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1.
Arq. bras. cardiol ; 110(1): 44-51, Jan. 2018. graf
Article in English | LILACS | ID: biblio-887998

ABSTRACT

Resumo Background: Melatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis. Objective: Demonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes. Methods: In vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats. Results: In vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059. Conclusion: melatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.


Resumo Fundamento: A melatonina é um hormônio neuroendócrino sintetizado principalmente pela glândula pineal que é indicado para prevenir efetivamente a lesão de reperfusão miocárdica. Não está claro se a melatonina protege a função cardíaca da lesão de reperfusão através da modulação da homeostase do cálcio intracelular. Objetivo: Demonstrar que a melatonina protege contra a lesão de reperfusão miocárdica através da modulação de IP3R e SERCA para manter a homeostase de cálcio por meio da ativação de ERK1 em cardiomiócitos. Métodos: Foram realizados experimentos in vitro usando células H9C2 submetidas a indução de hipoxia / reoxigenação simulada (H/R). O nível de expressão de ERK1, IP3R e SERCA foi avaliado por Western Blots. A apoptose de cardiomiócitos foi detectada por TUNEL. A coloração de faloidina foi utilizada para avaliar a alteração da organização de filamentos de actina dos cardiomiócitos. Fura-2 / AM foi utilizado para medir a concentração intracelular de Ca2+. Realizando experiências in vivo, a expressão miocárdica de IP3R e SERCA foi detectada por coloração com imunofluorescência usando modelo de isquemia miocárdica / reperfusão (I/R) em ratos. Resultados: resultados in vitro mostraram que a melatonina induz a ativação de ERK1 em cardiomiócitos contra H/R que foi inibida por PD98059 (inibidor de ERK1). Os resultados mostraram que a melatonina inibe a apoptose dos cardiomiócitos e melhora a organização do filamento de actina em cardiomiócitos contra H/R, pois ambas poderiam ser revertidas pela PD98059. A melatonina mostrou reduzir a sobrecarga de cálcio, além de inibir a expressão de IP3R e promover a expressão de SERCA através da via ERK1 em cardiomiócitos contra H/R. A melatonina induziu menor IP3R e maior expressão de SERCA no miocárdio que foram revertidas pela PD98059. Conclusão: a cardioproteção induzida pela melatonina contra lesão de reperfusão é pelo menos parcialmente através da modulação de IP3R e SERCA para manter a homeostase de cálcio intracelular via ativação de ERK1.


Subject(s)
Animals , Male , Rats , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/prevention & control , MAP Kinase Signaling System/drug effects , Myocytes, Cardiac/drug effects , Sarcoplasmic Reticulum Calcium-Transporting ATPases/drug effects , Inositol 1,4,5-Trisphosphate Receptors/drug effects , Melatonin/pharmacology , Myocardial Reperfusion Injury/pathology , Rats, Sprague-Dawley , Myocytes, Cardiac/pathology , Disease Models, Animal , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism
2.
Article in English | WPRIM | ID: wpr-764776

ABSTRACT

Calcium has versatile roles in diverse physiological functions. Among these functions, intracellular Ca²⁺ plays a key role during the secretion of salivary glands. In this review, we introduce the diverse cellular components involved in the saliva secretion and related dynamic intracellular Ca²⁺ signals. Calcium acts as a critical second messenger for channel activation, protein translocation, and volume regulation, which are essential events for achieving the salivary secretion. In the secretory process, Ca²⁺ activates K⁺ and Cl⁻ channels to transport water and electrolyte constituting whole saliva. We also focus on the Ca²⁺ signals from intracellular stores with discussion about detailed molecular mechanism underlying the generation of characteristic Ca²⁺ patterns. In particular, inositol triphosphate signal is a main trigger for inducing Ca²⁺ signals required for the salivary gland functions. The biphasic response of inositol triphosphate receptor and Ca²⁺ pumps generate a self-limiting pattern of Ca²⁺ efflux, resulting in Ca²⁺ oscillations. The regenerative Ca²⁺ oscillations have been detected in salivary gland cells, but the exact mechanism and function of the signals need to be elucidated. In future, we expect that further investigations will be performed toward better understanding of the spatiotemporal role of Ca²⁺ signals in regulating salivary secretion.


