Mechanistic study of endothelium independent vasodilation effects of wogonin

Scutellaria baicalensis Georgi, locally known as HuangQin, and commonly as Baikal or Chinese skullcap, is an important herb in Chinese traditional medicine. The flavonoids from this plant are main active substances responsible for its medicinal applications. Wogonin is one such active ingredient derived from this plant. Here, we investigated the mechanism of the vasodilation effect of wogonin on isolated rat thoracic aortas. For this study, endothelium intact and endothelium removed thoracic aortic rings were prepared from rats. Using a tension transducer, the tension of the rat thoracic aortic rings was recorded. Results showed that wogonin is able to relax the endothelium-intact aortic rings, but L-NAME, indomethacin (Indo), and methylene blue (MB) could not reduce the tension in these rings. Wogonin was also able to relax endotheliumremoved rings. However, treatment with tetraethylammonium (TEA), BaCl2, glibenclamide (Gly), 4-aminopyridine (4-AP), and verapamil (Ver) had no effect on vasodilation induced by wogonin. Using wogonin to pre-treat endothelium-removed aortic rings reduced the contraction induced by K+. Pre-treatment of endothelium-removed aortic rings with wogonin markedly reduced the contraction induced by 10-6 M PE in Ca2+-free solution. It could be concluded that L-type calcium channels and intracellular Ca2+ release is inhibited by wogonin.

Effects of subchronic fluoride exposure through drinking water on renal toxicity and the expressions of STIM1 and ORAI1 in renal tissues in mice / 环境与职业医学

Background It has been found that fluoride may cause cell damage by inducing intracellular calcium overload. Store-operated calcium entry (SOCE) plays an important role in maintaining intracellular calcium homeostasis, but the effect of fluoride on renal SOCE is unknown. Objective To explore the renal toxicity and the expression levels of the key proteins of SOCE, stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (ORAI1) in the kidney tissues of mice exposed to fluoride subchronically. Methods Twenty male ICR mice were randomly divided into four groups with five mice in each group, including 0 (control group), 0.3, 3, and 30 mg·L−1 fluoride groups. The mice were given drinking water containing designed fluoride for 12 weeks. Body weight and liver and kidney organ coefficients of the mice were measured after the exposure; histopathological changes of the mouse kidney were observed; 24 h urine was collected at the end of 12 weeks of exposure to determine the levels of urine creatinine (UCr), urine calcium (UCa), albumin (ALB), and β2-microglobulin (β2-MG); the protein expression levels of STIM1 and ORAI1 in the kidney were detected by Western blotting; the fluorescence co-localization of STIM1 and ORAI1 was used to further verify the expression levels of STIM1 and ORAI1. Results After the exposure, there were no differences in body weight as well as liver and kidney organ coefficients among the groups (P > 0.05). Under optical microscope, the renal tubular cell showed degeneration, apical protrusion, shedding, and dilation in the 3 and 30 mg·L−1 fluoride groups. There was no statistical difference in UCr among the mice in each group (P > 0.05). Compared with the control group, the levels of UCa adjusted by UCr in the 3 and 30 mg·L−1 fluoride groups were (0.075±0.014) and (0.081±0.012) mol·mol−1 (represent by UCr per mol), which had a rising trend but showed no statistical difference. No difference was identified in the level of ALB among the groups (P > 0.05). The levels of β2-MG showed difference in different exposure groups, and the level of urine β2-MG in the 30 mg·L−1 fluoride group was (0.077±0.014) g·mol−1, higher than that in the control group (P<0.05). Based on the results of Western blotting, the protein expression levels of STIM1 and ORAI1 showed significant differences among the groups (F=18.411, 6.853; P=0.001, 0.013); compared with the control group, the expression levels of STIM1 protein increased in the 3 and 30 mg·L−1 fluoride groups (P < 0.05), and the protein expression level of ORAI1 in the 30 mg·L−1 fluoride group was increased (P < 0.05). The fluorescence co-localization results of STIM1 and ORAI1 showed that the expressions of STIM1 and ORAI1 were up-regulated in the 3 and 30 mg·L−1 fluoride groups. Conclusion Subchronic exposure to fluoride through drinking water can up-regulate the expression levels of STIM1 and ORAI1 in renal tissues and induce renal injury.

