Regulation Of Calcium Influx Research Articles

Reduced calcium influx in the hypoxia-tolerant Spalax: The role of the erythropoietin receptor

Tissue hypoxia is a condition that induces calcium influx into living cells. Calcium is a major player in maintaining cell signaling and homeostasis, and mediates the regulation of gene transcription and cell proliferation; however, acute and aggressive calcium influx induced by hypoxia eventually leads to programmed cell death.The blind mole rat, Spalax, is a wild-spread burrowing mammal adapted to hypoxic environments. A tyrosine -to- phenylalanine (F481 in Spalax corresponding to Y485 in human full-length receptor; Y460 in human mature form) substitution is found in the erythropoietin receptor of Spalax and other species, which was previously shown to be strongly involved in the calcium channels activation and subsequent calcium influx.The current work aimed to explore the dynamics of calcium transport across Spalax nonhematopoietic cells’ membrane compared to above ground rat and mouse, and the role of the erythropoietin receptor of Spalax in the regulation of calcium influx under hypoxia.We show here that Epo-induced calcium influx in HEK293 cells transfected with Spalax EpoR is significantly lower than that of mouse; in hypoxia this difference was even more pronounced. Western blots confirmed a significant increase of Erk phosphorylation after stimulation with erythropoietin under hypoxia in cells transfected with mouse full length erythropoietin receptor compared to Spalax. Native primary fibroblasts showed lower cytosolic calcium concentrations in Spalax cells when compared to those of rats under normoxic and hypoxic conditions. Spalax EpoR appears to play an important role in preventing deleterious consequences of hypoxia and maintaining cellular homeostasis under stress.

Voltage-Gated Calcium Influx Modifies Cholinergic Inhibition of Inner Hair Cells in the Immature Rat Cochlea.

Until postnatal day (P) 12, inner hair cells of the rat cochlea are invested with both afferent and efferent synaptic connections. With the onset of hearing at P12, the efferent synapses disappear, and afferent (ribbon) synapses operate with greater efficiency. This change coincides with increased expression of voltage-gated potassium channels, the loss of calcium-dependent electrogenesis, and the onset of graded receptor potentials driven by sound. The transient efferent synapses include near-membrane postsynaptic cisterns thought to regulate calcium influx through the hair cell's α9-containing and α10-containing nicotinic acetylcholine receptors. This influx activates small-conductance Ca2+-activated K+ (SK) channels. Serial-section electron microscopy of inner hair cells from two 9-d-old (male) rat pups revealed many postsynaptic efferent cisterns and presynaptic afferent ribbons whose average minimal separation in five cells ranged from 1.1 to 1.7 μm. Efferent synaptic function was studied in rat pups (age, 7-9 d) of either sex. The duration of these SK channel-mediated IPSCs was increased by enhanced calcium influx through L-type voltage-gated channels, combined with ryanodine-sensitive release from internal stores-presumably the near-membrane postsynaptic cistern. These data support the possibility that inner hair cell calcium electrogenesis modulates the efficacy of efferent inhibition during the maturation of inner hair cell synapses.SIGNIFICANCE STATEMENT Strict calcium buffering is essential for cellular function. This problem is especially acute for compact hair cells where increasing cytoplasmic calcium promotes the opposing functions of closely adjoining afferent and efferent synapses. The near-membrane postsynaptic cistern at efferent synapses segregates synaptic calcium signals by acting as a dynamic calcium store. The hair cell serves as an informative model for synapses with postsynaptic cisterns (C synapses) found in central neurons.