Subject(s)
Calcium Signaling , Calcium , Chloride Channels , Inositol , Inositol 1,4,5-Trisphosphate Receptors , Protein Transport , Saliva , Salivary Glands , Salivation , Second Messenger Systems , Secretory Pathway , Water
3.
Article in English | WPRIM | ID: wpr-727826

ABSTRACT

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.


Subject(s)
Bacteria , Bacterial Toxins , Calcium , Calcium Signaling , Calcium-Transporting ATPases , Cytokines , Epithelial Cells , Epithelium , Gram-Positive Bacteria , Humans , Inflammation , Inositol 1,4,5-Trisphosphate Receptors , Interleukin-8 , Peptidoglycan , Periodontal Diseases , Phospholipases , Reticulum , Thapsigargin , Toll-Like Receptor 2 , Toll-Like Receptors , Type C Phospholipases
4.
Biol. Res ; 48: 1-5, 2015. graf
Article in English | LILACS | ID: biblio-950819

ABSTRACT

BACKGROUND: In the central nervous system, interleukin-10 (IL-10) provides trophic and survival effects directly on neurons, modulates neurite plasticity, and has a pivotal importance in the neuronal regeneration in neurodegenerative and neuroinflammatory conditions. This cytokine is primarily produced by glial cells and has beneficial effects on the neuronal viability. However, the mechanisms of IL-10-elicited neuroprotection are not clear. RESULTS: Membrane preparations, isolated from wild-type (Wt) and IL-10 knockout (KO) mice brain were used in this study. It has been shown that compared to wild-type mice, in IL-10 KO mice brain, the amount of immunoglobulin binding protein (BiP) is greatly increased, whereas the content of sigma receptor-1 (SigR1) is not changed significantly. Co-immunoprecipitation experiments have shown that the association of SigR1 with small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1), NR2B subunit of NMDA-receptor (NMDAR) and inositol-3-phosphate receptor (IP3R) is higher in the IL-10 KO mice brain than in the Wt mice brain. Besides, we have found that either glutamate or sigma ligands, separately or together, do not change glutamate-induced NADPH-oxidase (NOX) activity in Wt-type mice brain membrane preparations, whereas in IL-10 KO mice high concentration of glutamate markedly increases the NOX-dependent production of reactive oxygen species (ROS). Glutamate-dependent ROS production was decreased to the normal levels by the action of sigma-agonists. CONCLUSIONS: It has been concluded that IL-10 deprivation, at least in part, can lead to the induction of ER-stress, which causes BiP expression and SigR1 redistribution between components of endoplasmic reticulum (ER) and plasma membrane. Moreover, IL-10 deficiency can change the specific organization of NMDAR, increasing the surface expression of SigR1-sensitive NR2B-containing NMDAR. In these conditions, glutamate-dependent ROS production is greatly increased leading to the initiation of apoptosis. In this circumstances, sigma-ligands could play a preventive role against NMDA receptor-mediated excitotoxicity.


Subject(s)
Animals , Male , Mice , Brain/metabolism , Interleukin-10/genetics , Receptors, sigma/metabolism , Glutamic Acid/metabolism , NADPH Oxidases/metabolism , Cell Membrane/metabolism , Receptors, sigma/classification , Receptors, sigma/agonists , Reactive Oxygen Species/analysis , Reactive Oxygen Species/metabolism , Receptors, N-Methyl-D-Aspartate/classification , Receptors, N-Methyl-D-Aspartate/metabolism , rac1 GTP-Binding Protein/metabolism , Immunoprecipitation , Endoplasmic Reticulum/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Gene Knockdown Techniques , Heat-Shock Proteins/metabolism , Mice, Inbred C57BL , Neurons/metabolism
5.
Article in English | WPRIM | ID: wpr-727692

ABSTRACT

Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.