Diagnosis and treatment of postoperative malignant hyperthermia in a pediatric patient with congenital myopathy due to RyR1 gene mutations / 中华麻醉学杂志

A 14-year-old male pediatric patient was admitted to the hospital mainly because of neck and back deformity, with limited activity for 7 yr, dysphagia and short of breath for more than 10 months.He was diagnosed with cervical lordosis deformity, RyR1 gene-related myopathy, high possibility of multi-minicore disease and being susceptible to malignant hyperthermia.Posterior cervical orthopedic internal fixation surgery was successfully performed under total intravenous anesthesia with propofol.The vital signs were stable during anesthesia and operation which lasted for 10 h. The patient was admitted to intensive care unit after the uneventful operation.When emerging from general anesthesia, the patient suddenly presented with symptoms of muscular fasciculation in the head, face, trunk and limbs, along with elevated body temperature as high as 39.4℃, severe acidosis and hypercapnia, meanwhile, the blood creatine kinase, blood myoglobin and urinary myoglobin gradually increased.The patient was diagnosed with malignant hyperthermia based on the clinical grading scale score of 63.Dantrolene sodium was infused intravenously, combined with multiple treatments such as physical cooling, correction of acidosis and electrolyte disturbance, alkalization of urine, intermittent hemofiltration and plasma exchange.The arrhythmia and delirium were treated symptomatically.The pediatric patient was fully recovered and discharged with good outcomes.

Effect of sublingual immunotherapy on functions of effector T cells and regulatory T cells / 上海交通大学学报（医学版）

Objective • To investigate the effect of sublingual immunotherapy (SLIT) on the functions of effector T cells (Teff) and regulatory T cells (Treg) under allergic rhinitis (AR) condition. Methods • Peripheral blood mononuclear cells (PBMC) were obtained from 20 AR patients who were recruited from Department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital North of Fudan University before and after the 24-and 30-month treatments of SLIT. The percentages of Teff and Treg in CD4+ T cells in PBMC were examined and the contents of calcium release-activated calcium channel modulator 1(ORAI1) protein and Ca2+ mean fluorescence intensity (MFI) in PBMC were measured. Then, Teff and Treg after the 30-month treatment were cultured in vitro, and were evaluated for the expression of ORAI1 protein and concentrations of interleukin-4(IL-4) and IL-10 in the cultures. Lentivirus vector (lenti) that encoded short hairpin RNA against ORAI1(lenti-ORAI1) was prepared and transfected into Teff and Treg, and then Ca2+ MFI in Teff and Treg and concentrations of IL-4 and IL-10 in the cultures were assessed again. Results • The percentage of Teff in CD4+ T cells in PBMC reduced gradually after the 24-and 30-month treatments (P=0.000, P=0.027); however, the percentage of Treg was increased (P=0.000, P=0.008). The expression of ORAI1 protein (P=0.000, P=0.003) and Ca2+ MFI (P=0.000, P=0.000) in Teff were also decreased gradually after the 24-and 30-month treatments; however, the expression of ORAI1 protein (P=0.000, P=0.007) and Ca2+ MFI in Treg were enhanced (P=0.000, P=0.000). ORAI1 protein was expressed in these two types of cells cultured in vitro. Furthermore, Ca2+ MFI in them (P=0.004, P=0.000) and IL-4(P=0.009) and IL-10(P=0.000) in the cultures were all decreased after the transfection of lenti-ORAI1. Conclusion • SLIT can control functions of Teff and Treg through regulating the expression of ORAI1 protein.

Research progress of STIM1 in tumor development and clinical application / 天津医药

Studies have shown that stromal interaction molecule 1(STIM1) is closely related to the development of tumors, and which is involved in the regulation of apoptosis, proliferation, migration and invasion in many human cancers. Blocking or knockdown of STIM1 can significantly inhibit the proliferation and migration of cancer cells. Elucidation of the regulatory mechanism of STIM1 in cancer cells will be helpful for the identification of new therapeutic targets. This paper reviews the mechanism of STIM1 molecule in different tumors and its clinical application.

