Depletion Of Extracellular Calcium Research Articles

IP3R-Mediated Calcium Release Promotes Ferroptotic Death in SH-SY5Y Neuroblastoma Cells.

Ferroptosis is an iron-dependent cell death pathway that involves the depletion of intracellular glutathione (GSH) levels and iron-mediated lipid peroxidation. Ferroptosis is experimentally caused by the inhibition of the cystine/glutamate antiporter xCT, which depletes cells of GSH, or by inhibition of glutathione peroxidase 4 (GPx4), a key regulator of lipid peroxidation. The events that occur between GPx4 inhibition and the execution of ferroptotic cell death are currently a matter of active research. Previous work has shown that calcium release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels contributes to ferroptosis-induced cell death in primary hippocampal neurons. Here, we used SH-SY5Y neuroblastoma cells, which do not express RyR channels, to test if calcium release mediated by the inositol 1,4,5-trisphosphate receptor (IP3R) channel plays a role in this process. We show that treatment with RAS Selective Lethal Compound 3 (RSL3), a GPx4 inhibitor, enhanced reactive oxygen species (ROS) generation, increased cytoplasmic and mitochondrial calcium levels, increased lipid peroxidation, and caused cell death. The RSL3-induced calcium signals were inhibited by Xestospongin B, a specific inhibitor of the ER-resident IP3R calcium channel, by decreasing IP3R levels with carbachol and by IP3R1 knockdown, which also prevented the changes in cell morphology toward roundness induced by RSL3. Intracellular calcium chelation by incubation with BAPTA-AM inhibited RSL3-induced calcium signals, which were not affected by extracellular calcium depletion. We propose that GPx4 inhibition activates IP3R-mediated calcium release in SH-SY5Y cells, leading to increased cytoplasmic and mitochondrial calcium levels, which, in turn, stimulate ROS production and induce lipid peroxidation and cell death in a noxious positive feedback cycle.

RvD2 stimulates conjunctival goblet cell secretion by activating multiple Ca 2+ ‐dependent intracellular signaling pathways

Background Resolvin (Rv)D2 is biosynthesized from docosahexaenoic acid (DHA) and was identified from murine self-resolving exudates during the resolution phase of self-limited acute inflammation in vivo. RvD2 was identified in non-diseased human tears, which indicates the potential physiological role of RvD2 on the ocular surface. We determined the action of RvD2 on conjunctival goblet cells and the Ca2+- dependent signaling pathways involved. Method Goblet cells were cultured from male rat conjunctiva. Signaling pathways were studied by measuring intracellular [Ca2+] ([Ca2+]i) using fura 2/AM. Mucin secretion was determined using an enzyme-linked lectin assay (ELLA). Results Administration of RvD2 at 10-10M-10-7M to conjunctival goblet cells increased both the [Ca2+]i and the secretion of high molecular weight glycoprotein including the secretory mucin MUC5AC in a dose-dependent manner. The depletion of extracellular calcium significantly blocked the RvD2-induced [Ca2+]i increase. Pre-treatment with phospholipase (PL) C inhibitor U73122 (10-6M), significantly blocked the [Ca2+]i increase and mucin secretion induced by RvD2 10-8M, U73343 as the negative control failed to lower the RvD2-induced [Ca2+]i increase and mucin secretion. Treatment with 2APB 10-6M, the inositol trisphosphate (IP)3 receptor inhibitor, significantly reduced the RvD2-induced [Ca2+]i increase and the mucin secretion. Pre-treatment with 1-butanol 0.3%, the inhibitor of PLD, significantly blocked the [Ca2+]i increase induced by RvD2 10-8M, but not mucin secretion. The PLA2 inhibitor aristolochic acid 10-5M did not block the RvD2-induced [Ca2+]i increase, but successfully downregulated the RvD2-induced mucin secretion. Conclusion RvD2 induces an increase in [Ca2+]i level and mucin secretion in conjunctival goblet cells. The [Ca2+]i increase is dependent upon activation of PLC and PLD, but not PLA2 pathways. The mucin secretion is dependent on PLA2 and PLC, but not PLD pathways. RvD2 may act as a regulator of conjunctival goblet cells physiologically.

