Redox Stability Controls the Cellular Uptake and Activity of Ruthenium‐Based Inhibitors of the Mitochondrial Calcium Uniporter (MCU)

The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca²⁺ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca²⁺. The best‐known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X‐ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.     

A Glycosylated Covalent Organic Framework Equipped with BODIPY and CaCO3 for Synergistic Tumor Therapy

Ca²⁺, a ubiquitous but nuanced modulator of cellular physiology, is meticulously controlled intracellularly. However, intracellular Ca²⁺ regulation, such as mitochondrial Ca²⁺ buffering capacity, can be disrupted by ¹O₂. Thus, the intracellular Ca²⁺ overload, which is recognized as one of the important cell pro‐death factors, can be logically achieved by the synergism of ¹O₂ with exogenous Ca²⁺ delivery. Reported herein is a nanoscale covalent organic framework (NCOF)‐based nanoagent, namely CaCO₃@COF‐BODIPY‐2I@GAG ( 4 ), which is embedded with CaCO₃ nanoparticle (NP) and surface‐decorated with BODIPY‐2I as photosensitizer (PS) and glycosaminoglycan (GAG) targeting agent for CD44 receptors on digestive tract tumor cells. Under illumination, the light‐triggered ¹O₂ not only kills the tumor cells directly, but also leads to their mitochondrial dysfunction and Ca²⁺ overload. An enhanced antitumor efficiency is achieved via photodynamic therapy (PDT) and Ca²⁺ overload synergistic therapy.     

Release of Ca2+ and Mg2+ from yeast mitochondria is stimulated by increased ionic strength

Background  

Divalent cations are required for many essential functions of mitochondrial metabolism. Yet the transporters that mediate the flux of these molecules into and out of the mitochondrion remain largely unknown. Previous studies in yeast have led to the molecular identification of a component of the major mitochondrial electrophoretic Mg²⁺ uptake system in this organism as well as a functional mammalian homolog. Other yeast mitochondrial studies have led to the characterization of an equilibrative fatty acid-stimulated Ca²⁺ transport activity. To gain a deeper understanding of the regulation of mitochondrial divalent cation levels we further characterized the efflux of Ca²⁺ and Mg²⁺ from yeast mitochondria.     

Synthesis and Biological Assessment of 4,1-Benzothiazepines with Neuroprotective Activity on the Ca2+ Overload for the Treatment of Neurodegenerative Diseases and Stroke

In excitable cells, mitochondria play a key role in the regulation of the cytosolic Ca²⁺ levels. A dysregulation of the mitochondrial Ca²⁺ buffering machinery derives in serious pathologies, where neurodegenerative diseases highlight. Since the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) is the principal efflux pathway of Ca²⁺ to the cytosol, drugs capable of blocking NCLX have been proposed to act as neuroprotectants in neuronal damage scenarios exacerbated by Ca²⁺ overload. In our search of optimized NCLX blockers with augmented drug-likeness, we herein describe the synthesis and pharmacological characterization of new benzothiazepines analogues to the first-in-class NCLX blocker {"type":"entrez-protein","attrs":{"text":"CGP37157","term_id":"875406365","term_text":"CGP37157"}}CGP37157 and its further derivative ITH12575, synthesized by our research group. As a result, we found two new compounds with an increased neuroprotective activity, neuronal Ca²⁺ regulatory activity and improved drug-likeness and pharmacokinetic properties, such as clog p or brain permeability, measured by PAMPA experiments.     

Possible involvement of mitochondrial calcium transport in causing cell injury in experimental hepatic chronic iron overload

The functional state of isolated mitochondria and, specifically, the integrity of the inner membrane were investigated in the livers of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the mitochondrial fraction increased progressively up to nearly 40 days and it reached a steady-state level. When the iron content reached a threshold value (higher than 30 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by phosphorylative capability and membrane potential measurements. On the contrary, the induction of the lipoperoxidative reaction appeared to be associated with the activation of Ca²⁺ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca²⁺ release from iron treated mitochondria resulted in enhancement of Ca²⁺ cycling, a process which dissipates energy to reaccumulate the releaased Ca²⁺ into mitochondria. The perturbation in mitochondrial Ca²⁺ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic chronic iron overload.     

Redox Stability Controls the Cellular Uptake and Activity of Ruthenium-Based Inhibitors of the Mitochondrial Calcium Uniporter (MCU)

The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca²⁺ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca²⁺. The best-known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X-ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities.     