Impact of Valsartan on Sarcoplasmic Reticulum Ryanodine Receptor2 in Myocardiocyte of Heart Failure Rabbits / 中国循环杂志

Objective: To explore sarcoplasmic reticulum ryanodine receptor2 (RyR 2) expression and calcium releasing function in chronic heart failure (CHF) rabbits and to study the impact of long term valsartan treatment in relevant animals. Methods: HF model was established by volume overloading with pressure overloading in experimental rabbits. 27 rabbits were divided into 3 groups: Sham group, HF group and HF+valsartan group. n=9 in each group and the animals were treated for 7 weeks. Left ventricular structure, hemodynamic parameters, expression and functional changes of myocardiocyte sarcoplasmic reticulum RyR 2 were observed and compared among different groups. Results: Compared with Sham group, HF group had increased left ventricular mess index (LVMI), left ventricular end diastolic pressure (LVEDP) and decreased left ventricular shortening fraction, LVEF, all P<0.05. Compared with HF group, HF+valsartan group showed decreased LVMI, LVEDP and increased left ventricular shortening fraction, LVEF, all P<0.05. Sarcoplasmic reticulum RyR 2 expression and calcium releasing function were lower in HF group than Sham group, P<0.05; while they were both higher in HF+valsartan group than HF group, P<0.05. Conclusion: Long term application of valsartan could improve the cardiac function which might be related to increased myocardial sarcoplasmic reticulum RyR 2 expression and calcium releasing function in experimental CHF rabbits.

Interaction between STIM1 and Orai1 in calcium-sensing receptor-media-ted calcium influx and nitric oxide generation / 中国病理生理杂志

AIM:To investigate the interaction of Ca 2+-sensing proteins , stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1), in Ca2+-sensing receptor (CaSR)-mediated extracellular Ca2+ influx and production of nitric oxide ( NO).METHODS: Human umbilical vein endothlial cells (HUVECs) were incubated with CaSR agonist spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels ( ROC) ] alone or combined with CaSR negative allosteric modulator Calhex 231+ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro 31-8220, or PKCα/β1 selective inhibitor Go 6976 (activate SOC, blocking ROC).The protein expression of STIM1 and Orai1 was determined by the method of immu-nofluorescence .The interaction between STIM 1 and Orai1 was examined by co-immunoprecipitation .The second to third passages of HUVECs were divided into STIM 1 and Orai1 short hairpin RNA group ( shSTIM1+shOrai1 group ) , vehicle-STIM1+vehicle-Orai1 group and control group , and then incubated with the 4 different treatments above .The intracellular Ca2+concentration ( [ Ca2+] I ) was detected using the fluorescent Ca 2+indicator Fura-2/AM.The production of NO was also determined by DAF-FM DA fluorescent probe .RESULTS:The protein expression of STIM 1 and Orai1 was located in the cytoplasm.Compared with control group , the localization of STIM1 and Orai1 in the cytoplasm was reduced after the HUVECs were incubated with Calhex 231+TPA, Ro 31-8220 or Go 6976, and the interaction of STIM1 and Orai1 was de-creased significantly .The [ Ca2+] I and the net NO fluorescence intensity in shSTIM 1+shOrai1 group were significantly re-duced after the 4 different treatments (P<0.05).CONCLUSION:STIM1 and Orai1 are components of SOC and ROC in store-and receptor-operated Ca 2+entry and NO generation .

Interaction between STIM1 and Orai1 in calcium-sensing receptor-media-ted calcium influx and nitric oxide generation / 中国病理生理杂志

