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1.
Effects of extracellular magnesium ions ([Mg2+]o ) on intracellular free Mg2+ ([Mg2+]i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg2+ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg2+]o , [Mg2+]i in yeast cells was 0.91±0.08 mM. Elevation of [Mg2+]o to 1.97 mM induced rapid (within 5 min) increments in [Mg2+]i (2.18±0.11 mM). Lowering [Mg2+]o to 0.06 mM, however, exerted no significant effects on [Mg2+]i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg2+]o used, the subcellular distribution of [Mg2+]i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg2+] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg2+]i when placed in 1.97 mM [Mg2+]o . We conclude that [Mg2+]i in fission yeast is maintained at a physiologic level when [Mg2+]o is low, but intracellular free Mg2+ rapidly rises when [Mg2+]o is elevated. Like most eukaryotic cells, yeast may have a Mg2+ transport system(s) which functions to maintain gradients of Mg2+ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996  相似文献   

2.
The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg2+ homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other’s cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg2+-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg2+, but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect—a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg2+-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg2+-uptake is thus important knowledge.  相似文献   

3.
Changes in cytosolic Ca2+ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca2+]i) at a low level in the face of steep gradients of extracellular and sequestered Ca2+. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca2+ into the cytosol and elevation of [Ca2+]i. After such stimulation Ca2+ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca2+]i to its basal level. Such responses to elevation of [Ca2+]i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.  相似文献   

4.
Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca2+ concentration ([Ca2+]i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca2+]i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca2+]i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca2+]i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca2+]i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na+,K+ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca2+]i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002  相似文献   

5.
Summary The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.  相似文献   

6.
The alpha-kinase family: an exceptional branch on the protein kinase tree   总被引:1,自引:1,他引:0  
The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg2+ homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer.  相似文献   

7.
Summary The actions on amphibian embryos of UV-irradiation, exposure to Li+ or exposure to ouabain show interesting parallels with their effects on spontaneous release at the presynaptic terminals of the neuromuscular junction. It is suggested that these treatments serve to raise intracellular Ca2+ ([Ca2+ i) in these examples, and that UV-promoted abnormalities in embryogenesis are a consequence of changes in [Ca2+]i at critical stages in development.  相似文献   

8.
The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca2+]i ), and modulation of [Ca2+]i via treatment with IP 3R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca2+]i, followed by Ca2+-ERK and Ca2+-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008  相似文献   

9.
Calcium signaling in plants   总被引:9,自引:0,他引:9  
Changes in the cytosolic concentration of calcium ions ([Ca2+]i) play a key second messenger role in signal transduction. These changes are visualized by making use of either Ca2+-sensitive fluorescent dyes or the Ca2+-sensitive photoprotein, aequorin. Here we describe the advances made over the last 10 years or so, which have conclusively demonstrated a second messenger role for [Ca2+]i in a few model plant systems. Characteristic changes in [Ca2+]i have been seen to precede the responses of plant cells and whole plants to physiological stimuli. This has had a major impact on our understanding of cell signaling in plants. The next challenge will be to establish how the Ca2+ signals are encrypted and decoded in order to provide specificity, and we discuss the current understanding of how this may be achieved.  相似文献   

10.
Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC50 values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca2+-mobilization from intracellular Ca2+-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca2+]i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca2+]i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.  相似文献   

11.
Experiments with permeabilised platelets, and with intact platelets loaded with fluorescent Ca2+-indicators, over the past several years have greatly extended our knowledge and understanding of cytosolic Ca2+ as a platelet activator and its interactions with other cytosolic regulators. This article outlines insights, gained from the use of the fluorescent dyes, into maintenance and restoration of basal [Ca2+]i, mechanisms of receptor-mediated Ca2+-mobilisation and quantitation of [Ca2+]i/response relations in intact human platelets.  相似文献   

12.
Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133+ cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133+ stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca2+]i) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca2+]i is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133+ stem cells, supporting the assumption of a “CD20-related calcium impairment-affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133+ stem cells. Received 15 October 2008; received after revision 27 November 2008; accepted 05 December 2008  相似文献   

