Mediated Calcium Influx Research Articles

Metabotropic NMDAR Signaling Contributes to Sex Differences in Synaptic Plasticity and Episodic Memory.

NMDA receptor (NMDAR) - mediated calcium influx triggers the induction and initial expression of Long-Term Potentiation (LTP). Here we report that in male rodents ion flux-independent (metabotropic) NMDAR signaling is critical for a third step in the production of enduring LTP, i.e., cytoskeletal changes that stabilize the activity-induced synaptic modifications. Surprisingly, females rely upon estrogen receptor alpha (ERα) for the metabotropic NMDAR operations used by males. Blocking NMDAR channels with MK-801 eliminated LTP expression in hippocampal field CA1 of both sexes but left intact theta burst stimulation (TBS)-induced actin polymerization within dendritic spines. A selective antagonist (Ro25-6981) of the NMDAR GluN2B subunit had minimal effects on synaptic responses but blocked actin polymerization and LTP consolidation in males only. Conversely, an ERα antagonist thoroughly disrupted TBS-induced actin polymerization and LTP in females while having no evident effect in males. In an episodic memory paradigm, Ro25-6981 prevented acquisition of spatial locations by males but not females whereas an ERα antagonist blocked acquisition in females but not males. Sex differences in LTP consolidation were accompanied by pronounced differences in episodic memory in tasks involving a minimal (for learning) cue-sampling. Males did better on acquisition of spatial information whereas females had much higher scores than males on tests for acquisition of the identity of cues (episodic 'what') and the order in which the cues were sampled (episodic 'when'). We propose that sex differences in synaptic processes used to stabilize LTP result in differential encoding of the basic elements of episodic memory.Significance Statement Calcium influx through NMDARs has long been recognized as the initiating event for LTP. Results of the present studies call for a substantial revision to this fundamental observation about learning-related synaptic plasticity. Specifically, we show cytoskeletal mechanisms that consolidate field CA1 LTP and episodic memory are triggered not by NMDAR-mediated calcium but by ion flux-independent (metabotropic) signaling. Males used metabotropic functions of the NMDARs for this purpose whereas females relied upon synaptic estrogen receptors. This unprecedented instance of sex differences in synaptic function was accompanied by surprisingly large male/female differences in the acquisition of the three basic elements of episodic memory.

TRPV2 calcium channel promotes breast cancer progression potential by activating autophagy

Breast cancer, the most prevalent and aggressive tumor affecting women, requires identification of disease determinants to facilitate the development of effective therapeutic strategies. Transient receptor potential vanilloid 2 (TRPV2), an ion channel highly permeable for calcium (Ca2+), is implicated in physiological and pathological processes. Nevertheless, the role of TRPV2 in breast cancer remains poorly elucidated. In this study, we found high levels of TRPV2 expression associated with advanced malignancy, thereby suggesting its potential as a biomarker for breast cancer staging. We demonstrated that TRPV2 activation promotes breast cancer cell proliferation, migration, and invasion, while silencing of TRPV2 suppresses breast cancer progression, highlighting the oncogenic role of TRPV2. Moreover, we reveal that TRPV2 facilitates cancer progression by modulating the CaMKKβ/AMPK/ULK1-autophagic axis through mediating calcium influx, providing new insights into TRPV2 as a novel therapeutic target for breast cancer treatment.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

Inhibition of TRPA1-like alleviated unfolded protein response and apoptosis by regulating cytoplasmic Ca2+ in Yesso scallop Patinopecten yessoensis under high temperature stress