AIM:To investigate the interaction of Ca 2+-sensing proteins , stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1), in Ca2+-sensing receptor (CaSR)-mediated extracellular Ca2+ influx and production of nitric oxide ( NO).METHODS: Human umbilical vein endothlial cells (HUVECs) were incubated with CaSR agonist spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels ( ROC) ] alone or combined with CaSR negative allosteric modulator Calhex 231+ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro 31-8220, or PKCα/β1 selective inhibitor Go 6976 (activate SOC, blocking ROC).The protein expression of STIM1 and Orai1 was determined by the method of immu-nofluorescence .The interaction between STIM 1 and Orai1 was examined by co-immunoprecipitation .The second to third passages of HUVECs were divided into STIM 1 and Orai1 short hairpin RNA group ( shSTIM1+shOrai1 group ) , vehicle-STIM1+vehicle-Orai1 group and control group , and then incubated with the 4 different treatments above .The intracellular Ca2+concentration ( [ Ca2+] I ) was detected using the fluorescent Ca 2+indicator Fura-2/AM.The production of NO was also determined by DAF-FM DA fluorescent probe .RESULTS:The protein expression of STIM 1 and Orai1 was located in the cytoplasm.Compared with control group , the localization of STIM1 and Orai1 in the cytoplasm was reduced after the HUVECs were incubated with Calhex 231+TPA, Ro 31-8220 or Go 6976, and the interaction of STIM1 and Orai1 was de-creased significantly .The [ Ca2+] I and the net NO fluorescence intensity in shSTIM 1+shOrai1 group were significantly re-duced after the 4 different treatments (P<0.05).CONCLUSION:STIM1 and Orai1 are components of SOC and ROC in store-and receptor-operated Ca 2+entry and NO generation .

Regulation of autophagy by store-operated calcium channel in arterial smooth muscle cells / 中国药理学通报

Aim To investigate the effect of store-oper-ated calcium channel( SOCC) on autophagy in rat arte-rial smooth muscle cells A7 R5 . Methods Lentiviruses containing STIM1 or Orai1 gene were packaged in 293 T cells and then were used to infect rat arterial smooth muscle cells A7 R5 . The expression levels of STIM1 , Orai1 and Beclin 1 , a critical autophagy-regu-lating protein, of lentivirus-infected A7R5 cells, were detected by Western-blot. Autophagy in lentivirus-in-fected A7 R5 cells was induced by starvation or rapamy-cin, an inhibitor of mammalian target of rapamycin ( mTOR ) . Autophagy marker LC3 of these cells was detected by Western-blot. Results The constructions of vector pLV-STIM1 and pLV-Orai1 were confirmed by restriction enzymes digestion analysis. Compared with the control group, expressions of STIM1 or Orai1 protein was significantly increased after lentivirusLV-STIM1 and LV-Orai1infection, whereas the expressions of autophagy related protein Beclin-1 were down-regu-lated. Starvation or rapamycin stimulated A7R5 auto-phagy but overexpression of STIM1 or Orai1 significant-ly inhibited starvation or rapamycin induced autoph-agy. Conclusion Overexpression of store-operated calcium channel components STIM1 and/or Orai1 in rat arterial smooth muscle cells A7 R5 inhibit autoph-agy. This mechanism might contribute to the develop-ment of pulmonary arterial hypertension.

Dexmendetomidine decreases spontaneous contraction of duodenal smooth muscle of rabbits in vitro / 中国药理学通报

Aim To observe the influences of dexmen-detomidine on the spontaneous contraction of duodenal smooth muscle of rabbits in vitro and explore the mech-anisms.Methods The rabbits ( male or female ) were stunned and the duodenums were isolated .The sam-ples of duodenal segments were connected with tension transducer , which were then put into oxygen saturation Krebs-Henseleit ( K-H) solution .The influences of dex-mendetomidine on amplitude ( AM ) and frequency ( FR ) of duodenal smooth muscle were recorded by BL-420 F biological signal processing system .The cu-mulative dosing method was used to observe the differ-ent concentrations of dexmedetomidine on duodenal smooth muscle spontaneous contraction .Glibenclamide ( Gli) was added to K-H solution before dexmendeto-midine.In the calcium-free K-H solution, calcium chloride and rynodine were added before dexmendeto-midine.The mechanisms of dexmendetomidine were studied .Results ① Dexmendetomidine reduced the amplitude of spontaneous contraction of duodenal smooth muscle in rabbits in a dose-dependent manner ( P0.05 ) .② Gli ( P <0.05 ) partly abolished the inhibitory effects of dexmendetomi-dine on duodenal smooth muscle .③ Dexmendetomi-dine inhibited the contraction of duodenum smooth muscle induced by calcium chloride ( P <0.05 ) and rynodine ( P<0.05 ) application into calcium-free K-H solution.Conclusion Dexmendetomidine inhibits the spontaneous contraction of duodenal smooth muscle of rabbits in vitro.The mechanisms may be related to ac-tivating ATP sensitive potassium channels , inhibition of the extracellular calcium influx via cell membrane and intracellular calcium release via sarcoplasmic reticulum in duodenal smooth muscle .

