共查询到20条相似文献,搜索用时 26 毫秒
1.
Yang Chen Ashley L. Pitzer Yang Zhang Pin‐Lan Li 《Journal of cellular and molecular medicine》2014,18(11):2165-2175
Dynein-mediated autophagosome (AP) trafficking was recently demonstrated to contribute to the formation of autophagolysosomes (APLs) and autophagic flux process in coronary arterial myocytes (CAMs). However, it remains unknown how the function of dynein as a motor protein for AP trafficking is regulated under physiological and pathological conditions. The present study tested whether the dynein-mediated autophagy maturation is regulated by a redox signalling associated with lysosomal Ca2+ release machinery. In primary cultures of CAMs, reactive oxygen species (ROS) including H2O2 and O2−. (generated by xanthine/xanthine oxidase) significantly increased dynein ATPase activity and AP movement, which were accompanied by increased lysosomal fusion with AP and APL formation. Inhibition of dynein activity by (erythro-9-(2-hydroxy-3-nonyl)adenine) (EHNA) or disruption of the dynein complex by dynamitin (DCTN2) overexpression blocked ROS-induced dynein activation, AP movement and APL formation, and resulted in an accumulation of AP along with a failed breakdown of AP. Antagonism of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ signalling with NED-19 and PPADS abolished ROS-enhanced lysosomal Ca2+ release and dynein activation in CAMs. In parallel, all these changes were also enhanced by overexpression of NADPH oxidase-1 (Nox1) gene in CAMs. Incubation with high glucose led to a marked O2−. production compared with normoglycaemic CAMs, while Nox1 inhibitor ML117 abrogated this effect. Moreover, ML117 and NED-19 and PPADS significantly suppressed dynein activity and APL formation caused by high glucose. Taken together, these data suggest that ROS function as important players to regulate dynein-dependent AP trafficking leading to efficient autophagic maturation in CAMs. 相似文献
2.
Jing Xiong Min Xia Ming Xu Yang Zhang Justine M. Abais Guangbi Li Christopher R. Riebling Joseph K. Ritter Krishna M. Boini Pin‐Lan Li 《Journal of cellular and molecular medicine》2013,17(12):1598-1607
Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38‐mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3‐II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3‐II and the content of both APs detected by Cyto‐ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin‐induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors. A colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38‐mediated regulation by PPADS completely abolished rapamycin‐induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes. 相似文献
3.
《Cell calcium》2010,47(5-6):347-355
TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine) is a membrane-permeable heavy-metal ion chelator with a dissociation constant for Ca2+ comparable to the Ca2+ concentration ([Ca2+]) within the intracellular Ca2+ stores. It has been used as modulator of intracellular heavy metals and of free intraluminal [Ca2+], without influencing the cytosolic [Ca2+] that falls in the nanomolar range. In our previous studies, we gave evidence that TPEN modifies the Ca2+ homeostasis of striated muscle independent of this buffering ability. Here we describe the direct interaction of TPEN with the ryanodine receptor (RyR) Ca2+ release channel and the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA). In lipid bilayers, at negative potentials and low [Ca2+], TPEN increased the open probability of RyR, while at positive potentials it inhibited channel activity. On permeabilized skeletal muscle fibers of the frog, but not of the rat, 50 μM TPEN increased the number of spontaneous Ca2+ sparks and induced propagating events with a velocity of 273 ± 7 μm/s. Determining the hydrolytic activity of the SR revealed that TPEN inhibits the SERCA pump, with an IC50 = 692 ± 62 μM and a Hill coefficient of 0.88 ± 0.10. These findings provide experimental evidence that TPEN directly modifies both the release of Ca2+ from and its reuptake into the SR. 相似文献
4.
Intracellular Ca2 + levels are tightly regulated in the neuronal system. The loss of Ca2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca2 + release followed by a delayed Ca2 + release. The delayed Ca2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca2 + release, decreased ATP-evoked IP3R-mediated Ca2 + release, and impaired subsequent Ca2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP3R-mediated Ca2 + release in PC-12 cells. In schizophrenia, Ca2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca2 + levels as well as dopamine signaling. 相似文献
5.
