Increase In Cytosolic Calcium Research Articles

Sound waves alter the viability of tobacco cells via changes in cytosolic calcium, membrane integrity, and cell wall composition.

The effect of sound waves (SWs) on plant cells can be considered as important as other mechanical stimuli like touch, wind, rain, and gravity, causing certain responses associated with the downstream signaling pathways on the whole plant. The objective of the present study was to elucidate the response of suspension-cultured tobacco cells (Nicotiana tabacum L. cv Burley 21) to SW at different intensities. The sinusoidal SW (1,000 Hz) was produced through a signal generator, amplified, and beamed to the one layer floating tobacco cells inside a soundproof chamber at intensities of 60, 75, and 90 dB at the plate level for 15, 30, 45, and 60 min. Calibration of the applied SW intensities, accuracy, and uniformity of SW was performed by a sound level meter, and the cells were treated. The effect of SW on tobacco cells was monitored by quantitation of cytosolic calcium, redox status, membrane integrity, wall components, and the activity of wall modifying enzymes. Cytosolic calcium ions increased as a function of sound intensity with a maximum level of 90 dB. Exposure to 90 dB was also accompanied by a significant increase of H2O2 and membrane lipid peroxidation rate but the reduction of total antioxidant and radical scavenging capacities. The increase of wall rigidity in these cells was attributed to an increase in wall-bound phenolic acids and lignin and the activities of phenylalanine ammonia-lyase and covalently bound peroxidase. In comparison, in 60- and 75 dB, radical scavenging capacity increased, and the activity of wall stiffening enzymes reduced, but cell viability showed no changes. The outcome of the current study reveals that the impact of SW on plant cells is started by an increase in cytosolic calcium. However, upon calcium signaling, downstream events, including alteration of H2O2 and cell redox status and the activities of wall modifying enzymes, determined the extent of SW effects on tobacco cells.

Taurine Prevents Angiotensin II-Induced Human Endocardial Endothelium Morphological Remodeling and the Increase in Cytosolic and Nuclear Calcium and ROS.

Endocardial endothelium (EE) is a layer of cells covering the cardiac cavities and modulates cardiomyocyte function. This cell type releases several cardioactive factors, including Angiotensin II (Ang II). This octopeptide is known to induce cardiac hypertrophy. However, whether this circulating factor also induces EE hypertrophy is not known. Taurine is known to prevent cardiac hypertrophy. Whether this endogenous antioxidant prevents the effect of Ang II on human EE (hEE) will be verified. Using quantitative fluorescent probe imaging for calcium and reactive oxygen species (ROS), our results show that Ang II induces (10-7 M, 48 h treatment) an increase in hEE cell (hEEC) volume and its nucleus. Pretreatment with 20 mM of taurine prevents morphological remodeling and increases intracellular calcium and ROS. These results suggest that the reported Ang II induces cardiac hypertrophy is associated with hEEC hypertrophy. This later effect is prevented by taurine by reducing intracellular calcium and ROS overloads. Thus, taurine could be an excellent tool for preventing Ang II-induced remodeling of hEECs.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

Soluble Abeta produces an increase in cytosolic and mitochondrial calcium in astrocytes in vivo, in part by direct effect in astroglial cells

AbstractBackgroundBy using in vivo multiphoton microscopy, we have previously reported that astrocytic cytosolic resting calcium (Ca2+) is globally elevated in a transgenic mouse model of cerebral β‐amyloidosis (APPswe/PS1dE9) (PMID:19251629). However, this phenomenon is independent of the proximity of astrocytes to amyloid β (Aβ) plaques. In addition, soluble Aβ oligomers (Aβo) are thought to mediate neuronal toxicity by increasing neuronal calcium and causing synapse loss. For those reasons, we hypothesized that Aβo, rather than Aβ plaques, contribute to the Ca2+ insult in astroglial cytosol and mitochondria in vivo.MethodTo test this hypothesis, we topically applied naturally secreted soluble Aβo (conditioned medium from cultured transgenic primary neurons) onto the brain surface of 4‐6‐month old wild‐type mice. We investigated changes on astrocyte and mitochondrial Ca2+ levels relative to baseline via intravital multiphoton imaging, by using a ratiometric genetically‐encoded FRET‐based Ca2+ indicator (Yellow Cameleon 3.6) targeted to astroglial cytosol or mitochondria.ResultWe observed that Ca2+ levels dramatically increased upon topical soluble Aβo application, in all cytosolic astrocyte compartments (soma, branches and end feet), but also in astrocyte mitochondria. Aβ‐immunodepleted transgenic conditioned media and wild‐type conditioned media did not alter astrocyte cytosolic or mitochondrial Ca2+, supporting the specificity of the observed effects. Furthermore, we have also seen this effect in cultured astrocytes, suggesting that Aβo is directly interacting with astroglial cells, and not through an effect on other cell types, such as neurons or microglia. In addition, we previously reported that upon blocking the mitochondrial calcium uniporter (MCU) with Ru360, calcium levels in neuronal mitochondria were restored (PMID:32358564). However, there was still an increase of mitochondrial calcium in astrocytes in presence of soluble Aβo and Ru360 in vivo. Thus, soluble Aβo is disrupting calcium levels in astrocytes through a different mechanism.ConclusionUltimately, these results together support a detrimental role of Aβo which leads to astrocyte Ca2+ dyshomeostasis in vivo, implying that Aβo are involved in the astrocytic dysfunction observed in AD. Future studies will continue to explore the source of soluble Aβo‐induced Ca2+ increase, including intracellular Ca2+ stores and channels involved, which could have therapeutic value.

