unit counting, highlighting the sensitivity of the technique in detecting low cell numbers. By combining fluorescent staining and microspectroscopy with software-based spectral analysis, successful detection of vital cells from RCs was possible after 5 min of Calcein AM incubation. Application of this technology during RCT has the potential to reduce persistent infections through vital cell detection and additional treatment. Furthermore, this technique could be applied to antimicrobial research and disinfection control in clinical settings (ClinicalTrials.gov NCT03055975).

AMPA Receptors Are Involved in Store-Operated Calcium Entry and Interact with STIM Proteins in Rat Primary Cortical Neurons.

The process of store-operated calcium entry (SOCE) leads to refilling the endoplasmic reticulum (ER) with calcium ions (Ca2+) after their release into the cytoplasm. Interactions between (ER)-located Ca2+ sensors (stromal interaction molecule 1 [STIM1] and STIM2) and plasma membrane-located Ca2+ channel-forming protein (Orai1) underlie SOCE and are well described in non-excitable cells. In neurons, however, SOCE appears to be more complex because of the importance of Ca2+ influx via voltage-gated or ionotropic receptor-operated Ca2+ channels. We found that the SOCE inhibitors ML-9 and SKF96365 reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i amplitude by 80% and 53%, respectively. To assess the possible involvement of AMPA receptors (AMPARs) in SOCE, we used their specific inhibitors. As estimated by Fura-2 acetoxymethyl (AM) single-cell Ca2+ measurements in the presence of CNQX or NBQX, thapsigargin (TG)-induced Ca2+ influx decreased 2.2 or 3.7 times, respectively. These results suggest that under experimental conditions of SOCE when Ca2+ stores are depleted, Ca2+ can enter neurons also through AMPARs. Using specific antibodies against STIM proteins or GluA1/GluA2 AMPAR subunits, co-immunoprecipitation assays indicated that when Ca2+ levels are low in the neuronal ER, a physical association occurs between endogenous STIM proteins and endogenous AMPAR receptors. Altogether, our data suggest that STIM proteins in neurons can control AMPA-induced Ca2+ entry as a part of the mechanism of SOCE.

Design and Synthesis of a 4-Nitrobromobenzene Derivative Bearing an Ethylene Glycol Tetraacetic Acid Unit for a New Generation of Caged Calcium Compounds with Two-Photon Absorption Properties in the Near-IR Region and Their Application in Vivo.

Among biologically active compounds, calcium ions (Ca2+) are one of the most important species in cell physiological functions. Development of new calcium chelators with two-photon absorption (TPA) properties is a state-of-the-art challenge for chemists. In this study, we report the first and efficient synthesis of 5-bromo-2-nitrobenzyl-substituted ethylene glycol tetraacetic acid (EGTA) as a platform for a new generation of calcium chelators with TPA properties in the near-infrared region. New calcium chelators with high TPA properties, that is, a two-photon (TP) fragmentation efficiency of δu = 20.7 GM at 740 nm for 2-(4-nitrophenyl)benzofuran (NPBF)-substituted EGTA (NPBF-EGTA, Kd = 272 nM) and δu = 7.8 GM at 800 nm for 4-amino-4′-nitro-1,1′-biphenyl (BP)-substituted EGTA (BP-EGTA, Kd = 440 nM) derivatives, were synthesized using Suzuki–Miyaura coupling reactions of the bromide with benzofuran-2-boronic acid and 4-(dimethylamino)phenyl boronic acid, respectively. The corresponding acetoxymethyl (AM) esters were prepared and successfully applied to the Ca2+-uncaging reaction triggered by TP photolysis in vivo.

Synthesis of (–)-(3aR,4R,5S,6aS)-4-[(acetoxy)methyl]-1-oxohexahydro-1H-cyclopenta[c]furan-5-yl Acetate

A new 1,2,3,4-functionalized cyclopentane, (–)-(3aR,4R,5S,6aS)-4-[(acetoxy)methyl]-1-oxohexahydro-1H-cyclopenta[c]furan-5-yl acetate, has been synthesized by cleavage of a tricyclic bowl-shaped aminal, (2aR,3S,4aS,6bR)-3-hydroxy-6-[(1R)-1-phenylethyl]-octahydro-5H-1-oxa-6-azacyclopenta[cd]pentalen-5-one.