Research progress on the role of CaMKⅡin cardiovascular disease / 天津医药

Calcium/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) has multiple functions, which made it play a central role in cardiovascular disease. Especially it activates numerous downstream targets in various signaling pathways that promotes vascular disease, heart failure, myocardial hypertrophy and arrhythmias. CaMKⅡcan impact calcium balance and increase calcium leak in myocardial cell via phosphorylating L type calcium channel, Ryanodine receptor (RyR 2) and phos?pholamban (PLN), and regulate ATP sensitive potassium current (IKATP) and late sodium current by affecting sodium channels and potassium channels. In addition, It can directly regulate transcription via activating the silk crack the original activated protein kinases (MAPKs) and acetylation enzyme (HDAC). These mechanisms have important roles in myocardial hypertro?phy, heart failure and arrhythmia. So we focus to demonstrating the structure and action mechanism of CaMKⅡto improve a new therapy of cardiovascular disease.

Ryanodine Receptor-mediated Calcium Release Regulates Neuronal Excitability in Rat Spinal Substantia Gelatinosa Neurons

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium (BKca) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of Ca2+ that induces Ca2+-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective BKCa channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove Ca2+ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through BKca channels, which are activated by intracellular Ca2+ increase via activation of RyR of Ca2+ stores.

La liberación de calcio de los depósitos intracelulares promueve la secreción de serotonina en terminales sinápticas / Calcium release from intracellular stores promotes serotonin secretion at synaptic terminals

En este trabajo se estudió la participación que tiene la liberación de calcio del retículo endoplásmico en la liberación de serotonina en terminales sinápticas. Los experimentos se llevaron a cabo en sinapsis formadas en cultivo entre neuronas serotonérgicas de Retzius y neuronas mecanosensoriales sensibles a presión, aisladas del Sistema Nervioso Central de la sanguijuela. En esta preparación la estimulación con pares de impulsos produjo facilitación sináptica. La estabilización de los receptores de rianodina en un estado de sub-conductancia por la incubación con rianodina 100 μM produjo un alargamiento del potencial sináptico en respuesta a impulsos presinápticos, sugiriendo que el calcio liberado por estos canales puede alcanzar las vesículas y promover la secreción. En contraste, el vaciamiento de los depósitos intracelulares de calcio con tapsigargina 500 nM produjo una disminución gradual de la facilitación sináptica ante impulsos presinápticos pareados y abolió la liberación extrasináptica en el axón neuronal en respuesta a trenes de impulsos. Todo esto ocurrió sin cambios en las propiedades de la membrana postsináptica, lo cual sugiere que la liberación de calcio intracelular participa en un mecanismo de retroalimentación positiva que promueve la liberación presináptica y perisináptica en las neuronas serotonérgicas.

This work analyses the role of intracellular calcium pools in serotonin release from nerve terminals. Experiments were carried out in synapses formed in culture between serotonergic Retzius neurones and pressure mechanosensory neurons, isolated from the Central Nervous System of the leech. In this configuration, serotonin is released from clear vesicles at synapses or from extrasynaptic dense core vesicles. Locking ryanodine receptors in a subconductance state by incubation with 100 μM ryanodine caused an elongation of the synaptic potential in response to a presynaptic action potential or to trains of them, suggesting that calcium released from the endoplasmic reticulum through these channels reaches the synaptic vesicles and may promote their fusion with the plasma membrane. By contrast, depletion of intracellular calcium pools by incubation with 500 nM thapsigargin gradually decreased paired-pulse synaptic facilitation and abolished extrasynaptic axonal serotonin release in response to trains of impulses. All this occurred without changes in the properties of the postsynaptic membrane, indicating that intracellular calcium release participates in a feedback mechanism that enhances presynaptic and perisynaptic release in serotonergic neurons.

