Intracellular Calcium Signaling Research Articles

Vitamin D in cardiac fibroblasts: new evidence for a TRP-mediated non-genomic pathway

Abstract Background/Introduction Vitamin D’s (VD) role transcends phosphocalcic metabolism to affect different systems, notably the cardiovascular system in health and diseases. Beyond its genomic effect, VD act through non-genomic calcium (Ca2+) signaling pathways, hinting at a broader spectrum of physiological impacts. Despite the established interactions with cardiomyocytes via the Vitamin D Receptor (VDR), little is known about VD and cardiac fibroblast, a pivotal cell player in cardiac function and disease. Purpose This research aims to reveal Vitamin D's role in modulating cardiac fibroblast function through Transient Receptor Potential (TRP) channels. Methods Utilizing primary cardiac fibroblasts from human and rats, our investigation focused on intracellular calcium signaling. Through Ca2+ imaging techniques combined to synthetic fluorescent VD analogue, we explored Vitamin D's behavior within the cell. The study further employed biochemical assays to elucidate the interaction between VDR on the cell membrane and TRP channels signaling, providing a detailed map of the cellular choreography orchestrated by VD within cardiac fibroblasts. In addition, in vitro and ex vivo heart perfusion experiments were incorporated to assess cardiac cGMP production in response to VD. Results Our findings reveal a unique pathway wherein VD induces Ca2+ oscillations in human and rat cardiac fibroblasts specifically through the TRPC3 channel, sidelining other calcium sources. This process is regulated by the interaction between TRPC3 and the membrane-bound VDR, harmonizing with phospholipase C signaling to a crescendo of cGMP production in cardiac fibroblasts, and across the entire heart. This was associated with less pro-fibrotic potential of cardiac fibroblasts. Conclusion This study provides the first evidence of a TRP-mediated, VD non-genomic pathway within human and rat cardiac fibroblasts. Our findings herald a promising horizon for investigating vitamin D’s antifibrotic potential in heart disease.

Measuring Calcium Levels in Bone-Resorbing Osteoclasts and Bone-Forming Osteoblasts.

Bone remodeling is a crucial, dynamic process that renews bone and maintains mineral homeostasis. It consists of several steps, including osteoclastic bone resorption and osteoblastic bone formation and mineralization. Intracellular calcium signaling is essential for osteoclast and osteoblast differentiation and activity. Here, we describe the differentiation of human osteoclasts and osteoblasts in vitro and provide common methods to determine cell differentiation and activity. We then describe protocols for measuringintracellular calcium in these cells using Fura2-AM.

Monitoring Calcium-Sensing Receptor (CaSR)-Induced Intracellular Calcium Flux Using an Indo-1 Flow Cytometry Assay.

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca2+i) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca2+I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells.

Methods for Imaging Intracellular Calcium Signals in the Mouse Mammary Epithelium in Two and Three Dimensions.

The mammary gland has a central role in optimal mammalian development and survival. Contractions of smooth muscle-like basal (or myoepithelial) cells in the functionally mature mammary gland in response to oxytocin are essential for milk ejection and are tightly regulated by intracellular calcium (Ca2+). Using mice expressing a genetically encoded Ca2+ indicator (GCaMP6f), we present in this chapter a method to visualize at high spatiotemporal resolution changes in intracellular Ca2+ in mammary epithelial cells, both in vitro (2D) and ex vivo (3D). The procedure to optimally prepare mammary tissue and primary cells is presented in detail.

Measuring GPCR-Induced Intracellular Calcium Signaling Using a Quantitative High-Throughput Assay.

Alterations in intracellular calcium are integral to signal transduction pathways for many G-protein-coupled receptors, but this signaling is not well studied. This is mostly due to a lack of reliable, robust, high-throughput, quantitative methods to monitor intracellular calcium concentrations in live cells. Recently, we developed a reliable, robust, quantitative method to measure intracellular calcium levels in which HEK293 cell suspensions loaded with Fura-2/AM are placed in 96-well plates. Minimum and maximum intracellular calcium levels, which are required for converting fluorescence into calcium concentrations, are calibrated using EGTA to chelate calcium and ionomycin to load calcium into cells, respectively. Fluorescence is monitored with a PHERAstar FS plate reader. We provide a detailed method for this high-throughput assay that can be used to quantitate intracellular calcium in endogenous and exogenously (stable or transient) expressed GPCRs in HEK293 cells.