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca²⁺-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca²⁺-regulated potassium (mitoBK_Ca) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BK_Ca channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BK_Ca channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBK_Ca channel in a bronchial epithelial cell line were described.     

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

An abrupt increase of intracellular Ca²⁺ is observed in cells under hypoxic or oxidatively stressed conditions. The dysregulated increase of cytosolic Ca²⁺ triggers apoptotic cell death through mitochondrial swelling and activation of Ca²⁺-dependent enzymes. Transglutaminase 2 (TG2) is a Ca²⁺-dependent enzyme that catalyzes transamidation reaction producing cross-linked and polyaminated proteins. TG2 activity is known to be involved in the apoptotic process. However, the pro-apoptotic role of TG2 is still controversial. In this study, we investigate the role of TG2 in apoptosis induced by Ca²⁺-overload. Overexpression of TG2 inhibited the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-induced apoptosis through suppression of caspase-3 and -9 activities, cytochrome c release into cytosol, and mitochondria membrane depolarization. Conversely, down-regulation of TG2 caused the increases of cell death, caspase-3 activity and cytochrome c in cytosol in response to Ca²⁺-overload. Western blot analysis of Bcl-2 family proteins showed that TG2 reduced the expression level of Bax protein. Moreover, overexpression of Bax abrogated the anti-apoptotic effect of TG2, indicating that TG2-mediated suppression of Bax is responsible for inhibiting cell death under Ca²⁺-overloaded conditions. Our findings revealed a novel anti-apoptotic pathway involving TG2, and suggested the induction of TG2 as a novel strategy for promoting cell survival in diseases such as ischemia and neurodegeneration.     

QSPR Modeling of Potentiometric Mg2+/Ca2+ Selectivity for PVC‐plasticized Sensor Membranes

The development of novel ionophores for ion‐selective sensors is a time‐consuming and tedious process requiring synthesis of candidate substances, preparation of plasticized polymeric membranes, and their thorough characterization with traditional protocols to assess sensitivity, selectivity and detection limits for target ions. The vast amount of literature data accumulated on various ion‐selective sensors allows for significant facilitation of the development through in silico experiments. In this report, we performed the feasibility study on the prediction of potentiometric Mg²⁺/Ca²⁺ selectivity for various amide ligands using quantitative structure‐property relationship (QSPR) modeling. The approach proved to be promising for ionophore screening purposes with achieved precision in prediction of the selectivity coefficient logK(Mg²⁺/Ca²⁺) of 0.5 in the range from ?1.7 to +2.3. The study also shows a route for prediction of new potential ionophores with high selectivity values.     

Optogenetic Control of Voltage‐Gated Calcium Channels

Voltage‐gated Ca²⁺ (Ca_V) channels mediate Ca²⁺ entry into excitable cells to regulate a myriad of cellular events following membrane depolarization. We report the engineering of RGK GTPases, a class of genetically encoded Ca_V channel modulators, to enable photo‐tunable modulation of Ca_V channel activity in excitable mammalian cells. This optogenetic tool (designated optoRGK) tailored for Ca_V channels could find broad applications in interrogating a wide range of Ca_V‐mediated physiological processes.     

Calcium overload is essential for the acceleration of staurosporine-induced cell death following neuronal differentiation in PC12 cells

Differentiation of neuronal cells has been shown to accelerate stress-induced cell death, but the underlying mechanisms are not completely understood. Here, we find that early and sustained increase in cytosolic ([Ca²⁺]_c) and mitochondrial Ca²⁺ levels ([Ca²⁺]_m) is essential for the increased sensitivity to staurosporine-induced cell death following neuronal differentiation in PC12 cells. Consistently, pretreatment of differentiated PC12 cells with the intracellular Ca²⁺-chelator EGTA-AM diminished staurosporine-induced PARP cleavage and cell death. Furthermore, Ca²⁺ overload and enhanced vulnerability to staurosporine in differentiated cells were prevented by Bcl-X_L overexpression. Our data reveal a new regulatory role for differentiation-dependent alteration of Ca²⁺ signaling in cell death in response to staurosporine.     