Estudo clínico, histológico e molecular da miopatia centronuclear / A clinical, histological and molecular study of centronuclear myopathy

Introdução: A miopatia centronuclear é uma doença muscular congênita com apresentação clínica heterogênea, caracterizada histologicamente pela proeminência de fibras musculares com núcleos centralizados. Três formas são reconhecidas: neonatal grave, com herança ligada ao X e envolvimento do gene MTM1; autossômica dominante, com início geralmente tardio e curso mais leve, associada a mutações no gene DNM2; e autossômica recessiva, com gravidade intermediária entre as outras formas e envolvimento dos genes BIN1, RYR1 ou TTN. Apesar da identificação dos principais genes responsáveis pela doença, os métodos usuais de diagnóstico genético não encontram mutações em cerca da metade dos casos. Objetivo: O objetivo deste estudo foi a caracterização clínica, histológica e molecular de pacientes brasileiros portadores de miopatia centronuclear. Métodos: Laudos de dois bancos de biópsia muscular foram usados para identificar pacientes com diagnóstico de miopatia centronuclear nos últimos dez anos. As lâminas das biópsias foram revisadas e analisadas, e as famílias correspondentes convocadas para aplicação de protocolo clínico e coleta de sangue periférico para extração de DNA genômico. As famílias foram estudadas para os genes conhecidos por sequenciamento Sanger, MLPA, painel de genes implicados em doenças neuromusculares ou sequenciamento de exoma. Resultados: Foram convocados 24 pacientes provenientes de 21 famílias, em 16 das quais foi possível estabelecer o diagnóstico molecular. As 7 famílias com a forma neonatal grave constituíam um grupo homogêneo clínica e histologicamente, e mutações novas e conhecidas foram encontradas no gene MTM1 em 6 destas. Dois meninos deste grupo, com evolução estável, tiveram óbito súbito por choque hipovolêmico subsequente a rompimento de cisto hepático. O gene MTM1 também foi implicado em uma menina portadora manifestante, com quadro mais leve, na forma de uma macrodeleção em heterozigose, detectada por MPLA...

Introduction: Centronuclear myopathy is a heterogeneous congenital muscle disease, characterized by the prominence of centralized nuclei in muscle fibers. Three disease forms are recognized: a severe neonatal, X-linked form caused by mutations in the MTM1 gene; an autosomal dominant, late-onset milder form, associated to the DNM2 gene; and an autosomal recessive form, with intermediate severity, so far with the BIN1, RYR1 or TTN genes implicated. In spite of the identification of these genes, usual molecular diagnostic methods don't yield a molecular diagnosis in about half of cases. Objetives: The aim of this work was to study clinical, histological, and molecular aspects of centronuclear myopathy Brazilian patients. Methods: Reports taken from two muscle biopsy banks were used to identify centronuclear myopathy patients in the last ten years. Biopsy slides were reviewed and analyzed, and corresponding families recruited to apply a clinical protocol and to draw peripheral blood to extract genomic DNA. Families were studied for known genes via Sanger sequencing, MLPA, panel of genes implicated in neuromuscular diseases, or exome sequencing. Results: Twentyfour patients out of 21 families were recruited, and in 16 families molecular diagnosis was established. The 7 families with the severe neonatal form amounted to a clinically and histologically homogeneous group, and mutations, both known and novel, were found in the MTM1 gene in 6 of these. Two boys of this group, with a stable course, died suddenly of hypovolemic shock due to a hepatic cyst rupture. The MTM1 gene was also implicated in the case of a mild manifesting carrier girl with a heterozygous macrodeletion detected via MLPA...

Analysis of osmotic stress induced Ca2+ spark termination in mammalian skeletal muscle.

