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Division of Cell Signalling, University of Dundee, Dundee, Scotland, United Kingdom
Received May 13, 2004; accepted September 1, 2004
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Abstract |
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), chromatin remodelling (Shen et al., 2003; Steger et al., 2003), DNA repair (Hanakahi et al., 2000; Byrum et al., 2004), membrane trafficking (Ye et al., 1995; Saiardi et al., 2002), and control of cell proliferation (Orchiston et al., 2004).
The least studied members of all inositol phosphates are the diphosphoinositol polyphosphates (DIPs). These are phosphorylation products of the most abundant inositol phosphates found in cells, inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6) (Safrany et al., 1999). Until recently, InsP6 was believed to be the endpoint in the inositol phosphate kinase cascade. Three human InsP6 kinases has been identified, producing diphosphoinositol pentakisphosphate (PP-InsP5) (Saiardi et al., 1999; Schell et al., 1999), and a Saccharomyces cerevisiae homolog (KCS1) has also been characterized (Saiardi et al., 2000). InsP6 kinases can also phosphorylate InsP5, producing diphosphoinositol tetrakisphosphate. PP-InsP5 can be further phosphorylated by a kinase that has yet to be cloned (Huang et al., 1998). The structure of the product, bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4), remains to be determined in mammalian systems. Despite their low levels, these DIPs are the most rapidly turned over inositol phosphates in unstimulated cells.
It has been shown previously that PP-InsP5 can be regulated by the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase pump inhibitor thapsigargin (Glennon and Shears, 1993). The phosphatases involved in hydrolysis of [PP]2-InsP4 and PP-InsP5, termed diphosphoinositol polyphosphate phosphohydrolases, are sensitive to fluoride (Shears et al., 1995; Safrany et al., 1998). As such, it was not a surprise to observe that incubating cells with low levels (below millimolar) of fluoride increased levels of PP-InsP5 and [PP]2-InsP4. Higher levels caused a paradoxical decrease in [PP]2-InsP4 levels that was found to be mediated by cyclic nucleotide monophosphates (cNMPs) via an unidentified mechanism (Safrany and Shears, 1998).
The function of these DIPs remains a target for research. It is clear that they play a role in protein trafficking (Saiardi et al., 2000). In vitro experiments show that PP-InsP5 and [PP]2-InsP4 can inhibit formation of clathrin triskelia (Ye et al., 1995). Yeast strains in which the KCS1 gene has been removed show clear trafficking defects (Saiardi et al., 2002). The major interest is determining which of the DIPs plays a role in vivo.
In this article, I present, for the first time, new experimental protocols that alter levels of DIPs. Together with previously published data, these treatments will help determine the roles of the DIPs. To this end, I have determined that hyperosmotic stress, caused by treating the Syrian hamster vas deferens smooth muscle cell line DDT1 MF-2 with sorbitol or sucrose, a treatment that is known to inhibit protein trafficking, also raises [PP]2-InsP4 levels. These results identify a possible mechanism through which hyperosmotic stress acts and reveals that [PP]2-InsP4 is a regulator of protein trafficking in vivo. These data clearly show that these inositol phosphates are rapidly turned over and that their pools can be selectively regulated. The protocols developed can be used to further determine the roles played by these inositol phosphates in physiology.
