Accumulation of inositol phosphates in low-passage human meningioma cells following treatment with epidermal growth factor

1994 ◽  
Vol 80 (5) ◽  
pp. 890-896 ◽  
Author(s):  
Tomoki Todo ◽  
Rudolf Fahlbusch
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Human Meningioma ◽  
Phosphate Accumulation ◽  
Phospholipid Hydrolysis ◽  
Inositol Phosphate Accumulation ◽  
Epidermal Growth ◽  
Inositol Phospholipid

✓ In order to elucidate some of the signal transduction processes in human meningioma cells, the authors studied the effect of epidermal growth factor (EGF) and bromocriptine on inositol phospholipid hydrolysis, using low-passage human meningioma cells in culture. Epidermal growth factor is a well-studied mitogenic factor for meningioma cells, whereas bromocriptine is known to have an inhibitory effect on meningioma cell proliferation. The addition of EGF to meningioma cells caused stimulation of inositol phosphate accumulation in a dose-dependent manner at 60 minutes posttreatment, with the maximum effect (120% to 167% of control) achieved at a concentration of 10 ng/ml. Extraction of separate inositol phosphates revealed that inositol monophosphate (IP1) and inositol bisphosphate (IP2), but not inositol trisphosphate (IP3), accounted for the increase at 60 minutes. Kinetic analysis of EGF-stimulated inositol phospholipid hydrolysis showed that a sharp and transient increase in IP3 from 5 to 12 minutes post-EGF and a transient but more gradual increase in IP2 from 2 to 12 minutes post-EGF were followed by a gradual and steady increase in IP1, which was significantly greater than control after 5 minutes. On the other hand, long-term studies showed a down-regulation of inositol phosphate accumulation (a 64% decrease vs. control) after 7 days of treatment with EGF (10 ng/ml). Bromocriptine (5 µM) exhibited no significant effect on inositol phosphate accumulation at 60 minutes in four of five meningiomas studied. However, of two meningiomas studied with bromocriptine in combination with EGF, both showed a significant additive increase in inositol phosphate accumulation compared to those treated with EGF alone. The results suggest a close involvement of inositol phospholipid turnover in human meningioma cells in response to mitogenic stimulation by EGF.

scholarly journals Epidermal-growth-factor-induced formation of inositol phosphates in human A431 cells. Differences from the effect of bradykinin

1988 ◽  
Vol 252 (3) ◽  
pp. 857-863 ◽  
Author(s):  
B C Tilly ◽  
P A van Paridon ◽  
I Verlaan ◽  
S W de Laat ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cell System ◽  
Ionophore A23187 ◽  
A431 Cells ◽  
Inositol Phosphate Metabolism ◽  
Absolute Requirement ◽  
Epidermal Growth

In human A431 epidermoid carcinoma cells, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids and raises cytoplasmic free [Ca2+]. In this paper, we investigate the action of EGF on inositol phosphate metabolism, and we compare it with the previously described effects of bradykinin on the same cell system [Tilly, van Paridon, Verlaan, Wirtz, de Laat & Moolenaar (1987) Biochem. J. 244, 129-135]. In cells prelabelled with [3H]inositol, EGF slowly but persistently (for at least 30 min) stimulates the formation of [3H]inositol phosphates, whereas bradykinin causes an immediate but transient release of inositol phosphates, which lasts for only a few minutes. The EGF effect is additive to bradykinin stimulation and does not require extracellular Ca2+. In contrast, inositol phosphate formation induced by Ca2+-ionophore A23187 has an absolute requirement for external Ca2+. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate completely abolishes the response to EGF and to sub-optimal doses of bradykinin, suggesting a negative-feedback function of protein kinase C. Pretreatment of the cells with pertussis toxin has no effect on inositol phosphate formation induced by either EGF or bradykinin. Unlike bradykinin, EGF stimulates very little accumulation of inositol 1,4,5-trisphosphate, with only a small and rather variable release of Ca2+ from intracellular stores. EGF rapidly but transiently increases inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate, but the effects are much smaller than those of bradykinin. In addition, EGF increases both inositol mono- and bis-phosphate. At 10 min after EGF addition, inositol monophosphate, unlike the other inositol phosphates, is still increasing. It is concluded that the EGF-dependent pattern of stimulation is different from that observed in bradykinin-stimulated A431 cells, suggesting that there are separate mechanisms of inositol-lipid hydrolysis involved.

