Calcium Signaling in the Photodamaged Skin: In Vivo Experiments and Mathematical Modeling

Function ◽

10.1093/function/zqab064 ◽

2021 ◽

Author(s):

Viola Donati ◽

Chiara Peres ◽

Chiara Nardin ◽

Ferdinando Scavizzi ◽

Marcello Raspa ◽

...

Keyword(s):

Mathematical Modeling ◽

Wave Propagation ◽

Atp Release ◽

P2y Receptors ◽

Extracellular Atp ◽

In Vivo Experiments ◽

Photodamaged Skin ◽

Bystander Cells ◽

Skin Conditions

Abstract The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration (${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ oscillations.

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00438.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C569-C578 ◽

Cited By ~ 91

Author(s):

Alexander A. Mongin ◽

Harold K. Kimelberg

Keyword(s):

Amino Acid ◽

Excitatory Amino Acid ◽

Atp Release ◽

Anion Channel ◽

P2y Receptors ◽

Cell Swelling ◽

Channel Blockers ◽

Medium Osmolarity ◽

Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

Dynamic regulation of extracellular ATP in Escherichia coli

Biochemical Journal ◽

10.1042/bcj20160879 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1395-1416 ◽

Cited By ~ 7

Author(s):

Cora Lilia Alvarez ◽

Gerardo Corradi ◽

Natalia Lauri ◽

Irene Marginedas-Freixa ◽

María Florencia Leal Denis ◽

...

Keyword(s):

Escherichia Coli ◽

Atpase Activity ◽

Atp Release ◽

Membrane Vesicles ◽

Fold Increase ◽

Extracellular Atp ◽

Outer Membrane Vesicles ◽

Dynamic Regulation ◽

E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

Effect of ATP on preadipocyte migration and adipocyte differentiation by activating P2Y receptors in 3T3-L1 cells

Biochemical Journal ◽

10.1042/bj20051037 ◽

2005 ◽

Vol 393 (1) ◽

pp. 171-180 ◽

Cited By ~ 33

Author(s):

Mariko Omatsu-Kanbe ◽

Kazuko Inoue ◽

Yusuke Fujii ◽

Takefumi Yamamoto ◽

Takahiro Isono ◽

...

Keyword(s):

Cell Line ◽

Adipocyte Differentiation ◽

P2y Receptors ◽

Extracellular Atp ◽

Dependent Manner ◽

Fat Cell ◽

Proliferating Cells ◽

Concentration Dependent Manner ◽

Membrane Ruffling

The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1–100 μM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

eLife ◽

10.7554/elife.57544 ◽

2020 ◽

Vol 9 ◽

Author(s):

Nami Kitajima ◽

Kenji Takikawa ◽

Hiroshi Sekiya ◽

Kaname Satoh ◽

Daisuke Asanuma ◽

...

Keyword(s):

In Vivo Imaging ◽

Fluorescent Sensor ◽

Atp Release ◽

Extracellular Atp ◽

Cysteine Residue ◽

Spatiotemporal Resolution ◽

Extracellular Signaling ◽

Ratiometric Measurement ◽

Atp Binding Protein

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Influence of the vessel shape and the equation of state on the formation of a pulse wave in the 1D hemodynamics model

Mathematical Modelling of Natural Phenomena ◽

10.1051/mmnp/2018054 ◽

2018 ◽

Vol 13 (5) ◽

pp. 46

Author(s):

Svetamira G. Davydova ◽

Ilya N. Kiselev ◽

Elina A. Biberdorf

Keyword(s):

Mathematical Modeling ◽

Experimental Data ◽

Wave Propagation ◽

Equation Of State ◽

Boundary Conditions ◽

Pulse Wave ◽

Filtration Coefficient

This paper is devoted to the problem of adequate mathematical modeling of the pulse wave using a 1D hemodynamics model. This is possible provided that the conditions of wave propagation and reflections are correctly established and translated into the model. We analyze how the propagation of the forward and backward waves in the model is affected by the boundary conditions, the choice of the filtration coefficient at the ends of terminal arteries, and the modeled vessel geometry. We also propose an equation of state based on experimental data acquired in vivo.