Ca2+ sparks represent synchronous opening of the ryanodine receptor (RyR) Ca2+ release channels located at the sarcoplasmic reticulum (SR) membrane. Whereas a quantal nature of Ca2+ sparks has been defined in cardiac muscle, the regulation of Ca2+ sparks in skeletal muscle has not been well-studied. Osmotic-stress applied to an intact skeletal muscle fiber can produce brief Ca2+ sparks and prolonged Ca2+ burst events. Here, we show that termination of Ca2+ bursts occurs in a step wise and quantal manner. Ca2+ burst events display kinetic features that are consistent with the involvement of both stochastic attrition and coordinated closure of RyR channels in the termination of SR Ca2+ release. Elemental unitary transition steps could be defined with a mean DF/F0 of ~0.28, corresponding to the gating of 1-2 RyR channels. Moreover, the amplitude of the elemental transition steps declines at the later stage of the burst event. In tandem Ca2+ burst events where two Ca2+ bursts occur at the same position within a fiber in rapid succession, the trailing event is consistently of lower amplitude than the initial event. These two complementary results suggest that SR Ca2+ release may be associated with local depletion of SR Ca2+ stores in mammalian skeletal muscle.

Advances of the relationship of asthma and store-operated calcium / 国际儿科学杂志

As one of the chronic diseases,asthma,plays a serious impact on human daily life.Asthma in children has showed an increasing trend in recent years,but the mechanisms of asthma are not yet clear.Studies have found that store-operated calcium entry(SOCE) plays an important role in the physiological activity of the body.The enhanced SOCE activity can promote cell growth,proliferation,and migration of a variety of cell types.SOCE important molecules STIM1 and ORAI1 may be involved in the asthmatic airway occurrence of hyperresponsiveness and airway remodeling,and closely to the asthmatic development.

Analysis of protease-activated receptor 2 expression and function in cultured human keratinocytes / 中华皮肤科杂志

Objective To assess the expression pattern of protease-activated receptor 2 (PAR2) in human keratinocytes and to characterize its biological functions in the regulation of skin barrier.Methods Primary human keratinocytes and human N/TERT keratinocytes were used as the subject of this study.The expression and distribution of PAR2 in the keratinocytes were analyzed by using immunoflorescence staining and Western blot.Two different PAR2 agonists,trypsin and a PAR2-activating peptide (AP),as well as a PAR2-antagonistic peptide (H2N-FSLLRY-COOH) and a control peptide were used to induce the activation of PAR2 in the keratinocytes.Then,a fluorescence-based calcium mobilization assay was performed to evaluate the biological function of PAR2.Data were statistically analyzed by one-factor analysis of variance.Results Under normal culture conditions,PAR2 was weakly expressed in keratinocytes,and the expression was unaffected by culture medium composition or culture duration.Calcium mobilization was induced by trypsin of 50-250 nmol/L and the PAR2-activating peptide in a dose-and time-dependent pattern.The maximal activation of PAR2 was observed in keratinocytes treated with the PAR2 agonist HAN-SLIGKV-COOH of 75-250 μmol/L.The PAR2-antagonistic peptide (H2N-FSLLRY-COOH) obviously suppressed the increase in calcium mobilization induced by trypsin,while the control peptide PAR-RAP showed no inductive effect on the PAR2 activation based on the absence of calcium mobilization.The substrate-induced calcium release was complete within 250 seconds,and peaked at 50 seconds after the initial trypsin or PAR-AP stimulation.Moreover,the activation of PAR2 was accompanied by an increase in ERK phosphorylation and elicitation of MAPK signaling pathway in keratinocytes.Conclusions Human keratinocytes positively express PAR2,which can be activated by trypsin and PAR2-activating peptides,and the activation of PAR2 may influence the physiological function of keratinocytes by inducing intracellular calcium release.