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

Inhibition of Ca2+-permeable TRPV3 and inflammatory cytokine release by honokiol and magnolol in human epidermal keratinocytes

Transient receptor potential vanilloid-3 (TRPV3) ion channels are prominently expressed in keratinocytes, playing a vital role in skin functions. Honokiol and magnolol (H&M) the primary bioactive constituents in Magnolia officinalis extract, demonstrate anti-inflammatory and skin-protective properties. Nevertheless, the underlying mechanism regarding their effect on Ca2+-permeable ion channels remain unclear. Our purpose in this study is to investigate the effect of H&M on TRPV3 and cytokine release in normal human epidermal keratinocytes (NHEKs), including its gain-of-function (GOF) mutants (G573S and G573C) associated with Olmstead syndrome. We performed whole-cell patch-clamp, fura-2 spectrofluorimetry to investigate channels activity, CCK-8 assay to analyze cell death and enzyme-linked immunosorbent assay to assess the cytokine release from NHEKs. H&M inhibited the TRPV3 current (ITRPV3) and cytosolic calcium increase in NHEKs, HEK293T cells overexpressing hTRPV3 and its GOF mutants. Moreover, the release of pro-inflammatory cytokines (interleukin-6 and -8) from keratinocytes stimulated by TRPV3 agonist was effectively suppressed by H&M. Our findings provide insights into the mechanism underlying the anti-inflammatory effects of H&M, highlighting their potential in treating skin diseases.

Isolation and Biological Activity of Iezoside and Iezoside B, SERCA Inhibitors from Floridian Marine Cyanobacteria.

Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside (1) and its C-31 O-demethyl analogue, iezoside B (2), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC50 of 1.5 and 3.0 μΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 μΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC50 values of 0.3 and 0.6 μΜ in A549 cells and 0.1 and 0.5 μΜ, respectively, in HeLa cells, near the IC50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.

Butyrate inhibits LPC-induced endothelial dysfunction by regulating nNOS-produced NO and ROS production

Lipids oxidation is a key risk factor for cardiovascular diseases. Lysophosphatidylcholine (LPC), the major component of oxidized LDL, is an important triggering agent for endothelial dysfunction and atherogenesis. Sodium butyrate, a short-chain fatty acid, has demonstrated atheroprotective properties. So, we evaluate the role of butyrate in LPC-induced endothelial dysfunction. Vascular response to phenylephrine (Phe) and acetylcholine (Ach) was performed in aortic rings from male mice (C57BL/6J). The aortic rings were incubated with LPC (10 μM) and butyrate (0.01 or 0.1 Mm), with or without TRIM (an nNOS inhibitor). Endothelial cells (EA.hy296) were incubated with LPC and butyrate to evaluate nitric oxide (NO) and reactive oxygen species (ROS) production, calcium influx, and the expression of total and phosphorylated nNOS and ERK½. We found that butyrate inhibited LPC-induced endothelial dysfunction by improving nNOS activity in aortic rings. In endothelial cells, butyrate reduced ROS production and increased nNOS-related NO release, by improving nNOS activation (phosphorylation at Ser1412). Additionally, butyrate prevented the increase in cytosolic calcium and inhibited ERk½ activation by LPC. In conclusion, butyrate inhibited LPC-induced vascular dysfunction by increasing nNOS-derived NO and reducing ROS production. Butyrate restored nNOS activation, which was associated with calcium handling normalization and reduction of ERK½ activation.

Changes in K+ Concentration as a Signaling Mechanism in the Apicomplexa Parasites Plasmodium and Toxoplasma.

