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1.
Xiong W Liu T Wang Y Chen X Sun L Guo N Zheng H Zheng L Ruat M Han W Zhang CX Zhou Z 《PloS one》2011,6(10):e24573
Aim
Neurotransmitter release is elicited by an elevation of intracellular Ca2+ concentration ([Ca2+]i). The action potential triggers Ca2+ influx through Ca2+ channels which causes local changes of [Ca2+]i for vesicle release. However, any direct role of extracellular Ca2+ (besides Ca2+ influx) on Ca2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.Results
Using photolysis of caged Ca2+ and caffeine-induced release of stored Ca2+, we found that extracellular Ca2+ inhibited exocytosis following moderate [Ca2+]i rises (2–3 µM). The IC50 for extracellular Ca2+ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca2+ concentration ([Ca2+]o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca2+]o. The calcimimetics Mg2+, Cd2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca2+-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.Conclusion/Significance
As an extension of the classic Ca2+ hypothesis of synaptic release, physiological levels of extracellular Ca2+ play dual roles in evoked exocytosis by providing a source of Ca2+ influx, and by directly regulating quantal size and release probability in neuronal cells. 相似文献2.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(7):1218-1229
Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. 相似文献
3.
Hannah MJ Skehel P Erent M Knipe L Ogden D Carter T 《The Journal of biological chemistry》2005,280(24):22827-22830
The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis of individual Weibel-Palade bodies (WPBs) from single human endothelial cells was analyzed. Chimeras of enhanced green fluorescent protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular patches was 323.5 +/- 146.2 s (+/-S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 +/- 1.88 s (+/-S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface. 相似文献
4.
Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis. 相似文献
5.
Varga Z Juhász T Matta C Fodor J Katona É Bartok A Oláh T Sebe A Csernoch L Panyi G Zákány R 《PloS one》2011,6(11):e27957
Background
Understanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.Methodology/Principal Findings
Using patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation.Conclusions/Significance
These data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca2+-oscillations, which are modulated by KV-driven membrane potential changes. KV1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use. 相似文献6.
Djamiatun K van der Ven AJ de Groot PG Faradz SM Hapsari D Dolmans WM Sebastian S Fijnheer R de Mast Q 《PLoS neglected tropical diseases》2012,6(5):e1628
Background
Thrombocytopenia, bleeding and plasma leakage are cardinal features of severe dengue. Endothelial cell activation with exocytosis of Weibel-Palade bodies (WPBs) may play an etiological role in this condition.Methods and Principal Findings
In a cohort of 73 Indonesian children with dengue hemorrhagic fever (DHF), of which 30 with dengue shock syndrome (DSS), we measured plasma levels of the WPB constituents von Willebrand factor antigen (VWF:Ag), VWF propeptide and osteoprotegerin (OPG), together with activity levels of the VWF-cleaving enzyme ADAMTS-13 and the amount of VWF in a platelet binding conformation (VWF activation factor). Compared with healthy controls (n = 17), children with DHF/DSS had significantly higher levels of VWF:Ag, VWF propeptide and OPG and decreased ADAMTS-13 activity. The VWF activation factor was also significantly higher in DHF/DSS and highest in children who died. There were significant differences in the kinetics of the various WPB constituents: VWF propeptide and OPG levels decreased toward discharge, while VWF:Ag levels were lower than expected at enrollment with plasma levels increasing toward discharge. Moreover, VWF propeptide levels correlated better with markers of disease severity (platelet count, liver enzymes, serum albumin and pleural effusion index) than corresponding VWF levels. Together, these findings suggest that there is consumption of VWF in DHF/DSS. In 4 out of 15 selected children with low ADAMTS-13 levels on admission, we found a remarkable reduction in the large and intermediate VWF multimers in the discharge blood samples, consistent with an acquired von Willebrand disease.Conclusion
These findings suggest that severe dengue is associated with exocytosis of WPBs with increased circulating levels of VWF:Ag, VWF propeptide and OPG. High circulating levels of VWF in its active conformation, together with low ADAMTS-13 activity levels, are likely to contribute to the thrombocytopenia and complications of dengue. During the convalescence phase, qualitative defects in VWF with loss of larger VWF multimers may develop. 相似文献7.