13.
Summary Internal longitudinal resistance (ri), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H+ and Ca2+ may modulate ri is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure ri during various interventions. In some experiments, intracellular pH (pHi) was recorded simultaneously to study the pHi-ri relation. Both intracellular Ca2+ and H+ independently modified ri. However, internal resistance of cardiac fibers was insensitive to pHi changes compared to other tissues. A latent period preceded the pHi-related changes in ri and the amount of change depended upon methodology. The results suggest that direct action of protons on ri may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H+- or Ca2+-dependent phosphorylation of junctional proteins.  相似文献   

14.
The Ca2+ ionophore ionomycin induced cytosolic [Ca2+]i elevation as well as strong activation of Cl efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl efflux was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A2 had no effect, indicating the involvement of Ca2+-dependent Cl- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A2. These results indicate that phospholipase A2 activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999  相似文献   

15.
Store-operated Ca2+ entry is a pathway that is remodelled in a variety of cancers, and altered expression of the components of store-operated Ca2+ entry is a feature of breast cancer cells of the basal molecular subtype. Studies of store-operated Ca2+ entry in breast cancer cells have used non-specific pharmacological inhibitors, complete depletion of intracellular Ca2+ stores and have mostly focused on MDA-MB-231 cells (a basal B breast cancer cell line). These studies compared the effects of the selective store-operated Ca2+ entry inhibitors Synta66 and YM58483 (also known as BTP2) on global cytosolic free Ca2+ ([Ca2+]CYT) changes induced by physiological stimuli in a different breast cancer basal cell line model, MDA-MB-468. The effects of these agents on proliferation as well as serum and epidermal growth factor (EGF) induced migration were also assessed. Activation with the purinergic receptor activator adenosine triphosphate, produced a sustained increase in [Ca2+]CYT that was entirely dependent on store-operated Ca2+ entry. The protease activated receptor 2 activator, trypsin, and EGF also produced Ca2+ influx that was sensitive to both Synta66 and YM58483. Serum-activated migration of MDA-MB-468 breast cancer cells was sensitive to both store-operated Ca2+ inhibitors. However, proliferation and EGF-activated migration was differentially affected by Synta66 and YM58483. These studies highlight the need to define the exact mechanisms of action of different store-operated calcium entry inhibitors and the impact of such differences in the control of tumour progression pathways.  相似文献   

16.
The exposure of phosphatidylserine (PS) at the cell surface plays a critical role in blood coagulation and serves as a macrophage recognition moiety for the engulfment of apoptotic cells. Previous observations have shown that a high extracellular [K+] and selective K+ channel blockers inhibit PS exposure in platelets and erythrocytes. Here we show that the rate of PS exposure in erythrocytes decreases by ~50% when the intracellular [K+] increases from 0 to physiological concentrations. Using resealed erythrocyte membranes, we further show that lipid scrambling is inducible by raising the intracellular [Ca2+] and that K+ ions have a direct inhibitory effect on this process. Lipid scrambling in resealed ghosts occurs in the absence of cell shrinkage and microvesicle formation, processes that are generally attributed to Ca2+-induced lipid scrambling in intact erythrocytes. Thus, opening of Ca2+-sensitive K+ channels causes loss of intracellular K+ that results in reduced intrinsic inhibitory effect of these ions on scramblase activity. Received 11 September 2008; received after revision 17 October 2008; accepted 27 October 2008  相似文献   

17.
The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca2+ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca2+ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca2+ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca2+ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca2+ concentration ([Ca2+]c), albeit with different kinetics and capacity. Reduction in the ER Ca2+ level following the P2Y receptor activation can further induce store-operated Ca2+ entry as a distinct Ca2+ influx pathway that contributes in ATP-induced increase in the [Ca2+]c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca2+ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.  相似文献   

18.
Summary Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump.Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity.The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.  相似文献   

19.
Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca2+]i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-0-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca2+]o-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca2+]o-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.  相似文献   

20.
Summary Both Mg2+-ATPase and HCO 3 -stimulated ATPase activity were inhibited by sodium azide and to a lesser extent ethacrynic acid and amiloride. 1 mM DNP stimulated Mg2+-ATPase activity by 22% and HCO 3 -stimulated ATPase activity by 7%.  相似文献   

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