Increase In Intracellular Calcium Concentration Research Articles

Diagnostic Ultrasound-Based Investigation of Central vs. Peripheral Arterial Changes Consequent to Low-Dose Caffeine Ingestion.

Caffeine is present in various foods and medicines and is highly accessible through various routes, regardless of age. However, most studies on caffeine have focused on the effects of high-dose caffeine ingestion based on the recommended daily amount for adults. In this study, we examined the physiological changes in the central and peripheral vessels that may occur when ingesting low-dose caffeine due to its high accessibility, with the aim of creating an environment of safe caffeine ingestion. This study included 26 healthy participants in their 20s. Peak systolic velocity (PSV), heart rate (HR), and pulse wave velocity (PWV) for vascular stiffness assessment were measured at 0, 30, and 60 min after caffeine ingestion using diagnostic ultrasound to determine the physiological changes in the blood vessels, common carotid artery (CCA) and radial artery (RA). In addition, percutaneous oxygen saturation (SpO2), blood pressure (BP), and accelerated photoplethysmography (APG) were measured. In comparison with before ingestion, the HR tended to decrease and showed a significant difference at 30 and 60 min (p = 0.014 and p = 0.031, respectively). PSV significantly decreased in both vessels at 30 and 60 min (p < 0.001 and p < 0.001, respectively). APG showed a decreasing trend until 60 min after ingestion, with a significant difference at 30 and 60 min (p = 0.003 and p = 0.012, respectively). No significant difference was observed in SpO2, BP, or PWV; however, they showed a tendency to increase after ingestion. Decreased HR may occur because of the baroreflex caused by an increase in BP. The RA has many branches and a smaller diameter; therefore, the PSV was lower in the RA than that in the CCA. This effect can occur because of the difficulty in the smooth expansion of blood vessels, which leads to a decrease in blood flow. In addition, an increase in intracellular calcium concentration can prevent vasodilation and increase the propagation velocity of pulse waves. The reflected waves can increase systolic blood pressure but reduce PWV and vascular elasticity. These results suggest that even low-dose caffeine can improve blood vessel health by providing temporary stimulation to the blood vessels; however, it can also cause changes in blood flow and blood vessel elasticity, which can lead to serious diseases such as stroke and high blood pressure. Therefore, caution should be exercised when caffeine consumption is indiscriminate.

Phytol and (−)-α-bisabolol Synergistically trigger intrinsic apoptosis through redox and Ca2+ imbalance in non-small cell lung cancer

This study aimed to identify a combination of natural compounds that could enhance lung cancer treatment by targeting multiple pathways and reducing toxicity. The Chow-Talay method's CI value of 0.75 demonstrated that the synergistic combination of phytol and (−)-α-bisabolol (Phy + Bis) exhibited a significant anti-proliferative effect on A549 cells. After 24 h of treatment, Phy + Bis significantly altered the morphology of A549 cells and activated apoptosis. Increased levels of reactive oxygen species (ROS) are evidence that the combination treatments induced oxidative stress. Consequently, there was an increase in intracellular calcium concentration ([Ca2+]i), leading to perturbations in the membrane potential (ΔΨm). Intrinsic apoptosis was triggered by these effects, which resulted in depolarization of the mitochondrial membrane, activation of Cas 9 and Cas 3, upregulation of Bax and Cyt-C, and downregulation of Bcl-2. Phy + Bis also caused G0/G1 phase arrest and cell senescence, with senescent cells expressing more p53. The combined therapy prevented the migration of A549 cells, possibly by interfering with the polymerization of the actin cytoskeleton. Importantly, Phy + Bis did not have any harmful effects on human lung L-132 cells, red blood cells, peripheral blood mononuclear cells, and an in vivo artemia model. Overall, phytol and (−)-α-bisabolol appear to be a promising combination as a safe and effective therapy choice for non-small cell lung cancer.

Novel Calcium-Binding Peptide from Bovine Bone Collagen Hydrolysates and Its Potential Pro-Osteogenic Activity via Calcium-Sensing Receptor (CaSR).