Role of Cavβ subunits in cardiac differentiation

Calcium entering the cell through voltage-gated calcium channels (VGCC) activates multiple signaling pathways controlling gene expression. By regulating both plasma membrane targeting and biophysical properties of VGCC, VGCC β-subunits actively regulate calcium influx and thus gene transcription. Emerging studies have reported alternative pathways through which Cavβ translocates to the nucleus and directly regulates gene expression. These studies have highlighted several partners of Cavβ which mediate its role in gene transcription. Several genes regulated by Cavβ have been previously shown to be involved in cell differentiation suggesting its role in this process. However, how this alternative feature of Cavβ impacts cell differentiation remains unclear and Cavβ partners and mechanisms involved are still to be discovered. Here, we investigate how nuclear Cavβ function regulates cardiomyocytes differentiation using iPS-derived cardiomyocyte model. Preliminary experiments show nuclear localization for Cavβ and expression of several Cavβ isoforms in iPS-derived cardiomyocytes. First part of the project will consist in identifying Cavβ partners involved in its nuclear targeting in iPS-derived cardiomyocytes and in its role in gene transcription regulation. This will be achieved using protein-protein interaction screening methods, such as immunoprecipitation of tagged-Cavβ and mass spectrometry analysis of co-immunoprecipitated proteins. Additionally, using ChIP sequencing, we aim at identifying what part of DNA interacts with those complexes and which gene expressions are regulated. Finally, we will decipher how this regulates cardiac differentiation through specific Cavb knockout approach, using CRISPR technology.

Magnesium sulfate for postoperative analgesia after surgery under spinal anesthesia

BackgroundMagnesium has been proven to have antinociceptive effects in animal and human models of pain. Its effect is primarily based on the regulation of calcium influx into the cell, which is natural physiological calcium antagonism and N-methyl-d-aspartate (NMDA) receptor antagonism. MethodsOne hundred and eight patients undergoing surgery with spinal anesthesia received either 250 mg of intravenous magnesium sulfate followed by an infusion of 500 mg magnesium sulfate (25 mg/mL) at the rate of 20 mL/hour; or the same volume of normal saline (control group) as bolus and infusion. The primary end-points in the study were to evaluate the analgesic effect and duration of sensory and motor blockade. The secondary end-points included assessment of hemodynamic effects of intravenous magnesium sulfate and rescue analgesia requirement. ResultsSensory and motor blockade, respectively, were 25 minutes and 34 minutes shorter in the control group. Less patients in the magnesium group (33% vs. 53.7%) than in control group required rescue analgesia in the postoperative period. The control group required rescue analgesia nearly 3 hours earlier than the magnesium group. Only one patient in the control group experienced bradycardia. There was no event of intraoperative hypotension in either of the groups. ConclusionIntravenous magnesium sulfate when given as a bolus, followed by an infusion, delayed and decreased the need of rescue analgesics after spinal anesthesia.

Chemical Genetic Analysis Of Mast‐Cell Activation

Mast cells play critical roles in allergic responses, and calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. Using chemical genetic approaches, we identify the actin reorganizing protein Drebrin as a target of the immunosuppressant BTP, which inhibits calcium influx into cells and IgE‐mediated allergic responses. We also show that Drebrin−/− mice exhibit reduced IgE‐mediated allergic responses. Furthermore, Drebrin−/− mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcɛRI, and agents that relieve actin reorganization rescue mast cell FcɛRI induced degranulation. Drebrin thus represents a novel regulator of mast cell function by regulating the actin cytoskeleton.Support or Funding InformationAI108598

Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

Perioperative Adjunct Magnesium Decreases Postoperative Opioid Requirements—A Meta-Analysis

