NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Coupled action potential and calcium dynamics underlie robust spontaneous firing in dopaminergic neurons.

Dopaminergic neurons are specialized cells in the substantia nigra, tasked with dopamine secretion. This secretion relies on intracellular calcium signaling coupled to neuronal electrical activity. These neurons are known to display spontaneous calcium oscillationsin-vitroandin-vivo, even in synaptic isolation, controlling the basal dopamine levels. Here we outline a kinetic model for the ion exchange across the neuronal plasma membrane. Crucially, we relax the assumption of constant, cytoplasmic sodium and potassium concentration. We show that sodium-potassium dynamics are strongly coupled to calcium dynamics and are essential for the robustness of spontaneous firing frequency. The model predicts several regimes of electrical activity, including tonic and 'burst' oscillations, and predicts the switch between those in response to perturbations. 'Bursting' correlates with increased calcium amplitudes, while maintaining constant average, allowing for a vast change in the calcium signal responsible for dopamine secretion. All the above traits provide the flexibility to create rich action potential dynamics that are crucial for cellular function.

Time-frequency cross-coupling between cortical low-frequency neuronal calcium oscillations and blood oxygen metabolism evoked by ultrasound stimulation.

Low-intensity transcranial ultrasound stimulation (TUS) can modulate the coupling of high-frequency (160-200 Hz) neural oscillations and cerebral blood oxygen metabolism (BOM); however, the correlation of low-frequency (0-2 Hz) neural oscillations with BOM in temporal and frequency domains under TUS remains unclear. To address this, we monitored the TUS-evoked neuronal calcium oscillations and BOM simultaneously in the mouse visual cortex by using multimodal optical imaging with a high spatiotemporal resolution. We demonstrated that TUS can significantly increase the intensity of the neuronal calcium oscillations and BOM; the peak value, peak time, and duration of calcium oscillations are functionally related to stimulation duration; TUS does not significantly increase the neurovascular coupling strength between calcium oscillations and BOM in the temporal domain; the time differences of the energy peaks between TUS-induced calcium oscillations and BOM depend on their spectral ranges; the frequency differences of the energy peaks between TUS-induced calcium oscillations and BOM depend on their time ranges; and TUS can significantly change the phase of calcium oscillations and BOM from uniform distribution to a more concentrated region. In conclusion, ultrasound stimulation can evoke the time-frequency cross-coupling between the cortical low-frequency neuronal calcium oscillations and BOM in mouse.

Improved bounds on entropy production in living systems.

Living systems maintain or increase local order by working against the second law of thermodynamics. Thermodynamic consistency is restored as they consume free energy, thereby increasing the net entropy of their environment. Recently introduced estimators for the entropy production rate have provided major insights into the efficiency of important cellular processes. In experiments, however, many degrees of freedom typically remain hidden to the observer, and, in these cases, existing methods are not optimal. Here, by reformulating the problem within an optimization framework, we are able to infer improved bounds on the rate of entropy production from partial measurements of biological systems. Our approach yields provably optimal estimates given certain measurable transition statistics. In contrast to prevailing methods, the improved estimator reveals nonzero entropy production rates even when nonequilibrium processes appear time symmetric and therefore may pretend to obey detailed balance. We demonstrate the broad applicability of this framework by providing improved bounds on the energy consumption rates in a diverse range of biological systems including bacterial flagella motors, growing microtubules, and calcium oscillations within human embryonic kidney cells.

The Multifaceted Functions of TRPV4 and Calcium Oscillations in Tissue Repair.

The transient receptor potential vanilloid 4 (TRPV4) specifically functions as a mechanosensitive ion channel and is responsible for conveying changes in physical stimuli such as mechanical stress, osmotic pressure, and temperature. TRPV4 enables the entry of cation ions, particularly calcium ions, into the cell. Activation of TRPV4 channels initiates calcium oscillations, which trigger intracellular signaling pathways involved in a plethora of cellular processes, including tissue repair. Widely expressed throughout the body, TRPV4 can be activated by a wide array of physicochemical stimuli, thus contributing to sensory and physiological functions in multiple organs. This review focuses on how TRPV4 senses environmental cues and thereby initiates and maintains calcium oscillations, critical for responses to organ injury, tissue repair, and fibrosis. We provide a summary of TRPV4-induced calcium oscillations in distinct organ systems, along with the upstream and downstream signaling pathways involved. In addition, we delineate current animal and disease models supporting TRPV4 research and shed light on potential therapeutic targets for modulating TRPV4-induced calcium oscillation to promote tissue repair while reducing tissue fibrosis.