This paper aims to explore the osteogenic activity and potential mechanism of the peptide-calcium chelate, and provides a theoretical basis for peptide-calcium chelates as functional foods to prevent or improve osteoporosis. In this research, a novel peptide (Phe-Gly-Leu, FGL) with a high calcium-binding capacity is screened from bovine bone collagen hydrolysates (CPs), calcium binding sites of which mainly included carbonyl, amino and carboxyl groups. The FGL-Ca significantly enhances the osteogenic activity of MC3T3-E1 cells (survival rate, differentiation, and mineralization). The results of calcium fluorescence labeling and molecular docking show that FGL-Ca may activate calcium-sensing receptor (CaSR), leading to an increase in intracellular calcium concentration, then enhancing osteogenic activity of MC3T3-E1 cells. Further research found that FGL-Ca significantly promotes the mRNA and protein expression levels of CaSR, transforming growth factor β (TGF-β1), TGF-β-type II receptor (TβRII), Smad2, Smad3, osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegrin (OPG), and collagen type I (COLI). Subsequently, in the signal pathway intervention experiment, the expression levels of genes and proteins related to the TGF-β1/Smad2/3 signaling pathway that are promoted by FGL-Ca are found to decrease. These results suggest that FGL-Ca may activate CaSR, increase intracellular calcium concentration, and activate TGF-β1/Smad2/3 signaling pathway, which may be one of the potential mechanisms for enhancing osteogenic activity.

Inorganic polyphosphate regulates functions of thymocytes via activation of P2X purinoreceptors

Inorganic polyphosphate (polyP) is an ancient polymer, which was proven to be a signalling molecule in the mammalian brain, mediating the communication between astrocytes via activation of P2Y1 purinoreceptors and modulating the activity of neurons. There is very limited information regarding the ability of polyP to transmit the information as an agonist of purinoreceptors in other cells and tissues. Here, we show that application of polyP to the suspension of primary thymocytes increases the concentration of intracellular calcium. PolyP evoked calcium signal was dependent on the presence of P2X inhibitors but not P2Y1 inhibitor. PolyP dependent increase in intracellular calcium concentration caused mild mitochondrial depolarization, which was dependent on inhibitors of purinoreceptors, extracellular calcium and inhibitor of mitochondrial calcium uniporter but wasn't dependent on cyclosporin A. Application of polyP modulated cell volume regulation machinery of thymocytes in calcium dependent manner. Molecular docking experiments revealed that polyP can potentially bind to several types of P2X receptors with binding energy similar to ATP - natural agonist of P2X purinoreceptors. Further molecular dynamics simulations with P2X4 showed that binding of one molecule of polyP dramatically increases permeability of this receptor-channel for water molecules. Thus, in this research we for the first time showed that polyP can interact with P2X receptors in thymocytes and modulate physiological processes.

Regulating Striated Muscle Contraction: Through Thick and Thin.

Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.

Dependence of myosin filament structure on intracellular calcium concentration in skeletal muscle.

Contraction of skeletal muscle is triggered by an increase in intracellular calcium concentration that relieves the structural block on actin-binding sites in resting muscle, potentially allowing myosin motors to bind and generate force. However, most myosin motors are not available for actin binding because they are stabilized in folded helical tracks on the surface of myosin-containing thick filaments. High-force contraction depends on the release of the folded motors, which can be triggered by stress in the thick filament backbone, but additional mechanisms may link the activation of the thick filaments to that of the thin filaments or to intracellular calcium concentration. Here, we used x-ray diffraction in combination with temperature-jump activation to determine the steady-state calcium dependence of thick filament structure and myosin motor conformation in near-physiological conditions. We found that x-ray signals associated with the perpendicular motors characteristic of isometric force generation had almost the same calcium sensitivity as force, but x-ray signals associated with perturbations in the folded myosin helix had a much higher calcium sensitivity. Moreover, a new population of myosin motors with a longer axial periodicity became prominent at low levels of calcium activation and may represent an intermediate regulatory state of the myosin motors in the physiological pathway of filament activation.

The role of the HERG channel in the secretion of glucagon-like peptide-1 (GLP-1) from murine intestinal L-cells

The HERG ion channel belongs to the voltage-gated potassium (Kv) channel family and is involved in potassium efflux during cellular repolarization. Mutations in HERG have been linked to long QT syndrome, which is associated with elevated secretion of glucagon-like peptide-1 (GLP-1). However, the precise contribution of HERG to GLP-1 secretion remains unclear. In this study, we demonstrate the expression of HERG in GLP-1-producing L-cells within the intestinal epithelium of rodents. Using a mouse L-cell model (GLUTag cell line), we observed that downregulation of HERG led to a significant prolongation of action potential duration, an increase in intracellular calcium concentration, and a stimulation of GLP-1 secretion following exposure to nutrients. These findings provide evidence that HERG plays a direct role in regulating GLP-1 secretion in the intestine and may hold promise as a potential target for the treatment of diabetes.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

The Role of Ryanodine Receptors in Regulating Neuronal Activity and Its Connection to the Development of Alzheimer's Disease.