Background
Heat shock protein 22 is a member of small heat shock proteins with molecular chaperone activity. Though their multiple functions have been well characterized, there is no report about the association between the polymorphisms of HSP22 and heat tolerance.Methodology
Three single nucleotide polymorphisms were identified in HSP22 from scallop Chlamys farreri (CfHSP22), and the +94 C-A locus was found to be nonsynonymous. Three genotypes at locus +94, A/A, A/C and C/C, were revealed by using Bi-PASA PCR analysis, and their frequencies were 19.5%, 27.6% and 52.9% in the heat resistant stock, while 9.3%, 17.4% and 73.3% in the heat susceptible stock, respectively. The frequency differences of the three genotypes were significant (P<0.05) between the two stocks. After incubating at 30°C for 84 h, the cumulative mortality of scallops with +94 C/C genotype and +94 A/C genotypes was 95% and 90%, respectively, which was significantly higher (P<0.01) than that of scallops with +94 A/A genotype (70%). The molecular chaperone activity of two His-tagged fusion proteins, rCfHSP22Q with +94 C/C genotype and rCfHSP22K with +94 A/A genotype were analyzed by testing the ability of protecting citrate synthase (CS) against thermal inactivation in vitro. After incubated with rCfHSP22Q or rCfHSP22K at 38°C for 1 h, the activity of CS lost 50% and 45%, and then recovered to 89% and 95% of the original activity following 1 h restoration at 22°C, respectively, indicating that the mutation from Gln to Lys at this site might have an impact on molecular chaperone activities of CfHSP22.Conclusions
These results implied that the polymorphism at locus +94 of CfHSP22 was associated with heat tolerance of scallop, and the +94 A/A genotype could be a potential marker available in future selection of Zhikong scallop with heat tolerance. 相似文献8.
de Boer OJ Teeling P Jansen M Ploegmakers H van der Loos CM Kummer JA Florquin S van der Wal AC 《PloS one》2011,6(4):e18656
Background
Allograft vasculopathy (AV) and native atherosclerosis (NA) share the presence of a T-cell mediated inflammatory response, but differ in overall plaque morphology and growth rate. We studied the distribution and frequency of regulatory- and cytotoxic T cells in the arterial intima lesions in both conditions.Methodology/Principal Findings
The study is based on vessels of 15 explanted human renal allografts with AV and 10 carotid artery plaques obtained at surgery. Distribution and frequency of cytotoxic- and regulatory T cells, as identified by the expression of Granzyme B (GrB) and FOXP3 was established in NA and AV. Furthermore, we compared the distribution of these cells in AV with the perivascular, interstitial renal tissue using immunohistochemistry. The total number of T cells was much higher in AV than in NA lesions (711±135 and 37±8 CD3/mm2 respectively, p<0.005, mean, ± SEM). Total numbers of FOXP3+ regulatory cells were also significantly increased in AV (36±10 and 0.9±0.3 FOXP3+/mm2 p<0.05), but relative numbers, expressed as a percentage of the total number of CD3+ T cells ((FOXP3+/CD3+) ×100), were not significantly different (4.6%±0.9 and 2.7%±0.6). GrB+ cells were rare in NA, but significantly increased numbers of GrB+ cells were found in AV lesions (85±24 and 0.2±0.1 GrB+/mm2, p<0.05). Perivascular tissues in the allografts showed a higher relative frequency of FOXP3+ cells than adjacent intimal lesions (14.0%±2.7 and 4.6%±0.9, respectively, p<0.05), but a lower frequency of GrB+ cytotoxic T cells (16.1%±2.7 and 22.6%±3.6, p<0.05).Conclusions
Similar to NA, AV is characterized by a low frequency of intimal FOXP3+ regulatory T cells. Moreover, significant spatial differences exist in the distribution of functional T cell subsets between the intra- and extravascular micro-environments of the graft. 相似文献9.
Background
High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated.Objective
The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting.Methods and Results
The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca2+ handling were evaluated under physiological conditions (37°C, 2 mM Ca2+, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca2+ transient delay induced by exposure to 2 DG (time to peak Ca2+ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR.Conclusion
This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function. 相似文献10.
Objectives
Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.Methods
Murine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis.Results
In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.Conclusions
Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca2+ through a NADPH oxidase-dependent mechanism. 相似文献11.
Background
Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.Methodology/Principal Findings
Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K+ channel blockade with glibenclamide (KATP), 4-aminopyradine (KV), BaCl2 (KIR) or tetraethylammonium (TEA, KCa) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca2+ fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca2+ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca2+ responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.Conclusion
In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca2+ responses, independent of muscarinic receptor, NO or K+ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs. 相似文献12.
