Regulación del calcio por SERC-A antes de la enfermedad de Alzheimer y durante la misma / Calcium regulation by SERC-A before and during Alzheimer disease

Hay muchos factores implicados en la incidencia de la enfermedad de Alzheimer que, en combinación, terminan por impedir o dificultar las funciones neuronales normales. Actualmente, poco se conoce sobre la regulación del calcio, antes de la enfermedad y durante la misma. La inestabilidad interna de los niveles de calcio se asocia a un mayor riesgo vascular, condición prevalente en un gran número de individuos ya comprometidos por la enfermedad de Alzheimer. Esta revisión proporciona una reevaluación de los mecanismos moleculares de la ATPasa dependiente de Ca2+ del retículo sarcoendoplásmico (SERC-A) en la enfermedad y analiza los aspectos más destacados de la función de los canales de calcio dependientes de voltaje; de esta manera, se podrán abrir nuevas alternativas de tratamiento. Estos mecanismos de regulación son clínicamente relevantes, ya que se ha implicado la función irregular de SERC-A en diversas alteraciones de la función cerebral.

There are many factors involved in the incidence of Alzheimer's disease that, in combination, impede or hinder normal neuronal functions. Little is currently known about calcium regulation before and during the disease. Internal instability of calcium levels is associated with increased vascular risk, a prevalent condition in a high number of individuals already compromised by Alzheimer's disease. This review provides a reevaluation of the molecular mechanism of the sarcoendoplasmic reticulum calcium ATPase (SERC-A) in the disease and discusses salient aspects of voltage-gated calcium channel function; in these way new alternatives could be open for its treatment. These regulation mechanisms are clinically relevant since the irregular functions of SERC+A has been implicated in pathologies of brain function.

Ultrashort wave alleviates oxygen -glucose deprivation/reoxygenation injury via up -regulation of SPCA1 expression in N2a cells / 中南大学学报(医学版)

OBJECTIVES@#Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca2+-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level.@*METHODS@#N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting.@*RESULTS@#Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001).@*CONCLUSIONS@#USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

Myricetin Protects Against High Glucose-Induced β-Cell Apoptosis by Attenuating Endoplasmic Reticulum Stress via Inactivation of Cyclin-Dependent Kinase 5

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.

Effect of paeoniflorin and menthol on membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity during transport of puerarin in Calu-3 cell / 中国中药杂志

The aim of this research is to investigate the effects of paeoniflorin and menthol on the physiological function of Calu-3 cell membrane during the transport of puerarin. Calu-3 cell was used as the cell model to simulate nasal mucosa tissues, and the cell membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity were detected by fluorescence recovery after photobleaching(FRAP) and ultramicro enzyme activity testing, in order to explore the mechanism of compatible drugs on promoting puerarin transport. The results showed that when puerarin associated with low, middle and high concentration of menthol or both paeoniflorin and menthol, the fluorescence recovery rate was increased significantly, while Na⁺-K⁺-ATPase activity had no significant change and Ca²⁺-ATPase activity was enhanced significantly as compared with puerarin alone. Therefore, it was concluded that menthol had the abilit of promoting the transport and the mechanism might be related to increasing membrane fluidity and activating Ca²⁺-ATPase.

Characterization and pathophysiological changes of cerebral infarction rat model with qi-deficiency and blood-stasis Syndrome / 中国中药杂志

