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1.
Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca2+ homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca2+ homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca2+ homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca2+ homeostasis. Here, we developed a system in which cellular Ca2+ homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca2+ entry pathway, to generate cells with constitutive and store depletion-induced Ca2+ entry. We found striking effects of Ca2+ entry induced by overexpression of the constitutively active STIM1D76A mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca2+ entry by expression of STIM1D76A significantly reduced Aβ secretion. Our results suggest that disruptions in Ca2+ homeostasis may influence AD pathogenesis directly through the modulation of Aβ production.  相似文献   

2.
The β-defensins, expressed in epithelial cells of multiple tissues including intestine, play a critical role in the mammalian innate immunity. However, it is little known about the role of functional nutrients in the regulation of porcine β-defensins’ expressions in intestinal epithelial cells. The present study was conducted to determine the hypothesis that zinc and l-isoleucine regulate the expressions of porcine β-defensins in IPEC-J2 cells. Cells were cultured in DMEM/F12 medium containing supplemental 0–500 μg/mL l-isoleucine or 0–500 μmol/mL zinc sulfate that was used to increase the concentration of Zn2+ in the medium. At 12 h after the treatment by the appropriate concentrations of l-isoleucine or Zn2+, the mRNA and protein expressions of porcine β-defensin 1, 2 and 3 were increased (P < 0.05), and reached their maximum after treatment with 25 or 100 μmol/mL zinc sulfate and 25 or 50 μg/mL isoleucine (P < 0.05). These results suggested that both Zn2+ and l-isoleucine could induce β-defensins’ expressions in porcine intestinal epithelial cells.  相似文献   

3.
The aim of this study was to clarify the frequency of patients with type 1 diabetes that have serum that increases pancreatic β-cell cytoplasmic free Ca2+ concentration, [Ca2+]i, and if such an effect is also present in serum from first-degree relatives. We also studied a possible link between the serum effect and ethnic background as well as presence of autoantibodies. Sera obtained from three different countries were investigated as follows: 82 Swedish Caucasians with newly diagnosed type 1 diabetes, 56 Americans with different duration of type 1 diabetes, 117 American first-degree relatives of type 1 diabetic patients with a mixed ethnic background and 31 Caucasian Finnish children with newly diagnosed type 1 diabetes. Changes in [Ca2+]i , upon depolarization, were measured in β-cells incubated overnight with sera from type 1 diabetic patients, first-degree relatives or healthy controls. Our data show that there is a group constituting approximately 30% of type 1 diabetic patients of different gender, age, ethnic background and duration of the disease, as well as first-degree relatives of type 1 diabetic patients, that have sera that interfere with pancreatic β-cell Ca2+-handling. This effect on β-cell [Ca2+]i could not be correlated to the presence of autoantibodies. In a defined subgroup of patients with type 1 diabetes and first-degree relatives a defect Ca2+-handling may aggravate development of β-cell destruction.  相似文献   

4.
The existence of a Na+-dependent mechanism for Ca2+ efflux from isolated rat liver mitochondria was confirmed. The activity of this system is decreased by 60% in mitochondria isolated from perfused livers. The Na+-dependent activity is fully restored by infusion of either 1μm-adrenaline or 1μm-isoprenaline, but the α-adrenergic agonist phenylephrine is ineffective.  相似文献   

5.
High voltage-activated Ca2+ (CaV) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating CaV channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on CaV2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed CaV2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of CaV2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of CaV2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in CaV channel gating. The phospholipid–protein interaction observed here provides structural insight into mechanisms of membrane–protein association and the role of phospholipids in ion channel regulation.  相似文献   

6.
Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca2+ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca2+ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca2+ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.  相似文献   

7.
The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.  相似文献   

8.
9.
During perifusion with medium deprived of Ca2+, addition of glucose or omission of Na+ resulted in prompt and quantitatively similar inhibitions of 45Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na+ was isoosmotically replaced by sucrose or choline+. When K+ was used as a substitute for Na+, the inhibitory effect of Na+ removal on 45Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting 45Ca efflux in the presence or absence of Na+ is difficult to reconcile with the postulate that the Na+-Ca2+ countertransport mechanism is a primary site of action for glucose.  相似文献   

10.
Numerous studies have demonstrated the effects of Tβ4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which Tβ4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with Tβ4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that Tβ4 interacts with Ku80, which may operate as a novel receptor for Tβ4 and mediates its intracellular activity. In this paper, we provide evidence that Tβ4 induces cellular processes without changes in the intracellular Ca(2+) concentration. External treatment of HUVECs with Tβ4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (Tβ4(AcSDKPT/4A)) or the actin-binding sequence KLKKTET (Tβ4(KLKKTET/7A)) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by Tβ4 was not associated with the intracellular Ca(2+) elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added Tβ4 induces HUVEC migration via the surface membrane receptors known to generate Ca(2+) influx. Our data confirm the concept that externally added Tβ4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.  相似文献   