Transient receptor potential ankyrin subtype 1 (TRPA1) is a nonselective cation channel protein typically forms ion channels that regulate intracellular calcium homeostasis, and can be induced by temperature and various chemicals. In the present study, the involvement of PyTRPA1-like in regulating unfolded protein response (UPR) and apoptosis in Yesso Scallop Patinopecten yessoensis under high temperature stress was investigated. The mRNA transcripts of PyTRPA1-like were detected in haemocytes and all the examined tissues with the highest expression level in mantle. After TRPA1 activator (allyl-isothiocyanate, AITC) and high temperature (25°C) treatment, the expression level of PyTRPA1-like mRNA and the Ca2+ content in haemocytes increased significantly (p < 0.05) at 3 h, and then recovered to the normal level at 12 h, and the expression level of PyGRP78, PyIRE1, PyATF6β, PyPERK and PyCaspase-3 mRNA in haemocytes, and Caspase-3 activity and apoptosis rate were also significantly upregulated (p < 0.05). After TRPA1 antagonist (HC-030031) and high temperature (25°C) treatment, the intracellular Ca2+ content, the transcripts of PyGRP78, PyIRE1 and PyCaspase-3 in haemocytes, as well as the Caspase-3 activity and apoptosis rate decreased significantly compared to the control group (p < 0.05), while the Ca2+ distribution in haemocytes showed no difference with that in control group. These results collectively suggest that PyTRPA1-like plays important roles in regulating UPR and apoptosis by mediating calcium influx under high temperature stress in scallop P. yessoensis.

Ahf-Caltide, a Novel Polypeptide Derived from Calpastatin, Protects against Oxidative Stress Injury by Stabilizing the Expression of CaV1.2 Calcium Channel.

Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVβ2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 μM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.

TRPA1-dependent and -independent activation by commonly used preservatives.

Background and purpose: Addition of preservatives ensures microbial stability, especially in multidose containers of parenterally administered pharmaceuticals. These compounds can cause side effects, and particularly at the site of application, might elicit or facilitate pain. TRPA1 is a cation channel expressed in peripheral neurons which contributes to pain and inflammation and is sensitive to many irritants. The most commonly used preservatives, in particular with a focus on parenteral formulations, were investigated for their potential to activate TRPA1. Experimental approach: Sixteen preservatives were screened for mediating calcium influx in human TRPA1-transfected HEK293t cells. Untransfected cells served as control, results were further validated in mouse sensory neurons. In addition, proinflammatory mediators serotonin, histamine and prostaglandin E2 were co-administered to probe a potential sensitisation of preservative-induced TRPA1 activation. Key results: Butylparaben, propylparaben, ethylparaben, bronopol, methylparaben, phenylethyl alcohol and phenol induced a TRPA1-dependent calcium influx in transfected HEK293t cells at concentrations used for preservation. Other preservatives increased calcium within the used concentration ranges, but to a similar degree in untransfected controls. Serotonin, histamine, and prostaglandin enhanced TRPA1 activation of phenylethyl alcohol, bronopol, ethylparaben, propylparaben and butylparaben. Conclusion and implications: Systematic screening of common preservatives applied for parenterally administered drugs resulted in identifying several preservatives with substantial TRPA1 channel activation. This activation was enhanced by the addition of proinflammatory meditators. This allows selecting a preservative without TRPA1 activation, particularly in case of pharmaceuticals that could act proinflammatory.

Extracellular fluid viscosity enhances cell migration and cancer dissemination

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

Blockage of Orai1-Nucleolin interaction meditated calcium influx attenuates breast cancer cells growth

As an important second messenger, calcium (Ca2+) regulates a wide variety of physiological processes. Disturbance of intracellular calcium homeostasis implicated in the occurrence of multiple types of diseases. Orai1 is the major player in mediating store-operated calcium entry (SOCE) and regulates calcium homeostasis in non-excitable cells. Over-expression and activation of Orai1 have been reported in breast cancer. However, its molecular mechanisms are still not very clear. Here, we demonstrated that Nucleolin (NCL) was a novel interacting partner of Orai1. NCL is a multifunctional nucleocytoplasmic protein and is upregulated in human breast tumors. The binding of C-termini of NCL (NCL-CT) to N-termini of Orai1 (Orai1-NT) is critical for mediating calcium influx and proliferation of breast cancer cells. Blocking the NCL-Orai1 interaction by synthesized Orai1 peptide can effectively reduce the intracellular calcium influx and suppress the proliferation of breast cancer cells in vitro and in vivo. Our findings reveal a novel activation mechanism of Orai1 via direct interaction with NCL, which may lead to calcium homeostasis imbalance and promote the proliferation of breast cancer cells. Blocking NCL-Orai1 interaction might be an effective treatment of breast cancer.