Roth EK Hirtz S Duerr J Wenning D Eichler I Seydewitz HH Amaral MD Mall MA 《PloS one》2011,6(8):e24445
Background
The identification of strategies to improve mutant CFTR function remains a key priority in the development of new treatments for cystic fibrosis (CF). Previous studies demonstrated that the K+ channel opener 1-ethyl-2-benzimidazolone (1-EBIO) potentiates CFTR-mediated Cl− secretion in cultured cells and mouse colon. However, the effects of 1-EBIO on wild-type and mutant CFTR function in native human colonic tissues remain unknown.Methods
We studied the effects of 1-EBIO on CFTR-mediated Cl− secretion in rectal biopsies from 47 CF patients carrying a wide spectrum of CFTR mutations and 57 age-matched controls. Rectal tissues were mounted in perfused micro-Ussing chambers and the effects of 1-EBIO were compared in control tissues, CF tissues expressing residual CFTR function and CF tissues with no detectable Cl− secretion.Results
Studies in control tissues demonstrate that 1-EBIO activated CFTR-mediated Cl− secretion in the absence of cAMP-mediated stimulation and potentiated cAMP-induced Cl− secretion by 39.2±6.7% (P<0.001) via activation of basolateral Ca2+-activated and clotrimazole-sensitive KCNN4 K+ channels. In CF specimens, 1-EBIO potentiated cAMP-induced Cl− secretion in tissues with residual CFTR function by 44.4±11.5% (P<0.001), but had no effect on tissues lacking CFTR-mediated Cl−conductance.Conclusions
We conclude that 1-EBIO potentiates Cl−secretion in native CF tissues expressing CFTR mutants with residual Cl− channel function by activation of basolateral KCNN4 K+ channels that increase the driving force for luminal Cl− exit. This mechanism may augment effects of CFTR correctors and potentiators that increase the number and/or activity of mutant CFTR channels at the cell surface and suggests KCNN4 as a therapeutic target for CF. 相似文献13.
Victor Babich Laura Knipe Lindsay Hewlett Athinoula Meli John Dempster Matthew J. Hannah Tom Carter 《The Journal of biological chemistry》2009,284(18):12459-12468
Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in
regulating inflammatory and hemostatic responses. Inflammation is associated
with the extracellular acidification of tissues and blood, conditions that can
alter the behavior of secreted proteins. The effect of extracellular pH
(pHo) on the release of von Willebrand factor (VWF), the
VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63
from WPBs was investigated using biochemical approaches and by direct optical
analysis of individual WPB fusion events in human endothelial cells expressing
green or red fluorescent fusions of these different cargo proteins. Between
pHo 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized
by the adhesion of VWF to the cell surface and the formation of long
filamentous strands. The rapid dispersal of Proregion, interleukin-8, and
eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane,
was unaltered over this pHo range. At pHo 6.8 or lower,
Proregion remained associated with VWF, in many cases WPB failed to collapse
fully and VWF failed to form filamentous strands. At pHo 6.5
dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained
unaltered compared with that at pHo 7.4; however, P-selectin
dispersal into the plasma membrane was significantly slowed. Thus,
extracellular acidification to levels of pHo 6.8 or lower
significantly alters the behavior of secreted VWF, Proregion, and P-selectin
while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3
is essentially unaltered. Together, these data suggest that WPB exocytosis
during extracellular acidosis may favor the control of inflammatory
processes.Local acidosis is associated with inflammation and ischemia and can have
significant effects on the normal function of cells, tissues, cellular, and
blood components, in particularly those associated with the immune, vascular,
and hemostatic systems
(1-8).
Endothelial cells regulate inflammatory, vascular and hemostatic responses
through the secretion of a wide range of bioactive molecules from specialized
secretory organelles, the Weibel-Palade bodies
(WPBs).3The major WPB core proteins are von Willebrand factor (VWF) and the
VWF-propolypeptide (Proregion). VWF is synthesized as a pre-proprotein
comprising an N-terminal signal peptide (pre-), and several distinct repeating
structural domains (termed A, B, C, and D) arranged as
D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK
(9). During translation, the
signal peptide is removed to yield proVWF, which then undergoes
disulfide-linked dimerization to produce proVWF dimers
(10). The Proregion domains
(D1-D2) are cleaved from the main peptide in the Golgi apparatus, and further
disulfide bond formation produces VWF multimers. The two resulting proteins,
VWF and Proregion are co-packaged into the WPB where they noncovalently
associate to form ordered tubules in a pH- and Ca2+-dependent
fashion (11). Other
physiologically important WPB proteins include P-selectin, interleukin-8
(IL-8), eotaxin-3, osteoprotegerin, and angiopoietin-2, (reviewed in Ref.