Subject(s)
Calcium , Cell Death , Colorectal Neoplasms , Cytochromes c , DNA Damage , Homeostasis , Humans , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , Mitochondria
6.
Protein & Cell ; (12): 456-466, 2013.
Article in English | WPRIM | ID: wpr-757795

ABSTRACT

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.


Subject(s)
Animals , Ascaris suum , Metabolism , Calcineurin , Metabolism , Calcium , Metabolism , Calmodulin , Metabolism , Cytoskeleton , Metabolism , Cytosol , Metabolism , Egtazic Acid , Pharmacology , Helminth Proteins , Metabolism , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , Male , Membrane Potential, Mitochondrial , Physiology , Mitochondria , Metabolism , Pseudopodia , Metabolism , Signal Transduction , Sperm Motility , Spermatids , Physiology , Spermatogenesis , Type C Phospholipases , Metabolism
7.
Korean Circulation Journal ; : 581-591, 2013.
Article in English | WPRIM | ID: wpr-78989

ABSTRACT

Previously, we reviewed biological evidence that mercury could induce autoimmunity and coronary arterial wall relaxation as observed in Kawasaki syndrome (KS) through its effects on calcium signaling, and that inositol 1,4,5-triphosphate 3-kinase C (ITPKC) susceptibility in KS would predispose patients to mercury by increasing Ca2+ release. Hg2+ sensitizes inositol 1,4,5-triphosphate (IP3) receptors at low doses, which release Ca2+ from intracellular stores in the sarcoplasmic reticulum, resulting in delayed, repetitive calcium influx. ITPKC prevents IP3 from triggering IP3 receptors to release calcium by converting IP3 to inositol 1,3,4,5-tetrakisphosphate. Defective IP3 phosphorylation resulting from reduced genetic expressions of ITPKC in KS would promote IP3, which increases Ca2+ release. Hg2+ increases catecholamine levels through the inhibition of S-adenosylmethionine and subsequently catechol-O-methyltransferase (COMT), while a single nucleotide polymorphism of the COMT gene (rs769224) was recently found to be significantly associated with the development of coronary artery lesions in KS. Accumulation of norepinephrine or epinephrine would potentiate Hg2+-induced calcium influx by increasing IP3 production and increasing the permeability of cardiac sarcolemma to Ca2+. Norepinephrine and epinephrine also promote the secretion of atrial natriuretic peptide, a potent vasodilator that suppresses the release of vasoconstrictors. Elevated catecholamine levels can induce hypertension and tachycardia, while increased arterial pressure and a rapid heart rate would promote arterial vasodilation and subsequent fatal thromboses, particularly in tandem. Genetic risk factors may explain why only a susceptible subset of children develops KS although mercury exposure from methylmercury in fish or thimerosal in pediatric vaccines is nearly ubiquitous. During the infantile acrodynia epidemic, only 1 in 500 children developed acrodynia whereas mercury exposure was very common due to the use of teething powders. This hypothesis mirrors the leading theory for KS in which a widespread infection only induces KS in susceptible children. Acrodynia can mimic the clinical picture of KS, leading to its inclusion in the differential diagnosis for KS. Catecholamine levels are often elevated in acrodynia and may also play a role in KS. We conclude that KS may be the acute febrile form of acrodynia.


Subject(s)
Acrodynia , Arterial Pressure , Autoimmunity , Calcium , Calcium Signaling , Catechol O-Methyltransferase , Catecholamines , Child , Coronary Vessels , Diagnosis, Differential , Epinephrine , Heart Rate , Humans , Hydrazines , Hypertension , Inositol , Inositol 1,4,5-Trisphosphate , Inositol 1,4,5-Trisphosphate Receptors , Inositol Phosphates , Mucocutaneous Lymph Node Syndrome , Norepinephrine , Permeability , Phosphorylation , Polymorphism, Single Nucleotide , Powders , Relaxation , Risk Factors , S-Adenosylmethionine , Sarcolemma , Sarcoplasmic Reticulum , Tachycardia , Thimerosal , Thrombosis , Tooth , Tooth Eruption , Vaccines , Vasoconstrictor Agents , Vasodilation
8.
Article in Korean | WPRIM | ID: wpr-45681

