Calcium Ion Channel Activity Research Articles

Transcriptomics reveals dynamic changes in the “gene profiles” of rat supraspinatus tendon at three different time points after diabetes induction

ObjectiveThere is increasing evidence that type 2 diabetes mellitus (T2DM) is an independent risk factor for the occur of tendinopathy. Therefore, this study is the first to explore the dynamic changes of the “gene profile” of supraspinatus tendon in rats at different time points after T2DM induction through transcriptomics, providing potential molecular markers for exploring the pathogenesis of diabetic tendinopathy.MethodsA total of 40 Sprague-Dawley rats were randomly divided into normal (NG, n = 10) and T2DM groups (T2DM, n = 30) and subdivided into three groups according to the duration of diabetes: T2DM-4w, T2DM-8w, and T2DM-12w groups; the duration was calculated from the time point of T2DM rat model establishment. The three comparison groups were set up in this study, T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG. Differentially expressed genes (DEGs) in 3 comparison groups were screened. The intersection of the three comparison groups’ DEGs was defined as key genes that changed consistently in the supraspinatus tendon after diabetes induction. Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto encyclopedia of genes and genomes (KEGG) functional annotation and enrichment analysis were performed for DEGs.ResultsT2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG detected 519 (251 up-regulated and 268 down-regulated), 459 (342 up-regulated and 117 down-regulated) and 328 (255 up-regulated and 73 down-regulated) DEGs, respectively. 103 key genes of sustained changes in the supraspinatus tendon following induction of diabetes, which are the first identified biomarkers of the supraspinatus tendon as it progresses through the course of diabetes.The GO analysis results showed that the most significant enrichment in biological processes was calcium ion transmembrane import into cytosol (3 DEGs). The most significant enrichment in cellular component was extracellular matrix (9 DEGs). The most significant enrichment in molecular function was glutamate-gated calcium ion channel activity (3 DEGs). The results of KEGG pathway enrichment analysis showed that there were 17 major pathways (p < 0.05) that diabetes affected supratinusculus tendinopathy, including cAMP signaling pathway and Calcium signaling pathway.ConclusionsTranscriptomics reveals dynamic changes in the“gene profiles”of rat supraspinatus tendon at three different time points after diabetes induction. The 103 DEGs identified in this study may provide potential molecular markers for exploring the pathogenesis of diabetic tendinopathy, and the 17 major pathways enriched in KEGG may provide new ideas for exploring the pathogenesis of diabetic tendinopathy.

Modulation of calcium signaling and metabolic pathways in endothelial cells with magnetic fields.

Calcium signaling plays a crucial role in various physiological processes, including muscle contraction, cell division, and neurotransmitter release. Dysregulation of calcium levels and signaling has been linked to a range of pathological conditions such as neurodegenerative disorders, cardiovascular disease, and cancer. Here, we propose a theoretical model that predicts the modulation of calcium ion channel activity and calcium signaling in the endothelium through the application of either a time-varying or static gradient magnetic field (MF). This modulation is achieved by exerting magnetic forces or torques on either biogenic or non-biogenic magnetic nanoparticles that are bound to endothelial cell membranes. Since calcium signaling in endothelial cells induces neuromodulation and influences blood flow control, treatment with a magnetic field shows promise for regulating neurovascular coupling and treating vascular dysfunctions associated with aging and neurodegenerative disorders. Furthermore, magnetic treatment can enable control over the decoding of Ca signals, ultimately impacting protein synthesis. The ability to modulate calcium wave frequencies using MFs and the MF-controlled decoding of Ca signaling present promising avenues for treating diseases characterized by calcium dysregulation.

L-type calcium ion channel-mediated activation of autophagy in vascular smooth muscle cells via thonningianin A (TA) alleviates vascular calcification in type 2 diabetes mellitus