The phosphatidylinositol signalling system in elongating bovine blastocysts; formation of phosphoinositides, inositol phosphates and stimulation by growth factors

2002 ◽  
Vol 14 (8) ◽  
pp. 515 ◽  
Author(s):  
A. C. Hynes ◽  
J. M. Sreenan ◽  
M. T. Kane
Keyword(s):  
Signal Transduction ◽  
Growth Factors ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Signal Transduction System ◽  
Signalling System ◽  
Sodium Dependent ◽  
Epidermal Growth

The uptake of myo-inositol and its incorporation into the phosphoinositides and inositol phosphates of the phosphatidylinositol (PtdIns) signal transduction system by in vivo elongating cattle blastocysts was investigated using [3H]myo-inositol. Uptake was examined in 13-, 14- and 16-day-old blastocysts and was largely sodium-dependent throughout (P<0.001), indicating the presence of a sodium-dependent inositol transporter. Incorporation of inositol into the three phosphoinositides, PtdIns, PtdInsP and PtdInsP2, and the inositol phosphates of the phosphatidylinositol signal transduction system was examined at Days 14 and 16; incorporation into the three phosphoinositides and into the inositol phosphate species, InsP1, InsP2, InsP3 (including the second messenger, Ins(1,4,5)P3) and InsP4 was detected in both blastocyst stages. The effects of the peptide growth factor, epidermal growth factor (EGF), and the lipid growth factors, lysophosphatidic acid (LPA) and platelet activating factor (PAF), on the activity of the phosphatidylinositol signalling system in 14- and 16-day-old blastocysts were examined. All growth factors significantly stimulated phosphatidylinositol signalling activity. Epidermal growth factor was stimulatory (P<0.001) only in 16-day-old blastocysts, whereas LPA and PAF were active in both 14- (P<0.005 for LPA and P<0.001 for PAF) and 16-day-old blastocysts (P<0.001 for LPA and PAF). These results indicate that the phosphatidylinositol signalling system is present in cattle blastocysts at the elongation stage and is responsive to stimulation by growth factors.

scholarly journals A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

2004 ◽  
Vol 9 (4) ◽  
pp. 343-353 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Sarah L. Haftl ◽  
Dimitris N. Xanthos ◽  
Gregg Crumley ◽  
Mohamed Hachicha ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Column Chromatography ◽  
Large Volume ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Assay Method ◽  
Signal To Noise ◽  
Chromatography Method ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells

1986 ◽  
Vol 236 (1) ◽  
pp. 171-175 ◽  
Author(s):  
M E Monaco ◽  
R A Mufson
Keyword(s):  
Binding Affinity ◽  
Phorbol Ester ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Tumour Cells ◽  
Mammary Tumour ◽  
Phosphate Accumulation ◽  
Phosphoinositide Cycle ◽  
Inositol Phosphate Accumulation

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.

scholarly journals Anti-insulin antiserum increases inositol phosphate accumulation in rat pancreatic islets

1990 ◽  
Vol 267 (2) ◽  
pp. 339-342 ◽  
Author(s):  
E J Verspohl ◽  
H P Ammon ◽  
M Klosa
Keyword(s):  
Pancreatic Islets ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Indirect Evidence ◽  
Maximal Effect ◽  
Biologically Relevant ◽  
Rat Pancreatic Islets ◽  
Phosphate Accumulation ◽  
Control Serum ◽  
Inositol Phosphate Accumulation

The role of insulin in modulating phosphoinositide breakdown and accumulation of inositol phosphates was investigated in isolated rat pancreatic islets by using GPAIS (guinea-pig anti-insulin antiserum) that neutralizes effects of insulin in the medium. At either 3.0 mM- or 16.7 mM-glucose or 3.0 mM-glucose plus 10 microM-arecaidine propargyl ester (muscarinic receptor agonist), GPAIS (but not control serum) was able to increase InsP2 and InsP3, but not InsP, in myo-[3H] inositol-prelabelled islets. The effect of GPAIS on 3H incorporation into InsP3 was dose-dependent, with a half-maximal effect at a concentration able to bind 4004 +/- 163 microunits of insulin. A specific mass assay of the biologically relevant isomer Ins (1,4,5)P3 revealed a huge increase (greater than 3-folf). Formation of PtdIns, PtdInsP and PtdInsP2 was not affected by GPAIS. This is indirect evidence for an effect of insulin on inositide metabolism, and therefore endogenously released insulin may have led to an underestimation in earlier studies of effects of insulinotropic substances on inositol phosphate accumulation.

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