Dorothée Gilbert Marzia Lecchi Serge Arnaudeau Daniel Bertrand Nicolas Demaurex 《Cell calcium》2009,45(2):198-207
Neuronal nicotinic acetylcholine receptors (nAChRs) are Ca2+-permeable ligand-gated channels widely expressed in the central and peripheral nervous system. One of the most Ca2+ selective isoform is the homopentameric α7-nAChR implicated in schizophrenia. The activity of α7-nAChRs is usually recorded electrophysiologically, which limits the amount of information obtained. Here, we used fluorescence imaging to record Ca2+ transients associated with activation of the α7-nAChR in neuroblastoma cells stably expressing human α7-nAChRs. Application of nicotine (50 μM) consistently evoked transient (30 s), stereotyped Ca2+ responses that were inhibited by the selective α7-nAChRs antagonists methyllycaconitine (MLA) and α-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 μM). Unexpectedly, brief (1–5 s), repetitive Ca2+ transients of sub-micrometric dimension were observed in filopodia of cells expressing α7-nAChR. PNU-120596 increased the frequency and slowed the decay kinetics of these miniature Ca2+ elevations, which were insensitive to ryanodine, preserved during hyperpolarisation, and prevented by MLA, α-bungarotoxin, or Ca2+ removal. Global Ca2+ responses were also recorded in ganglion cells of embryo chicken retina during co-application of PNU-120596 and nicotine, together with whole-cell currents and brief current bursts. These data demonstrate that Ca2+ signals generated by α7-nAChRs can be recorded optically both in cell lines and in intact tissues. The possibility to image miniature Ca2+ signals enables to map the location of functional α7-nAChR channel clusters within cells and to analyze their single channel properties optically. Deciphering the rich pattern of intracellular Ca2+ signals generated by the activity of the α7-nAChRs will reveal the physiological role of these receptor-channels. 相似文献
6.
Michel Ronjat José Pablo Finkelstein Paola Llanos Luis Montecinos Hicham Bichraoui Michel De Waard Cecilia Hidalgo Ricardo Bull 《Cell calcium》2013,53(5-6):357-365
The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca2+ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca2+ uptake completion prevented this response. Maurocalcine enhanced equilibrium [3H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg2+ inhibition of [3H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca2+] from 10 μM to 0.1 μM or at cytoplasmic [Mg2+] ≥ 30 μM. At 0.1 μM [Ca2+], only channels that displayed poor activation by Ca2+ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions. 相似文献
7.
Eudoxie Dulary Shin-Yi Yu Marine Houdou Geoffroy de Bettignies Valérie Decool Sven Potelle Sandrine Duvet Marie-Ange Krzewinski-Recchi Anne Garat Gert Matthijs Yann Guerardel François Foulquier 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(3):394-402
The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca2 +/Mn2 + homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca2 + concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn2 + confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca2 +. The use of Pmr1p mutants either defective for Ca2 + or Mn2 + transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca2 + requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn2 + inside the Golgi lumen when Pmr1p exclusively transports Ca2 +. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn2 + sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn2 +/Ca2 + transport. 相似文献
8.
《Cytokine》2011,53(3):210-214
To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca2+ concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca2+]i was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1 h after RANKL-inducing, and the increased response of [Ca2+]i was completely abolished and sustained to at least 8 h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca2+]i which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors. 相似文献
9.
Maolong Dong Nan Hu Yinan Hua Xihui Xu Machender R. Kandadi Rui Guo Shasha Jiang Sreejayan Nair Dahai Hu Jun Ren 《生物化学与生物物理学报:疾病的分子基础》2013,1832(6):848-863
Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism. 相似文献
10.