Effect of trimebutine maleate on ion channels in colon smooth muscle cell of cold restraint stress induced rats / 中华消化杂志

Objective To investigate the effects of trimebutine maleate (TM) on the expression of large conductance calcium-activated potassium channel (BKCa) and ryanodine receptors (RyR)channels at mRNA and protein level in colonic smooth muscle cell of cold restraint stress(CRS)induced rats.Methods A total of 24 Wistar rats were divided into CRS group,CRS with TM group and control group equally.The rats of CRS group were gavaged with 0.9％NaCl (6 ml/kg) daily; the rats of CRS with TM group were gavaged with 15 g/L TM (6 ml/kg) daily and activity was restricted in wire cage at 4 ℃ for two hours,continuously for five days.The rats of control group were gavaged with 0.9 ％ NaCl (6 ml/kg) once without CRS.The amount and characteristics of stool of rats in each group were observed.The colonic smooth muscle was isolated to detect the expression of BKCa and RyR at mRNA and protein level by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results The median of rats defecation particles of CRS group was six,control group was one and CRS with TM group was five.Compared with control group,the defecation appearance of CRS group and CRS with TM group was looser and wetter observed by naked eyes.Compared with control group,there was no obvious pathological changes in CRS and CRS with TM group.There was no significant difference in the mRNA expression of BKCa and RyR channels between control group and CRS group.Compared with control group,the BKCa expression at mRNA level of CRS with TM group increased 1.45 fold.Compared with control group,the RyR2 expression at mRNA level of CRS with TM group increased 1.32 fold.Compared with control group,the BKCa expression at protein level of CRS with TM group increased 1.39 fold,and there was no RyR2 expression band at protein level.Conclusion TM might affect colonic smooth muscle contraction through the upregulation of BKCa expression at mRNA and protein level and RyR expression at mRNA level.

Ca(2+)-induced Ca2+ Release from Internal Stores in INS-1 Rat Insulinoma Cells

The secretion of insulin from pancreatic beta-cells is triggered by the influx of Ca2+ through voltage-dependent Ca2+ channels. The resulting elevation of intracellular calcium ([Ca2+]i) triggers additional Ca2+ release from internal stores. Less well understood are the mechanisms involved in Ca2+ mobilization from internal stores after activation of Ca2+ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic beta-cell line, INS-1 cells. To measure cytosolic and stored Ca2+, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. [Ca2+]i was repetitively increased by caffeine stimulation in normal Ca2+ buffer. However, peak [Ca2+]i was only observed after the first caffeine stimulation in Ca2+ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in [Ca2+]i were reduced by pretreatment with ruthenium red, as well as by depletion of internal Ca2+ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced Ca2+ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells, Ca2+ release from internal stores was activated by caffeine, Ca2+, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in permeabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic beta-cells.

Differential Expression of Ryanodine Receptors in the Developing Rat Cochlea / 听力学及言语疾病杂志

Objective Ryanodine receptor (RyR) is one of the Ca2+ release channels on the intracellular Ca2+ stores. RyR induced-Ca2+ release is activated by the voltage-dependent Ca2+ entry, that is, calcium-induced calcium release (CICR). Intracellular free Ca2+ concentration (ECa2+]i) plays a key role on cochlear function. Our study is to investigate the differential expression of RyR in the developing rat cochlea, and to analyze the relationship between the expression of RyR and auditory functional development. Methods Immature SD rats, which were 1 day (P1), 5 days (P5), 10 days (P10), 14 days (P14), 28 days (P28) after parturition, and adutl rats(5 rats for each age) were included in the study. Frozen cochlea sectioning and immunofluorescence were applied to observe the differential expression of RyR. Results The RyR expression in the Corti's organ increased during the cochlear development. It's not significant that the stain was observed on the hair cells and supporting cells in the Corti's organ of P1 and P5 rats. The appearance of the Corti's organ of P10 rats trended to maturity. In P14 rats the RyR expression on hair cells located in the synaptic area against the afferent or efferent nerve, and the strain on supporting cells was extensive. There was little different between the strain on cochlea of P14 and P28 rats. In postmature rat spiral ganglion neurons (SGN), the RyR expression verged gradually from extensive whole cell soma to the synaptic area near to the plasma membrane. Conclusion The RyR expression peaked the 14th day after parturition, which was close to that in the mature cochlea. The time course of the RyR expression during the cochlear development was coincident with that of the auditory functional development. The RyR expression on both hair cells and SGNs located in the synaptic area near to the plasmolemma, implying that RyR induced-CICR was related to the auditory functions such as neurotransmission. Extensive RyR expression in the soma of SGNs at the early stage possibly involved in apoptosis of SGNs during neuron development.