ACKR3 agonism induces heterodimerization with chemokine receptor CXCR4 and attenuates platelet function.

Platelet receptors ACKR3 and CXCR4 play a crucial role in a variety of cardiovascular diseases. Like most chemokine receptors, CXCR4 is a G protein coupled receptor that induces platelet activation. In contrast, the atypical chemokine receptor 3 (ACKR3) lacks the ability to activate heterotrimeric G proteins and its activation leads to platelet inhibition and attenuates thrombus formation. In nucleated cells, heterodimerization of ACKR3 with CXCR4 regulates CXCL12-dependent signalling. The aim of our study was to investigate the formation of ACKR3/CXCR4 heterodimers in platelets and the subsequent consequences for platelet function. Using a proximity ligation assay (PLA, Duolink®) to screen for CXCR4/ACKR3 heterodimerization inducing compounds, we found that ACKR3 agonism but not conventional platelet agonists or endogen ligands lead to heterodimer formation. To further characterize the formation of ACKR3/CXCR4 heterodimers, we studied the CXCL12-dependent platelet activation via CXCR4. Both, CXCL12-dependent platelet aggregation and collagen-dependent exvivo thrombus formation were significantly downregulated by ACKR3 agonism. Moreover, platelet intracellular calcium and Akt signalling were increased by CXCL12 and again suppressed by ACKR3-specific agonists. Previously, CXCL12 was shown to decrease platelet cAMP levels via CXCR4. Treatment with a specific ACKR3 agonist counteracted this CXCL12/CXCR4-dependent cAMP decrease. Our results reveal that the formation of platelet ACKR3/CXCR4 heterodimers is dependent on ACKR3 rather than CXCR4. Furthermore, ACKR3 agonism induced heterodimerization is associated with mitigating CXCL12/CXCR4-dependent platelet activation possibly by modulating CXCR4-dependent G protein signalling. Our results indicate possible ACKR3 agonist functions and reinforce the potential therapeutic applications of ACKR3 agonists.

FAHFA promotes intracellular calcium signaling via activating the fat taste receptor, CD36 and Src protein kinases in mice taste bud cells

Two lipid sensors, CD36 and GPR120, are crucial for the orosensory detection of fat taste and for mediating fat preference. However, the mechanism by which endogenous lipid (FAHFA) binds to CD36 to initiate intracellular signaling remains unexplained. Hence, the primary objective of this study is to investigate the binding mechanism of FAHFA to CD36 and its role in isolated mouse taste bud cells (mTBCs). The Schrodinger platform was used to assess the molecular dynamics of protein and ligand interactions, and an in vitro experiment was used to validate the findings. Based on the docking score of the ligand, the molecular mechanistic activities of the targeted complexes, CD36–5-POHSA (−8.2 kcal/mol), were investigated using the dynamic simulation. In comparison to linoleic acid (LA), POHSA rapidly increased [Ca2+]i via acting on CD36, and 5-POHSA treatment in mTBCs activated src-kinase at 20 μM. CD36 siRNA transfection in TBCs downregulate the CD36 protein expression as well as [Ca2+]i flux. This study suggests that 5-POHSA may help combat taste abnormalities and the adverse effects of obesity by binding to the lingual CD36 receptor and activating the tongue-brain axis.

Mathematical model for IP$_{3}$ dependent calcium oscillations and mitochondrial associate membranes in non-excitable cells

Theoretical studies on calcium oscillations within the cytosolic [Ca$^{2+}$], and mitochondria [Ca$^{2+}$]$_{mit}$ have been conducted using a mathematical model-based approach. The model incorporates the mechanism of calcium-induced calcium release (CICR) through the activation of inositol-trisphosphate receptors (IPR), with a focus on the endoplasmic reticulum (ER) as an internal calcium store. The production of 1,4,5 inositol-trisphosphate (IP$_{3}$) through the phospholipase \(C\) isoforms and its degradation via Ca$^{2+}$ are considered, with IP$_{3}$ playing a crucial role in modulating calcium release from the ER. The model includes a simple kinetic mechanism for mitochondrial calcium uptake, release and physical connections between the ER and mitochondria, known as mitochondrial associate membrane complexes (MAMs), which influence cellular calcium homeostasis. Bifurcation analysis is used to explore the different dynamic properties of the model, identifying various regimes of oscillatory behavior and how these regimes change in response to different levels of stimulation, highlighting the complex regulatory mechanisms governing intracellular calcium signaling.