《Cellular signalling》2014,26(5):968-978
Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca2+ channels (Ca1.3−/−) and maxiK Ca2+-dependent K+ channels (BK−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca2+ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca2 + channel in RPE cells, varied at different times of day. CaV1.3−/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK−/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK−/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca2 + channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding. 相似文献
11.
Activation of NAD(P)H oxidase has been reported to produce superoxide (O2??) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use an NAD(P)H oxidase product, NAD+ or NADP+, to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate, which mediates intracellular Ca2 + signaling. This study was designed to test a hypothesis that the CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2?? in mouse coronary arterial myocytes (CAMs). By fluorescence microscopic imaging, we simultaneously monitored extra- and intracellular O2?? production in wild-type (CD38+/+) and CD38 knockout (CD38?/?) CAMs in response to oxotremorine (OXO), a muscarinic type 1 receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2?? production in CAMs. Consistently, the OXO-induced intracellular O2?? production was markedly inhibited by CD38 shRNA or the CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2?? production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2?? production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2 + and O2?? production in CD38?/? CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that the CD38/cADPR pathway is an important controller of Nox4-mediated intracellular O2?? production and that CD38-dependent intracellular O2?? production is augmented in an autocrine manner by CD38-independent Nox1-derived extracellular O2?? production in CAMs. 相似文献
12.
《Cell calcium》2015,58(5-6):366-375
In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca2+]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca2+]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca2+]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca2+]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca2+]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca2+]i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca2+]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca2+]i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion. 相似文献
13.
14.
A.C. Celotto V.K. Capellini C.B.A. Restini C.F. Baldo L.M. Bendhack P.R.B. Evora 《Nitric oxide》2010,23(4):88-274
AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10?5 M) and methyl-β-cyclodextrin (10?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca2+/calmodulin and Na+/Ca2+ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca2+ concentration and activating the Ca2+/calmodulin-dependent NOS. In turn, NO is released promoting relaxation. 相似文献
15.
Mohammad Vatanparast Vahid Hosseininaveh Mohammad Ghadamyari Seyede Minoo Sajjadian 《Journal of Asia》2012,15(4):555-561
The elm leaf beetle, Xanthogaleruca luteola, is a serious pest of elm trees in urban areas. Partial biochemical characterization of pectinases and cellulases was conducted using the larval digestive system of the pest. Midgut extracts from larvae showed optimum activity for pectinase and cellulase against pectin and carboxymethyl cellulose, respectively, under acidic conditions (pH 6). Pectinases and cellulases were respectively more stable under acidic conditions (pH 4–7) and slightly acidic conditions (pH 5–7) than under highly acidic and alkaline conditions. However, the enzymes were more stable in slightly acidic conditions (pH 6) when incubation time was increased. Maximum activity for the pectinases and cellulases incubated at different temperatures was observed at 45 and 50 °C, respectively. Mg2+ remarkably increased pectinase activity, and cellulase activity increased significantly in the presence of Ca2+ and Mg2+. Sodium dodecyl sulfate significantly decreased pectinase and cellulase activity. The Michaelis–Menten constant (KM) and the maximal reaction velocity (Vmax) values for pectinase were 2 mg·mL? 1 and 0.017 mmol·min? 1·mg? 1 protein toward pectin, respectively. Zymogram analyses revealed the presence of one and five bands of pectinase and cellulase activity, respectively, in the larval midgut extract. 相似文献
16.
Na+- Ca2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca2 + pool along with the SER Ca2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na+-loaded and then placed in either a Na+-containing or in a Na+-substituted solution. Subsequently, the difference in Ca2 + entry between the two groups was examined and defined as the NCX mediated Ca2 + entry. The NCX mediated Ca2 + entry in the smooth muscle cells was monitored using two methods: Ca2 +sensitive fluorescence dye Fluo-4 and radioactive Ca2 +. Ca2 +-entry was greater in the Na+-substituted cells than in the Na+-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na+-substituted solution with or without thapsigargin. SER Ca2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca2 + entry may protect the cells against Ca2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged. 相似文献
17.