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 μM and 5 mM (but not 50 μM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

The mechanism by which ATP regulates alcoholic steatohepatitis through P2X4 and CD39

Alcoholic liver disease caused by chronic excessive drinking has become one of the most common types of liver disease. Alcohol-induced inflammatory immune responses play a central role in the development of alcohol-associated steatohepatitis. The content and expression of ATP and P2X4 in the livers of alcoholic steatohepatitis mice are significantly increased. The content of ATP increased by 20 percent and the expression of P2X4 receptor protein was 1.3 times higher than that in the livers of normal mice. Treatment with 5-BDBD, a P2X4 receptor-specific inhibitor, significantly reduced alcohol-induced liver inflammation and lipid deposition. In RAW264.7 cell experiments, 5-BDBD inhibited the expression of P2X4 and alleviated alcohol-induced inflammation, while the CD39-specific inhibitor POM-1 reduced extracellular ATP degradation and promoted the expression of P2X4, thereby exacerbating inflammation. After treatment with 5-BDBD, P2X4 receptor protein expression decreased by 0.2 times and after treatment with POM-1, P2X4 receptor protein expression increased by 0.1 times compared to the alcohol-stimulated group. In addition, inhibition of P2X4 expression in RAW264.7 cells reduced calcium influx in RAW264.7 cells. P2X4 may induce the activation of NLRP3 inflammasomes by mediating calcium influx, thus exacerbating the inflammatory response, and inhibition of P2X4 expression can effectively block this process. Conclusion: These results suggest that the ATP-P2X4 signaling pathway promotes the inflammatory response in alcoholic steatohepatitis and that CD39 may play a protective role in regulating P2X4 expression by hydrolyzing ATP. In conclusion, the CD39 and ATP-P2X4 signaling pathways may be potential therapeutic targets for alcoholic steatohepatitis.

Structural Basis of Functional Transitions in Mammalian NMDA Receptors

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that mediate calcium influx in excitatory synapses and play a crucial role in synaptic plasticity, learning, and memory function. In mammalian brains, the majority of NMDARs are heterotetramers composed of two copies each of GluN1 and GluN2 subunits to form a functional channel. Activation of NMDARs requires simultaneous binding of glycine and glutamate due to its heterotetrameric assembly. Conversely, competitive inhibition of NMDARs can be evoked by binding of either a glycine antagonist on GluN1 or a glutamate antagonist on GluN2. Despite the extensive efforts on unraveling the mechanism of channel gating and competitive inhibition of NMDARs, it remains elusive due to the lack of detailed structural information. Here, we present multiple cryo-EM structures in distinct ligand states at 4 Å or better. The structures elucidate conformational transitions and inter-subunit/domain reorientations across different ligand binding states, providing great insights of ligand-gating and subunit dependent competitive inhibition. Also, the structures reveal that activation and competitive inhibition of NMDARs is controlled by the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition.

A human relevant mixture of persistent organic pollutants (POPs) and perfluorooctane sulfonic acid (PFOS) differentially affect glutamate induced excitotoxic responses in chicken cerebellum granule neurons (CGNs) in vitro

Primary cultures of cerebellar granule neurons (CGNs) derived from chicken embryos were used to explore the effects on developmental neurotoxicity by a complex defined mixture of persistent organic pollutants (POPs). Its chemical composition and concentrations were based on blood levels in the Norwegian/Scandinavian population. Perfluorooctane sulfonic acid (PFOS) alone, its most abundant compound was also evaluated. Different stages of CGNs maturation, between day in vitro (DIV) 1, 3, and 5 were exposed to the POP mixture, or PFOS alone. Their combination with glutamate, an excitatory endogenous neurotransmitter important in neurodevelopment, also known to cause excitotoxicity was evaluated. Outcomes with the mixture at 500x blood levels were compared to PFOS at its corresponding concentration of 20 μM. The POP mixture reduced tetrazolium salt (MTT) conversion at earlier stages of maturation, compared to PFOS alone. Glutamate-induced excitotoxicity was enhanced above the level of that induced by glutamate alone, especially in mature CGNs at DIV5. Glutathione (GSH) concentrations seemed to set the level of sensitivity for the toxic insults from exposures to the pollutants. The role of N-methyl-D-aspartate receptor (NMDA-R) mediated calcium influx in pollutant exposures was investigated using the non-competitive and competitive receptor antagonists MK-801 and CGP 39551. Observations indicate a calcium-independent, but still NMDA-R dependent mechanism in the absence of glutamate, and a calcium- and NMDA-R dependent one in the presence of glutamate. The outcomes for the POP mixture cannot be explained by PFOS alone, indicating that other chemicals in the mixture contribute its overall effect.