12).At physiological extracellular pH (pHo 7.4) the majority of
agonist-evoked WPB fusion events (∼75-90%) result in complete exocytosis
and release of the stored molecules
(13,
14). Secreted VWF adheres to
the cell surface and can form long filamentous strands, particularly under
flow conditions, that are essential for the efficient capture of platelets
from solution both in vitro and in vivo (e.g. Ref.
15). Imaging individual WPB
exocytotic events in live endothelial cells expressing fluorescent WPB cargo
molecules has shown that Proregion, along with IL-8, disperse quickly into
solution, while P-selectin rapidly diffuses into the plasma membrane at sites
of fusion (14,
16).Under acidic conditions, however, the behavior of VWF and Proregion, are
reported to change. At pHo 6.4 or lower Proregion remains
associated with VWF, either at the cell surface after stimulated exocytosis of
WPBs (17) or in the cell
following removal of the WPB membrane by detergent treatment
(15). In addition, the
unfurling of VWF to form long filamentous strands is attenuated
(15,
17), a situation that impairs
its capacity to efficiently support platelet adhesion
(15). Retention of Proregion
at the cell surface has led to speculation that it might play some biological
role at such sites (17).
In vitro Proregion is a ligand for the integrins α4β1
(VLA-4) (18) and
α9β1 (VLA-9) (19)
present on monocytes, leukocytes, eosinophils (VLA-4), and neutrophils
(VLA-9). Thus it is possible that at sites of local acidosis produced during
inflammation or ischemia, Proregion could play a role, along with other
secreted factors, in regulating inflammatory responses. The influence of
extracellular acidification on the release of these other secreted factors
(e.g. IL-8, eotaxin-3, and P-selectin) is not known.pHo values of 6.4 or lower represent extreme conditions of
acidosis, with decreases in pHo between 7.2 and 6.5 more typically
reported during inflammation or ischemia
(7,
20-26).
The aim of this study was to determine the influence of extracellular
acidification in this range (pHo 7.4-6.5) on the release of a
variety of soluble and membrane proteins of the WPB involved in coagulant and
inflammatory responses. Specifically, we define more precisely the value of
pHo at which VWF fails to form filamentous strands and remains
associated with Proregion at the cell surface, demonstrate the rapid
reversibility of this pH-dependent association at individual sites of WPB
fusion, and examine in detail the influence of extracellular acidification on
the release and dispersal of fluorescent fusion proteins of the soluble
mediators IL-8, eotaxin-3, and the membrane proteins P-selectin and CD63 from
individual WPBs.The data show that the external conditions into which WPB deliver their
cargo has significant effects on the dispersal and behavior of some but not
other secreted molecules. Under acidic conditions, these changes could lead to
a subtle shift from a coagulant to a more inflammatory phenotype. The data
also highlight a more general problem of interpreting biochemical data of
soluble secreted proteins in terms of underlying secretory granule
exocytosis. 相似文献
14.
Purpose
To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.Methods
Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-13C] glucose during an infusion of 6, 6-[2H2] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.Results
Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L−1∶120 min, respectively) compared with CON (90.6±4.1 mmol·L−1∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.Conclusions
Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals. 相似文献15.
Background
Oxaliplatin, a platinum-based chemotherapy utilised in the treatment of colorectal cancer, produces two forms of neurotoxicity- acute sensorimotor neuropathic symptoms and a dose-limiting chronic sensory neuropathy. Given that a Na+ channelopathy has been proposed as the mechanism underlying acute oxaliplatin-induced neuropathy, the present study aimed to determine specific mechanisms of Na+ channel dysfunction.Methodology/Principal Findings
Specifically the function of transient and persistent Na+ currents were followed during treatment and were investigated in relation to oxaliplatin dose level. Eighteen patients were assessed before and after a single oxaliplatin infusion with motor and sensory axonal excitability studies performed on the median nerve at the wrist. While refractoriness (associated with Na+ channel inactivation) was significantly altered post-oxaliplatin infusion in both motor (Pre: 31.7±6.4%; Post: 68.8±14.5%; P≤.001) and sensory axons (Pre: 31.4±5.4%; Post: 21.4±5.5%; P<.05), strength-duration time constant (marker of persistent Na+ conductances) was not significantly altered post-infusion (Motor Pre: 0.395±0.01 ms; Post: 0.394±0.02 ms; NS; Sensory Pre:0.544±0.03 ms; Post: 0.535±0.05 ms; NS). However, changes in strength-duration time constant were significantly correlated with changes in refractoriness in motor and sensory axons (Motor correlation coefficient = −.65; P<.05; Sensory correlation coefficient = .67; P<.05).Conclusions/Significance
It is concluded that the predominant effect of acute oxaliplatin exposure in human motor and sensory axons is mediated through changes in transient rather than persistent Na+ conductances. These findings are likely to have implications for the design and trial of neuroprotective strategies. 相似文献16.