Objectives: Magnesium (Mg) is the fourth most common cation in the body and has numerous physiological activities and anti-nociceptive effects. The anti-nociceptive effects are primarily mediated by regulation of calcium influx into the cell and antagonism of the N-Methyl-D-aspartate glutamate receptors. Opioids are widely used as analgesics to minimize postoperative pain, but their use is associated with various side effects as well as the potential for addiction and tolerance. Systemic Mg has been proposed as an adjunct to minimize postoperative pain in numerous clinical studies. This meta-analysis aims to critically examine the ability of perioperative intravenous (IV) Mg to reduce opioid use and its’ side effects. Methods: A comprehensive literature search of Pub Med, Cochrane Central Registry of Controlled Trials, and Google Scholar (1966-2016) was performed to identify all randomized control trials (RCTs) assessing the use of perioperative IV Mg in the reduction of postoperative opioid consumption. Keywords searched included combinations of “magnesium”, “pain”, “postoperative”, “preoperative”, “analgesia” and “opioid”. Inclusion criteria included RCTs comparing the use of perioperative IV Mg with a control group in adult patients (>18 yrs) undergoing elective surgery. Cumulative opioid consumption within the first 24 hours (hrs) postoperative period and the incidence of nausea and vomiting were analyzed. Results: 14 RCTs involving 910 patients were identified (455 patients received Mg and 455 patients received placebo or no therapy). Opioid consumption was significantly decreased in the systemic Mg group (standard mean difference [SMD]: 1.39, 95% CI 1.83 to -0.96; p < 0.001) at 24 hrs postoperatively. Subgroup analysis revealed a significant reduction in the morphine consumption (SMD: -1.37, 95% CI: -1.79 to -0.95; p < 0.001) with the use of IV Mg. There was a decrease in tramadol consumption; however, this did not reach statistical significance (SMD: -1.74, 95% CI: -4.62 to 1.13; p = 0.234). Systemic Mg adjunct had no significant effect on postoperative nausea and vomiting (RR = 0.63; 95% CI 0.38 to 1.04; p = 0.07). Conclusion: Perioperative IV Mg administration reduces opioid use in the first 24 hours postoperatively without any serious adverse events. The decreased need for postoperative opioids in the Mg group was not associated with a decrease in opioid-related side effects such as nausea and vomiting. Mg is an efficacious adjunct for postoperative analgesia and should be considered in multimodal analgesic treatment plans. Additional studies are required to optimize the Mg dose and timing, and to address whether specific opioids display unique benefit or resistance to adjunct Mg therapy.

Anti-allergic and anti-inflammatory effects of aqueous extract of Pogostemon cablin.

Allergic disease is caused by exposure to normally innocuous substances that activate mast cells. Mast cell-mediated allergic inflammation is closely related to a number of allergic disorders, such as anaphylaxis, allergic rhinitis, asthma and atopic dermatitis. The discovery of drugs for treating allergic disease is an interesting subject and important to human health. The aim of the present study was to investigate the anti‑allergic and anti-inflammatory effects of the aqueous extract of Pogostemon cablin (Blanco) Benth (AEPC) (a member of the Labiatae family) using mast cells, and also to determine its possible mechanisms of action. An intraperitoneal injection of compound 48/80 or a serial injection of immunoglobulin E and antigen was used to induce anaphylaxis in mice. We found that AEPC inhibited compound 48/80‑induced systemic and immunoglobulin E-mediated cutaneous anaphylaxis in a dose-dependent manner. The release of histamine from mast cells was reduced by AEPC, and this suppressive effect was associated with the regulation of calcium influx. In addition, AEPC attenuated the phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated expression of pro-inflammatory cytokines in mast cells. The inhibitory effects of AEPC on pro-inflammatory cytokines were dependent on the activation of nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). AEPC blocked the PMACI-induced translocation of NF-κB into the nucleus by hindering the degradation of IκBα and the phosphorylation of p38 MAPK. Our results thus indicate that AEPC inhibits mast cell‑mediated allergic inflammation by suppressing mast cell degranulation and the expression of pro-inflammatory cytokines caused by reduced intracellular calcium levels and the activation of NF-κB and p38 MAPK.

GABA(B) receptors couple to Gαq to mediate increases in voltage-dependent calcium current during development.

Metabotropic GABA(B) receptors are known to modulate the activity of voltage-dependent calcium channels. Previously, we have shown that GABA(B) receptors couple to a non-Gi/o G-protein to enhance calcium influx through L-type calcium channels by activating protein kinase C in neonatal rat hippocampal neurons. In this study, the components of this signaling pathway were investigated further. Gαq was knocked down using morpholino oligonucleotides prior to examining GABA(B) -mediated enhancement of calcium influx. When Gαq G-proteins were eliminated using morpholino-mediated knockdown, the enhancing effects of the GABA(B) receptor agonist baclofen (10 μM) on calcium current or entry were eliminated. These data suggest that GABA(B) receptors couple to Gαq to regulate calcium influx. Confocal imaging analysis illustrating colocalization of GABA(B) receptors with Gαq supports this hypothesis. Furthermore, baclofen treatment caused translocation of PKCα (protein kinase C α) but not PKCβ or PKCε, suggesting that it is the α isoform of PKC that mediates calcium current enhancement. Inhibition of calcium/calmodulin-dependent kinase II did not affect the baclofen-mediated enhancement of calcium levels. In summary, activation of GABA(B) receptors during development leads to increased calcium in a subset of neurons through Gαq signaling and PKCα activation without the involvement of calcium/calmodulin-dependent kinase II. Activation of GABA(B) receptors in the neonatal rat hippocampus enhances voltage-dependent calcium currents independently of Gi/o . In this study, knockdown of Gαq with morpholino oligonucleotides abolished enhancement of calcium influx and protein kinase Cα was activated by GABA(B) receptors. Therefore, we hypothesize that GABA(B) receptors couple to Gq to activate PKCα leading to enhancement of L-type calcium current.

