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Jammes F  Hu HC  Villiers F  Bouten R  Kwak JM 《The FEBS journal》2011,278(22):4262-4276
Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.  相似文献   

3.
Phospholipase C-epsilon (PLC-epsilon) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (Galpha(12), Galpha(13), and Gbetagamma) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-epsilon as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-epsilon encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; approximately 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-beta isozymes, the purified fragment of PLC-epsilon maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of approximately 10 mumol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-epsilon fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique approximately 65 amino acid-insert within the catalytic core of PLC-epsilon. Although Rac1 activated purified PLC-beta2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-epsilon. These results indicate that PLC-epsilon is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC-epsilon depends on a unique insert within the catalytic core of the phospholipase.  相似文献   

4.
The effects of galanin (7-70 nM) on ATP-sensitive K+ channels (KATP channels), membrane potential and the release of insulin have been studied in the insulinoma cell line, RINm5F. Single-channel currents have been recorded from excised outside-out membrane patches as well as intact insulin-secreting cells and it is shown that galanin, added to the outside of the membrane, specifically activates KATP channels. Studies carried out using the fluorescent probe bisoxonol demonstrate that galanin hyperpolarizes RINm5F cells. Galanin was also found to abolish glyceraldehyde-stimulated immunoreactive insulin release from the insulinoma cells. Both the galanin-evoked hyperpolarization and inhibition of insulin release were abolished in cells pre-exposed to pertussis toxin. The possibility that the gating of KATP channels could be mediated by a G-protein was studied in patch-clamp experiments by adding F- to the solution bathing the inside of the cell membranes (open-cell), in order to generate the alumino-fluoride complex AlF4-. F- (1-10 mM) evoked dose-dependent activation of KATP channels and this effect was fully reversible. F- was also able to activate K+ channels inhibited by ATP. That the fluoride activation of KATP channels is mediated by the complex AlF4- was indicated by experiments in which AlCl3 (10 microM) was found to enhance further the activation of K+ channels evoked by 1 mM F- and by results showing that F(-)-stimulation of KATP channels was (i) abolished in the continued presence of F- by the Al3+ chelator deferoxamine (0.5 mM) and (ii) could be mimicked by VO4(3-) which has a structure similar to that of the AlF4- complex.  相似文献   

5.
Compelling evidence supports contributions of glutamate receptor overactivation ('excitotoxicity') to neurodegeneration in both acute conditions, such as stroke, and chronic neurodegenerative conditions, such as amyotrophic lateral sclerosis. However, anti-excitotoxic therapeutic trials, which have generally targeted highly Ca2+ permeable NMDA-type glutamate channels, have to date failed to demonstrate impressive efficacy. Whereas most AMPA type glutamate channels are Ca2+ impermeable, an evolving body of evidence supports the contention that relatively unusual Ca2+ permeable AMPA channels might be crucial contributors to injury in these conditions. These channels are preferentially expressed in discrete neuronal subpopulations, and their numbers appear to be upregulated in amyotrophic lateral sclerosis and stroke. In addition, unlike NMDA channels, Ca2+ permeable AMPA channels are not blocked by Mg2+, but are highly permeable to another potentially harmful endogenous cation, Zn2+. The targeting of these channels might provide efficacious new avenues in the therapy of certain neurological diseases.  相似文献   

6.
Calcium-permeable ion channels in cerebellar neurons from mdx mice.   总被引:2,自引:0,他引:2  
Recordings of single-channel activity were made from cell-attached patches on cerebellar granule cells from normal and mdx mice. Recordings from mdx granule cells show the activity of ion channels that are open for seconds at negative holding potentials near rest. These channels are permeable to divalent cations and have a conductance of 8-10 pS with either Ca2+ or Ba2+ as the charge carrier in the patch electrode. Under similar recording conditions, channel activity is virtually absent from normal mouse granule cells. The absence of dystrophin in neurons, as well as in skeletal muscle, is associated with an increase in the activity of Ca(2+)- permeable ion channels. Increased channel activity may be an early event leading to pathophysiological accumulation of intracellular Ca2+ in Duchenne muscular dystrophy.  相似文献   

7.
We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATP gamma S stimulated the GTP-dependent formation of inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP greater than ATP gamma S much greater than TTP greater than CTP much greater than beta, gamma-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P2-purinergic receptors expressed by human phagocytic leukocytes from the P2 gamma-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.  相似文献   

8.
The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.  相似文献   

9.
Cisplatin is a widely used platinum-based anticancer drug in the chemotherapy of numerous human cancers. However, cancer cells acquire resistance to cisplatin. So far, functional loss of volume-sensitive outwardly rectifying (VSOR) Cl channels has been reported to contribute to cisplatin resistance of cancer cells. Here, we analyzed protein expression patterns of human epidermoid carcinoma KB cells and its cisplatin-resistant KCP-4 cells. Intriguingly, KB cells exhibited higher β-actin expression and clearer actin filaments than KCP-4 cells. The β-actin knockdown in KB cells decreased VSOR Cl currents and inhibited the regulatory volume decrease (RVD) process after cell swelling. Consistently, KB cells treated with cytochalasin D, which depolymerizes actin filaments, showed smaller VSOR Cl currents and slower RVD. Cytochalasin D also inhibited cisplatin-triggered apoptosis in KB cells. These results suggest that the disruption of actin filaments cause the dysfunction of VSOR Cl channels, which elicits resistance to cisplatin in human epidermoid carcinoma cells.  相似文献   