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Materials and Methods |
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200,000 cells/well (16-mm diameter, 24-well multiplates) in the DMEM-based culture medium described above, supplemented with 50 µCi/ml [3H]inositol. On the fourth day, cell monolayers were washed (2 x 250 µl) and then incubated (250 µl) in [3H]inositol-free, HEPES-buffered Krebs-like media (115 mM NaCl, 5 mM KCl, 1 mM NaH2PO4, 0.5 mM MgSO4, 11 mM glucose, 1.36 mM CaCl2, 25 mM HEPES, pH 7.4 with NaOH). Cells were maintained at 37°C for 3 h before the beginning of any experiments. Otherwise, overnight treatments were performed in DMEM containing [3H]inositol. All experimental protocols were time-matched with control incubations. Experiments were quenched by rapid aspiration of the Krebs-like (or DMEM) media, followed by addition of 250 µl of ice-cold 0.6 M perchloric acid and neutralized by the addition of 70 µl of 1 M K2CO3 containing 5 mM Na2EDTA. After being kept at 4°C for 30 min, the perchlorate precipitate was removed by centrifugation (10,000g, 2 min). The supernatants were finally diluted with 3 volumes of 1 mM Na2EDTA. Samples were stored at -20°C before being loaded onto a 4.6 x 125-mm Partisphere 5 µm SAX HPLC column. Inositol phosphates were eluted at 1 ml/min by the following gradient generated by mixing buffer A (1 mM Na2EDTA) and buffer B [buffer A plus 1.3 M (NH4)2HPO4, pH 3.85 with H3PO4; total [Pi] = 2.6 M] as follows: 0 to 5 min, 0% B; 5 to 10 min, 0 to 50% B; 10 to 60 min, 50 to 100% B; 60 to 70 min, 100% B. Fractions were collected at 1-min intervals, mixed with 4.2 volumes of Flo-Scint IV scintillant, and radioactivity was determined using liquid scintillation spectrometry. EC50 values were derived using Prism (GraphPad, San Diego, CA). Inositol lipids were extracted from the perchloric acid pellet, prepared, and analyzed as described previously (Batty and Downes, 1994). Peaks were ascribed by coelution of standards in parallel runs. ATP levels were determined using a luciferase-based assay kit (Merck Biosciences, Nottingham, UK), following the manufacturer's recommended protocol. Samples were either prepared as described above (omitting the addition of [3H]inositol) or, for the determination of extracellular ATP, snap-frozen and assayed directly. Trypan blue exclusion was determined by incubating cells for 20 to 30 min in the presence of trypan blue (Sigma-Aldrich, Gillingham, UK) and determining a ratio of trypan blue-excluding (alive) cells to blue (dead) cells.
Materials. [myo-3H]inositol (10-25 Ci/mmol; 10 mCi/ml; in sterile water), was provided by Amersham Biosciences UK Ltd. (Chalfont, Bucks). Tissue culture reagents were purchased from Invitrogen Ltd., (Paisley, UK). Thapsigargin was purchased from Alexis Corporation UK (Nottingham, UK), and ZD7288 was purchased from Tocris Cookson Ltd. (Bristol, UK). All other reagents used in this study were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK) or Merck Biosciences Ltd. (Nottingham, UK).
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Results |
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). Although [PP]2-[3H]InsP4 was present at low levels, they were of sufficient magnitude to exceed those of all the InsP4 isomers combined. No more polar 3H-labeled material was detected in these or any further experiments described in this study, even when [Pi] in the HPLC eluate was increased to 2.6 M. It is noted that levels of [PP]2-InsP4 accumulated during incubation of the cells in Krebs-like media, reaching a plateau after 2 to 3 h. The reasons for this have yet to be determined. In an attempt to understand the roles played by DIPs, I have tested a number of reagents that regulate key cellular functions. The finding, some 5 years ago, that cNMPs lowered [PP]2-InsP4 still awaits explanation. It is still unclear why cyclic AMP and cyclic GMP lower [PP]2-InsP4, and what the consequences of this regulation are.