Cutting Edge: Drebrin-Regulated Actin Dynamics Regulate IgE-Dependent Mast Cell Activation and Allergic Responses.

Mast cells play critical roles in allergic responses. Calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. We have previously identified the actin reorganizing protein Drebrin as a target of the immunosuppressant 3,5-bistrifluoromethyl pyrazole, which inhibits calcium influx into cells. In this study, we show that Drebrin(-/-) mice exhibit reduced IgE-mediated histamine release and passive systemic anaphylaxis, and Drebrin(-/-) mast cells also exhibit defects in FcεRI-mediated degranulation. Drebrin(-/-) mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcεRI, and agents that relieve actin reorganization rescue mast cell FcεRI-induced degranulation. Our results indicate that Drebrin regulates the actin cytoskeleton and calcium responses in mast cells, thus regulating mast cell function in vivo.

Inhibition of Calcium Influx Reduces Dysfunction and Apoptosis in Lipotoxic Pancreatic β-Cells via Regulation of Endoplasmic Reticulum Stress.

Lipotoxicity plays an important role in pancreatic β-cell failure during the development of type 2 diabetes. Prolonged exposure of β-cells to elevated free fatty acids level could cause deterioration of β-cell function and induce cell apoptosis. Therefore, inhibition of fatty acids-induced β-cell dysfunction and apoptosis might provide benefit for the therapy of type 2 diabetes. The present study examined whether regulation of fatty acids-triggered calcium influx could protect pancreatic β-cells from lipotoxicity. Two small molecule compounds, L-type calcium channel blocker nifedipine and potassium channel activator diazoxide were used to inhibit palmitic acid-induced calcium influx. And whether the compounds could reduce palmitic acid-induced β-cell failure and the underlying mechanism were also investigated. It was found that both nifedipine and diazoxide protected MIN6 pancreatic β-cells and primary cultured murine islets from palmitic acid-induced apoptosis. Meanwhile, the impaired insulin secretion was also recovered to varying degrees by these two compounds. Our results verified that nifedipine and diazoxide could reduce palmitic acid-induced endoplasmic reticulum stress to generate protective effects on pancreatic β-cells. More importantly, it suggested that regulation of calcium influx by small molecule compounds might provide benefits for the prevention and therapy of type 2 diabetes.

The Ca(2+)-activated cation channel TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy.

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin–NFAT pathway and the development of pathological hypertrophy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-015-0501-x) contains supplementary material, which is available to authorized users.

AP2M1 induced internalization of TRPC6 regulates calcium influx

Transient receptor canonical channel (TRPC)‐6, activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. We recently showed that deletion of TRPC6 in mice prevented lung endothelial permeability and inflammation in response to thrombin and LPS indicating targeting TRPC6 will be valuable in preventing uncontrolled increase in endothelial permeability. Here we determine the pathway that inhibits TRPC6 channel activity under physiological setting. Amino acid sequence analyses of TRPC6 reveals the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions and activity. Thus, we performed mutational analysis on these repeats and assess channel activity. We found that deletion of Ist ankyrin repeat in TRPC6 blocked calcium influx in HEK cells. Further mutational analysis identified that 96‐111 residues within Ist ankyrin repeat was required for TRPC6 cell surface retention and calcium influx. Sequence analysis using ELM database showed that this domain has consensus site for AP2M1 (Adaptor‐related protein complex 2, mu 1 subunit) binding. AP2M1 is known to internalize transmembrane proteins via clathrin dependent endocytosis. Overexpression of AP2M1 with wt‐TRPC6 in HEK cells decreased calcium influx as well as expression level of TRPC6. Thus, designing a peptide against AP2M1 binding motif in TRPC6 will help in identifying targets for inhibiting TRPC6 activity.