Vascular calcification (VC) is associated with increased morbidity and mortality, especially among people with type 2 diabetes mellitus (T2DM). The pathogenesis of vascular calcification is incompletely understood, and until now, there have been no effective therapeutics for vascular calcification. The L-type calcium ion channel in the cell membrane is vital for Ca2+ influx. The effect of L-type calcium ion channels on autophagy remains to be elucidated. Here, the natural compound thonningianin A (TA) was found to ameliorate vascular calcification in T2DM via the activation of L-type calcium ion channels. The results showed that TA had a concentration-dependent ability to decrease the transcriptional and translational expression of the calcification-related proteins runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteopontin (OPN) (P < 0.01) via ATG7-dependent autophagy in β-glycerophosphate (β-GP)- and high glucose (HG)-stimulated primary mouse aortic smooth muscle cells (MASMCs) and alleviate aortic vascular calcification in VitD3-stimulated T2DM mice. However, nifedipine, an inhibitor of L-type calcium ion channels, reversed TA-induced autophagy and Ca2+ influx in MASMCs. Molecular docking analysis revealed that TA was located in the hydrophobic pocket of Cav1.2 α1C and was mainly composed of the residues Ile, Phe, Ala and Met, which confirmed the efficacy of TA in targeting the L-type calcium channel of Cav1.2 on the cell membrane. Moreover, in an in vivo model of vascular calcification in T2DM mice, nifedipine reversed the protective effects of TA on aortic calcification and the expression of the calcification-related proteins RUNX2, BMP2 and OPN (P < 0.01). Collectively, the present results reveal that the activation of cell membrane L-type calcium ion channels can induce autophagy and ameliorate vascular calcification in T2DM. Thonningianin A (TA) can target and act as a potent activator of L-type calcium ion channels. Thus, this research revealed a novel mechanism for autophagy induction via L-type calcium ion channels and provided a potential therapeutic for vascular calcification in T2DM.

Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase

The dual functions of TMEM16F as Ca2+-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca2+-activated Cl- channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.

Abstract A010: ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype?

Abstract Introduction The expression level and single nucleotide polymorphisms of the prostate-specific gene ANO7 have been shown to correlate with prostate cancer (PrCa) outcome. Very little is known about ANO7, apart from its expression profile in tissues and that the protein is either a calcium-activated ion channel or a lipid scramblase. Our results suggest that ANO7 likely has a role in PrCa progression and that loss of ANO7 is associated with malignant PrCa. Materials and methods Sections of a prostate cancer formalin-fixed paraffin-embedded tissue microarray were used for ANO7 mRNA and protein detection. mRNA was detected with fluorescent in situ hybridization (FISH). Protein was detected with immunohistochemistry (IHC) using a polyclonal anti-ANO7 antibody (HPA035730). mRNA from fresh radical prostatectomy primary tumor samples were sequenced and subsequent gene expression levels were analyzed for co-expression with ANO7. The sample set consisted of a matched tumor and benign tissue sample (15+15) from each patient. Results The FISH images indicated explicitly that ANO7 mRNA is exclusively expressed in the luminal cells of the prostatic glandular epithelium. ANO7 mRNA is expressed in all cancerous lumina-forming glandular structures, but not in poorly-formed glands or sheets of cancer, in which ANO7 mRNA expression is very low or non-existent. Morphologically benign glands amidst cancerous tissue showed no expression, in contrast to tissue in which benign glands are predominating. The IHC showed a similar staining pattern to FISH.ANO7 co-expression gene set enrichment analysis resulted in 19 positively correlating and 77 negatively correlating pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes) terms. Most of the positively correlating terms relate to metabolism, whereas among the negatively correlating terms, there were many signal transduction and cell growth and death-related pathways, such as Hippo, Ras, and p53 signaling pathways. Conclusions ANO7 mRNA and protein expression correlate spatially in the tissue, showing that the expression is specific to differentiated luminal cells and highly reduced in high-grade cancer. This notion is supported by the co-expressed gene-set enrichment analysis, which is showing a negative correlation of ANO7 with many pathways that are associated with aggressive PrCa. Mainly metabolism-related processes were positively correlated with ANO7, which could indicate that ANO7 is linked to the reprogramming of metabolism in the cells during cancer progression. Further research aims to identify the pathways in which ANO7 plays a significant role and to elucidate how the ANO7 protein functionally alters signaling related to PrCa progression. Citation Format: Olli Metsälä, Gudrun Wahlström, Pekka Taimen, Johanna Schleutker. ANO7 expression changes in prostate cancer progression — Association with an aggressive phenotype? [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A010.

A TMEM16J variant leads to dysregulated cytosolic calcium which may lead to renal disease.