There is some evidence that DIPs are involved in protein trafficking. Much of this information has been obtained using in vitro binding assays, and some preliminary information is now available on protein trafficking in yeast lacking the IP6 kinase, KCS1. No studies have yet been published on whether DIPs are involved in protein trafficking in mammalian cells. A protocol in which hyperosmotic stress, afforded by treating cells with sorbitol or sucrose, is known to block such trafficking was used to determine whether such treatment altered levels of DIPs. Treatment of DDT1 MF-2 cells with either reagent caused a rapid and dose-dependent decrease in PP-InsP5, accompanied by an increase in [PP]2-InsP4 levels. The effects of sucrose were short-lived, but sorbitol gave a robust and prolonged increase in [PP]2-InsP4 (Fig. 1). This is consistent with in vitro binding assays showing that [PP]2-InsP4 was able to inhibit clathrin triskelion formation and block protein trafficking. The ability of sorbitol to raise [PP]2-InsP4 was not blocked by U0126 (10 µM), or by butan-1-ol (2%), excluding a role for extracellular signal-regulated kinase and phospholipase D.
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I have also identified that genistein, a broad-range protein tyrosine kinase inhibitor (Akiyama et al., 1987), is a potent regulator of [PP]2-InsP4 levels. Treatment of DDT1 MF-2 cells with 100 µM genistein caused a rapid reduction in the levels of [PP]2-InsP4 without affecting the levels of other HIPs. The decline in [PP]2-InsP4 levels was both time- (Fig. 2) and dose- (Fig. 3) dependent. Genistein lowered [PP]2-InsP4 levels in a dose-dependent manner, with an EC50 of 28 µM. This is in close agreement with the published IC50 for pp60v-src of 26 µM (Akiyama et al., 1987). These effects were not mimicked by a similar protein kinase inhibitor, herbimycin A (10 µM, data not shown), therefore excluding the role of a pp60v-src -like kinase in regulating PP-InsP5 phosphorylation. Protein tyrosine phosphatase inhibitors dephostatin (100 µM) and mpV(pic) (100 µM), like the broad inhibitor okadaic acid (1 µM), were without effect. mpV(pic) did, however, greatly raise levels of InsP4, by more than 15-fold over control levels (data not shown). This is the first observation of mpV(pic) raising inositol phosphate levels, presumably by activating phospholipase C. The mechanism by which genistein lowered [PP]2-InsP4 is unclear. The roles of MEK, LCK, p70S6 kinase, phosphoinositide 3-kinase, and smooth muscle myosin light chain kinase were excluded by the lack of effect of SB203580 (10 µM), PD98059 (1 mM), rapamycin (100 nM), and wortmannin (100 nM) (data not shown).
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That InsP3 3-kinases are regulated by calmodulin-dependent kinase (Dewaste et al., 2000), suggested that the gateway to all the HIPs could be regulated by calmodulin kinase inhibitors. Low levels of W-7, a calmodulin inhibitor, were without effect. Higher levels (>10 µM), however, caused a marked and rapid decline of HIPs and DIPs, InsP5 to [PP]2-InsP4, although the ratios between each of these remained constant, identifying the presence of a specific block at the level of InsP5 production (Fig. 4). Similar data were obtained with chlorpromazine (Byrum et al., 2004), whereas KN-93 (500 µM) and K-252a (50 µM) calmodulin kinase inhibitors had no effect. Because it has been proposed that W-7 is cytotoxic (Jan et al., 2000), its effects were more systematically assessed. Cells treated with W-7 or chlorpromazine (up to 1 mM) for 1 h excluded trypan blue (>97% exclusion), even after a 24-h recovery period in DMEM. At this highest dose, a morphological change occurred. Cells lost their usual stellate shapes and became rounded. This is consistent with a significant decrease in cellular phosphatidylinositol 4,5-bisphosphate levels, which is accompanied by an increase in phosphatidylinositol 4-phosphate (Fig. 5a). ATP levels were also determined. These, like inositol phosphate levels, were seriously compromised at higher doses of both W-7 and chlorpromazine (Fig. 5b). Measurement of extracellular ATP and inositol phosphate levels indicated that much of that lost was recoverable from outside the cells. This indicates that the cell membranes, despite their impermeability to trypan blue, allowed the passage of ATP and inositol phosphates.