Shear-Induced Platelet Activation is Sensitive to Age and Calcium Availability: A Comparison of Adult and Cord Blood.

Antiplatelet therapy for neonates and infants is often extrapolated from the adult experience, based on limited observation of agonist-induced neonatal platelet hypoactivity and poor understanding of flow shear-mediated platelet activation. Therefore, thrombotic events due to device-associated disturbed flow are inadequately mitigated in critically ill neonates with indwelling umbilical catheters and infants receiving cardiovascular implants. Whole blood (WB), platelet-rich plasma (PRP), and gel-filtered platelets (GFP) were prepared from umbilical cord and adult blood, and exposed to biochemical agonists or pathological shear stress of 70 dyne/cm2. We evaluated α-granule release, phosphatidylserine (PS) scrambling, and procoagulant response using P-selectin expression, Annexin V binding, and thrombin generation (PAS), respectively. Activation modulation due to depletion of intracellular and extracellular calcium, requisite second messengers, was also examined. Similar P-selectin expression was observed for sheared adult and cord platelets, with concordant inhibition due to intracellular and extracellular calcium depletion. Sheared cord platelet Annexin V binding and PAS activity was similar to adult values in GFP, but lower in PRP and WB. Annexin V on sheared cord platelets was calcium-independent, with PAS slightly reduced by intracellular calcium depletion. Increased PS activity on purified sheared cord platelets suggest that their intrinsic function under pathological flow conditions is suppressed by cell-cell or plasmatic components. Although secretory functions of adult and cord platelets retain comparable calcium-dependence, PS exposure in sheared cord platelets is uniquely calcium-independent and distinct from adults. Identification of calcium-regulated developmental disparities in shear-mediated platelet function may provide novel targets for age-specific antiplatelet therapy.

CRHR2 (Corticotropin-Releasing Hormone Receptor 2) in the Nucleus of the Solitary Tract Contributes to Intermittent Hypoxia-Induced Hypertension.

This study tested the hypothesis that CRHRs (corticotropin-releasing hormone receptors) in the nucleus of the solitary tract (NTS) contribute to the hypertension induced by intermittent hypoxia (IH) exposure in rats. Initial studies using in situ hybridization revealed low mRNA level of CRHR1 (CRH type 1 receptor) but high mRNA level of CRHR2 (CRH type 2 receptor) in the NTS. Calcium imaging studies on NTS slice preparations using Fura-2-acetoxymethyl ester demonstrated that CRH induced a transient increase of intracellular calcium level. The CRH-induced calcium response was reproduced in the presence of TTX (tetrodotoxin) but was abolished by depletion of extracellular calcium or by the L-type calcium channel blocker Nifedipine. The CRH-induced calcium influx was attenuated by the CRHR2 antagonist K41498 but not by the CRHR1 antagonist NBI-35 965. Calcium influx can be induced by the CRHR2 agonist Urocortin II but not by the CRHR1 agonist Stressin 1. IH exposure did not affect CRHR1 mRNA level but significantly decreased CRHR2 mRNA level and the CRH-induced calcium influx in the NTS. Further in vivo studies showed that intra-fourth ventricle infusion of K41498 did not affect the basal blood pressure but significantly attenuated the IH-induced hypertension; intra-fourth ventricle infusion of Urocortin II significantly increased basal blood pressure and exacerbated the IH-induced hypertension. Collectively, these results suggest that CRHR2 in the NTS contributes to the IH-induced hypertension; downregulation of CRHR2 and CRHR2-mediated calcium influx in the NTS may serve as an adaptive response to protect against the IH-induced hypertension.