ABSTRACT

Pancreatic acinar cells exhibit a polarity that is characterized by the localization of secretory granules at the apical membrane. However, the factors that regulate cellular polarity in these cells are not well understood. In this study, we investigated the effect of Mist1, a basic helix-loop-helix transcription factor, on the cellular architecture of pancreatic acinar cells. Mist1-null mice displayed secretory granules that were diffuse throughout the pancreatic acinar cells, from the apical to basolateral membranes, whereas Mist1 heterozygote mice showed apical localization of secretory granules. Deletion of the Mist1 gene decreased the expression of type 3 inositol 1,4,5-triphosphate receptors (IP3R) but did not affect apical localization and expression of IP3R2. Mist1-null mice also displayed an increase in luminal areas and an increase in the expression of zymogen granules in pancreatic acinar cells. These results suggest that Mist1 plays a critical role in polar localization of cellular organelles and in maintaining cellular architecture in mouse pancreatic acinar cells.


Subject(s)
Acinar Cells , Animals , Cell Polarity , Heterozygote , Inositol 1,4,5-Trisphosphate Receptors , Membranes , Mice , Organelles , Phenobarbital , Secretory Vesicles , Transcription Factors
9.
Article in English | WPRIM | ID: wpr-727561

ABSTRACT

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.


Subject(s)
Animals , Blood Proteins , Boron Compounds , Calcium-Transporting ATPases , Cell Membrane , Dentin , Estrenes , Inositol , Inositol 1,4,5-Trisphosphate , Inositol 1,4,5-Trisphosphate Receptors , Macrophages , Mice , NF-kappa B , Osteoclasts , Phosphoproteins , Proteins , Pyrrolidinones , Receptor Activator of Nuclear Factor-kappa B , Reticulum , Signal Transduction , Tumor Necrosis Factor-alpha , Type C Phospholipases
10.
Protein & Cell ; (12): 990-996, 2011.
Article in English | WPRIM | ID: wpr-757320

ABSTRACT

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.


Subject(s)
Adenosine Triphosphate , Pharmacology , Amino Acid Substitution , Biological Transport , Physiology , Blotting, Western , Calcium , Metabolism , Calcium Signaling , Physiology , HeLa Cells , Humans , Immunoprecipitation , Inositol 1,4,5-Trisphosphate , Metabolism , Inositol 1,4,5-Trisphosphate Receptors , Physiology , Membrane Potentials , Physiology , Membrane Proteins , Genetics , Metabolism , Microscopy, Confocal , Molecular Chaperones , Genetics , Metabolism , Mutation , Plasmids , Transfection
11.
Acta Physiologica Sinica ; (6): 442-452, 2011.
Article in English | WPRIM | ID: wpr-335969

ABSTRACT

Cytosolic Ca(2+) ions play an important role in the regulation of numerous aspects of cellular activity in virtually all cell types. There is a complex interaction between the neuronal electrical signals on plasma membrane and the chemical signals of intracellular calcium. Each neuron can be considered as a binary membrane system with plasma membrane and endoplasmic reticulum membrane, and the neuronal endoplasmic reticulum can be regarded as a neuron-within-a-neuron. This review explores the simulation modeling of neuronal dynamics mutually coupled with the intracellular calcium signaling released from endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor calcium channels. We show that a current trend is to include the intracellular calcium dynamics into the neuronal models, and the frontier of this research is now shifting to spatial neuronal models with diffusing intracellular calcium. It is expected that more important results will be obtained with the neuronal models incorporating the intracellular calcium dynamics, especially the spatial models considering the calcium diffusion both in soma and dendritic branches.