SIGIRR (single immunoglobulin IL-1 related receptor), PKP3 (plakophilin 3), and TMEM16J (anoctamin 9), a putative calcium-activated ion channel and phospholipid scramblase, control the immune response and the extent of inflammation. Variants of SIGIRR/PKP3/TMEM16J lead to severe inflammatory diseases such as pneumonia, enterocolitis, and kidney graft rejection. Meta-analysis of genome-wide association studies identified TMEM16J-T604A as a promotor for chronic kidney disease (CKD), but the disease mechanism and function of TMEM16J remain unknown. Here, we demonstrate TMEM16J as a calcium-activated calcium-permeable channel, which is expressed in the endoplasmic reticulum (ER). TMEM16J controls the intracellular distribution of calcium, and inhibits intracellular receptor-mediated Ca2+ signals and Ca2+ -dependent activation of ion channels, but augments transcription and release of pro-inflammatory cytokines. Renal epithelial cells expressing the variant TMEM16J-T604A show enhanced calcium signals when compared to cells expressing wt-TMEM16J, and demonstrate spontaneous transcription and release of cytokines. This study identifies TMEM16J as an important regulator of intracellular Ca2+ signals, ion channel activity, and cytokine release. TMEM16J may therefore affect immune response in renal tissue and immune cells.

Impaired Bestrophin Channel Activity in an iPSC-RPE Model of Best Vitelliform Macular Dystrophy (BVMD) from an Early Onset Patient Carrying the P77S Dominant Mutation.

Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.

Exome sequencing of Japanese schizophrenia multiplex families supports the involvement of calcium ion channels.

BackgroundMost sequencing studies of schizophrenia (SCZ) have focused on de novo genetic variants due to interpretability. However, investigating shared rare variants among patients in the same multiplex family is also important. Relatively large-scale analyses of SCZ multiplex families have been done in Caucasian populations, but whether detected variants are also pathogenic in the Japanese population is unclear because of ethnic differences in rare variants.Materials and methodsWe performed whole-exome sequencing (WES) of 14 Japanese SCZ multiplex families. After quality control and filtering, we identified rare variants shared among affected persons within the same family. A gene ontology (GO) analysis was performed to identify gene categories possibly affected by these candidate variants.ResultsWe found 530 variants in 486 genes as potential candidate variants from the 14 SCZ multiplex families examined. The GO analysis demonstrated significant enrichment in calcium channel activity.ConclusionThis study provides supporting evidence that calcium ion channel activity is involved in SCZ. WES of multiplex families is a potential means of identifying disease-associated rare variants for SCZ.

Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer.

Simple SummaryWhole-genome sequencing and bioinformatics analysis on unique colorectal cancer families revealed two attractive candidate predisposition genes, CYBA and TRPM4, each with a loss-of-function variant. Supported by our functional studies, we suggest that the two gene defects mechanistically involve intestinal barrier integrity through reactive oxygen species and mucus biology, which converges in chronic bowel inflammation, a known risk factor for colorectal cancer.Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25–1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48–12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

Regulation of Hippocampal Gamma Oscillations by Modulation of Intrinsic Neuronal Excitability.

Ion channels activated around the subthreshold membrane potential determine the likelihood of neuronal firing in response to synaptic inputs, a process described as intrinsic neuronal excitability. Long-term plasticity of chemical synaptic transmission is traditionally considered the main cellular mechanism of information storage in the brain; however, voltage- and calcium-activated channels modulating the inputs or outputs of neurons are also subjects of plastic changes and play a major role in learning and memory formation. Gamma oscillations are associated with numerous higher cognitive functions such as learning and memory, but our knowledge of their dependence on intrinsic plasticity is by far limited. Here we investigated the roles of potassium and calcium channels activated at near subthreshold membrane potentials in cholinergically induced persistent gamma oscillations measured in the CA3 area of rat hippocampal slices. Among potassium channels, which are responsible for the afterhyperpolarization in CA3 pyramidal cells, we found that blockers of SK (KCa2) and KV7.2/7.3 (KCNQ2/3), but not the BK (KCa1.1) and IK (KCa3.1) channels, increased the power of gamma oscillations. On the contrary, activators of these channels had an attenuating effect without affecting the frequency. Pharmacological blockade of the low voltage-activated T-type calcium channels (CaV3.1–3.3) reduced gamma power and increased the oscillation peak frequency. Enhancement of these channels also inhibited the peak power without altering the frequency of the oscillations. The presented data suggest that voltage- and calcium-activated ion channels involved in intrinsic excitability strongly regulate the power of hippocampal gamma oscillations. Targeting these channels could represent a valuable pharmacological strategy against cognitive impairment.

Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

Re-analysis of Genetic Risks for Chronic Fatigue Syndrome From 23andMe Data Finds Few Remain.