Attenuation of mechanical pain hypersensitivity by treatment with Peptide5, a connexin-43 mimetic peptide, involves inhibition of NLRP3 inflammasome in nerve-injured mice

Connexin43 (Cx43) hemichannels in spinal cord astrocytes are implicated in the maintenance of neuropathic pain following peripheral nerve injury. Peptide5 is a Cx43 mimetic peptide that blocks hemichannels. In this study, we investigated the effects of spinal delivery of Peptide5 on mechanical pain hypersensitivity in two mouse models of neuropathic pain, peripheral nerve injury and chemotherapy-induced peripheral neuropathy (CIPN). We demonstrated that 10days following a chronic constriction injury (CCI) of the sciatic nerve, Cx43 expression, co-localised predominantly with astrocytes, was increased in the ipsilateral L3–L5 lumbar spinal cord. An intrathecal injection of Peptide5 into nerve-injured mice, on day 10 when pain was well-established, caused significant improvement in mechanical pain hypersensitivity 8h after injection. Peptide5 treatment resulted in significantly reduced Cx43, and microglial and astrocyte activity in the dorsal horn of the spinal cord, as compared to control saline-treated CCI mice. Further in vitro investigations on primary astrocyte cultures showed that 1h pre-treatment with Peptide5 significantly reduced adenosine triphosphate (ATP) release in response to extracellular calcium depletion. Since ATP is a known activator of the NOD-like receptor protein 3 (NLRP3) inflammasome complex, a key mediator of neuroinflammation, we examined the effects of Peptide5 treatment on NLRP3 inflammasome expression. We found that NLRP3, its adaptor apoptosis-associated spec-like protein (ASC) and caspase-1 protein were increased in the ipsilateral spinal cord of CCI mice and reduced to naïve levels following Peptide5 treatment. In the models of oxaliplatin- and paclitaxel-induced peripheral neuropathy, treatment with Peptide5 had no effect on mechanical pain hypersensitivity. Interestingly, in these CIPN models, although spinal Cx43 expression was significantly increased at day 13 following chemotherapy, NLRP3 expression was not altered. These results suggest that the analgesic effect of Peptide5 is specifically achieved by reducing NLRP3 expression. Together, our findings demonstrate that blocking Cx43 hemichannels with Peptide5 after nerve injury attenuates mechanical pain hypersensitivity by specifically targeting the NLRP3 inflammasome in the spinal cord.

Depletion of extracellular calcium increases cadmium toxicity in barley root tip via enhanced Cd uptake-mediated superoxide generation and cell death

Whereas severe Cd stress (150 µM Cd) causes root growth arrest as a consequence of marked superoxide generation leading to extensive cell death in the root tips, mild Cd stress (15 µM Cd) evokes morphogenic responses, such as reduced root elongation and radial root expansion, resulting in shorter and thicker roots. Similar to the low Cd concentration-caused mild stress, treatment of roots with either Ba to remove exchangeable or EDTA to remove both exchangeable and tightly bound cations, including Ca and Mg, from the apoplast, induced root growth inhibition and swelling. However, pre-treatment of roots with Ba had a synergistic effect on the development of these mild Cd stress-induced morphogenic responses, but without the development of any other symptoms in the root tips. In turn, EDTA pre-treatment markedly increased the toxicity of Cd in barley root tips via enhanced Cd uptake-mediated superoxide generation, which evoked extensive cell death in the transition zone of root tips identically to the high Cd concentration-induced severe stress. While the mild stress-induced responses were alleviated by the inhibition of auxin signalling pathway, the severe stress-induced symptoms were prevented by Ca, but not Mg, supplementation or by the inhibition of Cd uptake into the root symplasm. Therefore, the appropriate concentration of Ca in the apoplast is crucial to prevent the rapid accumulation of Cd in the symplasm, which above a certain threshold level leads to the huge superoxide generation and cell death.

Increase of Calcium Sensing Receptor Expression Is Related to Compensatory Insulin Secretion during Aging in Mice.