A Highly Selective Fluorescence Turn‐On Sensor for Extracellular Calcium Ion Detection

A highly water‐soluble, fluorescence turn‐on sensor for Ca²⁺ is reported. The sensor affords high selectivity in sensing Ca²⁺ over other biologically important metal cations. The dissociation constant of the sensor in binding Ca²⁺ is 0.92 mm . Fluorescence microscopy experiments demonstrate that the sensor is cell‐impermeable and capable of detecting extracellular Ca²⁺.     

Synthesis and binding studies ofN-(2-Hydroxy-l-phenoxyacetyl) glycylglycine

The synthesis ofN-(2-hydroxy-l-phenoxyacetyl)glycylglycine3 from 2-acetoxyphenoxyacetic acid is described. Compound3, a simple model for the carboxy-containing ionophore, Lasalocid, binds cations in methanol in the order: Ca²⁺ Ba²⁺ > Sr²⁺ 2>> Li⁺ > Na⁺, K⁺.     

Relation of Photochemical Internalization to Heat,pH and Ca2+ Ions

The inefficient endosomal escape of drugs or macromolecules is a major obstacle to achieving successful delivery to therapeutic targets. An efficient approach to circumvent this barrier is photochemical internalization (PCI), which uses light and photosensitizers for endosomal escape of the delivered macromolecules. The PCI mechanism is related to photogenerated singlet oxygen, but the mechanism is still unclear. In this study, we examined the relation of PCI to heat, pH and Ca²⁺ ions using cell penetrating peptide (CPP)‐cargo‐photosensitizer (Alexa546 or Alexa633) conjugates. A cell temperature changing experiment demonstrated that heat (thermal mechanism) does not significantly contribute to the photoinduced endosomal escape. Inhibition of V‐ATPase proton pump activity and endosomal pH upregulation indicated that PCI‐mediated endosomal escape needs endosomal acidification prior to photoirradiation. Imaging of the CPP‐cargo‐photosensitizer and Ca²⁺ ions during photostimulation showed that intracellular calcium increase is not the cause of the endosomal escape of the complex. The increment is mainly due to Ca²⁺ influx. These findings show the importance of extra‐ and intracellular milieu conditions in the PCI mechanism and enrich our understanding of PCI‐related changes in cell.     

New Calcium‐Selective Smart Contrast Agents for Magnetic Resonance Imaging

Calcium plays a vital role in the human body and especially in the central nervous system. Precise maintenance of Ca²⁺ levels is very crucial for normal cell physiology and health. The deregulation of calcium homeostasis can lead to neuronal cell death and brain damage. To study this functional role played by Ca²⁺ in the brain noninvasively by using magnetic resonance imaging, we have synthesized a new set of Ca²⁺‐sensitive smart contrast agents (CAs). The agents were found to be highly selective to Ca²⁺ in the presence of other competitive anions and cations in buffer and in physiological fluids. The structure of CAs comprises Gd³⁺‐DO3A (DO3A=1,4,7‐tris(carboxymethyl)‐1,4,7,10‐tetraazacyclododecane) coupled to a Ca²⁺ chelator o‐amino phenol‐N,N,O‐triacetate (APTRA). The agents are designed to sense Ca²⁺ present in extracellular fluid of the brain where its concentration is relatively high, that is, 1.2–0.8 mM . The determined dissociation constant of the CAs to Ca²⁺ falls in the range required to sense and report changes in extracellular Ca²⁺ levels followed by an increase in neural activity. In buffer, with the addition of Ca²⁺ the increase in relaxivity ranged from 100–157 %, the highest ever known for any T₁‐based Ca²⁺‐sensitive smart CA. The CAs were analyzed extensively by the measurement of luminescence lifetime measurement on Tb³⁺ analogues, nuclear magnetic relaxation dispersion (NMRD), and ¹⁷O NMR transverse relaxation and shift experiments. The results obtained confirmed that the large relaxivity enhancement observed upon Ca²⁺ addition is due to the increase of the hydration state of the complexes together with the slowing down of the molecular rotation and the retention of a significant contribution of the water molecules of the second sphere of hydration.     