Erythrocytes: Oxygen Sensors and Modulators of Vascular Tone

Physiology ◽

10.1152/physiol.00038.2008 ◽

2009 ◽

Vol 24 (2) ◽

pp. 107-116 ◽

Cited By ~ 175

Author(s):

Mary L. Ellsworth ◽

Christopher G. Ellis ◽

Daniel Goldman ◽

Alan H. Stephenson ◽

Hans H. Dietrich ◽

...

Keyword(s):

Vascular Tone ◽

Computational Models ◽

Control Mechanism ◽

Atp Release ◽

Oxygen Demand ◽

Oxygen Sensors ◽

Feedback Mechanisms ◽

In Vivo Experiments ◽

The Impact

Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.

An ultrasensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo

10.1101/2021.02.24.432680 ◽

2021 ◽

Author(s):

Zhaofa Wu ◽

Kaikai He ◽

Yue Chen ◽

Hongyu Li ◽

Sunlei Pan ◽

...

Keyword(s):

Hippocampal Neurons ◽

Atp Release ◽

Cell Types ◽

Extracellular Atp ◽

Zebrafish Model ◽

Mouse Cortex ◽

Versatile Tool ◽

Subcellular Resolution

SUMMARYThe purinergic transmitter ATP (adenosine 5’-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. We developed an ultrasensitive GPCRActivation‒Based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including primary hippocampal neurons, a zebrafish model of injury-induced ATP release, and LPS-induced ATP-release events in individual astrocytes in the mouse cortex measured using in vivo two-photon imaging. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

P2X-GCaMPs as versatile tools for imaging extracellular ATP signaling

10.1101/2020.04.23.057513 ◽

2020 ◽

Author(s):

Matthias Ollivier ◽

Juline Beudez ◽

Nathalie Linck ◽

Thomas Grutter ◽

Vincent Compan ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Atp Release ◽

Cell Types ◽

Extracellular Atp ◽

P2x Receptor ◽

High Signal ◽

Calcium Sensors ◽

Extracellular Signaling

AbstractAdenosine 5’ triphosphate (ATP) is an extracellular signaling molecule involved in numerous physiological and pathological processes. Yet, in situ characterization of the spatiotemporal dynamic of extracellular ATP is still challenging due to the lack of sensor with appropriate specificity, sensitivity and kinetics. Here we report the development of biosensors based on the fusion of cation permeable ATP receptors (P2X) to genetically encoded calcium sensors (GECI). By combining the features of P2X receptors with the high signal to noise ratio of GECIs, we generated ultrasensitive green and red fluorescent sniffers that detect nanomolar ATP concentrations in situ and also enable the tracking of P2X receptor activity. We provide the proof of concept that these sensors can dynamically track ATP release evoked by neuronal depolarization or by extracellular hypotonicity. Targeting these P2X-based biosensors to diverse cell types should advance our knowledge of extracellular ATP dynamics in vivo.

Calcium signaling in the photodamaged skin

10.1101/2021.05.14.443726 ◽

2021 ◽

Author(s):

Viola Donati ◽

Chiara Peres ◽

Chiara Nardin ◽

Ferdinando Scavizzi ◽

Marcello Raspa ◽

...