Neurovascular coupling and CO2 interrogate distinct vascular regulations

Neurovascular coupling (NVC), which mediates rapid increases in cerebral blood flow in response to neuronal activation, is commonly used to map brain activation or dysfunction. Here we tested the reemerging hypothesis that CO2 generated by neuronal metabolism contributes to NVC. We combined functional ultrasound and two-photon imaging in the mouse barrel cortex to specifically examine the onsets of local changes in vessel diameter, blood flow dynamics, vascular/perivascular/intracellular pH, and intracellular calcium signals along the vascular arbor in response to a short and strong CO2 challenge (10 s, 20%) and whisker stimulation. We report that the brief hypercapnia reversibly acidifies all cells of the arteriole wall and the periarteriolar space 3–4 s prior to the arteriole dilation. During this prolonged lag period, NVC triggered by whisker stimulation is not affected by the acidification of the entire neurovascular unit. As it also persists under condition of continuous inflow of CO2, we conclude that CO2 is not involved in NVC.

TRPM7 controls skin keratinocyte senescence by targeting intracellular calcium signaling.

Cellular senescence is described as an irreversible cell cycle arrest for proliferating cells and is associated with the secretion of senescence associated secretory phenotype factors. It has been known to accumulate with age and is regarded as a key driver of aging-associated skin pathologies. However, the lack of markers of skin senescence and partially understood skin cellular senescence mechanisms has limited the exploration of skin aging and anti-skin aging strategies. Recently, intracellular calcium signaling has emerged as an important regulator of cellular senescence and aging. However, little is known about the modulation of skin cellular senescence by calcium-associated factors. Here, we found that the expression of calcium channel transient receptor potential melastatin 7 (TRPM7) is elevated during skin keratinocyte senescence and aging. Importantly, TRPM7 promotes skin keratinocyte senescence by triggering intracellular calcium transfer from the endoplasmic reticulum to the mitochondria; accumulation of mitochondrial calcium then induces a drop in mitochondrial membrane potential and reactive oxygen species production, leading to subsequent nuclear enlargement and DNA damage. Altogether, these findings indicate that TRPM7 controls skin keratinocyte senescence through regulating intracellular calcium signaling, and thus, shed light on novel strategies for anti-skin aging therapy.

Rotenone-Induced Optic Nerve Damage and Retinal Ganglion Cell Loss in Rats.

Rotenone is a mitochondrial complex I inhibitor that causes retinal degeneration. A study of a rat model of rotenone-induced retinal degeneration suggested that this model is caused by indirect postsynaptic N-methyl-D-aspartate (NMDA) stimulation triggered by oxidative stress-mediated presynaptic intracellular calcium signaling. To elucidate the mechanisms by which rotenone causes axonal degeneration, we investigated morphological changes in optic nerves and the change in retinal ganglion cell (RGC) number in rats. Optic nerves and retinas were collected 3 and 7 days after the intravitreal injection of rotenone. The cross-sections of the optic nerves were subjected to a morphological analysis with axon quantification. The axons and somas of RGCs were analyzed immunohistochemically in retinal flatmounts. In the optic nerve, rotenone induced axonal swelling and degeneration with the incidence of reactive gliosis. Rotenone also significantly reduced axon numbers in the optic nerve. Furthermore, rotenone caused axonal thinning, fragmentation, and beading in RGCs on flatmounts and decreased the number of RGC soma. In conclusion, the intravitreal injection of rotenone in rats induced morphological abnormities with a reduced number of optic nerve axons and RGC axons when the RGC somas were degenerated. These findings help elucidate the pathogenesis of optic neuropathy induced by mitochondrial dysfunction.

Evaluation of mechanisms involved in regulation of intrinsic excitability by extracellular calcium in CA1 pyramidal neurons of rat.