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One is also able to exclude any roles played by Rho-associated, coiled-coil-forming protein kinase II, protein kinase C-related protein kinase 2, mitogen- and stress-activated protein kinase 1, mitogen-activated protein kinase-activated protein kinase-1b, and p70 ribosomal protein S6 kinase, because HA1077 (3 mM) and H-89 (1 µM), which are known not to affect [PP]2-InsP4 levels (Safrany and Shears, 1998), have been subsequently found to inhibit these kinases in addition to PKA and cyclic GMP-dependent protein kinase (Davies et al., 2000; Bain et al., 2003).
Potassium depletion of cells has also been shown to inhibit protein trafficking, and so potassium regulation of DIPs has also been investigated. Amiloride (1 mM), ouabain (100 µM), Cs+ (10 mM), Ba+ (1 mM), and tetraethylammonium (20 mM) were all found to have no effect. Oxidative stress afforded by treatment of cells with hydrogen peroxide (100 µM) also had no effect. Arachidonic acid (30 µM) did not alter levels of DIPs. No effect on levels was observed after treating cells with histamine [acting on H1 receptors (Cowlen et al., 1990)], lipopolysaccharide (10 µM), ZD7288 (1 mM), cycloheximide (100 µg/ml), pertussis toxin (50 ng/ml), or dexamethasone (1 nM). Also tested without effect were 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 µM), and its control 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (10 µM), and platelet-derived growth factor (50 ng/ml).
Using these data, along with those previously published, we can now address the roles of DIPs and HIPs in a cellular context. cNMPs can lower [PP]2-InsP4 levels selectively, these effect are not mediated by PKA or cyclic GMP-dependent protein kinase, nor are they mediated by cyclic nucleotide-gated cation channels. The mechanism and consequences of this regulation await to be determined. Hyperosmotic stress, which inhibits protein trafficking, significantly lowers PP-InsP5 levels, and raises [PP]2-InsP4 levels, whereas treatment of cells with high (
10 mM) levels of fluoride causes an increase in PP-InsP5 and decrease in [PP]2-InsP4 levels. Lower levels of fluoride (<1 mM) cause a selective increase in PP-InsP5 levels.
The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase pump inhibitors thapsigargin and cyclopiazonic acid show a selective effect on PP-InsP5, lowering this inositol phosphate (Glennon and Shears, 1993), whereas W-7, a calmodulin inhibitor, lowers all HIPs and DIPs from InsP5 to [PP]2-InsP4, although this reagent had greater effects on inositol lipids and ATP. In contrast, overnight treatment of cells with thapsigargin allow for PP-InsP5 levels to rebound. Levels of PP-InsP5 were doubled by overnight treatment with thapsigargin (10 µM) (Fig. 6). Interferons 
, 
, and 
were unable to affect levels of inositol phosphates, despite interferon having been shown to up-regulate InsP6 kinase (Morrison et al., 2001).
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These effects were not limited to DDT1 MF-2 cells. Treatment of human embryonic kidney 293 and human cervix epithelioid carcinoma HeLa cells afforded similar results. This suggests that these treatments can be used in a wide variety of cell types to allow the study of DIPs.
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Discussion |
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In attempting to understand the mechanism by which cNMPs, I have tested a number of treatments implying that cNMPs act through novel pathways. cNMPs cause activation of the glucocorticoid receptor; these effects may be partly PKA-independent (Eickelberg et al., 1999). The ability of dexamethasone to alter [PP]2-InsP4 was tested. The lack of effects suggests that GR activation by cNMPs is not upstream of the effects on DIPs. A report that cNMPs can also regulate Na+ channels (Niisato et al., 1999) was also investigated. Amiloride, a Na+ transport blocker was found to be without effect, as was ouabain, a Na+/K+ ATPase inhibitor, suggesting, again, that cNMP-mediated Na+ transport is not upstream from DIPs. The ability of genistein to mimic cNMPs on lowering [PP]2-InsP4 also suggests that the effects on [PP]2-InsP4 mediated by both cNMPs and genistein are not related to Na+ channels, because genistein blocks cAMP-mediated Na+ channel flux (Niisato et al., 1999). Because cNMPs are known to activate Ih currents (Bosmith et al., 1993), the effects of ZD7288, an effective and selective blocker of Ih channels, were tested. ZD7288 had no reproducible effect alone, and it did not alter the ability of cNMPs to lower [PP]2-InsP4.