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling.

Endotoxin-induced vascular endothelial cell migration is dependent on TLR4/NF-κB pathway, NAD(P)H oxidase activation, and transient receptor potential melastatin 7 calcium channel activity

Endothelial dysfunction is decisive and leads to the development of several inflammatory diseases. Endotoxemia-derived sepsis syndrome exhibits a broad inflammation-induced endothelial dysfunction. We reported previously that the endotoxin, lipopolysaccharide (LPS), induces the conversion of endothelial cells (ECs) into activated fibroblasts, showing a myofibroblast-like protein expression profile. Enhanced migration is a hallmark of myofibroblast function. However, the mechanism involved in LPS-induced EC migration is no totally understood. Some studies have shown that the transient receptor potential melastatin 7 (TRPM7) ion channel is involved in fibroblast and tumor cell migration through the regulation of calcium influx. Furthermore, LPS modulates TRPM7 expression. However, whether TRPM7 is involved in LPS-induced EC migration remains unknown.Here, we study the participation of LPS as an inducer of EC migration and study the mechanism underlying evaluating the participation of the TRPM7 ion channel.Our results demonstrate that LPS induced EC migration in a dose-dependent manner. Furthermore, this migratory process was mediated by the TLR-4/NF-κB pathway and the generation of ROS through the PKC-activated NAD(P)H oxidase. In addition, LPS increased the intracellular calcium level and the number of focal adhesion kinase (FAK)-positive focal adhesions in EC. Finally, we demonstrate that using TRPM7 blockers or suppressing TRPM7 expression through siRNA successfully inhibits the calcium influx and the LPS-induced EC migration.These results point out TRPM7 as a new target in the drug design for several inflammatory diseases that impair vascular endothelium function.

Phospholipase Cγ1 Connects the Cell Membrane Pathway to the Nuclear Receptor Pathway in Insect Steroid Hormone Signaling

In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.

Alveolar bone loss: mechanisms, potential therapeutic targets, and interventions.

This article reviews recent research into mechanisms underlying bone resorption and highlights avenues of investigation that may generate new therapies to combat alveolar bone loss in periodontitis. Several proteins, signaling pathways, stem cells, and dietary supplements are discussed as they relate to periodontal bone loss and regeneration. RGS12 is a crucial protein that mediates osteoclastogenesis and bone destruction, and a potential therapeutic target. RGS12 likely regulates osteoclast differentiation through regulating calcium influx to control the calcium oscillation-NFATc1 pathway. A working model for RGS10 and RGS12 in the regulation of Ca(2+) oscillations during osteoclast differentiation is proposed. Initiation of inflammation depends on host cell-microbe interactions, including the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Oral p38 inhibitors reduced lipopolysaccharide (LPS)-induced bone destruction in a rat periodontitis model but showed unsatisfactory safety profiles. The p38 substrate MK2 is a more specific therapeutic target with potentially superior tolerability. Furthermore, MKP-1 shows anti-inflammatory activity, reducing inflammatory cytokine biosynthesis and bone resorption. Multipotent skeletal stem cell (SSC) populations exist within the bone marrow and periosteum of long bones. These bone-marrow-derived SSCs and periosteum-derived SSCs have shown therapeutic potential in several applications, including bone and periodontal regeneration. The existence of craniofacial bone-specific SSCs is suggested based on existing studies. The effects of calcium, vitamin D, and soy isoflavone supplementation on alveolar and skeletal bone loss in post-menopausal women were investigated. Supplementation resulted in stabilization of forearm bone mass density and a reduced rate of alveolar bone loss over 1 yr, compared with placebo. Periodontal attachment levels were also well-maintained and alveolar bone loss suppressed during 24 wk of supplementation.