The impact of CRMP4 SUMOylation on the Cav1.2 interaction, neurite outgrowth and thermal pain sensitivity.

Collapsin response mediator protein 4 (CRMP4) is critical for neuronal development. However, whether CRMP4 could be SUMOylated and how the SUMOylation regulates the interaction with the L-type voltage-gated calcium channel (Cav1.2), neurite outgrowth, and thermal pain sensitivity remain to be elucidated. To determine the SUMOylation of CRMP4, Glutathione S-transferase (GST) - Small Ubiquitin-like Modifier 1 (-SUMO1), -SUMO2, and -SUMO3 proteins were purified for GST-pulldown. Immunofluorescence staining was performed to observe colocalization of CRMP4 and SUMOs. Co-immunoprecipitation (co-IP) was performed to assess the interaction between CRMP4 and SUMO2. GST-pulldown and co-IP were performed to verify the interaction between CRMP4 and Cav1.2. The impact of SUMOylation of CRMP4 on its interaction with Cav1.2 was determined. Then, the effect of CRMP4 SUMOylation on neurite outgrowth was observed. Whole-cell patch clamping revealed the effect of CRMP4 SUMOylation on Cav1.2 mediated calcium influx. Paw withdrawal latency was measured to assess the impact of CRMP4 SUMOylation on thermal pain sensitivity in rats. The data revealed that CRMP4 K374 is a potential site for SUMO modification. SUMO1, SUMO2, and SUMO3 can all interact with CRMP4. SUMO2 interacts with CRMP4, but not a variant of CRMP4 harboring a mutation of K374. CRMP4 and SUMO proteins colocalized in neurites, and CRMP4 deSUMOylation promoted neurite outgrowth. CRMP4 interacted with Cav1.2, and deSUMOylation of CRMP4 strengthened this interaction. CRMP4 promoted calcium influx via Cav1.2, and overexpression of CRMP4 significantly increased thermal pain sensitivity in rats, which CRMP4 deSUMOylation strengthened. In conclusion, these data demonstrate the SUMOylation of CRMP4, elucidate the impacts of SUMOylation on the interaction with Cav1.2 on neurite outgrowth and thermal pain sensitivity.

Songorine promotes cardiac mitochondrial biogenesis via Nrf2 induction during sepsis

Septic cardiomyopathy is characterized by impaired contractive function with mitochondrial dysregulation. Songorine is a typical active C20-diterpene alkaloid from the lateral root of Aconitum carmichaelii, which has been used for the treatment of heart failure. This study investigated the protective role of songorine in septic heart injury from the aspect of mitochondrial biogenesis. Songorine (10, 50 mg/kg) protected cardiac contractive function against endotoxin insult in mice with Nrf2 induction. In cardiomyocytes, lipopolysaccharide (LPS) evoked mitochondrial reactive oxygen species (ROS) production and redistributed STIM1 to interact with Orai1 for the formation of calcium release-activated calcium (CRAC) channels, mediating calcium influx, which were prevented by songorine, likely due to ROS suppression. Songorine activated Nrf2 by promoting Keap1 degradation, having a contribution to enhancing antioxidant defenses. When LPS shifted metabolism away from mitochondrial oxidative phosphorylation (OXPHOS) in cardiomyocytes, songorine upregulated mitochondrial genes involved in fatty acid β-oxidation, tricarboxylic acid (TCA) cycle and electron transport chain in a manner dependent on Nrf2, resultantly protecting the capability of OXPHOS. Songorine increased luciferase report gene activities of nuclear respiratory factor-1 (Nrf1) and mitochondrial transcription factor A (Tfam) dependently on Nrf2, indicative of the regulation of Nrf2/ARE and NRF1 signaling cascades. Songorine promoted PGC-1α binding to Nrf2, and the cooperation was required for songorine to activate Nrf2/ARE and NRF1 for the control of mitochondrial quality and quantity. In support, the beneficial effects of songorine on cardioprotection and mitochondrial biogenesis were diminished by cardiac Nrf2 deficiency in mice subjected to LPS challenge. Taken together, these results showed that Nrf2 transcriptionally promoted mitochondrial biogenesis in cooperation with PGC-1α. Songorine activated Nrf2/ARE and NRF1 signaling cascades to rescue cardiomyocytes from endotoxin insult, suggesting that protection of mitochondrial biogenesis was a way for pharmacological intervention to prevent septic heart injury.

