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  • 刊频： Twice monthly, 2012-
  • NLM标题： Am J Physiol Cell Physiol
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  • 1.

    【2hr】Retraction

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    机译 缩回
    • 作者：
    • 刊名：American Journal of Physiology - Cell Physiology
    • 2012年第9期
    摘要：
  • 2.

    【2hr】Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells

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    机译 Mg2 +抑制的K +电流和TRPM7样电流在人红白血病细胞中的组成型表达
    摘要：Whole cell patch-clamp experiments were undertaken to define the basal K+ conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K+ current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg2+ concentration. Activation or inactivation of the Mg2+-inhibited K+ current (MIP) was paralleled by activation or inactivation of a Mg2+-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K+ equilibrium potential, indicative of a highly K+-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg2+-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg2+, as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg2+. The MIP current was blocked in a voltage-dependent fashion by extracellular Cs+ and, to a lesser degree, by Ba2+ and was blocked by extracellular La3+ and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K+ channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca2+-activated K+ channels. In addition, the MIP current displayed characteristics distinct from conventional inwardly rectifying K+ channels. A similar current was detected in the leukemic cell line CHRF-288-11, consistent with this current being more generally expressed in cells of leukemic origin.
  • 3.

    【2hr】RESCUE OF THE MUTANT CFTR CHLORIDE CHANNEL BY PHARMACOLOGICAL CORRECTORS AND LOW TEMPERATURE ANALYZED BY GENE EXPRESSION PROFILING

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    机译 药物突变体对CFTR氯化物突变通道的挽救及基因表达谱分析的低温。
    摘要：The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27 °C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o-, to determine the efficacy of various known treatments and to discover new correctors. Compared to other cell types, CFBE41o- shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325.A screening of a small molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor SAHA. However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells.
  • 4.

    【2hr】Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

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    机译 肌无盐小鼠骨骼肌中Ca2 +转运增强和肌肉松弛
    摘要：Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (−dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (−dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca2+ regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca2+-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.
  • 5.

    【2hr】Extracellular signal regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase

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    机译 细胞外信号调节激酶和GEF-H1介导去极化诱导的Rho激活和细胞旁通透性增加
    摘要：Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular [K+], the lipophilic cation tetraphenylphosphonium or L-alanine, which is taken up by electrogenic Na+-cotransport) all provoke robust phosphorylation of Extracellular Signal Regulated Kinase (ERK) in LLC-PK1 and MDCK cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified GEF-H1 as the ERK-regulated critical exchange factor, responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that a) depolarization activated GEF-H1, but not p115RhoGEF; b) siRNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway; c) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na+/K+ pump inhibitor ouabain also caused ERK, GEF-H1 and Rho activation, partially due to its depolarizing effect. Regarding functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells, and this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation-incompetent) MLC. Taken together, we propose, that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium.
  • 6.

    【2hr】Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

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    机译 高渗胁迫诱导肾小管细胞中Rho / Rho激酶/ LIM激酶介导的cofilin磷酸化：在渗透性触发的F-肌动蛋白应答中的关键作用
    摘要：Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation. We then examined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.
  • 7.

    【2hr】Shear Stress Influences Spatial Variations in Vascular Mn-SOD Expression

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    机译 剪应力影响血管中Mn-SOD表达的空间变化。
    摘要：Fluid shear stress modulates vascular production of endothelial superoxide anion (O2˙) and nitric oxide (˙NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well-defined. We constructed a 3-D Computational Fluid Dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohisto-localization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the athero-protective regions, but was nearly absent in the lateral wall of arterial bifurcation. Pulsatile shear stress (PSS: mean shear stress τave= 23 dyn·cm−2) up-regulated Mn-SOD mRNA expression at a higher level than did oscillatory shear stress (OSS: τave= 0.02 dyn·cm−2 ± 3.0 dyn·cm−2·s−1 at 1 Hz) in cultured bovine aortic endothelial cells (PSS by 11.3±0.4-fold versus OSS by 5.0±0.5-fold. p < 0.05, n=4). Furthermore, PSS decreased the extent of Low Density Lipoprotein (LDL) nitration, whereas OSS increased by Liquid chromatography and tandem mass spectrometry (P < 0.05, n=4). Treatment with Mn-SOD siRNA significantly increased intracellular nitrotyrosine level in presence of LDL (n=4, p < 0.5). Our findings indicate that shear stress in the athero-prone versus athero-protective regions regulates spatial variations in mitochondrial Mn-SOD expression. Shear stress modulated LDL protein nitration via Mn-SOD expression.
  • 8.