Research into the early impacts of Alzheimer's disease (AD) on synapse function is one of the most promising approaches to finding a treatment. In this context, we have recently demonstrated that the Abeta42 peptide, which builds up in the brain during the processing of the amyloid precursor protein (APP), targets the ryanodine receptors (RyRs) of mouse hippocampal neurons and potentiates calcium (Ca2+) release from the endoplasmic reticulum (ER). The uncontrolled increase in intracellular calcium concentration ([Ca2+]i), leading to the development of Ca2+ dysregulation events and related excitable and synaptic dysfunctions, is a consolidated hallmark of AD onset and possibly other neurodegenerative diseases. Since RyRs contribute to increasing [Ca2+]i and are thought to be a promising target for AD treatment, the goal of this review is to summarize the current level of knowledge regarding the involvement of RyRs in governing neuronal function both in physiological conditions and during the onset of AD.

Extracellular Histone-Induced Protein Kinase C Alpha Activation and Troponin Phosphorylation Is a Potential Mechanism of Cardiac Contractility Depression in Sepsis.

Reduction in cardiac contractility is common in severe sepsis. However, the pathological mechanism is still not fully understood. Recently it has been found that circulating histones released after extensive immune cell death play important roles in multiple organ injury and disfunction, particularly in cardiomyocyte injury and contractility reduction. How extracellular histones cause cardiac contractility depression is still not fully clear. In this work, using cultured cardiomyocytes and a histone infusion mouse model, we demonstrate that clinically relevant histone concentrations cause significant increases in intracellular calcium concentrations with subsequent activation and enriched localization of calcium-dependent protein kinase C (PKC) α and βII into the myofilament fraction of cardiomyocytes in vitro and in vivo. Furthermore, histones induced dose-dependent phosphorylation of cardiac troponin I (cTnI) at the PKC-regulated phosphorylation residues (S43 and T144) in cultured cardiomyocytes, which was also confirmed in murine cardiomyocytes following intravenous histone injection. Specific inhibitors against PKCα and PKCβII revealed that histone-induced cTnI phosphorylation was mainly mediated by PKCα activation, but not PKCβII. Blocking PKCα also significantly abrogated histone-induced deterioration in peak shortening, duration and the velocity of shortening, and re-lengthening of cardiomyocyte contractility. These in vitro and in vivo findings collectively indicate a potential mechanism of histone-induced cardiomyocyte dysfunction driven by PKCα activation with subsequent enhanced phosphorylation of cTnI. These findings also indicate a potential mechanism of clinical cardiac dysfunction in sepsis and other critical illnesses with high levels of circulating histones, which holds the potential translational benefit to these patients by targeting circulating histones and downstream pathways.

Calcium/Calmodulin-Stimulated Protein Kinase II (CaMKII): Different Functional Outcomes from Activation, Depending on the Cellular Microenvironment.

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a family of broad substrate specificity serine (Ser)/threonine (Thr) protein kinases widely expressed in many tissues that is capable of mediating diverse functional responses depending on its cellular and molecular microenvironment. This review briefly summarises current knowledge on the structure and regulation of CaMKII and focuses on how the molecular environment, and interaction with binding partner proteins, can produce different populations of CaMKII in different cells, or in different subcellular locations within the same cell, and how these different populations of CaMKII can produce diverse functional responses to activation following an increase in intracellular calcium concentration. This review also explores the possibility that identifying and characterising the molecular interactions responsible for the molecular targeting of CaMKII in different cells in vivo, and identifying the sites on CaMKII and/or the binding proteins through which these interactions occur, could lead to the development of highly selective inhibitors of specific CaMKII-mediated functional responses in specific cells that would not affect CaMKII-mediated responses in other cells. This may result in the development of new pharmacological agents with therapeutic potential for many clinical conditions.