TRESK is a key regulator of nocturnal suprachiasmatic nucleus dynamics and light adaptive responses

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.

Abstract 15: The Role Of Opioid Receptors In Podocytes In The Development Of Hypertension In Dahl Salt-sensitive Rats

The rise in opioid use underscores the importance to better understand the direct and indirect effects of opioids on renal function and blood pressure. Although opioid use is associated with predictors of cardiovascular diseases, these drugs are common analgesics for hypertensive patients. We hypothesize that stimulation of opioid receptors (ORs) leads to elevated intracellular calcium level in podocytes ultimately leading to cell apoptosis, development of albuminuria and consequent progression of hypertension. Live calcium imaging experiments on freshly isolated glomeruli from rat and human kidneys, as well as human immortalized podocyte cell line, was performed to test the effect of specific ORs agonists. Following experiments assessed the effect of opioid signaling on the development of hypertension and kidney function in Dahl salt-sensitive (SS) rats, which were fed a 0.4% (LS) or 8% (HS) NaCl diets for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Stimulation of kappa-ORs, but not mu-ORs or delta-ORs, mediated calcium influx in podocytes through activation of TRPC6 channels. The effect of BRL52537 was completely abolished when we used the 0 mM calcium media or when a TRPC6 channel inhibitor (SAR7334) was applied. Triggering the kappa-OR/TRPC6 pathway induced podocyte cell shape changes via actin cytoskeleton remodeling. In vivo studies revealed that rats chronically treated with BRL52537 exhibited augmented blood pressure (MAP was 179 ± 15 vs. 151 ± 11 mmHg), albuminuria, and elevation in podocyte calcium. Western blot analysis revealed elevated levels of nephrin in urine samples and pro-caspase-3 in renal cortex. Moreover, TRPC6 expression was elevated under hypertensive conditions and further promoted pathological increase in calcium influx in response to kappa-OR stimulation. Summarizing the data, the opioid-induced increase in the calcium flux in podocytes is expected to contribute to kidney injury leading to progression of salt-induced hypertension. These data demonstrate that the kappa-OR/TRPC6 signaling pathway directly influences podocyte calcium handling, provoking the development of kidney injury in the opioid treated hypertensive cohort.

Pulsed radiofrequency for chronic pain: In vitro evidence of an electroporation mediated calcium uptake

Pulsed radiofrequency (PRF) treatments for chronic pain consist in the delivery of a train of sinusoidal electric bursts to the targeted nerve. Despite numerous clinical evidence of its efficiency, the mechanism of action of PRF remains unclear. Since most of the reported biological effects of PRF can be initiated by a calcium influx into the neurons, we hypothesized that PRF may induce a mild electroporation effect causing a calcium uptake. To test this hypothesis, HEK-293 cells were exposed to PRF bursts and cytosolic calcium and Yo-Pro-1 uptake were monitored. After a single burst, calcium peaks were observed for electric fields above 480 V/cm while the uptake of Yo-pro-1 was insignificant. After a train of 120 bursts, the electric fields required to induce a calcium and Yo-pro-1 uptake decreased to 330 V/cm and 880 V/cm respectively. Calcium peaks were not detected when cells were treated in calcium free media. The temperature increase during the treatments was lower than 5 °C in all cases. Finally, the cell response for different burst frequencies and extracellular media conductivities correlated with the induced transmembrane voltage calculated with a numerical model. Our results support the hypothesis of an electroporation mediated calcium influx.

Tunable Fast Relaxation in Imine-Based Nanofibrillar Hydrogels Stimulates Cell Response through TRPV4 Activation.