Keyword(s):

Wave Propagation ◽

Plasma Membrane ◽

Laser Radiation ◽

Connexin 43 ◽

Membrane Integrity ◽

Basal Layer ◽

P2y Receptors ◽

Receptor Activation ◽

Photodamaged Skin ◽

Molecular Components

BACKGROUND: The mammalian skin, the body's largest single organ, is a highly organized tissue that forms an essential barrier against dehydration, pathogens, light and mechanical injury. Damage triggers perturbations of the cytosolic free Ca2+ concentration ([Ca2+]c) that spread from cell to cell (known as intercellular Ca2+ waves) in different epithelia, including epidermis. Ca2+ waves are considered a fundamental mechanism for coordinating multicellular responses, however the mechanisms underlying their propagation in the damaged epidermis are incompletely understood. AIM OF THE PROJECT: To dissect the molecular components contributing to Ca2+ wave propagation in murine model of epidermal photodamage. METHODS: To trigger Ca2+ waves, we used intense and focused pulsed laser radiation and targeted a single keratinocyte of the epidermal basal layer in the earlobe skin of live anesthetized mice. To track photodamage-evoked Ca2+ waves, we performed intravital multiphoton microscopy in transgenic mice with ubiquitous expression of the sensitive and selective Ca2+ biosensor GCaMP6s. To dissect the molecular components contributing to Ca2+ wave propagation, we performed in vivo pharmacological interference experiments by intradermal microinjection of different drugs. EXPERIMENTAL RESULTS: The major effects of drugs that interfere with degradation of extracellular ATP or P2 purinoceptors suggest that Ca2+ waves in the photodamaged epidermis are primarily due to release of ATP from the target cell, whose plasma membrane integrity was compromised by laser irradiation. The limited effect of the Connexin 43 (Cx43) selective inhibitor TAT-Gap19 suggests ATP-dependent ATP release though connexin hemichannels (HCs) plays a minor role, affecting Ca2+ wave propagation only at larger distances, where the concentration of ATP released from the photodamaged cell was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases. The ineffectiveness of probenecid suggests pannexin channels have no role. As GCaMP6s signals in bystander keratinocytes were augmented by exposure to the Ca2+ chelator EGTA in the extracellular medium, the corresponding transient increments of the [Ca2+]c should be ascribed primarily to Ca2+ release from the ER, downstream of ATP binding to P2Y purinoceptors, with Ca2+ entry through plasma membrane channels playing a comparatively negligible role. The effect of thapsigargin (a well-known inhibitor of SERCA pumps) and carbenoxolone (a recently recognized inhibitor of Ca2+ release through IP3 receptors) support this conclusion. CONCLUSIONS: The one presented here is an experimental model for accidental skin injury that may also shed light on the widespread medical practice of laser skin resurfacing, used to treat a range of pathologies from photodamage and acne scars to hidradenitis suppurativa and posttraumatic scarring from basal cell carcinoma excision. The results of our experiments support the notion that Ca2+ waves reflect chiefly the sequential activation of bystander keratinocytes by the ATP released through the compromised plasma membrane of the cell hit by laser radiation. We attributed the observed increments of the [Ca2+]c chiefly to signal transduction through purinergic P2Y receptors. Several studies have highlighted fundamental roles of P2Y receptors during inflammatory and infectious diseases, and the initial phase of wound healing involves acute inflammation. In addition, hyaluronan is a major component of the extracellular matrix and its synthesis is rapidly upregulated after tissue wounding via P2Y receptor activation. It is tempting to speculate that response coordination after injury in the epidermis occurs via propagation of the ATP-dependent intercellular Ca2+ waves described in this work.

Airway Exposure to Polyethyleneimine Nanoparticles Induces Type 2 Immunity by a Mechanism Involving Oxidative Stress and ATP Release

International Journal of Molecular Sciences ◽

10.3390/ijms22169071 ◽

2021 ◽

Vol 22 (16) ◽

pp. 9071

Author(s):

Yotesawee Srisomboon ◽

Noriyuki Ohkura ◽

Koji Iijima ◽

Takao Kobayashi ◽

Peter J. Maniak ◽

...

Keyword(s):

Oxidative Stress ◽

Immune Responses ◽

Atp Release ◽

Bronchial Epithelial ◽

Pannexin 1 ◽

In Vivo Experiments ◽

Atp Efflux ◽

Deficient Mice

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 μM), the VDAC-1 inhibitor, DIDS (100 μM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 μM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.