It is well recognized that changes in the extracellular concentration of calcium ions influence the excitability of neurons, yet what mechanism(s) mediate these effects is still a matter of debate. Using patch-clamp recordings from rat hippocampal CA1 pyramidal neurons, we examined the contribution of G-proteins and intracellular calcium-dependent signaling mechanisms to changes in intrinsic excitability evoked by altering the extracellular calcium concentration from physiological (1.2 mM) to a commonly used experimental (2 mM) level. We find that the inhibitory effect on intrinsic excitability of calcium ions is mainly expressed as an increased threshold for action potential firing (with no significant effect on resting membrane potential) that is not blocked by either the G-protein inhibitor GDPβS or the calcium chelator BAPTA. Our results therefore argue that in the concentration range studied, G-protein coupled calcium-sensing receptors, non-selective cation conductances, and intracellular calcium signaling pathways are not involved in mediating the effect of extracellular calcium ions on intrinsic excitability. Analysis of the derivative of the action potential, dV/dt versus membrane potential, indicates a current shift towards more depolarized membrane potentials at the higher calcium concentration. Our results are thus consistent with a mechanism in which extracellular calcium ions act directly on the voltage-gated sodium channels by neutralizing negative charges on the extracellular surface of these channels to modulate the threshold for action potential activation.

Agrimonia coreana Extract Exerts Its Therapeutic Effect through CRAC Channel Inhibition for Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (ACext) can be used in treating AD-related dermatologic symptoms. ACext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon ACext treatment. In a mouse model of AD, ACext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.

Inhibition of transglutaminase 2 inhibits ionizing radiation-induced cellular senescence in skin keratinocytes in vitro.

Senescent cells are typically characterized by a stable proliferation arrested in dividing cells accompanied with a senescence-associated secretory phenotype (SASP). Skin cellular senescence is the primary cause of skin aging, whereas the lack of identified skin senescence markers limits our understanding of the mechanisms involved in skin aging. Recent studies have revealed that intracellular calcium signaling has emerged as a key player in regulating cellular senescence and aging. However, the implication and roles of calcium signaling in skin keratinocyte senescence remain only partially understood. In this study, we developed a model for skin keratinocyte senescence using ionizing radiation (I/R) stimulation and found that the calcium-associated gene transglutaminase 2 (TGM2) was significantly induced compared with normal control. Interestingly, inhibition of TGM2 was found to delay skin keratinocyte senescence by suppressing I/R-promoted intracellular calcium signaling, accumulation of reactive oxygen species (ROS), DNA damage, as well as NF-κB-mediated SASP secretion. Taken together, our findings demonstrate that inhibition of TGM2 contributes to bypassing I/R-induced skin keratinocyte senescence and sheds light on novel strategies against skin stresses caused by I/R.

A spatial threshold for astrocyte calcium surge.

There is a spatial threshold for the astrocyte intracellular calcium signal propagation that is determined by astrocyte intrinsic properties and controls gliotransmission.

Enhancing mitotane efficacy in adrenocortical carcinoma by calcineurin inhibition with cyclosporine A

Aims: The aim of this study is to determine the effect of calcineurin (CaN) in adrenocortical cancer (ACC) cells, which is a rare but aggressive type of cancer resistant to mitotane therapy. The intracellular calcium signaling pathway is one of the most important mechanisms for cells. The effect of intracellular calcium concentration [(Ca2+i)] on the function of cancer cells is also known. CaN, activated by the binding of calmodulin and Ca2+, is critical in this pathway. Methods: H295 adrenocortical cancer cells were treated with mitotane, cyclosporine A (CsA), and a combination of both. Cell viability, apoptosis, cell cycle, and gene expression levels of apoptosis-related genes (BCL2, BAX, TP53) were analyzed. Western blotting was used to measure CaN protein levels, and wound healing assays assessed cell migration. Results: CsA significantly suppressed CaN protein levels in a dose-dependent manner, reducing cell viability and increasing apoptosis in H295 cells. Mitotane alone also suppressed CaN protein, but the combination of mitotane and CsA had a synergistic effect, further decreasing cell viability and increasing apoptosis. The combination treatment led to significant suppression of the BCL2 gene and upregulation of TP53. Cell cycle analysis showed increased arrest in the G0/G1 phase with combination treatment. Conclusion: Suppression of CaN by CsA enhances the cytotoxic effects of mitotane on ACC cells, suggesting a potential therapeutic strategy to improve ACC treatment outcomes. This study highlights the importance of targeting intracellular calcium signaling pathways to overcome resistance and enhance the efficacy of existing cancer therapies.

