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1.
Abstract: The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.  相似文献   

2.
Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 μM) for 1–60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway. J. Cell. Physiol. 172:373–381, 1997. © 1997 Wiley-Liss, Inc.  相似文献   

3.
Abstract: We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast, to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.  相似文献   

4.
Inhibitors of signaling enzymes such as guanosine-5-O-2-thiodiphosphate, aristolochic acid, aspirin, indomethacin, and trifluoperazine block H2O2-induced platelet aggregation and H2O2-induced increase in the intracellular concentration of Ca2+. These findings suggest that the effect of H2O2 on platelets is associated with activation of signal pathways responsible for increase in the concentration of intracellular Ca2+. On H2O2-induced platelet aggregation, the concentration of cAMP in the cytoplasm decreases and that of cGMP increases.  相似文献   

5.
Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca2+-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30 °C to 37 °C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca2+ ([Ca2+]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe2+-accumulation following pretreatment with FeSO4. Thus intracellular Fe2+-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe2+-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe2+-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37 °C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe2+-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe2+-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2 channels by Fe2+ may implicated in hemorrhagic brain injury via aggravation of inflammation, since Fe2+ is released by heme degradation under intracerebral hemorrhage.  相似文献   

6.
To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H2O2-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H2O2 at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H2O2-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H2O2-treated cells in Western blot analysis. An H2O2-induced increase of the intracellular Ca2+ concentration ([Ca2+]i) was also observed and an intracellular Ca2+ chelator (BAPTA-AM) significantly inhibited the H2O2-induced increase of permeability. However, it showed no inhibitory effects on the H2O2-induced phosphorylation of p38 MAPK and ERK. The H2O2-induced increase of [Ca2+]i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca2+]i are essential for the H2O2-induced increase of endothelial permeability and that ERK is not.  相似文献   

7.
Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H2O2) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H2O2. In this study, the involvement of calcium (Ca2+) and calmodulin (CaM) in NO- and H2O2-induced adventitious rooting in marigold was investigated. Exogenous Ca2+ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl2. Ca2+ chelators and CaM antagonists prevented NO- and H2O2-induced adventitious rooting, indicating that both endogenous Ca2+ and CaM may play crucial roles in the adventitious rooting induced by NO and H2O2. NO and H2O2 treatments increased the endogenous content of Ca2+ and CaM, suggesting that NO and H2O2 enhanced adventitious rooting by stimulating the endogenous Ca2+ and CaM levels. Moreover, treatment with Ca2+ enhanced the endogenous levels of NO and H2O2. Additionally, Ca2+ might be involved as an upstream signaling molecule for CaM during NO- and H2O2-induced rooting. Altogether, the results suggest that both Ca2+ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H2O2.  相似文献   

8.
Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP2) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP2, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP2 for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP2. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP2. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP2 in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP2, inhibited the PIP2-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP2, owing to its specific interaction with PIP2, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP2 effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP2 serves as an anchor to localize PLD on plasma membranes in vivo.  相似文献   

9.
Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca2+-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca2+-triggered fusion were unknown. Ca2+-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca2+-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca2+-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca2+ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca2+-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.  相似文献   

10.
Pulsatile insulin release from glucose-stimulated β-cells is driven by oscillations of the Ca2+ and cAMP concentrations in the subplasma membrane space ([Ca2+]pm and [cAMP]pm). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP]pm, [Ca2+]pm, and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 β-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca2+]pm, [cAMP]pm, and PIP3. Inhibitors of protein kinase A (PKA) markedly diminished the PIP3 response when applied before glucose stimulation, but did not affect already manifested PIP3 oscillations. The reduced PIP3 response could be attributed to accelerated depolarization causing early rise of [Ca2+]pm that preceded the elevation of [cAMP]pm. However, the amplitude of the PIP3 response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP3 oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP3 oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca2+]pm and amplifying [cAMP]pm signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.  相似文献   

11.
Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H2O2 production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H2O2 generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H2O2 production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H2O2 production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.  相似文献   

12.
Oscillations in cytoplasmic Ca2+ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca2+ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca2+-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca2+ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li+, an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li+-treated cells, cytoplasmic Ca2+ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca2+ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca2+ oscillations, and these could not be rescued by inositol or PI4P. In Li+-treated cells, recovery of the cytoplasmic Ca2+ oscillations in the presence of inositol or PI4P was suppressed when Ca2+ influx through store-operated Ca2+ channels was inhibited. After rundown of the Ca2+ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca2+ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca2+ entry is needed to sustain cytoplasmic Ca2+ signaling following leukotriene receptor activation both by refilling the Ca2+ stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.  相似文献   

13.
The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol‐4,5‐bisphosphate (PIP2) antagonist, strongly repressed both physiological cytokinin effect and cytokinin‐dependent PLD activation. N‐acylethanolamine (NAE 12), an inhibitor of α‐class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP2‐dependent non‐class α‐PLD in the molecular mechanism of cytokinin action.  相似文献   

14.
The phospholipid requirement for Ca2+-stimulated, Mg2+-dependent ATP hydrolysis (Ca2+/Mg2+-ATPase) and Mg2+-stimulated ATP hydrolysis (Mg2+-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A2 (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A2 caused an increase in the activities of both Ca2+/Mg2+-ATPase and Mg2+-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca2+/Mg2+-ATPase but Mg2+-ATPase activity was inhibited. Inhibition of Mg2+-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP2) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP2 brought about the reactivation of Ca2+/Mg2+-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg2+-ATPase activity.K ms for Ca2+ (0.47 M) and Mg2+ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.  相似文献   

15.
The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca2+ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca2+ ionophore, appeared to parallel the decrease in Ca2+ uptake activity. Phospholipase A2 activity, monitored by the production of [3H]lysophosphatidylcholine and [3H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca2+ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca2+ content.  相似文献   

16.
Abstract: The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65–70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca2+-free buffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca2+-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.  相似文献   

17.
The effect of GTP on the hydrolysis of [3H]phosphatidyinositol (PI), [3H]phosphatidylinositol-4-phosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP2 was actively degradted, opposite to PI and PIP which require Ca2+ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP2 exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca2+ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP2. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP3 through the activation of phospholipase C acting on PIP2 exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP2 due to stimulation of PIP kinase.  相似文献   

18.
Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane‐cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca2+‐independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia‐induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca2+cyt.], hydrogen peroxide (H2O2) and PA. The concentrations of cytosolic Ca2+ and H2O2 were determined by fluorescence microscopy and the fluorescent dyes Fura 2‐AM and CM‐H2DCFDA, specific for calcium and H2O2, respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca2+ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.  相似文献   

19.
Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H2O2 and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H2O2 levels as the wild type in response to ABA. However, ABA- or H2O2-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H2O2 in ABA-induced stomatal closure.  相似文献   

20.
Large-conductance calcium and voltage-dependent potassium (BKCa) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H2O2) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTENwt, PTENC124S, PTENG129E in HEK 293 cells. Typical BKCa channel currents could be recorded in cell-attached configurations. We found that PTENwt reduced the H2O2-induced BKCa channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTENC124S/PTENG129E mutants that lack lipid phosphatase activity produced no regulation on the H2O2-induced BKCa channel activation. These results demonstrated that PTEN regulated the H2O2-induced BKCa channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H2O2-induced BKCa channel activation was attenuated when cells were treated with H2O2 at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTENwt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca2+]i) induced by H2O2 was also inhibited. These findings may elucidate a new mechanism for H2O2-induced BKCa channel activation and provide some evidences for the role of PTEN on vasodilation induced by H2O2.  相似文献   

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