Genome-Wide Association Study of Proneness to Anger

BackgroundCommunity samples suggest that approximately 1 in 20 children and adults exhibit clinically significant anger, hostility, and aggression. Individuals with dysregulated emotional control have a greater lifetime burden of psychiatric morbidity, severe impairment in role functioning, and premature mortality due to cardiovascular disease.MethodsWith publically available data secured from dbGaP, we conducted a genome-wide association study of proneness to anger using the Spielberger State-Trait Anger Scale in the Atherosclerosis Risk in Communities (ARIC) study (n = 8,747).ResultsSubjects were, on average, 54 (range 45–64) years old at baseline enrollment, 47% (n = 4,117) were male, and all were of European descent by self-report. The mean Angry Temperament and Angry Reaction scores were 5.8±1.8 and 7.6±2.2. We observed a nominally significant finding (p = 2.9E-08, λ = 1.027 - corrected pgc = 2.2E-07, λ = 1.0015) on chromosome 6q21 in the gene coding for the non-receptor protein-tyrosine kinase, Fyn.ConclusionsFyn interacts with NDMA receptors and inositol-1,4,5-trisphosphate (IP3)-gated channels to regulate calcium influx and intracellular release in the post-synaptic density. These results suggest that signaling pathways regulating intracellular calcium homeostasis, which are relevant to memory, learning, and neuronal survival, may in part underlie the expression of Angry Temperament.

Using Exofacially Tagged Functional Cav2.2 to Investigate the Modulation of Pore Subunit Trafficking by Auxiliary Calcium Channel Subunits

N type Calcium Channels are voltage gated channels composed of a pore forming α1B (Cav2.2) subunit and 2 auxiliary subunits α2δ and β. N type calcium channels regulate calcium influx and exocytosis in presynaptic terminals of neurons. Tight control of Cav2.2 trafficking is important as small changes in channel density at the cell surface can result in large changes in calcium ion influx and therefore neurotransmitter release. The auxiliary subunits α2δ and β modulate the biophysical properties and surface channel density of Cav2.2 through mechanisms that are not fully defined. To investigate the contribution of these subunits to Cav2.2 trafficking we have created a fully functional exofacially tagged Cav2.2. Using this tool we are able to quantify the contribution of each subunit on Cav2.2 trafficking to the surface of N2a cells (mouse neuroblastoma derived cell line). Our results support previous electrophysiological data indicating the absolute requirement for the β subunit and additional enhancement by the α2δ subunit. Our data also suggest an intracellular site of interaction between Cav2.2 and α2δ. Epitope obstruction of α2δ bound to Cav2.2 may indicate the region of α2δ involved in interaction with Cav2.2.

Different Calcium Sources Control Somatic versus Dendritic SK Channel Activation during Action Potentials

Small-conductance calcium-activated potassium (SK) channels play an important role in regulating neuronal excitability. While SK channels at the soma have long been known to contribute to the medium afterhyperpolarization (mAHP), recent evidence indicates they also regulate NMDA receptor activation in dendritic spines. Here we investigate the activation of SK channels in spines and dendrites of rat cortical pyramidal neurons during action potentials (APs), and compare this to SK channel activation at the soma. Using confocal calcium imaging, we demonstrate that the inhibition of SK channels with apamin results in a location-dependent increase in calcium influx into dendrites and spines during backpropagating APs (average increase, ~40%). This effect was occluded by block of R-type voltage-dependent calcium channels (VDCCs), but not by inhibition of N- or P/Q-type VDCCs, or block of calcium release from intracellular stores. During these experiments, we noticed that the calcium indicator (Oregon Green BAPTA-1) blocked the mAHP. Subsequent experiments using low concentrations of EGTA (1 mm) produced the same result, suggesting that somatic SK channels are not tightly colocalized with their calcium source. Consistent with this idea, all known subtypes of VDCCs except R-type were calcium sources for the apamin-sensitive mAHP at the soma. We conclude that SK channels in spines and dendrites of cortical pyramidal neurons regulate calcium influx during backpropagating APs in a distance-dependent manner, and are tightly coupled to R-type VDCCs. In contrast, SK channels activated by APs at the soma of these neurons are weakly coupled to a variety of VDCCs.