Type 2 diabetes is caused by both insulin resistance and relative insulin deficiency. To investigate age-related changes in glucose metabolism and development of type 2 diabetes, we compared glucose homeostasis in different groups of C57BL/6J mice ranging in age from 4 months to 20 months (4, 8, 12, 16 and 20 months). Interestingly, we observed that non-fasting glucose levels were not significantly changed, but glucose tolerance gradually increased by 20 months of age, whereas insulin sensitivity declined with age. We found that the size of islets and glucose-stimulated insulin secretion increased with aging. However, mRNA expression of pancreatic and duodenal homeobox 1 and granuphilin was decreased in islets of older mice compared with that of 4-month-old mice. Serum calcium (Ca2+) levels were significantly decreased at 12, 20 and 28 months of age compared with 4 months and calcium sensing receptor (CaSR) mRNA expression in the islets significantly increased with age. An extracellular calcium depletion agent upregulated CaSR mRNA expression and consequently enhanced insulin secretion in INS-1 cells and mouse islets. In conclusion, we suggest that decreased Ca2+ levels and increased CaSR expression might be involved in increased insulin secretion to compensate for insulin resistance in aged mice.

Gingerol activates noxious cold ion channel TRPA1 in gastrointestinal tract

TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.

Extracellular calcium influx promotes antibacterial autophagy in Escherichia coli infected murine macrophages via CaMKKβ dependent activation of ERK1/2, AMPK and FoxO1

Autophagy induction has been found as an alternative mechanism for ultimate elimination of invaded bacteria in innate immune cells. However, underlying mechanisms for the regulation of antibacterial autophagy require further elucidation. The present study mainly explores calcium dependent regulation of autophagy and its contribution to bactericidal activity in Escherichia coli (E. coli) infected murine macrophages. In this study, E. coli was shown to increase cellular calcium levels by triggering extracellular calcium influx in murine bone marrow derived macrophages. The elevated calcium was required for autophagy and bactericidal activity against E. coli, as extracellular calcium depletion or inhibition of calcium influx suppressed E. coli induced Beclin1 and LC3B expression, dampened LC3B puncta or LC3I to LC3II conversion and impaired intracellular E. coli degradation. Then CaMKKβ was identified as activated by E. coli induced calcium influx and chemical inhibition or RNAi knockdown of CaMKKβ abolished calcium mediated antibacterial autophagy. CaMKKβ was demonstrated to activate signaling pathways involving ERK, AMPK and FoxO1 and RNAi knockdown of these molecules also dampened the antibacterial autophagy against E. coli. In summary, we demonstrate a new mechanism of calcium dependent antibacterial strategy in E. coli infected macrophages, which requires autophagy enhancement mediated by activation of CaMKKβ, ERK, AMPK and FoxO1.

ERK-Nrf2 pathway regulates the anti-oxidant response after in vitro diesel exhaust particles treatment

Previous studies established an essential role for small conductance calcium-activated potassium (SK) channels in neuronal cell death pathways induced by glutamate excitotoxicity in cortical neurons in vitro and after cerebral ischemia in vivo. In addition to the intracellular calcium deregulation, glutamate-induced cell death also involves mechanisms of oxidative stress and mitochondrial dysfunction. Therefore, we sought to investigate whether SK channel activation might also affect mechanisms of intrinsic death pathways induced by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Exposure of immortalized hippocampal HT-22 cells to H2O2 imposed activation of a cascade of intracellular toxic events resulting in intracellular ROS production, mitochondrial loss of function, and ultimately cell death. Using a pharmacological approach to activate SK channels with CyPPA, we demonstrated a reduction of H2O2-mediated intracellular ROS production and cell death. Interestingly, CyPPA mediated neuroprotection in conditions of extracellular calcium and/or pyruvate depletion, pointing to a neuroprotective role of mitochondrial SK channels. Moreover, CyPPA partially inhibited H2O2-induced mitochondrial superoxide production, but did not prevent mitochondrial membrane depolarization. CyPPA treatment resulted in slight ATP depletion and a reduction of mitochondrial respiration/oxygen consumption. These findings postulate that SK channels mediate a protective effect by preventing neuronal death from subsequent oxidative stress through an adaptive metabolic response at the level of mitochondria. Therefore, SK channel activation may serve as a therapeutic target, where mitochondrial dysfunction and related mechanisms of oxidative stress contribute to progressive degeneration and death of neurons.

Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation.

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199

Angiotensin II에 의해 유도되는 활성산소발생 기전에 대한 연구

Angiotensin II (AngII) is an essential hormone that affects vascular physiology. For example, stimulation of vascular smooth muscle cells (VSMCs) rapidly induces vasoconstriction and results in the up-regulation of blood pressure. Chronic stimulation of VSMCs with AngII also results in hypertrophy. In this study, we confirmed an involvement of phosphatidylinositol 3-kinase (PI3K)-dependent calcium mobilization in AngII-induced generation of reactive oxygen species (ROS). Stimulation of rat aortic smooth muscle cells (RASMCs) with AngII significantly induced the generation of ROS in a dose- and time-dependent manner. AngII-induced generation of ROS was completely abolished by pharmacological inhibition of PI3K (with LY294002), but inhibition of the ERK signaling pathway had no effect. AngII-induced calcium mobilization was completely blocked by inhibition of PI3K, whereas inhibition of the ERK signaling pathway by PD98059 was ineffective. Depletion of extracellular calcium or inhibition of the L-type calcium channel by nifedipine completely blocked AngII-induced calcium mobilization. Depletion of extracellular calcium by EGTA and incubation of RASMCs with calcium- free medium both significantly blocked AngII-induced ROS generation. Inhibition of the L-type calcium channel also significantly blocked AngII-induced ROS generation. These results suggest that AngII-induced ROS generation is regulated by calcium mobilization, which, in turn, is modulated by a PI3K/L-type calcium channel signaling pathway.

SK channel activation modulates mitochondrial respiration and attenuates neuronal HT-22 cell damage induced by H2O2

Previous studies established an essential role for small conductance calcium-activated potassium (SK) channels in neuronal cell death pathways induced by glutamate excitotoxicity in cortical neurons in vitro and after cerebral ischemia in vivo. In addition to the intracellular calcium deregulation, glutamate-induced cell death also involves mechanisms of oxidative stress and mitochondrial dysfunction. Therefore, we sought to investigate whether SK channel activation might also affect mechanisms of intrinsic death pathways induced by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Exposure of immortalized hippocampal HT-22 cells to H2O2 imposed activation of a cascade of intracellular toxic events resulting in intracellular ROS production, mitochondrial loss of function, and ultimately cell death. Using a pharmacological approach to activate SK channels with CyPPA, we demonstrated a reduction of H2O2-mediated intracellular ROS production and cell death. Interestingly, CyPPA mediated neuroprotection in conditions of extracellular calcium and/or pyruvate depletion, pointing to a neuroprotective role of mitochondrial SK channels. Moreover, CyPPA partially inhibited H2O2-induced mitochondrial superoxide production, but did not prevent mitochondrial membrane depolarization. CyPPA treatment resulted in slight ATP depletion and a reduction of mitochondrial respiration/oxygen consumption. These findings postulate that SK channels mediate a protective effect by preventing neuronal death from subsequent oxidative stress through an adaptive metabolic response at the level of mitochondria. Therefore, SK channel activation may serve as a therapeutic target, where mitochondrial dysfunction and related mechanisms of oxidative stress contribute to progressive degeneration and death of neurons.