Ca2+ release or Ca2+ entry, that is the question: what governs Ca2+ oscillations in pancreatic ß cells?

The standard model for Ca2+ oscillations in insulin-secreting pancreatic ß cells centers on Ca2+ entry through voltage-activated Ca2+ channels. These work in combination with ATP-dependent K+ channels, which are the bridge between the metabolic state of the cells and plasma membrane potential. This partnership underlies the ability of the ß cells to secrete insulin appropriately on a minute-to-minute time scale to control whole body plasma glucose. Though this model, developed over more than 40 years through many cycles of experimentation and mathematical modeling, has been very successful, it has been challenged by a hypothesis that calcium-induced calcium release from the endoplasmic reticulum through ryanodine or inositol trisphosphate (IP3) receptors is instead the key driver of islet oscillations. We show here that the alternative model is in fact incompatible with a large body of established experimental data and that the new observations offered in support of it can be better explained by the standard model.

SKF96365 modulates activity of CatSper channels in human sperm.

Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.

Extracellular ATP-induced calcium oscillations regulating the differentiation of osteoblasts through aerobic oxidation metabolism pathways.

INTRODUCTION: The increase of ATP concentration in the extracellular space represents one of the effective signals that stimulate the physiological activities of cells when the bone is exposed to external mechanical stimulation such as stretching and shear stress force throughout life. However, the effects of ATP on osteoblast differentiation and related mechanisms are not well understood. MATERIALS AND METHODS: In this study, the roles of extracellular ATP on osteoblast differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. RESULTS: Our results showed that 100 µM extracellular ATP initiated intracellular calcium ([Ca2+]i) oscillations via the calcium-sensing receptor (P2R) and promoted the differentiation of MC3T3-E1 cells. Metabolomics analysis showed that the differentiation of MC3T3-E1 cells depended on aerobic oxidation, but little glycolysis. Moreover, the differentiation of MC3T3-E1 cells and aerobic oxidation were suppressed with the inhibition of AMP-activated protein kinase (AMPK). CONCLUSION: These results indicate that calcium oscillations triggered by extracellular ATP can activate aerobic oxidation through AMPK-related signaling pathways and thus promote osteoblast differentiation.

Endoplasmic reticulum in oocytes: spatiotemporal distribution and function.

ENDOPLASMIC RETICULUM IN OOCYTES: The storage and release of calcium ions (Ca2 +) in oocyte maturation and fertilization are particularly noteworthy features of the endoplasmic reticulum (ER). The ER is the largest organelle in the cell composed of rough ER, smooth ER, and nuclear envelope, and is the main site of protein synthesis, transport and folding, and lipid and steroid synthesis. An appropriate calcium signaling response can initiate oocyte development and embryogenesis, and the ER is the central link that initiates calcium signaling. The transition from immature oocytes to zygotes also requires many coordinated organelle reorganizations and changes. Therefore, the purpose of this review is to generalize information on the function, structure, interaction with other organelles, and spatiotemporal localization of the ER in mammalian oocytes. Mechanisms related to maintaining ER homeostasis have been extensively studied in recent years. Resolving ER stress through the unfolded protein response (UPR) is one of them. We combined the clinical problems caused by the ER in in vitro maturation (IVM), and the mechanisms of ER have been identified by single-cell RNA-seq. This article systematically reviews the functions of ER and provides a reference for assisted reproductive technology (ART) research.

The Mitochondrial Ca2+ uniporter is a central regulator of interorganellar Ca2+ transfer and NFAT activation.

Mitochondrial Ca2+ uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release-activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.

Spontaneous Ca2+ events are linked to the development of neuronal firing during maturation in mice primary hippocampal culture cells.

Calcium is one of the most vital intracellular secondary messengers that tightly regulates a variety of cell physiology processes, especially in the brain. Using a fluorescent Ca2+-sensitive Oregon Green probe, we revealed three different amplitude distributions of spontaneous Ca2+ events (SCEs) in neurons between 15 and 26 days in vitro (DIV) culture maturation. We detected a series of amplitude events: micro amplitude SCE (microSCE) 25% increase from the baseline, intermediate amplitude SCE (interSCE) as 25-75%, and macro amplitude SCE (macroSCE) - over 75%. The SCEs were fully dependent on extracellular Ca2+ and neuronal network activity and vanished in the Ca2+-free solution, 10 mM Mg2+-block, or in the presence of voltage-gated Na+-channel blocker, tetrodotoxin. Combined patch-clamp and Ca2+-imaging techniques revealed that microSCE match single action potential (AP), interSCE - burst of 3-12 APs, and macroSCE - 'superburst' of 10+ APs. MicroSCEs were blocked by a common α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) receptor antagonist, CNQX. The γ-aminobutyric acid (GABA) A-type receptor (GABAAR) picrotoxin blockade and L-type voltage-dependent Ca2+-channel inhibitor diltiazem significantly reduced microSCE frequency. InterSCEs were inhibited by CNQX, but picrotoxin treatment significantly increased its amplitude. The N-methyl-d-aspartate (NMDA) receptor antagonist, D-APV, voltage-gated K+-channel blocker, tetraethylammonium, noticeably suppressed interSCE amplitude. We also demonstrate that macroSCEs were AMPA/KA receptor-independent.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