The potent effects of genistein in lowering [PP]2-InsP4 levels and subsequently PP-InsP5 levels, and raises further concerns regarding the specificity of this drug as a protein kinase inhibitor. One could suggest that the recorded effects of genistein now need reanalysis to determine whether some of its effects can be attributed to its regulation of [PP]2-InsP4. The mechanism of action of genistein and herbimycin differ greatly. Herbimycin acts via binding to protein sulfhydryl groups, and genistein competes with ATP at the active site (Simonson and Herman, 1993). These data suggest that the active site of PP-InsP5 kinase resembles a protein tyrosine kinase ATP-binding pocket.
The ability of sorbitol to raise [PP]2-InsP4 levels suggests that the effects of hyperosmotic stress on protein trafficking (Oka et al., 1989) may be mediated by these effects, in that [PP]2-InsP4 has been previously shown to inhibit clathrin triskelion formation (Ye et al., 1995). Much discussion has taken place as to whether inositol phosphates or inositol lipids are the key ligands for proteins involved in the inhibition of clathrin triskelia (see Gaidarov et al., 1996; Hao et al., 1997), yet based on this and earlier (Jones et al., 1999) studies, it is apparent that levels of 3-phosphorylated inositides are decreased and only levels of [PP]2-InsP4 are increased. In contrast, K+ depletion and cytosolic acidification, other treatments known to inhibit protein trafficking (Hansen et al., 1993), did not mimic sorbitol in its ability to raise [PP]2-InsP4. This suggests that these reagents have their effects by different mechanisms; it will be interesting to determine how these other treatments alter trafficking. It is noted that in Saccharomyces cerevisiae, a reduction in levels of DIPs leads to inhibited trafficking, and it is proposed that inositol phosphate-binding proteins could be regulated in a fashion analogous to heterotrimeric G-proteins (Saiardi et al., 2002). A recent article by Sajan et al. (2002) showed that sorbitol activated atypical protein kinase C and GLUT4 glucose transporter translocation and that these events were mediated by extracellular signal-regulated kinase and phospholipase D. That U0126 and butan-1-ol failed to inhibit sorbitol-mediated increases in [PP]2-InsP4 excludes these components from being upstream of [PP]2-InsP4. It is possible that [PP]2-InsP4 is upstream from these cellular components.
We have recently used W-7 and chlorpromazine to study the effects of depleting HIPs on Ku70 function (Byrum et al., 2004). Under conditions in which InsP5 to [PP]2-InsP4 are reduced, Ku70 mobility is perturbed. These data suggest that some of these inositol phosphates represent physiological regulators of DNA-dependent protein kinase, whereby Ku70 requires inositol phosphates to relocate to areas of DNA damage (Byrum et al., 2004). W-7 and chlorpromazine also affected levels of inositol lipids and depleted ATP. These observations mirror those made previously in human platelets (Strunecka et al., 1987; Tharmapathy et al., 2000). Phosphatidylinositol 4,5-bisphosphate is required for cytoskeletal integrity (Niebuhr et al., 2002), its depletion leading to a change in cellular morphology. Significant loss of ATP and inositol phosphates to the media would suggest that these cells are permeable to these small molecules (Holmsen and Rygh, 1990) and could explain how exogenous InsP6 could effect Ku70 mobilization in W-7-treated cells (Byrum et al., 2004). The effects observed suggest that the cationic amphiphilic nature of these compounds allows shielding of the negative charge of ATP and inositol phosphates (among other components), thus allowing them to pass through the membrane down their concentration gradients. The effects on adenine nucleotides could be mimicked by the cationic detergent cetyl-trimethylammonium bromide, whereas in contrast, the anionic detergent dioctyl sulfosuccinate was without effect (Tharmapathy et al., 2000). The complex nature of the effects of W-7 and chlorpromazine may bring into question their usefulness in further studies of HIP and DIP function. It is clear that they have a number of other effects above that of calmodulin antagonists, although the ability of extracellular inositol phosphates to rescue a response can act as a good control.