Subject(s)
Animals , Calcium , Metabolism , Calcium Signaling , Physiology , Cytosol , Metabolism , Endoplasmic Reticulum , Metabolism , Physiology , Humans , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , Models, Neurological , Neurons , Metabolism , Physiology
12.
Article in English | WPRIM | ID: wpr-727334

ABSTRACT

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) modulate Ca2+ release from intracellular Ca2+ store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP3Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca2+ release from intracellular Ca2+ stores and Ca2+ entry through store-operated Ca2+ (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca2+ entry, but significantly inhibited carbamylcholine (CCh)-induced Ca2+ release. In contrast, 2-APB did not block CCh-induced Ca2+ release, but remarkably blocked SOC-mediated Ca2+ entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP3-induced Ca2+ release, but 2-APB at lower concentration, which effectively blocked Ca2+ entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP3-induced Ca2+ release and direct stimulation of Ca2+ release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP3R, and 2-APB at lower concentration is considered a blocker of Ca2+ entry through SOC channels in the pancreatic acinar cell.


Subject(s)
Acinar Cells , Animals , Boron Compounds , Caffeine , Calcium , Carbachol , Inositol 1,4,5-Trisphosphate Receptors , Membranes , Mice , Reticulum
13.
Yonsei Medical Journal ; : 311-321, 2008.
Article in English | WPRIM | ID: wpr-30669

ABSTRACT

PURPOSE: The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca(2+) signaling. However, whether the changes in Ca(2+) signaling and Ca(2+) signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2(+/-) mouse parotid gland acinar cells, Ca(2+) signaling, expression levels of Ca(2+) signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2(+/-) mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca(2+) ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP(3)Rs), but the localization and activities of IP3Rs were not altered. In SERCA2(+/-) mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca(2+) signaling proteins in the parotid gland acini, however, overall Ca(2+) signaling is unchanged.


Subject(s)
Amylases/metabolism , Animals , Blotting, Western , Calcium/metabolism , Calcium Signaling/drug effects , Carbachol/pharmacology , Immunohistochemistry , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice , Mice, Knockout , Parotid Gland/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Signal Transduction/drug effects
14.
Chinese Medical Journal ; (24): 341-346, 2008.
Article in English | WPRIM | ID: wpr-287736

ABSTRACT

<p><b>BACKGROUND</b>Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in primary rat cortical neurons of rat in a concentration- and time-dependent manner by an unknown mechanism. We hypothesized that isoflurane induced apoptosis by causing abnormal calcium release from the endoplasmic reticulum (ER) via activation of inositol 1, 4, 5-trisphosphate (IP(3)) receptors. Sevoflurane has a reduced ability to disrupt intracellular calcium homeostasis and is a less potent cytotoxic agent. This study examined and compared the cytotoxic effects of isoflurane and sevoflurane on rat primary cortical neurons and their relationship with disruption of intracellular calcium homeostasis and production of reactive oxygen species (ROS).</p><p><b>METHODS</b>Primary rat cortical neurons were treated with the equivalent of 1 minimal alveolar concentration (MAC) of isoflurane and sevoflurane for 12 hours. MTT reduction and LDH release assays were performed to evaluate cell viability. Changes of calcium concentration in the cytosolic space, [Ca(2+)](c), and production of ROS were determined after exposing primary rat cortical neurons to isoflurane and sevoflurane. We also determined the effects of IP(3) receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in primary rat cortical neurons.</p><p><b>RESULTS</b>Isoflurane at 1 MAC for 12 hours induced cytotoxicity in primary rat cortical neurons, which was also associated with a high and fast elevation of peak [Ca(2+)](c). Xestospongin C significantly ameliorated isoflurane cytotoxicity in primary cortical neurons, as well as inhibited the calcium release from the ER in primary cortical neurons. Isoflurane did not induce significant changes of ROS production in primary rat cortical neurons. Sevoflurane, at equivalent exposure to isoflurane, did not induce similar cytotoxicity or elevation of peak [Ca(2+)](c) in primary rat cortical neurons.</p><p><b>CONCLUSION</b>These results suggested that isoflurane induced elevation in [Ca(2+)](c), partially via elevated activity of IP(3) receptors, which rendered cells vulnerable to isoflurane neurotoxicity. ROS production was not involved in isoflurane-induced neurotoxicity. Sevoflurane, at an equivalent exposure to isoflurane, did not induce similar elevations of [Ca(2+)](c) or neurotoxicity in primary cortical neurons of rat.</p>