EFFECT OF CALCIUM AND VITAMIN D SUPPLEMENTATION IN PREGNANT WOMAN WITH HYPERTENSION AT SENTANI COMMUNITY HEALTH CENTER

Low calcium intake causes an increase in high blood pressure by stimulating the release of parathyroid hormone and renin, which causes an increase in intracellular calcium concentration in the smooth muscle cells of blood vessels and results in vasoconstriction. This study aims to determine the effect of giving calcium and vitamin D supplements to pregnant hypertension women on the anthropometric outcomes of newborns. The research design was quasi-experimental with two groups of post-test-only design. A respondent of 30 hypertension pregnant women at 28-32 weeks of gestation was selected by purposive sampling—data analysis using Mann Whitney. Calcium supplements (2 x 500 mg/day) and vitamin D3 (400 IU/day) were administered and monitored for eight weeks to 15 pregnant women as the intervention group and 15 pregnant women as the control group who were assumed to receive calcium supplements from the Health Service Program. Data on blood pressure and calcium levels were taken before and after eight weeks of intervention. The results showed that there was a significant difference in mean blood pressure between the control and intervention groups, with a p-value of systolic blood pressure (0.002) and a p-value of diastolic blood pressure (0.014), and the average decrease in blood pressure was more significant in the intervention group. There were differences in anthropometric results between the intervention group and the control group, with p-value s ​​for body weight (0.000), body length (0.000), and head circumference (0.000). The intervention group's average weight, length, and head girth were higher than the control group's. Thus, for eight weeks, calcium and vitamin D supplementation in hypertensive pregnant women can reduce blood pressure and result in better baby weight, body length, and head circumference. Keywords: Anthropometry, Calcium, Hypertension, Vitamin D

TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 μM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Role of GABAA receptor depolarization-mediated VGCC activation in sevoflurane-induced cognitive impairment in neonatal mice.

BackgroundIn neonatal mice, anesthesia with sevoflurane depolarizes the GABA Type A receptor (GABAAR), which leads to cognitive impairment. Calcium accumulation in neurons can lead to neurotoxicity. Voltage-gated calcium channels (VGCCs) can increase intracellular calcium concentration under isoflurane and hypoxic conditions. The underlying mechanisms remain largely unknown.MethodsSix-day-old mice were anesthetized with 3% sevoflurane for 2 h/day for 3 days. The Y-Maze, new object recognition (NOR) test, the Barnes maze test, immunoassay, immunoblotting, the TUNEL test, and Golgi–Cox staining were used to assess cognition, calcium concentration, inflammatory response, GABAAR activation, VGCC expression, apoptosis, and proliferation of hippocampal nerve cells in mice and HT22 cells.ResultsCompared with the control group, mice in the sevoflurane group had impaired cognitive function. In the sevoflurane group, the expression of Gabrb3 and Cav1.2 in the hippocampal neurons increased (p < 0.01), the concentration of calcium ions increased (p < 0.01), inflammatory reaction and apoptosis of neurons increased (p < 0.01), the proliferation of neurons in the DG area decreased (p < 0.01), and dendritic spine density decreased (p < 0.05). However, the inhibition of Gabrb3 and Cav1.2 alleviated cognitive impairment and reduced neurotoxicity.ConclusionsSevoflurane activates VGCCs by inducing GABAAR depolarization, resulting in cognitive impairment. Activated VGCCs cause an increase in intracellular calcium concentration and an inflammatory response, resulting in neurotoxicity and cognitive impairment.

Sensing Mechanisms: Calcium Signaling Mediated Abiotic Stress in Plants.

Plants are exposed to various environmental stresses. The sensing of environmental cues and the transduction of stress signals into intracellular signaling are initial events in the cellular signaling network. As a second messenger, Ca2+ links environmental stimuli to different biological processes, such as growth, physiology, and sensing of and response to stress. An increase in intracellular calcium concentrations ([Ca2+]i) is a common event in most stress-induced signal transduction pathways. In recent years, significant progress has been made in research related to the early events of stress signaling in plants, particularly in the identification of primary stress sensors. This review highlights current advances that are beginning to elucidate the mechanisms by which abiotic environmental cues are sensed via Ca2+ signals. Additionally, this review discusses important questions about the integration of the sensing of multiple stress conditions and subsequent signaling responses that need to be addressed in the future.

DEVELOPMENT OF AN ADRENOCORTICAL CELL MODEL OF CALCIUM SIGNALING MODULATION TO DECIPHER THE MOLECULAR MECHANISMS RESPONSIBLE FOR PRIMARY ALDOSTERONISM

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis.The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA.We have developed an adrenocortical H295R_S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties.Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around -60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around -10 mV.This cell line, in which we can modulate the intracellular calcium concentration "on demand", is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Development of an adrenocortical cell model of calcium signaling modulation to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels and defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand-binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose-dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around −60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around −10 mV. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Modulation of Purinergic Signaling in Keratinocytes in Spared Nerve Injury Model of Neuropathic Pain