Constitutive IP3R1-mediated Ca2+ release reduces Ca2+ store content and stimulates mitochondrial metabolism in mouse GV oocytes.

In mammals, fertilization initiates Ca2+ oscillations in metaphase II oocytes, which are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca2+ oscillations, although these unfold spontaneously in the absence of any known agonist(s) and their function remains unclear. We found that the main intracellular store of Ca2+ in GV oocytes, the endoplasmic reticulum ([Ca2+]ER), constitutively 'leaks' Ca2+ through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca2+]ER leak ceases around the resumption of meiosis, the GV breakdown (GVBD) stage, which coincides with the first noticeable accumulation of Ca2+ in the stores. It also concurs with downregulation of the Ca2+ influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca2+ channels. Lastly, we demonstrate that mitochondria take up Ca2+ during the Ca2+ oscillations, mounting their own oscillations that stimulate the mitochondrial redox state and increase the ATP levels of GV oocytes. These distinct features of Ca2+ homeostasis in GV oocytes are likely to underpin the acquisition of both maturation and developmental competence, as well as fulfill stage-specific cellular functions during oocyte maturation.

Research progress in the mechanism of calcium ion on contraction and relaxation of airway smooth muscle cells.

lntracellular calcium ion is the key secondary messenger system of the cellular processes in airway smooth muscle cells(ASMc). The treatment and regulation of Ca2+ in airway smooth muscle (ASM) is, in part, to associated with many airway diseases such as asthma, COPD and pulmonary fibrosis. The mechanism of contraction and relaxation of ASM is a concerned aspect in airway diseases. This review emphasizes established and recent discoveries whice show the research progress of Ca2+ on cell contraction and relaxation in ASM in recent years, to provide theoretical support and new targets for clinical prevention and treatment of perioperative bronchospasm and variousrespiratory related diseases.

A Tale of two receptors.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Calcium Dynamics and Water Transport in Salivary Acinar Cells.

Saliva is secreted from the acinar cells of the salivary glands, using mechanisms that are similar to other types of water-transporting epithelial cells. Using a combination of theoretical and experimental techniques, over the past 20 years we have continually developed and modified a quantitative model of saliva secretion, and how it is controlled by the dynamics of intracellular calcium. However, over approximately the past 5 years there have been significant developments both in our understanding of the underlying mechanisms and in the way these mechanisms should best be modelled. Here, we review the traditional understanding of how saliva is secreted, and describe how our work has suggested important modifications to this traditional view. We end with a brief description of the most recent data from living animals and discuss how this is now contributing to yet another iteration of model construction and experimental investigation.

Maturation conditions, post-ovulatory age, medium pH, and ER stress affect [Ca2+]i oscillation patterns in mouse oocytes.

Insufficiency of oocyte activation impairs the subsequent embryo development in assisted reproductive technology (ART). Intracellular Ca2+ concentration ([Ca2+]i) oscillations switch the oocytes to resume the second meiosis and initiate embryonic development. However, the [Ca2+]i oscillation patterns in oocytes are poorly characterized. In this study, we investigated the effects of various factors, such as the oocytes age, pH, cumulus cells, in vitro or in vivo maturation, and ER stress on [Ca2+]i oscillation patterns and pronuclear formation after parthenogenetic activation of mouse oocytes. Our results showed that the oocytes released to the oviduct at 17 h post-human chorionic gonadotrophin (hCG) displayed a significantly stronger [Ca2+]i oscillation, including higher frequency, shorter cycle, and higher peak, compared with oocytes collected at earlier or later time points. [Ca2+]i oscillations in acidic conditions (pH 6.4 and 6.6) were significantly weaker than those in neutral and mildly alkaline conditions (pH from 6.8 to 7.6). In vitro-matured oocytes showed reduced frequency and peak of [Ca2+]i oscillations compared with those matured in vivo. In vitro-matured oocytes from the cumulus-oocyte complexes (COCs) showed a significantly higher frequency, shorter cycle, and higher peak compared with the denuded oocytes (DOs). Finally, endoplasmic reticulum stress (ER stress) severely affected the parameters of [Ca2+]i oscillations, including elongated cycles and lower frequency. The pronuclear (PN) rate of oocytes after parthenogenetic activation was correlated with [Ca2+]i oscillation pattern, decreasing with oocyte aging, cumulus removal, acidic pH, and increasing ER stress. These results provide fundamental but critical information for the mechanism of how these factors affect oocyte activation.