This study aimed to observe the general state and changes in pathophysiological indexes of multiple cerebral infarction rat model with Qi-deficienty and Blood-stasis syndrome. Rats were randomly divided into 4 groups(with 30 in each group): the normal group, the sham group, the model group and the Yiqi Huoxue recipe group. Rats in the model group and Yiqi Huoxue group were provided with interruptable sleep deprivation for 7 days before the multiple cerebral infarction operation, and followed by another 4 weeks of sleep deprivation; rats in the Yiqi Huoxue group were intragastrically administrated with drug at a dose of 26 g·kg⁻¹, once a day for 4 weeks. The general state was observed, and the pathophysiological indexes were measured at 48 h, 2 weeks and 4 weeks after administration. The results showed that rats in the normal group and the sham group represented a good general state and behaviors, with a normal morphological structure of brain tissues; rats in the model group featured yellow fur, depression, accidie, loose stools and movement disorder, with obvious brain histomorphological damage, which became aggravated with the increase of modeling time; rats in the Yiqi Huoxue group showed release in the general state and above indexes. Compared with the sham group at three time points, rats in the model group showed decrease in body weight, exhaustive swimming time and RGB value of tongue surface image, and increase in whole blood viscosity of the shear rate under 5, 60 and 150 S⁻¹, reduction in cerebral cortex Na⁺-K⁺-ATPase, Ca²⁺-ATPase activity and contents of 5-HT, rise in TXB2 levels and decline in 6-keto-PGF1a in serum(<0.05, <0.01). Compared with the model group, rats in the Yiqi Huoxue group showed alleviations in the above indexes at 2 w and 4 w(<0.05, <0.01). The results showed that the characterization and pathophysiological indexes in the multiple cerebral infarction rat model with Qi-deficiency and blood-stasis syndrome were deteriorated; Yiqi Huoxue recipe could significantly alliviate the abnormal conditions, which suggested of the model was stable and reliable and the pathophysiologic evolutionary mechanism might be related to energy metabolism dysfunction, vasoactive substance abnormality and changes in neurotransmitters.

Peptidoglycan Induces the Production of Interleukin-8 via Calcium Signaling in Human Gingival Epithelium

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.

Effects of benzo(a)pyrene exposure on the ATPase activity and content of Ca²⁺ in the hippocampus of neonatal SD rats / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of benzo(α)pyrene on the ATPase activity and content of Ca²⁺ in the hippocampus of neonatal SD rats.@*METHODS@#Sixty male and 60 female 4-days-old neonatal SD rats were randomly divided into 5 groups (n=24): a blank control group, a vehicle control group (peanut oil), 3 benzo(α)pyrene groups (0.02, 0.2 and 2 mg/kg, respectively). SD rats were given benzo(α)pyrene (dissolved in peanut oil) by gavage daily from postnatal day 4 (PND4) to PND20. The nerve reflex, the condition of neuro-muscle development and motion function were examined in the period of treatment. The colorimetric technique was used to detect the activity of Ca²⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in hippocampus after the treatment. The concentration of Ca²⁺ of synapse in the hippocampus of rats was detected by fluorescent labeling.@*RESULTS@#The results from the behavior tests showed that duration of surface reflex latency in rats with medium dose of benzo(α)pyrene was longer compared with that in the control group in PND12. The duration of surface reflex latency in rats with high dose of benzo(α) pyrene is longer in PND 14 and PND 16 compared with that in the control group (P<0.05). Compared with the rats in the control group, the activities of Ca²⁺-Mg²⁺-ATPase and Ca²⁺-ATPase in hippocampus in rats with high dose benzo(α) pyrene were significantly decreased, and the degree in the decrease of Ca²⁺-ATPase activity was dose-dependent (P<0.05). The contents of Ca²⁺ in the hippocampus in rats with medium or high dose of benzo(α) pyrene were significantly increased compared with that in the control group (P<0.05), which showed a dose-dependent manner (P<0.05).@*CONCLUSION@#Benzo(α)pyrene exposure led to the decrease in ATPase activity as well as Ca²⁺ overload of the synapse in the hippocampal tissue, which in turn results in the nerve damage of newborn SD rats.