Subject(s)
Anesthetics, Inhalation , Toxicity , Animals , Calcium , Metabolism , Cell Survival , Cells, Cultured , Inositol 1,4,5-Trisphosphate Receptors , Physiology , Isoflurane , Toxicity , Methyl Ethers , Toxicity , Rats , Reactive Oxygen Species , Metabolism
15.
Article in Korean | WPRIM | ID: wpr-650022

ABSTRACT

BACKGROUND AND OBJECTIVES: Elevated intracellular calcium level is known to play important roles in the apoptotic pathway. IP3 receptor (ligand-gated channels that release Ca2+ from intracellular stores) is emerging as a key site for regulation of apoptosis. 2-Aminoethoxydiphenyl borate (2-APB) is one of the reliable IP3 receptor antagonists. We examined the effect of 2-APB on gentamicin ototoxicity in vitro, using the HEI-OC1 cell line. MATERIALS AND METHOD: HEI-OC1 cells were trWWeated with 100micrometer gentamicin. Using a CaspACE assay, we measured the caspases-3 activity in the gentamicin treated hair cells with and without 2-APB pre-incubation. We also observed intra-cellular calcium concentrations in HEI-OC1 cells using a confocal microscopy (calcium green-1 stain). Live cell imaging was performed by using fluorescence video-time lapse system. RESULTS: Cytosolic calcium elevation by gentamicin was remarkably inhibited by 2-APB. Caspases-3 activities of gentamicin treated cells were higher than those of the control. After incubation with 2-APB, caspases-3 activities and cell death of gentamicin treated cells were shown to decrease. CONCLUSION: 2-APB reduces Caspases-3 activity in the gentamicin treated HEI-OC1 cells by inhibition of cytosolic calcium increase.


Subject(s)
Apoptosis , Calcium , Caspase 3 , Cell Death , Cell Line , Cytosol , Fluorescence , Gentamicins , Hair , Inositol 1,4,5-Trisphosphate Receptors , Microscopy, Confocal
16.
Article in Chinese | WPRIM | ID: wpr-298687

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of 11, 12-epoxyeicosatrienoic acid (11, 12-EET) preconditioning and postconditioning on Ca(2+)-handling proteins in myocardial ischemia/reperfusion (IR) injury in rats and reveal the effects and mechanism of 11, 12-EET on cardioprotection. METHODS The IR injury model was built by stopping perfusion for 40 minutes followed by reperfusion for 30 minutes. The isolated Langendorff-perfused rat hearts were divided into 4 groups: control group, IR group, EET preconditioning (Pre-EET) group and EET postconditioning (Post-EET) group. The computer-based electrophysiological recorder system was used to measure the changes of the maximal rate of pressure increased in the contraction phase (+dp/dt(max)), the maximal rate of pressure decreased in the diastole phase (-dp/dt(max)), the left ventricular end diastolic pressure (LVEDP) and the difference of left ventricular pressure (delta LVP). The activity of Ca(2+)-ATPase in sarcoplasmic reticulum was measured with colorimetric method. Reverse transcription-polymerase chain reaction was used to assess the gene expression of C(a2+)-handling protein [sarcoplasic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), ryanodine receptor type 2 (RyR,), and 1, 4, 5-trisphosphate inositol receptor type 2 (IP3 R2) ] mRNAs level.</p><p><b>RESULTS</b>Compared with IR group, the myocardial functions, the value of Ca(2+)-ATPase, and the expressions of IP3 R2 mRNA were significantly increased and the expression of PLB mRNA was significantly decreased in both Pre-EET group and Post-EET group (P < 0.05, P < 0.01). And the expression of SERCA mRNA was significantly increased in Pre-EET group (P < 0. 05). However, no significant differences were detected between Pre-EET and Post-EET groups. Moreover, the expression of RyR2 mRNA was not significantly different among all groups.</p><p><b>CONCLUSIONS</b>11, 12-EET preconditioning and post-conditioning can protect myocardium from IR injury by elevating the activity of Ca(2+)-ATPase in sarcoplasmic reticulum, up-regulating the expression of IP3 R2 mRNA, and down-regulating the expression of PLB mRNA. Moreover, up-regulating the expression of SERCA mRNA maybe one of mechanisms of 11, 12-EET preconditioning on cardio protection against IR injury.</p>