As a key mechanical signal of natural extracellular matrix (ECM), stress relaxation plays an essential role in cell fate decision. However, the biomimetic matrix with fast stress relaxation and its cellular response mechanism have received little attention. Meanwhile, the nanofibrillar architecture which is conductive to mechanical transduction has invariably been ignored in the previous viscoelastic matrix design. Herein, by introducing a dynamic covalent imine bond into a physically cross-linked collagen hydrogel, we prepared bionic fast-relaxing nanofibrillar hydrogels with relaxation time less than 10 s. Through a single control of imine bond content, we realized fine-tuning of the relaxation rate while maintaining a constant initial modulus and fiber density. Using MC3T3-E1 cells as a model, we then proved that the nanofibrillar matrix with fast relaxation mechanics can effectively promote cell spreading and differentiation. In particular, TRPV4 as a molecular sensor of matrix viscoelasticity was demonstrated to regulate cell fate on the nanofibrillar hydrogels by mediating calcium influx. It is expected that the material design principle combining both nanofibrillar structure and tunable fast-relaxation can provide a more broadly adaptable materials platform for simulating natural ECM mechanical cues, and the investigation of the TRPV4 ion channel mediated cellular response will facilitate discovery of more fundamental mechanisms in tissue growth and development.

Distribution of Acid Sensing Ion Channels in Axonal Growth Cones and Presynaptic Membrane of Cultured Hippocampal Neurons.

Although acid-sensing ion channels (ASICs) are widely expressed in the central nervous system, their distribution and roles in axonal growth cones remain unclear. In this study, we examined ASIC localization and function in the axonal growth cones of cultured immature hippocampal neurons. Our immunocytochemical data showed that native and overexpressed ASIC1a and ASIC2a are both localized in growth cones of cultured young hippocampal neurons. Calcium imaging and electrophysiological assay results were utilized to validate their function. The calcium imaging test results indicated that the ASICs (primarily ASIC1a) present in growth cones mediate calcium influx despite the addition of voltage-gated Ca2+ channels antagonists and the depletion of intracellular calcium stores. The electrophysiological tests results suggested that a rapid decrease in extracellular pH at the growth cones of voltage-clamped neurons elicits inward currents that were blocked by bath application of the ASIC antagonist amiloride, showing that the ASICs expressed at growth cones are functional. The subsequent immuno-colocalization test results demonstrated that ASIC1a and ASIC2a are both colocalized with Neurofilament-H and Bassoon in mature hippocampal neurons. This finding demonstrated that after reaching maturity, ASIC1a and ASIC2a are both distributed in axons and the presynaptic membrane. Our data reveal the distribution of functional ASICs in growth cones of immature hippocampal neurons and the presence of ASICs in the axons and presynaptic membrane of mature hippocampal neurons, indicating a possible role for ASICs in axonal guidance, synapse formation and neurotransmitter release.

Label‐Free Quantitative Proteomic Profiling of LAD2 Mast Cell Releasates Reveals the Mechanism of Tween‐80‐Induced Anaphylactoid Reaction

Tween-80 is one of the most important causes resulting in anaphylactoid reaction. However, its mechanism remains unclear. Proteomic characterizations of mast cells' excreta in response to Tween-80 are assayed to investigate the mechanism of anaphylactoid reaction. A label-free LCMS/MS-based proteomics is used to analyze Tween-80-stimulated Laboratory of Allergic Diseases 2 (LAD2) mast cells releasates. The results of proteomic are analyzed by bioinformatics analysis. Western blotting is used to verify the expression of proteins. Overall, endocytosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and calcium signaling pathways play important roles in Tween-80-induced LAD2 cells activation by bioinformatics analysis. The expressions of relative proteins including actin-related protein 2/3 complexes, vacuolar protein sorting-associated protein, phosphorylation of transcription factor of P105 and P65, phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3 R), phosphoinositide phospholipase Cγ (PLCγ), and protein kinase C (PKC), are significantly increased in Tween-80 group compared to control. Tween-80 might be internalized via endocytosis, which induces degranulation by PLCγ/PKC pathways mediated calcium influx, and promotes the generation of inflammatory mediators via NF-κB pathway resulting in anaphylactoid reaction.