10.
The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.  相似文献   

11.
Treatment of intact human umbilical vein endothelial cells with NaF results in a dose-dependent biphasic response in both prostacyclin and inositol phosphate production: the stimulation observed with 10-20 mM NaF decreases with higher concentrations. High concentrations of NaF furthermore reduce thrombin- or A23187-stimulated prostacyclin production. Direct assay of phospholipase C activity in cell homogenates shows a similar biphasic response to NaF, also after chelation of Ca2+; addition of AlCl3 shifts the inhibition toward lower NaF concentrations. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) also causes a dose-dependent biphasic response in inositol phosphate formation in permeabilized cells and homogenates; a higher inhibitory concentration of GTP gamma S abolishes the stimulation of inositol phosphate production by low NaF concentrations. A high concentration of NaF furthermore inhibits the non-G-protein-dependent activation of phospholipase C by deoxycholate. NaF also induces a dose-dependent biphasic response in cyclic AMP formation in intact cells, indicating that the inhibition of phospholipase C at higher NaF concentrations does not result from a rise in cyclic AMP. The data are compatible with the existence of a guanine nucleotide-dependent, cyclic AMP-independent, phospholipase C-inhibitory pathway in endothelial cells.  相似文献   

12.
Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h51Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.  相似文献   

13.
A study was made of the Rb+ transport via activated sodium channels of clone N 18 phi 1 neuroblastoma cells cultured in the Eagle medium with 10% bovine serum. The time of population doubling was about 10 h. The cell differentiation was induced by adding bromdeoxyuridine in a concentration of 1-4 10(-5) M. The cells contained 172 +/- 12 and 340 +/- 35 micrograms of protein per 10(6) cells at the logarithmic growth phase and in differentiated state, respectively. It is shown that veratrin produced a 1.3-fold increase in the rate of 86Rb+ removal from undifferentiated cells and 2.5-fold increase in that from differentiated cells. Tetrodotoxin removed completely the effect of veratrin. A conclusion is made on the presence of a new clone of fast sodium channels in cell membranes.  相似文献   

14.
Summary Patch-clamp methods were used to search for and characterize channels that mediate calcium influx through the plasma membrane of human carcinoma A431 cells. Here we present four Ca2+-permeable channel types referred to as SG, G, I and BI. With 105mm Ca2+ as the charge carrier, at 30–33°C their mean unitary conductances (in pS) are: 1.3 (SG), 2.4 (G), 3.7 (I) and 12.8 (BI). SG and G channels are activated by nonhydrolyzable analogues of guanosine 5-triphosphate (GTP) applied to the inside of the membrane, suggesting an involvement of G-proteins in the control of their activity. I and BI channels are activated by inositol 1,4,5-trisphosphate (InsP3). G, I, BI and possibly SG channels are activated from the extracellular side of the membrane by epidermal growth factor (EGF) and histamine. It is assumed that all identified Ca2+ channels take part in the generation of the agonist-induced intracellular Ca2+ signal. The variety of Ca-channel types seems to be necessary to tune cell responses according to the respective type and level of an external signal, on the one hand, and to the functional state of the cell, on the other.  相似文献   

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Zhang WH  Ryan PR  Tyerman SD 《Plant physiology》2001,125(3):1459-1472
Aluminum (Al(3+))-dependent efflux of malate from root apices is a mechanism for Al(3+) tolerance in wheat (Triticum aestivum). The malate anions protect the sensitive root tips by chelating the toxic Al(3+) cations in the rhizosphere to form non-toxic complexes. Activation of malate-permeable channels in the plasma membrane could be critical in regulating this malate efflux. We examined this by investigating Al(3+)-activated channels in protoplasts from root apices of near-isogenic wheat differing in Al(3+) tolerance at a single locus. Using whole-cell patch clamp we found that Al(3+) stimulated an electrical current carried by anion efflux across the plasma membrane in the Al(3+)-tolerant (ET8) and Al(3+)-sensitive (ES8) genotypes. This current occurred more frequently, had a greater current density, and remained active for longer in ET8 protoplasts than for ES8 protoplasts. The Al(3+)-activated current exhibited higher permeability to malate(2-) than to Cl(-) (P(mal)/P(Cl) > or = 2.6) and was inhibited by anion channel antagonists, niflumate and diphenylamine-2-carboxylic acid. In ET8, but not ES8, protoplasts an outward-rectifying K(+) current was activated in the presence of Al(3+) when cAMP was included in the pipette solution. These findings provide evidence that the difference in Al(3+)-induced malate efflux between Al(3+)-tolerant and Al(3+)-sensitive genotypes lies in the differing capacity for Al(3+) to activate malate permeable channels and cation channels for sustained malate release.  相似文献   

17.
Summary Cell walls of Saccharomyces cerevisiae and S. uvarum were activated by periodate oxidation of vicinal diol groups in cell wall polysaccharides. The aldehyde groups thus generated allow the yeast cells to be covalently bound to modified bead cellulose or macroporous glycidyl methacrylate supports, or to enzymes such as glucose oxidase and catalase.  相似文献   

18.
A controversy exists for many years about the role of sex hormone binding globulin (SHBG) in the uptake of estradiol by the cells. Using the estradiol-sensitive human breast carcinoma cell line MCF-7 and SHBG isolated from human serum by a new method, we observed a strong inhibition of estradiol uptake. The inhibition was higher when the concentration of the hormone was low. On the other hand, there seemed to be a lag period in inhibition when the concentrations of SHBG were very low, followed by an exponential increase, when the concentration exceeded a critical value. The inhibitory activity was higher when SHBG was added before or along with estradiol in the cell culture, as well as when the incubation period was elongated, while was dramatically minimized by the presence of dihydrotestosterone. Despite the inhibition of estradiol uptake caused by SHBG, the distribution of the hormone in various cell components remained practically the same. In conclusion, all indications from experimental data seem to suggest a simple deprivative mechanism being responsible for the inhibitory activity of SHBG on estradiol uptake by MCF-7 cells in culture.  相似文献   

19.
Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.  相似文献   

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