    【2hr】Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells

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    机译 重复变形激活人Caco-2肠上皮细胞中Src依赖性FAK依赖性ERK致病信号
    摘要：Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr418, FAK-Tyr397-Tyr576-Tyr925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 (µmol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr397 and Tyr576 but not FAK-Tyr925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F–Y577F, and Y397F–Y576F–Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F–Y576F–Y577F FAK construct exhibited less basal FAK-Tyr925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.
  • 9.

    【2hr】Neuroinflammation facilitates LIF entry into brain: role of TNF and NFκB

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    机译 神经炎症促进LIF进入大脑：TNF和NFκB的作用
    摘要：Leukemia inhibitor factor (LIF) is a proinflammatory cytokine mediating a variety of CNS responses to inflammatory stimuli. During lipopolysaccharide (LPS)-induced inflammation, blood concentrations of LIF increase, correlating with lethality of sepsis. Circulating LIF crosses the blood-brain barrier (BBB) by a saturable transport system. Here we determine how this transport system is regulated in neuroinflammation. Using transport assays that quantify the influx rate and volume of distribution of LIF in mice, we show that LPS facilitated the permeation of LIF from blood to brain without compromising the paracellular permeability of the BBB as determined by co-administration of fluorescein. Concurrently, gp130 (shared by the interleukin-6 family of cytokines), but not gp190 (the specific receptor for LIF) or CNTF-Rα (unique receptor for cilliary neurotrophic factor which also uses gp130 and gp190), showed increased levels of mRNA and protein expression in cerebral microvessels from the LPS-treated mice. The upregulation of gp130 by LPS was at least partially mediated by vascular tumor necrosis factor receptor (TNFR)1 and TNFR2. This was shown by elevated TNFR1 and TNFR2 mRNA and protein in cerebral microvessels after LPS and by the absence of the LPS effect on gp130 in knockout mice lacking these receptors. The increase of gp130 was also abolished in mice lacking the p50 unit for nuclear factor (NF)-κB. This indicates a role for NFκB signaling in the upregulation of gp130 and increase of LIF transport across the BBB. The results show that neuroinflammation by LPS induces endothelial signaling and enhances cytokine transport across the BBB.
  • 10.

    【2hr】Glucosamine Protects Neonatal Cardiomyocytes from Ischemia-Reperfusion Injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

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    机译 葡萄糖胺通过增加蛋白O-GlcNAc和增加线粒体Bcl-2来保护新生儿心肌细胞免受缺血再灌注损伤。
    摘要：We have previously reported that glucosamine protected neonatal rat ventricular myocytes (NRVMs) against ischemia/reperfusion (I/R) injury, and this was associated to an increase in protein O linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus, the initial goal of this study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had similar protective effect as glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5mM) and OGT overexpression increased basal and I/R induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome C. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels, but had no effect on Bad on Bax levels. Compared to glucosamine and OGT overexpression, NButGT (100 µM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5 to 10 fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R induced increase in O-GlcNAc and mitochondrial Bcl-2 and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due in part to enhanced mitochondrial Bcl-2 translocation.
  • 11.

    【2hr】Calmodulin in adult mammalian skeletal muscle: localization and effect on sarcoplasmic reticulum Ca2+ release

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    机译 钙调蛋白在成年哺乳动物骨骼肌中的定位和对肌浆网Ca2 +释放的影响
    • 作者：George G. Rodney
    • 刊名：American Journal of Physiology - Cell Physiology
    • 2008年第5期
    摘要：Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (RyR) and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca2+ sparks as an assay of activation in saponin-permeabilized mouse myofibers. Control myofibers exhibited a time-dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time-dependent appearance of Ca2+ sparks and decreased the derived Ca2+ flux from the sarcoplasmic reticulum during a spark by ~37%. A recombinant Ca2+-insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca2+ flux by ~24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal muscle.
  • 12.