Abstract It is tempting to mine the abundance of DNA data that is now available from direct-to-consumer genetic tests but this approach also has its pitfalls A recent study put forth a list of 50 single nucleotide polymorphisms (SNPs) that predispose to Chronic Fatigue Syndrome (CFS), a potentially major advance in understanding this still mysterious condition. However, only the patient cohort data came from a commercial company (23andMe) while the control was from a genetic database. The extent to which 23andMe data agree with genetic reference databases is unknown. We reanalyzed the 50 purported CFS SNPs by comparing to control data specifically from 23andMe which are available through public platform OpenSNP. In addition, large high-quality database ALFA was used as an additional control. The analysis lead to dramatic change with the top of the leaderboard for CFS risk reduced and reversed from an astronomical 129,000 times to 0.8. Errors were found both within 23andMe data and the original study-reported Kaviar database control. Only 3 of 50 SNPs survived initial study criterion of at least twice as prevalent in patients, EFCAB4B, involved in calcium ion channel activation, LINC01171, and MORN2 genes. We conclude that the reported top-50 deleterious polymorphisms for Chronic Fatigue Syndrome were more likely the top-50 errors in the 23andMe and Kaviar databases. In general, however, correlation of 23andMe control with ALFA was a respectable 0.93, suggesting an overall usefulness of 23andMe results for research purposes but only if caution is taken with chips and SNPs.

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.

Janus 3D printed dynamic scaffolds for nanovibration-driven bone regeneration

The application of physical stimuli to cell cultures has shown potential to modulate multiple cellular functions including migration, differentiation and survival. However, the relevance of these in vitro models to future potential extrapolation in vivo depends on whether stimuli can be applied “externally”, without invasive procedures. Here, we report on the fabrication and exploitation of dynamic additive-manufactured Janus scaffolds that are activated on-command via external application of ultrasounds, resulting in a mechanical nanovibration that is transmitted to the surrounding cells. Janus scaffolds were spontaneously formed via phase-segregation of biodegradable polycaprolactone (PCL) and polylactide (PLA) blends during the manufacturing process and behave as ultrasound transducers (acoustic to mechanical) where the PLA and PCL phases represent the active and backing materials, respectively. Remote stimulation of Janus scaffolds led to enhanced cell proliferation, matrix deposition and osteogenic differentiation of seeded human bone marrow derived stromal cells (hBMSCs) via formation and activation of voltage-gated calcium ion channels.

Anoctamin 3: A Possible Link between Cluster Headache and Ca2+ Signaling

Cluster headache is a severe primary headache characterized by extremely painful attacks of unilateral headache. Verapamil is commonly used as a prophylactic treatment with good effect. In order to search for new pathways involved in the pathophysiology of cluster headache, we analyzed genetic variants that were previously linked to verapamil response in migraine in a Swedish cluster headache case-control sample. We used TaqMan qPCR for genetic screening and performed a gene expression analysis on associated genes in patient-derived fibroblasts, and further investigated which reference genes were suitable for analysis in fibroblasts from cluster headache patients. We discovered a significant association between anoctamin 3, a gene encoding a calcium-activated ion channel, and cluster headache. The association was not dependent on verapamil treatment since the associated variant, rs1531394, was also overrepresented in patients not using verapamil. No difference was found in the anoctamin 3 gene expression between controls and patients. Also, we determined that TBP, IPO8 and PDHB were suitable reference genes in cluster headache fibroblasts. This finding is the first report of an association between a variant in a gene encoding an ion-channel and cluster headache, and the first significant genetic evidence of calcium involvement in cluster headache pathophysiology.

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling

SUMMARYAs a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.

Elevation of cytosolic Ca2+ in response to energy deficiency in plants: the general mechanism of adaptation to low oxygen stress.

Ca2+ can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca2+ release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca2+ release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca2+ is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca2+ activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb-nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca2+- and low pH-activated external NADH and NADPH dehydrogenases participate. Ca2+ can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.