Emerging Role of TCA Cycle-Related Enzymes in Human Diseases.

The tricarboxylic acid (TCA) cycle is the main source of cellular energy and participates in many metabolic pathways in cells. Recent reports indicate that dysfunction of TCA cycle-related enzymes causes human diseases, such as neurometabolic disorders and tumors, have attracted increasing interest in their unexplained roles. The diseases which develop as a consequence of loss or dysfunction of TCA cycle-related enzymes are distinct, suggesting that each enzyme has a unique function. This review aims to provide a comprehensive overview of the relationship between each TCA cycle-related enzyme and human diseases. We also discuss their functions in the context of both mitochondrial and extra-mitochondrial (or cytoplasmic) enzymes.

Computational Analysis of Ca2+ Oscillatory Bio-Signals: Two-Parameter Bifurcation Diagrams.

Two types of bifurcation diagrams of cytosolic calcium nonlinear oscillatory systems are presented in rectangular areas determined by two slowly varying parameters. Verification of the periodic dynamics in the two-parameter areas requires solving the underlying model a few hundred thousand or a few million times, depending on the assumed resolution of the desired diagrams (color bifurcation figures). One type of diagram shows period-n oscillations, that is, periodic oscillations having n maximum values in one period. The second type of diagram shows frequency distributions in the rectangular areas. Each of those types of diagrams gives different information regarding the analyzed autonomous systems and they complement each other. In some parts of the considered rectangular areas, the analyzed systems may exhibit non-periodic steady-state solutions, i.e., constant (equilibrium points), oscillatory chaotic or unstable solutions. The identification process distinguishes the later types from the former one (periodic). Our bifurcation diagrams complement other possible two-parameter diagrams one may create for the same autonomous systems, for example, the diagrams of Lyapunov exponents, Ls diagrams for mixed-mode oscillations or the 0-1 test for chaos and sample entropy diagrams. Computing our two-parameter bifurcation diagrams in practice and determining the areas of periodicity is based on using an appropriate numerical solver of the underlying mathematical model (system of differential equations) with an adaptive (or constant) step-size of integration, using parallel computations. The case presented in this paper is illustrated by the diagrams for an autonomous dynamical model for cytosolic calcium oscillations, an interesting nonlinear model with three dynamical variables, sixteen parameters and various nonlinear terms of polynomial and rational types. The identified frequency of oscillations may increase or decrease a few hundred times within the assumed range of parameters, which is a rather unusual property. Such a dynamical model of cytosolic calcium oscillations, with mitochondria included, is an important model in which control of the basic functions of cells is achieved through the Ca2+ signal regulation.

Endoplasmic reticulum stress alters ryanodine receptor function in the murine pancreatic ß cell.

Alterations in endoplasmic reticulum (ER) calcium (Ca2+) levels diminish insulin secretion and reduce ß-cell survival in both major forms of diabetes. The mechanisms responsible for ER Ca2+ loss in ß cells remain incompletely understood. Moreover, a specific role for either ryanodine receptor (RyR) or inositol 1,4,5-triphosphate receptor (IP3R) dysfunction in the pathophysiology of diabetes remains largely untested. To this end, here we applied intracellular and ER Ca2+ imaging techniques in INS-1 ß cells and isolated islets to determine whether diabetogenic stressors alter RyR or IP3R function. Our results revealed that the RyR is sensitive mainly to ER stress-induced dysfunction, whereas cytokine stress specifically alters IP3R activity. Consistent with this observation, pharmacological inhibition of the RyR with ryanodine and inhibition of the IP3R with xestospongin C prevented ER Ca2+ loss under ER and cytokine stress conditions, respectively. However, RyR blockade distinctly prevented ß-cell death, propagation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin-treated INS-1 ß cells and mouse islets and Akita islets. Monitoring at the single-cell level revealed that ER stress acutely increases the frequency of intracellular Ca2+ transients that depend on both ER Ca2+ leakage from the RyR and plasma membrane depolarization. Collectively, these findings indicate that RyR dysfunction shapes ER Ca2+ dynamics in ß cells and regulates both UPR activation and cell death, suggesting that RyR-mediated loss of ER Ca2+ may be an early pathogenic event in diabetes.