I have also recently shown that InsP5 levels can be selectively lowered by overexpressing PTEN M-CBR3. Such treatment of U87-MG cells decreased their proliferative rate (Orchiston et al., 2004). Although no manipulation that selectively regulates InsP6 levels has yet been described, the overexpression of cytosolic multiple inositol polyphosphate phosphatase, achieved by removal of the C-terminal ER recycling signal (SDEL), has been shown previously to lower levels of InsP5 by 60% and InsP6 by 40%. This treatment, too, was found to cause a decrease in the rate of cell proliferation (Chi et al., 2000). It would be expected that such decreases in the levels of these DIP precursors would also lead to a decrease in the DIPs themselves, although this was not addressed in the above articles. A combination of these two approaches in parallel may yield insight into the roles of InsP6 in mammalian cells.
These results, complemented by earlier studies, identify means by which DIPs can be regulated. The calmodulin inhibitors W-7 and chlorpromazine lower all HIPs and DIPs, PTEN M-CBR3 selectively lowers InsP5, and cytosolic multiple inositol polyphosphate phosphatase lowers InsP5 and InsP6 levels. Short-term treatment with thapsigargin selectively raises PP-InsP5 levels, whereas long-term treatment selectively lowers PP-InsP5 levels. Low levels of fluoride raise PP-InsP5 and [PP]2-InsP4; higher levels raise PP-InsP5 and lower [PP]2 to InsP4. Genistein, along with cNMPs, lower [PP]2-InsP4 levels. Sucrose and sorbitol lower PP-InsP5 and raise [PP]2-InsP4. In summary, a number of treatments can alter levels of DIPs, either en masse or selectively. These tools can now be used to regulate the DIPs and determine their roles and functions in intact cells.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: DIP, diphosphoinositol polyphosphate; InsP5, inositol 1,3,4,5,6-pentakisphosphate; InsP6, inositol hexakisphosphate; PP-InsP5, diphosphoinositol pentakisphosphate; [PP]2-InsP4, bisdiphosphoinositol tetrakisphosphate; cNMP, cyclic nucleotide monophosphate; DMEM, Dulbecco's modified Eagle's medium; HPLC, high-performance liquid chromatography; ZD7288, 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyridinium chloride; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD98059, 2'-amino-3'-methoxyflavone; W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; HIP, higher inositol phosphates; KN-93, 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; mpV(pic), monoperoxo(picolinato)oxovanadate(V); K-252a, methyl 9-(S)-12-(R)-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-2,3,9,10,11,12-hexahydro-10-(R)-hydroxy-9-methyl-1-oxo-10-carboxylate; HA1077, (5-isoquinolinesulfonyl)homopiperazine; H-89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; PKA, protein kinase A.
Address correspondence to: S. T. Safrany, Division of Cell Signaling, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH. E-mail: s.t.safrany{at}dundee.ac.uk
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) and [PP]-InsP5 (
). [3H]Inositol-labeled cells were incubated for the indicated times with 100 µM genistein. Cells were quenched, extracted, and analyzed by HPLC as described under Materials and Methods. The levels of [PP]2-InsP4 and PP-InsP5 are shown as a percentage of vehicle-treated controls. Data are representative of three experiments.
) or treated with W-7 (1 mM, 60 min) (
), quenched, extracted, and analyzed by HPLC as described under Materials and Methods. The identity of each peak was ascertained by reference to previous data using this HPLC system (Glennon and Shears, 1993