Lanthanum Chloride Promoted Proliferation with Enhanced S‐phase Entry and Inhibited Potassium Currents of NIH 3T3 Cells

The effects of La³⁺ on proliferation, cell cycles, apoptosis and ion channels were investigated in mouse embryo fibroblast NIH 3T3 cells and its possible mechanisms were explored. Our data showed that La³⁺ promoted cell proliferation with increased S‐phase entry and inhibited the outward potassium currents in a concentration‐dependent manner in NIH 3T3 cells. La³⁺ and Ca²⁺ had synergistic effect on cell proliferation and cell cycles. It showed that Ca²⁺ was needed for La³⁺‐promoted cell cycle progression. Using the whole‐cell voltage‐clamp technique, we found that La³⁺ blocked the outward potassium current in a concentration‐dependent manner in NIH 3T3 cells. Lanthanum ions can increase intracellular Ca²⁺ concentration through inhibition of potassium currents, which induce a series of physiological changes and improve proliferation of cells. This may be one of the molecular mechanisms of lanthanum ions induced cell proliferation. The present work provides a new perspective for understanding the biological and toxicological effects of lanthanum.     

Design and Applications of Small Molecular Probes for Calcium Detection

The physiological significance of calcium ions such as the role in cellular signalling, cell growth, etc. have driven the development of methods to detect and monitor the level of Ca²⁺ ions, both in vivo and in vitro. Although various approaches for the detection of calcium ions have been reported, methods based on small molecular fluorescent probes have unique advantages including small probe size, easy monitoring of detection processes and applicability in biological systems. In this review article, we will discuss the progress in the development of Ca²⁺‐binding fluorescent probes by taking into account the types of chelating groups that have been employed for Ca²⁺ binding.     

Novel organometallic chalcones functionalized with a crown ether fragment as optical ion chemosensors

We previously reported the synthesis and characterization of new organometallic chalcones derived from ferrocene, cyrhetrene and cymantrene functionalized with a benzo‐15‐crown‐5 fragment. The ferrocene and cyrhetrene chalcones have been investigated as chemosensors for metal ions with optical response in acetonitrile. Several metal ions were selected considering the diameter of the cavity and the charge‐to‐radius ratio of the cation. The stoichiometry of the complexes was determined using Job's method. It was found that Na⁺ and Ca²⁺ complexes have a 1:1 stoichiometry while a 2:1 (metaloligand‐to‐cation) stoichiometry was determined for Ba²⁺ and Pb²⁺ complexes. The association constants were calculated according to the stoichiometry of the complex and the results showed that they are directly affected by the electron‐withdrawing nature of the organometallic fragment. Moreover, complexes of ferrocenyl chalcone have larger association constants than those of the cyrhetrenyl analogue. This experimental observation is consistent with the electronic properties of the ferrocenyl fragment.     

Cell protection from Ca2+-overloading by bioactive molecules extracted from olive pomace

We are reporting in the present study that molecules extracted from olive pomace prevent cell death induced by Ca²⁺-overloading in different cell types. Exposure of cells to these molecules counteracts the Ca²⁺-induced cell damages by reducing the activation of the Ca²⁺-dependent protease calpain, acting possibly through the modification of the permeability to Ca²⁺ of the plasma membrane. The purification step by RP-HPLC suggests that effective compound(s), differing from the main biophenols known to be present in the olive pomace extract, could be responsible for this effect. Our observations suggest that bioactive molecules present in the olive pomace could be potential candidates for therapeutic applications in pathologies characterised by alterations of intracellular Ca²⁺ homeostasis.     

Design,characterization and quantum chemical computations of a novel series of pyrazoles derivatives with potential anti-proinflammatory response

The synthesis and characterization of the full family of 11 pyrazoles were performed by means of UV–Vis, FTIR, ¹H NMR, ¹³C NMR, two-dimensional NMR experiments and DFT simulations. As pyrazoles are known for showing diverse biological actions, they were also tested in the NCI-60 cancer cell line panel, showing moderate to good activity against different cell lines. Furthermore, the anti-proinflammatory activity test of a set of pyrazoles of the form (E)-4-((4-bromophenyl)diazenyl)-3,5-dimethyl-1-R-phenyl-1H-pyrazole was performed, this is based on the study of the blockage of the increase in intracellular [Ca²⁺] observed in response to platelet-activating factor (PAF) treatment of four pyrazoles (i.e. 6, 8, 9 and 10), which successfully displayed [Ca²⁺] channel inhibition. Therefore, the obtained intracellular [Ca²⁺] signal results indicate that the pyrazole family characterized in this study, in particular compounds 6 and 10, are potent blockers of the PAF-initiated Ca²⁺ signaling that mediates the hyperpermeability typically observed during the development of inflammation.