Subject(s)
8,11,14-Eicosatrienoic Acid , Pharmacology , Animals , Calcium-Binding Proteins , Metabolism , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , Ischemic Preconditioning, Myocardial , Methods , Myocardial Reperfusion Injury , Metabolism , Rats , Ryanodine Receptor Calcium Release Channel , Metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Metabolism
17.
Chinese Journal of Hepatology ; (12): 403-407, 2007.
Article in Chinese | WPRIM | ID: wpr-230581

ABSTRACT

<p><b>OBJECTIVE</b>To study the changes of expression of type I inositol 1,4,5 triphosphate receptors (IP3RI) in the kidneys of mice with fulminant hepatic failure (FHF) in order to understand the role renal vasoconstriction plays in the development of hepatorenal syndrome (HRS).</p><p><b>METHODS</b>One hundred twenty male Balb/c mice were divided into 4 groups. In the fourth group (60 mice) lipopolysaccharide with D-galactosamine was injected intraperitoneally to induce acute liver necrosis. The first-third groups (20 mice in each group) served as controls and those mice were given NS, LPS or GalN intraperitoneally. At the end of 2 h, 6 h, and 9 h, mice were sacrificed and their livers and kidneys were removed and examined histologically. Immunohistochemistry, Western blot and reverse transcription PCR (RT-PCR) were used to detect the distribution and expression of IP3RI in the kidneys.</p><p><b>RESULTS</b>IP3RI protein was localized in the cytoplasma of glomerular mesangial cells and vascular smooth muscle cells in the kidneys. In the kidney tissues from mice with FHF at 6 h and 9 h, IP3RI-positive staining cells increased significantly (6 h: chi(2)=7.11, P less than 0.01; 9 h: (chi)2=9.15, P less than 0.01). Western blot demonstrated a consistent and significant increase of IP3RI expression in mice with FHF at 6 h and 9 h (6 h: t=3.16, P less than 0.05; 9 h: t=5.43, P less than 0.01). Using RT-PCR we observed that IP3RI mRNA in FHF samples at 2 h, 6 h and 9 h was markedly up-regulated in comparison to that of the controls (2 h: t=2.47, P less than 0.05; 6 h: t=4.42, P less than 0.01; 9 h: t=2.16, P less than 0.05).</p><p><b>CONCLUSION</b>The expression of IP3RI protein increased in glomerular mesangial cells and renal vascular smooth muscle cells of FHF mice. Perhaps this was caused by IP3RI mRNA up-regulation.</p>


Subject(s)
Animals , Disease Models, Animal , Inositol 1,4,5-Trisphosphate Receptors , Metabolism , Liver Failure, Acute , Metabolism , Male , Mice , Mice, Inbred BALB C , RNA, Messenger , Genetics
18.
Indian J Physiol Pharmacol ; 2006 Apr-Jun; 50(2): 99-113
Article in English | IMSEAR | ID: sea-108723

ABSTRACT

This is a concise review of important calcium-transporters on the sarcolemma and organellar membranes of myocardial cells, and their functional roles in cell physiology. It briefly addresses L and T type calcium channels, store-operated calcium channel (SOC), sodium-calcium exchanger (NCX), and the plasma membrane calcium ATPase (PMCA) on the sarcolemma, ryanodine receptor (RyR), IP3 receptor (IP3R) and the sarcoplasmic reticulum (SR) calcium ATPase (SAERCA) on the SR membrane and their contributions to contraction and rhythm-generation. Several agonists and blockers for every transporter that are commonly used in research, and those with therapeutic applications have also been discussed.