Effect of Hypoxic Paracrine Media on Calcium-Regulatory Proteins in Infarcted Rat Myocardium

BACKGROUND AND OBJECTIVES: An increase in intracellular calcium concentration due to loss of Ca2+ homeostasis triggers arrhythmia or cardiac cell death in the heart. Paracrine factors released from stem cells have beneficial cardioprotective effects. However, the mechanism of modulation of Ca2+ homeostasis by paracrine factors in ischemic myocardium remains unclear. MATERIALS AND METHODS: We isolated rat bone marrow-derived mesenchymal stem cells (MSCs), and prepared paracrine media (PM) from MSCs under hypoxic or normoxic conditions (hypoxic PM and normoxic PM). We induced rat myocardial infarction by left anterior descending ligation for 1 hour, and treated PM into the border region of infarcted myocardium (n=6/group) to identify the alteration in calcium-regulated proteins. We isolated and stained the heart tissue with specific calcium-related antibodies after 11 days. RESULTS: The hypoxic PM treatment increased Ca2+-related proteins such as L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ ATPase, Na+/K+ ATPase, and calmodulin, whereas the normoxic PM treatment increased those proteins only slightly. The sodium-calcium exchanger was significantly reduced by hypoxic PM treatment, compared to moderate suppression by the normoxic PM treatment. CONCLUSION: Our results suggest that hypoxic PM was significantly associated with the positive regulation of Ca2+ homeostasis in infarcted myocardium.

Role of calcium channels responsible for phenylephrine-induced contraction in rat aorta 3 days after acute myocardial infarction / 대한마취과학회지

BACKGROUND: Phenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI). METHODS: Endothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions. RESULTS: The effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group. CONCLUSIONS: Results suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

Protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells.</p><p><b>METHODS</b>Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 μg/mL quercetin and/or cadmium (2.5, 5.0 μmol/L), in a serum-free medium at 37 °C at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress.</p><p><b>RESULTS</b>Exposure of rPT cells to cadmium acetate (2.5, 5.0 µmol/L) induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na+, K+-ATPase, Ca2+-ATPase, glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 µg/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants (non-enzymatic and enzymic) levels.</p><p><b>CONCLUSION</b>The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.</p>

New insights into store-independent Ca(2+) entry: secretory pathway calcium ATPase 2 in normal physiology and cancer / 国际口腔科学杂志·英文版

Recent studies in secretory pathway calcium ATPases (SPCA) revealed novel functions of SPCA2 in interacting with store-operated Ca(2+) channel Orai1 and inducing Ca(2+) influx at the cell surface. Importantly, SPCA2-mediated Ca(2+) signaling is uncoupled from its conventional role of Ca(2+)-ATPase and independent of store-operated Ca(2+) signaling pathway. SPCA2-induced store-independent Ca(2+) entry (SICE) plays essential roles in many important physiological processes, while unbalanced SICE leads to enhanced cell proliferation and tumorigenesis. Finally, we have summarized the clinical implication of SICE in oral cancer prognosis and treatment. Inhibition of SICE may be a new target for the development of cancer therapeutics.

Therapeutic efficacy of artemisinin combination therapies and prevalence of S769N mutation in PfATPase6 gene of Plasmodium falciparum in Kolkata, India

OBJECTIVE@#To study the in vivo efficacy of these two ACTs in the treatment of Plasmodium falciparum (P. falciparum malaria) in Kolkata and to determine the prevalence of mutant S769N codon of the PfATPase6 gene among field isolates of P. falciparum collected from the study area.@*METHODS@#A total of 207P. falciparum positive cases were enrolled randomly in two study arms and followed up for 42 days as per WHO (2009) protocol. A portion of PfATPase6 gene spanning codon S769N was amplified and sequenced by direct sequencing method.@*RESULTS@#It was observed that the efficacy of both the ACT regimens were highly effective in the study area and no mutant S769N was detected from any isolate.@*CONCLUSIONS@#The used, combination AS+SP is effective and the other combination AM+LF might be an alternative, if needed.