    【2hr】VE-cadherin and β-catenin binding dynamics during histamine-induced endothelial hyperpermeability

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    机译 组胺诱导的内皮通透性过高时VE-钙黏着蛋白和β-catenin的结合动力学
    摘要：β-Catenin plays an important role in the regulation of vascular endothelial cell-cell adhesions and barrier function by linking the VE-cadherin junction complex to the cytoskeleton. The purpose of this study was to evaluate the effect of β-catenin and VE-cadherin interactions on endothelial permeability during inflammatory stimulation by histamine. We first assessed the ability of a β-catenin binding polypeptide known as inhibitor of β-catenin and T cell factor (ICAT) to compete β-catenin binding to VE-cadherin in vitro. We then overexpressed recombinant FLAG-ICAT in human umbilical vein endothelial cells (HUVECs) to study its impact on endothelial barrier function controlled by cell-cell adhesions. The binding of β-catenin to VE-cadherin was quantified before and after stimulation with histamine along with measurements of transendothelial electrical resistance (TER) and apparent permeability to albumin (Pa) under the same conditions. The results showed that ICAT bound to β-catenin and competitively inhibited binding of the VE-cadherin cytoplasmic domain to β-catenin in a concentration-dependent manner. Overexpression of FLAG-ICAT in endothelial cell monolayers did not affect their basal permeability properties, as indicated by unaltered TER and Pa; however, the magnitude and duration of histamine-induced decreases in TER were significantly augmented. Likewise, the increase in Pa in the presence of histamine was exacerbated. Overexpression of FLAG-ICAT also significantly decreased the level of β-catenin-associated VE-cadherin following histamine stimulation. Taken together, these data suggest that inflammatory agents like histamine cause a transient and reversible disruption of binding between β-catenin and VE-cadherin, during which endothelial permeability is elevated.
  • 13.

    【2hr】Apical maxi-K (KCa1.1) channels mediate K+ secretion by the mouse submandibular exocrine gland

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    机译 顶端maxi-K（KCa1.1）通道介导小鼠下颌外分泌腺的K +分泌
    摘要：The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3− efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3−, demonstrating that neither K+/HCO3− cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3− did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.
  • 14.

    【2hr】Two-dimensional kinetics of β2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

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    机译 剪切流中嗜中性粒细胞和黑色素瘤细胞之间β2-整合素和ICAM-1结合的二维动力学
    摘要：Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between β2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of β2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of β2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.
  • 15.

    【2hr】DIRECT INTERACTION BETWEEN RAB3D AND THE POLYMERIC IMMUNOGLOBULIN RECEPTOR AND TRAFFICKING THROUGH REGULATED SECRETORY VESICLES IN LACRIMAL GLAND ACINAR CELLS

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    机译 RAB3D与聚合物免疫球蛋白受体之间的直接相互作用以及通过调控的分泌性囊泡在泪腺腺癌细胞中的相互作用
    摘要：The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric immunoglobulin receptor (pIgR), the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGAC) revealed that the small GTPase rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGAC was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGAC. In pull-down assays from resting LGAC, recombinant wild-type rab3D (rab3DWT) or the GDP-locked mutant rab3DT36N both pull down pIgR, but the GTP-locked mutant rab3DQ81L does not. When the pull-down assays are performed in the presence of GTPγS, GTP, or GDPβS, binding of rab3DWT to pIgR is inhibited. In blot overlays, recombinant rab3DWT bound to immunoprecipitated pIgR, suggesting that rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant rab3DT36N in LGAC inhibited CCH-stimulated SC release, and, in CCH-stimulated LGAC, pull-down of pIgR with rab3DWT and co-localization of pIgR with endogenous rab3D were decreased relative to resting cells, suggesting that the pIgR-rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGAC and its secretion therefrom may be effected by its novel interaction with rab3D.
  • 16.

    【2hr】Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

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    机译 附睾透明细胞中蛋白激酶A介导碱性pH和cAMP诱导的V-ATPase膜积累
    摘要：In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H+-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N6-monobutyryl-3′-5′-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 µM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 µM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 µM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.
  • 17.