《生物化学与生物物理学报:生物膜》2014,1838(10):2547-2554
Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca2 + after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca2 + level from the nominal 2–5 μM to 2 mM for the first 60–90 min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca2 +, the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2 mM Ca2 + in the medium. With the nominal Ca2 +, most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2 mM Ca2 +, the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca2 +-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture. 相似文献
18.
《生物化学与生物物理学报:生物膜》2018,1860(4):943-952
BK channels are dually regulated by voltage and Ca2 +, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2 + concentration is sensed by a large cytoplasmic region in the channel known as “gating ring”, which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2 + coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2 +. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2 + in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2 + sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2 + coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2 + binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2 + binding sites regulating the Ca2 + dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. 相似文献
19.
Jian-An Xian Yu-Tao Miao Bin Li Hui Guo An-Li Wang 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2013,164(2):301-306
This study was aimed at investigating the toxicity mechanism of lipopolysaccharide (LPS) on Penaeus monodon haemocytes at a cellular level. Reactive oxygen species (ROS) production, nitric oxide (NO) production, non-specific esterase activity, cytoplasmic free-Ca2 + (CF-Ca2 +) concentration, DNA damaged cell ratio and apoptotic cell ratio of in vitro LPS-treated haemocytes were measured by flow cytometry. Two concentrations of Escherichia coli LPS (5 and 10 μg mL? 1) were used. Results showed that ROS production, NO production and CF-Ca2 + concentration were significantly induced in the LPS-treated haemocytes. Ratio of DNA damaged cell and apoptotic cell increased caused by LPS, while esterase activity increased at the initial 60 min and dropped later. The initial increase in esterase activity suggested that LPS activated the release of esterase, and the later decrease might result from apoptosis. These results indicated that LPS would induce oxidative stress on shrimp haemocytes, and cause Ca2 + release, DNA damage and subsequently cell apoptosis. This process of ROS/RNS-induced Ca2 +-mediated apoptosis might be one of the toxicity mechanisms of LPS on shrimp haemocytes. 相似文献
20.
Girish R. Bankar Gopalan Kutty Nampurath Pawan G. Nayak Shoumyo Bhattacharya 《Chemico-biological interactions》2010,183(2):327-331
We have previously demonstrated the vasorelaxant activity of 1,3,4-oxadiazole derivative (NOX-1) through L-type Ca2+ channel blockage. In the present study, we investigated whether the correction of endothelial dysfunction is dependent on the normalization of high blood pressure levels by 1,3,4-oxadiazole derivative (NOX-1) in deoxycorticosterone acetate (DOCA-salt) and NG-nitro-l-arginine (L-NNA) hypertensive rats. In DOCA-salt and L-NNA hypertensive rats, the mean systolic blood pressure (MSBB) was 185.3 ± 4.7 and 170.2 ± 4.1 mmHg, whereas after administration of NOX-1 to hypertensive rats, MSBB was 127.8 ± 4.5 and 120.2 ± 5.1 mmHg, respectively. To study the endothelial dysfunction, concentration–response curves of norepinephrine (NE) and acetylcholine (Ach) were constructed in rat aortic rings isolated from normotensive, hypertensive (DOCA and L-NNA) and NOX-1 treated rats. NE-induced contractions and Ach-induced relaxations were significantly (p < 0.05) decreased and increased, respectively in the aorta of NOX-1 treated rats. Vasorelaxant activity of NOX-1 was not abolished by pretreatment of aortic rings with L-NNA, 1H-[1,2,4] oxadiazolo [4,3-A] quinoxalin-1-one (ODQ), indomethacin or glibenclamide. The results suggest that the endothelial dysfunction can be corrected by the L-type Ca2+ channel blocker with endothelium-independent action and that is dependent on the normalization of high blood pressure levels. The antihypertensive and vasorelaxant effects of NOX-1 are mainly endothelial-independent and it can be used to treat hypertension, a state associated with endothelial dysfunction. 相似文献