Subject(s)
Animals , Calcium Channels/physiology , Calcium Channels, L-Type/physiology , Calcium Channels, T-Type/physiology , Calcium-Transporting ATPases/physiology , Cation Transport Proteins/physiology , Humans , Inositol 1,4,5-Trisphosphate Receptors , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Plasma Membrane Calcium-Transporting ATPases , Receptors, Cytoplasmic and Nuclear/physiology , Ryanodine Receptor Calcium Release Channel/physiology , Sarcolemma/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sodium-Calcium Exchanger/physiology
19.
Article in Chinese | WPRIM | ID: wpr-319053

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the intracellular calcium ion release and the system of calcium channel by 1,25 (OH)2D3 stimulus, and the effect of mechanical pressure on it in rabbit mandibular condylar chondrocytes (MCC) in vitro.</p><p><b>METHODS</b>In vitro cultured MCC from two-week-old New Zealand rabbits were incubated under 20 g/L heparin, 1 g/L procaine, continuous pressure of 90 kPa for 60 min and 360 min in a hydraulic pressure controlled cellular strain unit. With the Fluo-3/AM probe loaded, 1,25(OH)2D3 was added to the medium and then the intracellular calcium level was detected by a laser confocal scanning microscope.</p><p><b>RESULTS</b>Intracellular calcium concentration increased in MCC treated with 1,25(OH)2D3, 1,25(OH)2D3 and procaine, while it didn't change in heparin treated group. Calcium in group under continuous pressure of 90 kPa for 60 min was also increased, even higher than the group stimulated only with 1,25(OH)2D3. Intracellular calcium in group treated with continuous pressure of 90 kPa for 360 min showed no significant difference compared to the control and even decreased at the end of the recording period.</p><p><b>CONCLUSION</b>1,25(OH)2D3 could stimulate the intracellular calcium release channel of inositol triphosphate (IP3) receptor open in MCC in vitro and increases the level of intracellular calcium concentration. Pretreatment of definite mechanical pressure could modulate the sensitivity of IP3 channel to 1,25(OH)2D3 stimulus.</p>


Subject(s)
Animals , Calcium , Metabolism , Calcium Channels , Metabolism , Cells, Cultured , Chondrocytes , Cell Biology , Metabolism , Inositol 1,4,5-Trisphosphate Receptors , Mandibular Condyle , Cell Biology , Metabolism , Microscopy, Confocal , Pressure , Rabbits , Receptors, Cytoplasmic and Nuclear , Metabolism , Steroid Hydroxylases , Pharmacology
20.
Chinese Journal of Hepatology ; (12): 609-611, 2004.
Article in Chinese | WPRIM | ID: wpr-250133

ABSTRACT

<p><b>OBJECTIVE</b>To study the expression of type I inositol 1,4,5-triphosphate receptor in rat glomerular and afferent arterioles in a model of liver cirrhosis and study the role of cross-membrane message transduction in the pathogenesis of hepatorenal syndrome.</p><p><b>METHODS</b>In a rat model of carbontetrachloride liver cirrhosis, the expression of type I inositol 1,4,5-triphosphate receptor (IP3R) on glomerular and afferent arterioles was measured by immunohistochemical method.</p><p><b>RESULTS</b>In the experimental group, 30 rats were used to make a model of liver cirrhosis. 11 rats survived during the experiment. The expression of type I IP3R on glomerular and afferent arterioles was 4.97+/-1.34 and 4.09+/-1.14 in the liver cirrhosis group, and it was 2.43+/-1.67 and 1.83+/-1.32 in the normal control rats. The differences between these two groups are statistically significant (t = 2.28, P = 0.0458).</p><p><b>CONCLUSION</b>Expression of type I IP3 receptor on rat glomerular and afferent arterioles in a model of liver cirrhosis indicated that the mechanism of cross-membrane message transduction plays a very important role in the pathogenesis of hepatorenal syndrome.</p>


Subject(s)
Animals , Arterioles , Metabolism , Calcium Channels , Genetics , Carbon Tetrachloride , Carbon Tetrachloride Poisoning , Inositol 1,4,5-Trisphosphate Receptors , Kidney , Metabolism , Kidney Glomerulus , Metabolism , Liver Cirrhosis, Experimental , Metabolism , Male , Random Allocation , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear , Genetics , Renal Artery , Metabolism
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