TRPV4 Activation ‐ Implications in Hydrocephalic Neurodegenerative Disease

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and traumatic brain injury, often share similar pathologies, one of which is hydrocephalus. Hydrocephalus is characterized by an imbalance in cerebrospinal fluid (CSF) production and/or reabsorption or by a blockage in the CSF circulation resulting in enlarged ventricles and an increase in brain hydrostatic pressure leading to neuronal destruction. The choroid plexus (CP) is the main producer of CSF and is composed of a high resistance barrier epithelium that surrounds a network of capillaries. The CP epithelium regulates the transport of ions and water between the ventricles and capillaries, thus controlling the production of CSF. Transient Receptor Potential Vanilloid‐4 (TRPV4) is a mechano‐ and osmo‐sensitive, cation channel present in CP epithelia. TRPV4 activation leads to an influx of calcium, which can stimulate calcium activated ion channels leading to the potential for significant transepithelial ion flux. If TRPV4 is overstimulated, it is possible that excess ion movement can cause an increase in CSF volume, resulting in hydrocephalus. Current research in our laboratory has shown that treatment with a TRPV4 antagonist ameliorates hydrocephalus in the Wpk rodent model. We have also shown that TRPV4 expression is increased in the late‐stage hydrocephalic animal compared to normal. TRPV4 can be activated through a number of stimuli, including physical means osmotic changes, increases in temperature and chemical activators, such as cytokines and inflammatory mediators. In particular, the latter activators can be seen in the symptomologies of neurodegenerative diseases exhibiting hydrocephalus. Utilizing a high resistance porcine choroid plexus (PCP‐R) cell line, we investigated the effects of various cytokines, inflammatory mediators, and inflammation pathways on TRPV4 activity using immunohistochemistry and electrophysiology techniques. We found that pro‐inflammatory cytokines, such as Tumor Necrosis Factor (TNF)‐α and interleukin (IL)‐1β, had no effect on TRPV4 stimulated ion flux in vitro; pro‐inflammatory mediators, arachidonic acid and its metabolite 5,6‐epoxyeicosatrienoic acid (EET), had inhibitory effects. Furthermore, the hormone epinephrine, used to combat inflammatory anaphylactic reactions, potentiated TRPV4 activity. Immunofluorescence of the kinase SPAK, which plays a role in Nuclear Factor (NF)‐κB – dependent inflammation and epithelial barrier function, was increased in the CP of hydrocephalic rats compared to the wild type, and inhibitors of NF‐kB altered TRPV4‐mediated ion flux. Together these data imply that selected components of an inflammatory response can play a role in TRPV4 activation and can potentially contribute to the subsequent development of hydrocephalus.Support or Funding InformationDepartment of Defense office of the Congressionally Directed Medical Research Programs (CDMRP) and an Indiana Clinical and Translational Sciences AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Malignant hyperthermia in dogs during general anaesthesia

Malignant hyperthermia is a pharmacogenetic disorder of skeletal muscles developing as a hypermetabolic response to inhalation anaesthetics such as halothane, sevoflurane or isoflurane and depolarizing muscle relaxants such as succinylcholine. It is produced by mutation of the RYR1 gene in dogs. In anaesthetised dogs, regardless of the anaesthetic drugs used, calcium ion channel activity may change and malignant hyperthermia may develop. Clinical features are hyperthermia, tachypne, hypercarbia, increased oxygen consumption, severe metabolic acidosis, hyperkalemia, cardiac arrhytmias and muscle rigidity. For a diagnosis of malignant hyperthermia to be made, at least 3 of the clinical findings must be determined. Dantrolene sodium is specific antagonist of malignant hyperthermia. However, it is not usually preferred by veterinary practitioners due to its high cost as well as the low incidence of the occurence of this complication. Therefore it is useful for alternative drugs such as acepromazine, diazepam and alfentanil to be used for the treatment of clinical findings manifested during malignant hyperthermia. The purpose of this review is to share with colleagues the latest information regarding the treatment of canine malignant hyperthermia occurring in relation to general anaesthesia.

Transcriptomic analysis to uncover genes affecting cold resistance in the Chinese honey bee (Apis cerana cerana).

The biological activity and geographical distribution of honey bees is strongly temperature-dependent, due to their ectothermic physiology. In China, the endemic Apis cerana cerana exhibits stronger cold hardiness than Western honey bees, making the former species important pollinators of winter-flowering plants. Although studies have examined behavioral and physiological mechanisms underlying cold resistance in bees, data are scarce regarding the exact molecular mechanisms. Here, we investigated gene expression in A. c. cerana under two temperature treatments, using transcriptomic analysis to identify differentially expressed genes (DEGs) and relevant biological processes, respectively. Across the temperature treatments, 501 DEGs were identified. A gene ontology analysis showed that DEGs were enriched in pathways related to sugar and amino acid biosynthesis and metabolism, as well as calcium ion channel activity. Additionally, heat shock proteins, zinc finger proteins, and serine/threonine-protein kinases were differentially expressed between the two treatments. The results of this study provide a general digital expression profile of thermoregulation genes responding to cold hardiness in A. c. cerana. Our data should prove valuable for future research on cold tolerance mechanisms in insects, and may be beneficial in breeding efforts to improve bee hardiness.