Epidermal keratinocytes express various purinergic 2 receptors that play an essential role in cell growth, differentiation, and proliferation. In the conditions of injury, concentrations of extracellular adenosine triphosphate (ATP) may dramatically increase due to cell damage and inflammatory processes. In this situation activation of purinergic signaling in keratinocytes could act as a double-edged sword contributing to skin regeneration or cell apoptosis. As the role of keratinocytes in transducing and modulating nociceptive stimuli has been increasingly appreciated in recent years, the aim of the present study was to evaluate whether peripheral nerve injury affects purinergic signaling in keratinocytes. Spared nerve injury (SNI), a classical model of peripheral neuropathic pain, was induced in mice. The injury was induced by sparing of the tibial nerve, and ligation and cut of the sural and common peroneal nerves. Keratinocytes were isolated and cultured on Days 2-4 post-injury and ATP-mediated calcium responses in keratinocytes were examined by confocal imaging. On average, the number of keratinocytes that responded to ATP with an increase in intracellular calcium gradient as well as the magnitude of the peak response was not significantly different between sham and SNI groups. However, significantly less delay in ATP-induced increase in intracellular calcium concentration was observed in keratinocytes in SNI group compared to sham. Selective pharmacological inhibition of keratinocyte response to ATP indicated a major role of P2 × 4 receptors in the modulation of calcium homeostasis in SNI. Our results indicate that epidermal purinergic signaling undergoes dramatic changes following peripheral nerve injury that may contribute to injury-induced mechanical hypersensitivity.

Role of endoplasmic reticulum stress in apoptosis induced by HK2 inhibitor and its potential as a new drug combination strategy.

Compared with normal cells, tumor cells mainly obtain energy through aerobic glycolysis. Hexokinase 2 (HK2) plays a key role in the regulation of tumor cell aerobic glycolysis, and targeting HK2 has become a new strategy for cancer treatment. However, little is known about the role of HK2 in colon cancer and the regulation of its targeted inhibitors. In this study, we found that the expression of HK2 in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the expression level of HK2 in metastatic colorectal cancer was further increased. Meanwhile, the expression level of HK2 was closely related to clinical TNM stage and outcome of colorectal cancer patients. We provide here evidence that HK2 inhibitor 3-Bromopyruvate acid (3-BP) can significantly inhibit the survival and proliferation of colon cancer cells, and induce apoptosis through mitochondrial apoptosis signaling pathway. In addition, we found that 3-BP can also induce endoplasmic reticulum stress in colon cancer cells, the mechanism may be through the increase of intracellular calcium concentration. In vitro and in vivo experiments showed that inhibition of endoplasmic reticulum stress could further increase the proliferation inhibition and apoptosis induced by 3-BP. Collectively, our results show that HK2 is highly expressed in colorectal cancer. 3-BP, an inhibitor of HK2, can induce apoptosis and endoplasmic reticulum stress in colon cancer cells. Endoplasmic reticulum stress plays a protective role in cell death induced by 3-BP. This result suggested that targeting HK2 and endoplasmic reticulum stress may be a valuable strategy in targeted and combination therapy of colon cancer.

Melatonin Mediates Osteoblast Proliferation Through the STIM1/ORAI1 Pathway.

Based on the positive correlation between bone mineral density and melatonin levels in blood, this study confirmed that melatonin supplementation prevents postmenopausal osteoporosis. We further confirmed that melatonin promotes an increase in intracellular calcium concentrations through the STIM1/ORAI1 pathway, thereby inducing the proliferation of osteoblasts. Introduction: Osteoporosis (OP) is a progressive, systemic bone disease that is one of the main causes of disability and death in elderly female patients. As an amine hormone produced by the human pineal gland, melatonin plays an important role in regulating bone metabolism. This study intends to investigate the relationship between melatonin levels in human blood and bone density and to suggest the efficacy of melatonin in treating osteoporosis by performing in vivo and in vitro experiments. Methods: We used liquid chromatography-tandem mass spectrometry to determine the serum melatonin levels in postmenopausal women with osteoporosis and young women with a normal bone mass. The bone density, BV/TV, Tb.Th, Tb.Sp and other indicators of postmenopausal osteoporosis and mice with a normal bone mass were detected by measuring bone density and micro-CT. The intracellular calcium ion concentration was detected using fluorescence microscopy and a full-wavelength multifunctional microplate reader, and the expression of SOCE-related genes and STIM1/ORAI1 proteins was detected using PCR and WB. Results: This study confirmed that bone density positively correlates with the melatonin level in human blood. In the animal model, melatonin supplementation reverses postmenopausal osteoporosis. We explored the internal mechanism of melatonin treatment of osteoporosis. Melatonin promotes an increase in intracellular calcium ion concentrations through the STIM1/ORAI1 pathway to induce osteoblast proliferation. Conclusions: This study provides an important theoretical basis for the clinical application of melatonin in patients with osteoporosis and helps to optimize the diagnosis and treatment of postmenopausal osteoporosis.