11.
Latrunculin depolymerizes and jasplakinolide polymerizes β-cell actin microfilaments. Both increase insulin secretion when Ca(2+) enters β-cells during depolarization by glucose, sulfonylureas or potassium. Mouse islets were held hyperpolarized with diazoxide, and stimulated with acetylcholine to test the role of microfilaments in insulin secretion triggered by intracellular Ca(2+) mobilization and store-operated Ca(2+) entry (SOCE). Jasplakinolide slightly attenuated Ca(2+) mobilization and did not affect SOCE, but consistently inhibited the attending insulin secretion. Latrunculin did not affect Ca(2+) changes induced by acetylcholine, but consistently increased insulin secretion, its effect being larger in response to Ca(2+) entry than to Ca(2+) mobilization. Microfilaments have thus a distinct impact on exocytosis of insulin granules depending on the source of triggering Ca(2+).  相似文献   

12.
The large-conductance Ca2+-activated K+ (BKCa) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BKCa α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BKCa α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μm and 100 μm Ca2+. The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BKCa channel activity in cells, highlighting a novel mechanism of BKCa channel regulation.  相似文献   

13.
Lan TH  Kuravi S  Lambert NA 《PloS one》2011,6(2):e17361
G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β(2)-adrenergic receptors (β(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β(2)ARs between subcellular compartments. BRET between β(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β(2)ARs and endosome markers increases. Energy transfer between β(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β(2)ARs associate transiently with each other in the plasma membrane, or that β(2)AR dimers or oligomers are actively disrupted during internalization.  相似文献   

14.
15.

Objective

Beta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.

Methods and Design

Rat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.

Results

Insulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.

Conclusions

1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.  相似文献   

16.
Abstract

We report the isolation of the genes encoding the β1 and β2 adrenergic receptors from dog genomic DNA. Sequence analysis of both genes revealed intronless open reading frames of 473 and 415 amino acid residues, receptively. Heterologous expression of both receptors in CHO cells indicated that both receptors are functionally similar to the human homologs. Comparing the dog β1 and β2 adrenergic receptors, the β1 receptor appears to bind to G proteins more tightly than the β2 receptor. Heterologously expressed receptors provide a convenient system for evaluating novel receptor agonists and antagonists.  相似文献   

17.
In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca2+]i Ca2+ concentration inside proteoliposomes - [Ca2+]o Ca2+ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - Gs Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane  相似文献   

18.
淡水小球藻清除Cu2+、Cd2+、Zn2+污染能力的研究   总被引:3,自引:0,他引:3  
用接种密度(n/mL-1)为498 ×104的淡水小球藻对含Cu2+、Cd2+、Zn2+的水体分别处理,观察了藻细胞对上述3种离子的清除能力,并用金鱼存活检测了结果.结果表明(1)淡水小球藻对不同密度的Cu2+、Cd2+、Zn2+都有清除能力,Cu2+的密度在0.094~0.484 mol/L时,清除率为61%~84.5%;Cd2+的密度在0.053~0.32mol/L时,清除率为45.90%~78.20%;Zn2+的密度在0.077~0.466 mol/L时,清除率为61.80%~84.80%.(2)淡水小球藻Cu2+、Cd2+和Zn2+良好工作浓度分别为0.094~0.484 mol/L、0.053~0.32 mol/L和0.077~0.466mol/L.其中,Zn2+的清除能达到国家污染物排放的二级标准.  相似文献   

19.
The role of the Ca2+-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]0 from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca2+-calmodulin dependent protein kinase activity. 3×10–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol32P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]0. Low [Ca]0 did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca2+-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.  相似文献   

20.
Glucose induces insulin release from pancreatic β-cells by stimulating ATP synthesis, membrane depolarisation and Ca(2+) influx. As well as activating ATP-consuming processes, cytosolic Ca(2+) increases may also potentiate mitochondrial ATP synthesis. Until recently, the ability to study the role of mitochondrial Ca(2+) transport in glucose-stimulated insulin secretion has been hindered by the absence of suitable approaches either to suppress Ca(2+) uptake into these organelles, or to examine the impact on β-cell excitability. Here, we have combined patch-clamp electrophysiology with simultaneous real-time imaging of compartmentalised changes in Ca(2+) and ATP/ADP ratio in single primary mouse β-cells, using recombinant targeted (Pericam or Perceval, respectively) as well as entrapped intracellular (Fura-Red), probes. Through shRNA-mediated silencing we show that the recently-identified mitochondrial Ca(2+) uniporter, MCU, is required for depolarisation-induced mitochondrial Ca(2+) increases, and for a sustained increase in cytosolic ATP/ADP ratio. By contrast, silencing of the mitochondrial Na(+)-Ca(2+) exchanger NCLX affected the kinetics of glucose-induced changes in, but not steady state values of, cytosolic ATP/ADP. Exposure to gluco-lipotoxic conditions delayed both mitochondrial Ca(2+) uptake and cytosolic ATP/ADP ratio increases without affecting the expression of either gene. Mitochondrial Ca(2+) accumulation, mediated by MCU and modulated by NCLX, is thus required for normal glucose sensing by pancreatic β-cells, and becomes defective in conditions mimicking the diabetic milieu.  相似文献   

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