Abstract 385: TRPA1 Mediates Angiotensinogen Augmentation in Renal Proximal Tubular Cells

Overactivity of the intrarenal renin-angiotensin system contributes to the development of hypertension and renal injury. In this process, regulation of angiotensinogen (AGT) in renal proximal tubular cells (RPTC) is a key factor. Oxidative stress stimulates AGT expression accompanied by activation of p38 MAPK in RPTC. Transient receptor potential A1 (TRPA1) is known to be a sensor activated by a variety of noxious stimuli including hydrogen peroxide. Activated TRPA1 induces calcium influx across the plasma membrane leading to activation of AGT-inducible signal transducers. However, the presence and function of TRPA1 in the kidney have not been delineated. Therefore, this study was performed to demonstrate expression of intrarenal TRPA1 and its role in AGT augmentation in RPTC. Expression of TRPA1 in mouse kidney and cultured mouse RPTC were determined by RT-PCR, in situ hybridization, western blot analysis and immunocytochemistry. The RPTC were treated with 100 μM H2O2 for 1 hr with or without 10μM HC030031, a TRPA1 specific antagonist. EGTA was used to the culture medium for depletion of extracellular calcium. Thereafter, AGT expression levels were evaluated by real-time RT-PCR, and TRPA1 expression levels and p38 MAPK activity were determined by western blot analyses. TRPA1 expression was observed in tubules of renal cortex, and the cultured PRTC expressed TRPA1 mRNA and protein. H2O2 increased AGT expression (1.54 ± 0.08, ratio to control) in RPTC. The AGT augmentation was suppressed by HC030031 (1.19 ± 0.05, ratio to control). Calcium depletion also resulted in attenuation of the AGT augmentation induced by H2O2. Although H2O2 induced phosphorylation of p38 MAPK, HC030031 did not inhibit the p38 MAPK activation. TRPA1 expression was increased by H2O2 under the experimental condition (1.90 ± 0.06, ratio to control). These results suggest that TRPA1 is expressed in RPTC, which contributes to H2O2-induced AGT augmentation via calcium-dependent but p38 MAPK-independent pathways. Furthermore, increase in TRPA1 expression by H2O2 likely facilitates the stimulation of AGT expression. Therefore, TRPA1 may play an important role in the progression of oxidative stress-associated renal injury and hypertension via the stimulation of AGT expression in RPTC.

β-Adrenergic Inhibition of Contractility in L6 Skeletal Muscle Cells

The β-adrenoceptors (β-ARs) control many cellular processes. Here, we show that β-ARs inhibit calcium depletion-induced cell contractility and subsequent cell detachment of L6 skeletal muscle cells. The mechanism underlying the cell detachment inhibition was studied by using a quantitative cell detachment assay. We demonstrate that cell detachment induced by depletion of extracellular calcium is due to myosin- and ROCK-dependent contractility. The β-AR inhibition of L6 skeletal muscle cell detachment was shown to be mediated by the β2-AR and increased cAMP but was surprisingly not dependent on the classical downstream effectors PKA or Epac, nor was it dependent on PKG, PI3K or PKC. However, inhibition of potassium channels blocks the β2-AR mediated effects. Furthermore, activation of potassium channels fully mimicked the results of β2-AR activation. In conclusion, we present a novel finding that β2-AR signaling inhibits contractility and thus cell detachment in L6 skeletal muscle cells by a cAMP and potassium channel dependent mechanism.

Presynaptic Control of Rapid Estrogen Fluctuations in the Songbird Auditory Forebrain

Within the CNS of vertebrates, estrogens can directly modulate neural circuits that govern a wide range of behaviors, including feeding, spatial navigation, reproduction, and auditory processing. The rapid actions of estrogens in brain (seconds to minutes) have become well established, but it is unclear how estrogens are synthesized and released within restricted temporal and spatial domains in neural circuits. Anatomical localization of the estrogen synthesis enzyme (aromatase) within presynaptic terminals suggests that neuroestrogens can be synthesized directly at the neuronal synapse. A consequent prediction follows that synaptic estrogen production is controlled via classical electrochemical events in neurons. Here, we present evidence that acute fluctuations in local neuroestrogen levels in the forebrain of the zebra finch depend on calcium influx within presynaptic terminals. In vivo experiments using microdialysis linked to a sensitive estrogen ELISA showed that local forebrain neuroestrogens were both suppressed by potassium-evoked excitation and upregulated during 30 min periods of extracellular calcium depletion in a region enriched with presynaptic aromatase. Furthermore, potassium-evoked changes in local neuroestrogens were blocked by targeted delivery of the voltage-gated calcium channel blocker ω-conotoxin GVIA. Together, these experiments indicate that neuroestrogens are controlled by specific, depolarization-sensitive, calcium-dependent events within forebrain presynaptic terminals.