During their life cycle, apicomplexan parasites pass through different microenvironments and encounter a range of ion concentrations. The discovery that the GPCR-like SR25 in Plasmodium falciparum is activated by a shift in potassium concentration indicates that the parasite can take advantage of its development by sensing different ionic concentrations in the external milieu. This pathway involves the activation of phospholipase C and an increase in cytosolic calcium. In the present report, we summarize the information available in the literature regarding the role of potassium ions during parasite development. A deeper understanding of the mechanisms that allow the parasite to cope with ionic potassium changes contributes to our knowledge about the cell cycle of Plasmodium spp.

Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

S-18-3: SGLT2 INHIBITOR CANAGLIFLOZIN ANTAGONIZES SALT-SENSITIVE HYPERTENSION THROUGH MODIFYING TRPC3 MEDIATED VASCULAR CALCIUM HANDLING

Objectiv: Salt-sensitive hypertension is highly prevalent and associated with cardiorenal damage. Large clinical trials have demonstrated that Sodium-glucose cotransporter 2 (SGLT2) inhibitors exert hypotensive effect and cardiorenal protective benefits in hypertensive patients with and without diabetes. However, the underlying mechanism remains elusive. Methods: Dahl salt-sensitive (SS) rats and salt-insensitive controls were fed with 8% high salt diet to establish salt-sensitive hypertensive model, some of them were treated with canagliflozin. The blood pressure, urinary sodium excretion, vascular function and remodeling were detected. Rat aortic proteomics and TRPC3 knockout mice were used to explain the mechanism of the effect of canagliflozin on salt-sensitive hypertension. Results: Canagliflozin treatment significantly reduced high salt induced hypertension and this effect was not totally dependent on urinary sodium excretion in SS hypertensive rats. Assay of vascular function and proteomics showed that canagliflozin significantly inhibited vascular cytoplasmic calcium increase and vasoconstriction in response to high salt diet. High salt intake increased vascular expression of TRPC3 in salt-sensitive rats, which could be alleviated by canagliflozin treatment. Overexpression of TRPC3 mimicked salt-induced vascular cytosolic calcium increase in vitro and knockout of TRPC3 erased the anti-hypertensive effect of canagliflozin. Mechanistically, high-salt-induced activation of sodium/calcium exchanger 1 (NCX1) reverse mode increased cytoplasmic calcium level and vasoconstriction, which required TRPC3, and this process could be blocked by canagliflozin. Conclusions: We define a previously unrecognized role of TRPC3/NCX1 mediated vascular calcium dysfunction in the development of high salt induced hypertension, which can be improved by canagliflozin treatment. This pathway is potentially a novel therapeutic target to antagonize salt sensitive hypertension.

376 CARDIAC CONTRACTILITY MODULATION: A NEW THERAPY FOR HEART FAILURE

Abstract Introduction Cardiac Contractility Modulation is a therapy for chronic Heart Failure patients with EF 25-45% in sinus rhythm with QRS < 130 ms clinically symptomatic (NYHA Class > II) despite optimised medical therapy. It is made possible by the implantation of the OPTIMIZER SMART® device. The CCM works by delivering a non-excitatory high-energy bipolar signal, synchronised with local electrical activity (or cooperating with another pre-existing CIED), in the absolute ventricular refractory period, by means of two actively fixating leads, positioned in the interventricular septum and spaced at least 1-2 cm apart. Purpose Assessment and feedback of the CCM therapy process in patients with dilated cardiomyopathy. Educational update for health professionals. Patient education. Method A Pubmed search correlated with clinical data and field experience was performed. Results Patients with severe Dilated Cardiomyopathy already on optimised medical therapy. The mechanism of action demonstrated a remodelling of the left ventricle with a so-called 'metabolic' therapy. In CCM therapy, electrical stimulation is delivered to the heart muscle during the absolute refractory period. In this phase, electrical signals activate the mobilisation of calcium ions in cardiomyocytes. CCM signals applied during the absolute refractory period cause an increase in cytosolic calcium during systole, resulting in improved cardiac contraction without an increase in oxygen consumption. Conclusions CCM therapy has shown to improve contractility and cardiac performance improving the quality of life of patients undergoing this treatment by reducing symptoms of fatigue and dyspnoea; it has reduced the number of hospitalisations for moderate to severe heart failure. Healthcare professionals recognise The typical ECG of a patient implanted with a CCM device shows a 'spike' in the absolute refractory period of the cardiac cycle: the 'R wave' of the QRS complex without being alarmed. The patient has been trained to be autonomous in charging. OPTIMIZER SMART® is recharged externally with a weekly rechargeable battery through a mini induction charger, rechargeable by itself.