    【2hr】Increasing α7β1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression

    包量

    机译 增加α7β1-整合素可促进肌肉细胞增殖，粘附和对凋亡的抵抗力，而无需改变基因表达
    摘要：The dystrophin-glycoprotein complex maintains the integrity of skeletal muscle by associating laminin in the extracellular matrix with the actin cytoskeleton. Several human muscular dystrophies arise from defects in the components of this complex. The α7β1-integrin also binds laminin and links the extracellular matrix with the cytoskeleton. Enhancement of α7-integrin levels alleviates pathology in mdx/utrn−/− mice, a model of Duchenne muscular dystrophy, and thus the integrin may functionally compensate for the absence of dystrophin. To test whether increasing α7-integrin levels affects transcription and cellular functions, we generated α7-integrin-inducible C2C12 cells and transgenic mice that overexpress the integrin in skeletal muscle. C2C12 myoblasts with elevated levels of integrin exhibited increased adhesion to laminin, faster proliferation when serum was limited, resistance to staurosporine-induced apoptosis, and normal differentiation. Transgenic expression of eightfold more integrin in skeletal muscle did not result in notable toxic effects in vivo. Moreover, high levels of α7-integrin in both myoblasts and in skeletal muscle did not disrupt global gene expression profiles. Thus increasing integrin levels can compensate for defects in the extracellular matrix and cytoskeleton linkage caused by compromises in the dystrophin-glycoprotein complex without triggering apparent overt negative side effects. These results support the use of integrin enhancement as a therapy for muscular dystrophy.
  • 18.

    【2hr】NF-κB-dependent transcriptional regulation of the cardiac scn5a sodium channel by angiotensin II

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    机译 血管紧张素II对NF-κB依赖性的心脏scn5a钠通道的转录调控
    摘要：Angiotensin II (ANG II) increases oxidative stress and is associated with increased risk of sudden cardiac death. The cardiac Na+ channel promoter contains elements that confer redox sensitivity. We tested the hypothesis that ANG II-mediated oxidative stress may modulate Na+ channel current through altering channel transcription. In H9c2 myocytes treated for 48 h with ANG II (100 nmol/l) or H2O2 (10 µmol/l) showed delayed macroscopic inactivation, increased late current, and 59.6% and 53.8% reductions in Na+ current, respectively (P ≤ 0.01). By quantitative real-time RT-PCR, the cardiac Na+ channel (scn5a) mRNA abundance declined by 47.3% (P < 0.01) in H9c2 myocytes treated for 48 h with 100 nmol/l ANG II. A similar change occurred with 20 µmol/l H2O2 (46.9%, P < 0.01) after 48 h. Comparable effects were seen in acutely isolated ventricular myocytes. The effects of ANG II could be inhibited by prior treatment of H9c2 cells with scavengers of reactive oxygen species or an inhibitor of the NADPH oxidase. Mutation of the scn5a promoter NF-κB binding site prevented decreased activity in response to ANG II and H2O2. Gel shift and chromosomal immunoprecipitation assays confirmed that nuclear factor (NF)-κB bound to the scn5a promoter in response to ANG II and H2O2. Overexpression of the p50 subunit of NF-κB in H9c2 cells reduced scn5a mRNA (77.3%, P < 0.01). In conclusion, ANG II can decrease scn5a transcription and current. This effect appears to be through production of H2O2 resulting in NF-κB binding to the Na+ channel promoter.
  • 19.

    【2hr】Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

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    机译 人类嗜中性粒细胞的初级颗粒胞吐作用受Rac依赖性肌动蛋白重塑调控
    摘要：The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. Here, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (≤ 10 μM) of the actin depolymerizing drugs, latrunculin B (Lat B) or cytochalasin B (CB), enhanced both formyl peptide receptor and Ca2+ ionophore stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP) inhibited primary granule exocytosis measured as myeloperoxidase release, but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly prior to primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodelling was highly polarized when cells were primed with CB, however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis was blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor, NSC23766, also inhibited actin remodelling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not Ca2+ ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.
  • 20.

    【2hr】Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban

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    机译 心肌细胞内的神经元一氧化氮合酶信号转导靶向磷酰胺
    摘要：Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1−/−) have increased or decreased contractility, but consistently have found a slowed rate of intracellular Ca2+ ([Ca2+]i) decline and relengthening. Contraction and [Ca2+]i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1−/− and wild-type (WT) mice. We also simultaneously measured Ca2+ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1−/−, and PLB−/− mice. NOS1−/− trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-methyl-l-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca2+ transient and cell shortening amplitudes and prolonged decline of [Ca2+]i). Alternatively, NOS1 inhibition with SMLT in PLB−/− myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB serine16 phosphorylation. Furthermore, there was a decreased SR Ca2+ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. β-Adrenergic stimulation restored the slowed [Ca2+]i decline in NOS1−/− myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca2+]i decline, and this effect is absent in PLB−/− myocytes. Thus NOS1 signaling modulates PLB serine16 phosphorylation, in part, via peroxynitrite.
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