Rack1 promotes epithelial cell–cell adhesion by regulating E-cadherin endocytosis

E-cadherin and its cytoplasmic partners, catenins, mediate epithelial cell-cell adhesion. Disruption of this adhesion allows cancer cells to invade and metastasize. Aberrant activation of the Src tyrosine kinase disrupts cell-cell contacts through an E-cadherin/catenin-dependent mechanism. Previously we showed that Rack1 regulates the growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and in the intrinsic apoptotic and Akt cell survival pathways. Here we show that Rack1, partly by inhibiting Src, promotes cell-cell adhesion and reduces the invasive potential of colon cancer cells. Rack1 stabilizes E-cadherin and catenins at cell-cell contacts by inhibiting the Src phosphorylation of E-cadherin, the ubiquitination of E-cadherin by the E3 ligase Hakai and the endocytosis of E-cadherin. Upon depletion and restoration of extracellular calcium, Rack1 facilitates the re-assembly of E-cadherin-containing cell-cell contacts. Rack1 also blocks HGF-induced endocytosis of E-cadherin, disruption of cell-cell contacts and cell scatter. Our results uncover a novel function of Rack1 in maintaining the junctional homeostasis of intestinal epithelial cells by regulation of the Src- and growth factor-induced endocytosis of E-cadherin.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis

Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Comprehensive Study of Evodia rutaecarpa-induced Contraction on Blood Vascular in Vivo and in Vitro

Aim Evodiae Fructus (fruit of Evodia rutaecarpa), a blood vessel activator, has been used, with headache, migraine, shock etc., for a long history in Chinese medical practice. However, previous studies supported the vessel relaxation and the potential action on cardiac and cerebral blood circulation of vessel contraction. The present study was designed to explore it effects on contraction and the relative mechanism. Methods Evodiae Fructus was extracted by dialysis tubing (in diameter of 100 Dalton cutoff) and the effect of the evodia dialyzed (ED) (> 100 D) was studied in vivo and in vitro. Results ED could dose-dependently promote the micro-cerebral blood flow in mice and induced the contraction of rat thoracic aorta at rest and phenylephrine pre-evoked state. Compared with the vasocontraction under 50 mmol·L −1 KCl, 250 mg·L −1 ED increased by 113%. The effect was inhibited by a-adrenergic receptor blocker phentolamine, instead of β-adrenergic receptor blocker propranolol, M-adrenaline receptor blocker atropine, and angiotensin II receptor blocker losartan. The depletion of extracellular calcium decreased the contraction induced by ED and the addition of L-type calcium ion channel antagonist (nicardipine), calcium ion channel activator (BAY-K8644) and potassium ion channel opener (pinacidil) significantly affected the contraction induced by ED. Selected inhibitors were used to investigate the possible mechanism of ED-induced vasocontraction. Inhibitors of myosin light chain kinase (MLCK), Rho-kinase (ROK) and inositol 1, 4, 5-triphosphate (IP 3) receptor suppressed the effect of ED. Conclusion ED induced Ca 2+-dependent and Ca 2+-independent contraction, the Ca 2+ influx, a-adrenoceptor, ROK and IP 3 receptor signal pathways and MLCK activation might be involved in the contractile process. These results suggested that Evodiae Fructus could increase the blood vessel contraction by the effect on calcium channel on the membrane.

Numb is a negative regulator of HGF dependent cell scattering and Rac1 activation

Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial–mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell–cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell–cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell–cell adhesion to induce epithelial–mesenchymal transition (EMT) and cell dispersal.