Suppressing ERp57 diminishes osteoclast activity and ameliorates ovariectomy-induced bone loss via the intervention in calcium oscillation and the calmodulin/calcineurin/Nfatc1 pathway

BackgroundIncreased osteoclast activity constitutes the primary etiology of excessive bone erosion in postmenopausal osteoporosis. ERp57, otherwise referred to as protein disulfide isomerase A3 (PDIA3), plays a crucial role in the regulation of intracellular calcium signaling. This is documented to exert a profound impact on osteoclast differentiation and functionality. MethodsTo ascertain the potential role of ERp57 in disease progression, prevention, and treatment, network pharmacology and bioinformatics analyses were conducted in relation to postmenopausal osteoporosis and ERp57 inhibitor (Loc14). Then, subsequent experimental verifications were employed in vitro on osteoclast and osteoblast, and in vivo on ovariectomy (OVX) mice models. ResultsMultiple enrichment analyses suggested that the “calcium signaling pathway” may constitute a potential avenue for therapeutic intervention by Loc14 in the treatment of postmenopausal osteoporosis. In vitro experiments demonstrated inhibition of ERp57 could block osteoclast differentiation and function by interfering with the expression of osteoclast marker genes (Traf6, Nfatc1, and Ctsk). Further mechanisms studies based on calcium imaging, qPCR, and WB established that ERp57 inhibitor (Loc14) could obstruct calcium oscillation in osteoclast precursor cells (OPCs) by limiting the entry sources of cytosolic Ca2+ and interfering with calmodulin/calcineurin/Nfatc1 pathway. Evidence from Micro-CT scanning and double calcein labeling confirmed that the application of Loc14 in vivo could alleviate bone loss and partially reversed the osteogenic impairment caused by OVX in mice. ConclusionsOur findings proved the suppressive effects of Loc14 on osteoclastogenesis via attenuating calcium oscillation and associated singling pathways, providing ERp57 as a potential therapeutic target for postmenopausal osteoporosis.

Electromagnetic Fields Trigger Cell Death in Glioblastoma Cells through Increasing miR-126-5p and Intracellular Ca2+ Levels.

Electromagnetic fields create potential negative implications on biological systems, including modifications to DNA structure, nuclear condensation, cellular ion transport, and intracellular Ca2+ accumulation. To explore these effects on cancer cells, we exposed prostate, glioblastoma and cervix cancer cell lines to electromagnetic fields of wireless and assessed its anti-proliferative effects. PC3, A172, and HeLa cancer cells were cultured and exposed to electromagnetic fields for 24, 48, and 72 h. We used the MTT assay to detect cell viability and proliferation, Annexin V staining to determine apoptotic cells, and confocal microscopy to measure apoptosis-mediated intracellular calcium signals. Additionally, we performed profiling for apoptosis-related miRNAs. The results indicated that the electromagnetic field triggers apoptosis in the glioblastoma cell line A172 by increasing level of miR-129-5p, a known tumor suppressor. In contrast, the cervix cancer cell line and the prostate cancer cell line remained largely unaffected. In summary, our investigation underscores that electromagnetic fields at a 2.4 GHz frequency may adversely affect certain cancer cell lines, notably triggering apoptosis in the glioblastoma cancer cell line.

Mechanoactivation of Single Stem Cells in Microgels Using a 3D-Printed Stimulation Device.

In this study, the novel 3D-printed pressure chamber for encapsulated single-cell stimulation (3D-PRESS) platform is introduced for the mechanical stimulation of single stem cells in 3D microgels. The custom-designed 3D-PRESS, allows precise pressure application up to 400kPa at the single-cell level. Microfluidics is employed to encapsulate single mesenchymal stem cells within ionically cross-linked alginate microgels with cell adhesion RGD peptides. Rigorous testing affirms the leak-proof performance of the 3D-PRESS device up to 400kPa, which is fully biocompatible. 3D-PRESS is implemented on mesenchymal stem cells for mechanotransduction studies, by specifically targeting intracellular calcium signaling and the nuclear translocation of a mechanically sensitive transcription factor. Applying 200kPa pressure on individually encapsulated stem cells reveals heightened calcium signaling in 3D microgels compared to conventional 2D culture. Similarly, Yes-associated protein (YAP) translocation into the nucleus occurs at 200kPa in 3D microgels with cell-binding RGD peptides unveiling the involvement of integrin-mediated mechanotransduction in singly encapsulated stem cells in 3D microgels. Combining live-cell imaging with precise mechanical control, the 3D-PRESS platform emerges as a versatile tool for exploring cellular responses to pressure stimuli, applicable to various cell types, providing novel insights into single-cell mechanobiology.

Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.