Effects of NaOCl on Neuronal Excitability and Intracellular Calcium Concentration in Rat Spinal Substantia Gelatinosa Neurons

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca2+]i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate (H2DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca2+ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca2+ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca2+-sensitive fluorescence dye fura-2, the [Ca2+]i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.

alpha-Mangostin Reduced ER Stress-mediated Tumor Growth through Autophagy Activation

alpha-Mangostin is a xanthon derivative contained in the fruit hull of mangosteen (Garcinia mangostana L.), and the administration of alpha-Mangostin inhibited the growth of transplanted colon cancer, Her/CT26 cells which expressed Her-2/neu as tumor antigen. Although alpha-Mangostin was reported to have inhibitory activity against sarco/endoplasmic reticulum Ca2+ ATPase like thapsigargin, it showed different activity for autophagy regulation. In the current study, we found that alpha-Mangostin induced autophagy activation in mouse intestinal epithelial cells, as GFP-LC3 transgenic mice were orally administered with 20 mg/kg of alpha-Mangostin daily for three days. However, the activation of autophagy by alpha-Mangostin did not significantly increase OVA-specific T cell proliferation. As we assessed ER stress by using XBP-1 reporter system and phosphorylation of eIF2alpha, thapsigargin-induced ER stress was significantly reduced by alpha-Mangostin. However, coadministration of thapsigargin with alpha-Mangostin completely blocked the antitumor activity of alpha-Mangostin, suggesting ER stress with autophagy blockade accelerated tumor growth in mouse colon cancer model. Thus the antitumor activity of alpha-Mangostin can be ascribable to the autophagy activation rather than ER stress induction.

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

The receptor activator of NF-kappaB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-kappaB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) delta, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

Efecto de esfingolípidos y Agelosina B en la movilización de Ca2+ y la señalización celular en células de cáncer de mama (MCF-7) y su relación con la apoptosis / Effect of sphingolipids and Agelosin B on Ca2 + mobilization and cell signaling in breast cancer cells (MCF-7) and its relationship with apoptosis

Los esfingolípidos, como la ceramida (Cer), la ceramida-1-fosfato (C-1-P), la esfingosina (Sph) y la esfingosina-1-fosfato (S-1P) estan relacionados con la señalización intracelular en procesos como crecimiento celular, movilización intracelular de Ca+2 y apoptósis. En este trabajo se evaluó el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y en la apoptósis en células de cáncer de mama MCF-7. Se utilizaron fluoróforos específicos para el Ca+2 y microscopía confocal. Se demostró que en estas células, la Sph (20 uM), la Cer (10uM), la S-P (2uM) y la C--P (uM) aumentaron la concentración intracelular ce Ca+2, induciendo su liberación desde el retículo endoplasmático (RE). Además, se observo que la esfingosina abrioun canal de Ca2+ en la membrana plasmática. También se demostró que la Cer inhibe parcialmente la actividad de la Ca2+-ATPasa del RE (SERCA), de forma dosis dependiente, mientras que la ceramina, su análogo no hidrolisable la inhibe totalmente. La Sph también inhibe completamente la actividad de la SERCA, a la misma concentración que induce la liberación del Ca+2 del RE. Asimismo, se evaluó el efecto de estos esfingolípidos sobre la inducción de la apotósis en células MCF-7 evidenciando que el tratamiento con la Cer, la ceramida, la Sph inducen toxicidad. También se observo que mientras la ceramida activo la caspasa 7 y la caspasa 8, el esfingolipido natural, la Cer no tuvo ningún efecto. Por su parte, la Sph activa la caspasa 8 sin modificar la activdad de la caspasa 7. Tanto la Cer, como la ceramida y la Sph, disminuyeron la expresión de la proteína Bcl-2 amti-apoptótica, y también indujeron la fragmentación de ADN, visualizada mediante la técnica de TUNEL, demostrando que estos esfingolípidos inducen apoptósis en MCF-7. La agelasina B, toxina purificada a partir de la esponja marina Agelas clathrodes tiene un efecto citotóxico un orden de magnitud mayor en MCF-7, en comparación con fibroplastos humanos. La agelasina B induce la liberación del Ca+2 almacenado en el RE en celulas MCF-7, ademas de inhibir la actividad de la SERCA en un 100%. También se demostró que esta toxina induce apoptosis, ya que disminuye el potencial de membrana mitocondrial, activa la caspasa 8, disminuye la expresion de la proteina Bcl-2 e induce fragmentación del ADN de las células MCF-7. Este mecanismo es similar al efecto de la tapsigargina.