Neuronal Calcium Sensor-1 Protects Cortical Neurons from Hyperexcitation and Ca2+ Overload during Ischemia by Protecting the Population of GABAergic Neurons.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

Autophagy Mediates Escherichia Coli-Induced Cellular Inflammatory Injury by Regulating Calcium Mobilization, Mitochondrial Dysfunction, and Endoplasmic Reticulum Stress.

Bovine endometritis is a reproductive disorder that is induced by mucus or purulent inflammation of the uterine mucosa. However, the intracellular control chain during inflammatory injury remains unclear. In the present study, we found that E. coli activated the inflammatory response through the assembly of the NLRP3 inflammasome and activation of the NF-κB p65 subunit in primary bovine endometrial epithelial cells (bEECs). Infection with E. coli also led to an abnormal increase in cytoplasmic calcium and mitochondrial dysfunction. Additionally, live-cell imaging of calcium reporters indicated that the increase in cytosolic calcium mainly was caused by the release of Ca2+ ions stored in the ER and mitochondria, which was independent of extracellular calcium. Cytoplasmic calcium regulates mitochondrial respiratory chain transmission, DNA replication, and biogenesis. Pretreatment with NAC, BAPTA-AM, or 2-APB reduced the expression of IL-1β and IL-18. Moreover, ERS was involved in the regulation of bovine endometritis and cytosolic calcium was an important factor for regulating ERS in E. coli-induced inflammation. Finally, activation of autophagy inhibited the release of IL-1β and IL-18, cytochrome c, ATP, ERS-related proteins, and cytoplasmic calcium. Collectively, our findings demonstrate that autophagy mediated E. coli-induced cellular inflammatory injury by regulating cytoplasmic calcium, mitochondrial dysfunction, and ERS.

A mathematical model of calcium signals around laser-induced epithelial wounds.

Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound-healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium-signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. Here we have developed a mathematical model in the context of laser ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium-signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium-signaling events observed in experiments; it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP3 is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed here serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.

Kinetin induces microtubular breakdown, cell cycle arrest and programmed cell death in tobacco BY-2 cells

Plant cells can undergo regulated cell death in response to exogenous factors (often in a stress context), but also as regular element of development (often regulated by phytohormones). The cellular aspects of these death responses differ, which implies that the early signalling must be different. We use cytokinin-induced programmed cell death as paradigm to get insight into the role of the cytoskeleton for the regulation of developmentally induced cell death, using tobacco BY-2 cells as experimental model. We show that this PCD in response to kinetin correlates with an arrest of the cell cycle, a deregulation of DNA replication, a loss of plasma membrane integrity, a subsequent permeabilisation of the nuclear envelope, an increase of cytosolic calcium correlated with calcium depletion in the culture medium, an increase of callose deposition and the loss of microtubule and actin integrity. We discuss these findings in the context of a working model, where kinetin, mediated by calcium, causes the breakdown of the cytoskeleton, which, either by release of executing proteins or by mitotic catastrophe, will result in PCD.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

RationaleSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. ObjectivesWe examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. MethodsWe used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. ResultsSARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. ConclusionsCoronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Age-dependent effect of insulin in the regulation of intracellular calcium in ventricular cardiomyocytes.

In this study, we wanted to verify whether the effect of insulin on calcium homeostasis depends on the heart's development stage. Using a quantitative 3D confocal microscopy, we tested the effect of a high insulin concentration (100 µU) in freshly cultured ventricular cardiomyocytes from newborn and adult rats. Our results showed that the cytosolic basal level of calcium was higher in newborn cardiomyocytes with no change in the nuclear basal calcium level compared with the adult cardiomyocytes; in addition, insulin induced a slow increase of cytosolic and nuclear calcium in newborn ventricular cardiomyocytes, followed by two phases. However, the first phase of slow cytosolic and nuclear calcium increase was absent in adult rat ventricular cardiomyocytes. Furthermore, the time to the onset of increase of cytosolic and nuclear calcium was longer in newborn cardiomyocytes compared with adults. Moreover, the time to peak of the calcium transient was shorter in newborns than in adult cardiomyocytes. These results demonstrate that insulin differently regulates calcium homeostasis in newborns than in adult cardiomyocytes. Thus, newborn rat cardiomyocytes, commonly used in research as a model for adult cardiomyocytes, should be used with caution when dealing with insulin in normal and disease conditions.

Dynamic in Situ Confinement Triggers Ligand-Free Neuropeptide Receptor Signaling.

Membrane receptor clustering is fundamental to cell–cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.