17.
The immune cell composition in Barrett's metaplastic tissue resembles that in normal duodenal tissue
Background and Objective
Barrett''s esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum.Patients and Methods
Biopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry.Results
The high percentage of CD4+-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4+cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B+CD8+ cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4β7+ T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4β7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4+-T-cells were seen.Conclusion
The immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response. 相似文献18.
Duftner C Dejaco C Hengster P Bijuklic K Joannidis M Margreiter R Schirmer M 《PloS one》2012,7(3):e33939
Background
Pro-inflammatory, cytotoxic CD4+CD28− T-cells with known defects in apoptosis have been investigated as markers of premature immuno-senescence in various immune-mediated diseases. In this study we evaluated the influence of polyclonal antilymphocyte globulins (ATG-Fresenius, ATG-F) on CD4+CD28− T-cells in vivo and in vitro.Principal Findings
Surface and intracellular three colour fluorescence activated cell sorting analyses of peripheral blood mononuclear cells from 16 consecutive transplant recipients and short-term cell lines were performed. In vivo, peripheral levels of CD3+CD4+CD28− T-cells decreased from 3.7±7.1% before to 0±0% six hours after ATG-F application (P = 0.043) in 5 ATG-F treated but not in 11 control patients (2.9±2.9% vs. 3.9±3.0%). In vitro, ATG-F induced apoptosis even in CD4+CD28− T-cells, which was 4.3-times higher than in CD4+CD28+ T-cells. ATG-F evoked apoptosis was partially reversed by the broad-spectrum caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and prednisolon-21-hydrogensuccinate. ATG-F triggered CD25 expression and production of pro-inflammatory cytokines, and induced down-regulation of the type 1 chemokine receptors CXCR-3, CCR-5, CX3CR-1 and the central memory adhesion molecule CD62L predominately in CD4+CD28− T-cells.Conclusion
In summary, in vivo depletion of peripheral CD3+CD4+CD28− T-cells by ATG-F in transplant recipients was paralleled in vitro by ATG-F induced apoptosis. CD25 expression and chemokine receptor down-regulation in CD4+CD28− T-cells only partly explain the underlying mechanism. 相似文献19.
Inés Rojo PulidoReinhard Jahn Volker Gerke 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2011,1813(5):1038-1044
Weibel-Palade bodies (WPBs) are secretory organelles of endothelial cells that store the thrombogenic glycoprotein von Willebrand factor (vWF). Endothelial activation, e.g. by histamine and thrombin, triggers the Ca2+-dependent exocytosis of WPB that releases vWF into the vasculature and thereby initiates platelet capture and thrombus formation. Towards understanding the molecular mechanisms underlying this regulated WPB exocytosis, we here identify components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery associated with WPB. We show that vesicle-associated membrane protein (VAMP) 3 and VAMP8 are present on WPB and that VAMP3, but not VAMP8 forms a stable complex with syntaxin 4 and SNAP23, two plasma membrane-associated SNAREs in endothelial cells. By introducing mutant SNARE proteins into permeabilized endothelial cells we also show that soluble VAMP3 but not VAMP8 mutants comprising the cytoplasmic domain interfere with efficient vWF secretion. This indicates that endothelial cells specifically select VAMP 3 over VAMP8 to cooperate with syntaxin 4 and SNAP23 in the Ca2+-triggered fusion of WPB with the plasma membrane. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. 相似文献
20.
Liu J Kohler JE Blass AL Moncaster JA Mocofanescu A Marcus MA Blakely EA Bjornstad KA Amarasiriwardena C Casey N Goldstein LE Soybel DI 《PloS one》2011,6(6):e19638