Inhibitory effect of Suaeda asparagoides (Miq.) extract on the motility of rat gastric antrum is mediated by beta-adrenoceptor / 한국실험동물학회지

Suaeda asparagoides (Miq.) has long been used as a Korean folk herbal medicine for the treatment of functional gastrointestinal disorders. However, reports on its pharmacological activity on gastrointestinal motility are scarce. The present study investigated the effects of Suaeda asparagoides water fraction of the extract (SAWF) on antral motility in vitro. Muscle strips from rat gastric antrum were set up in an organ bath in a circular orientation. SAWF (100 microg/mL) inhibited the spontaneous contraction of antral circular muscle strips. These inhibitory effects were not significantly affected by tetrodotoxin (1 microM), N omega-Nitro-L-arginine methyl ester hydrochloride (100 microM), 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 microM), ryanodine (10 microM) and phentolamine (10 microM). SAWF-induced inhibition was mostly restored by cyclopiazonic acid (10 microM). Furthermore, the beta-adrenergic receptor antagonist, propranolol (10 microM), abolished SAWF-induced inhibition. These results suggest that SAWF may exert its activity on gastrointestinal smooth muscle via a-adrenergic receptors and sarcoplasmic reticulum Ca2+ ATPase.

Effect of ulinastatin donor-pretreatment on liver graft during cold preservation in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Donor-pretreatment with ulinastatin may influence the liver graft during cold preservation. The aim of this research was to determine whether pretreatment of donor liver with Ulinastatin can attenuate cold preservation injury, and to explore the mechanism by which Ulinastatin affects the donor liver graft.</p><p><b>METHODS</b>One hundred and forty-four Wistar rats were divided into the Ulinastatin treatment group (T group) pretreated with Ulinastatin 50 000 U/kg and control group (C group) treated with 0.9% normal saline via peritoneal injection prior to the anesthetization. After the abdominal cavity was opened and perfused with cold Ringer's lactate solution, the liver was harvested. The harvested liver was preserved in cold Ringer's lactate solution for 0, 2, 6, 24 hours, at which time the liver tissue was sampled for determination of dry weight and wet weight, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activity, lactic acid dehydrogenase (LDH) activity, lactic acid and malondialdehyde levels. Light microscopy and electron microscopy were used to observe liver morphology. The liver cold-preservation solution was taken for measurement of aspartate aminotransferase (AST) and alanine transaminase (ALT) levels. Correlation between ATPase activity and lactic acid level was analyzed by SPSS 13.0 for Windows.</p><p><b>RESULTS</b>The morphology in the T group had improved cell boundaries vs. the C group at each time point. Dry weight to wet weight in the T group was lower than in the C group at 6 hours (P < 0.05), but the difference was not significant at 24 hours. ALT levels in the T group were lower than that in the C group at 6 hours (P < 0.05) and 24 hours (P < 0.01). AST levels in the T group were lower than those in the C group at 2 hours (P < 0.05), 6 hours (P < 0.01) and 24 hours (P < 0.01). Na(+)-K(+)-ATPase activity in the T group was higher than in the C group and the mean difference between two groups was significant at 0 hour (P < 0.05) and 2 hours (P < 0.05). Ca(2+)-ATPase activity in the T group was higher than in the C group with the mean difference between two groups significant at 2 hours (P < 0.05). The T group had increased lactic acid levels at 0 hour (P < 0.01) and 2 hours (P < 0.05) compared with the C group, but there was no influence on the LDH activity at the same time. There were no obvious differences in the levels of malondialdehyde between the two groups at any time point. A linear correlation between Na(+)-K(+)-ATPase activity and lactic acid levels (r = 0.295, P < 0.05) was found.</p><p><b>CONCLUSIONS</b>Donor-pretreatment with ulinastatin may protect the cells in a liver graft from ischemia injury during cold preservation; the mechanism may be due to its promotion for cell glycolysis and its preservation of ATPase activity.</p>