scholarly journals Stimulation of phospholipid hydrolysis and arachidonic acid mobilization in human uterine decidua cells by phorbol ester

1987 ◽  
Vol 246 (3) ◽  
pp. 705-713 ◽  
Author(s):  
M P Schrey ◽  
A M Read ◽  
P J Steer
Keyword(s):  
Arachidonic Acid ◽  
Phorbol Ester ◽  
Inositol Phosphate ◽  
Fold Increase ◽  
Water Soluble ◽  
Phosphate Accumulation ◽  
Phospholipid Hydrolysis ◽  
Inositol Phosphate Accumulation ◽  
Cellular Water ◽  
Pi Hydrolysis

Vasopressin and oxytocin both stimulated inositol phosphate accumulation in isolated uterine decidua cells. Pretreatment of cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) prevented this agonist-induced phosphoinositide hydrolysis. TPA (0.1 microM) alone had no effect on basal inositol phosphate accumulation, but stimulated phosphoinositide deacylation, as indicated by a 2-fold increase in lysophosphatidylinositol and glycerophosphoinositol. TPA also stimulated a dose-related release of arachidonic acid from decidua-cell phospholipid [phosphatidylcholine (PC) much greater than phosphatidylinositol (PI) greater than phosphatidylethanolamine]. The phorbol ester 4 beta-phorbol 12,13-diacetate (PDA) at 0.1 microM had no effect on arachidonic acid mobilization. The TPA-stimulated increase in arachidonic acid release was apparent by 2 1/2 min (116% of control), maximal after 20 min (283% of control), and remained around this value (306% of control) after 120 min incubation. TPA also stimulated significant increases in 1,2-diacylglycerol and monoacylglycerol production at 20 and 120 min. Although the temporal increases in arachidonic acid and monoacylglycerol accumulation in the presence of TPA continued up to 120 min, that of 1,2-diacylglycerol declined after 20 min. In decidua cells prelabelled with [3H]choline, TPA also stimulated a significant decrease in radiolabelled PC after 20 min, which was accompanied by an increased release of water-soluble metabolites into the medium. Most of the radioactivity in the extracellular pool was associated with choline, whereas the main cellular water-soluble metabolite was phosphorylcholine. TPA stimulated extracellular choline accumulation to 183% and 351% of basal release after 5 and 20 min respectively and cellular phosphorylcholine production to 136% of basal values after 20 min. These results are consistent with a model in which protein kinase C activation by TPA leads to arachidonic acid mobilization from decidua-cell phospholipid by a mechanism involving phospholipase A-mediated PI hydrolysis and phospholipase C-mediated PC hydrolysis, coupled with further hydrolysis of the 1,2-diacylglycerol product.

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.

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 Effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on islet-cell responsiveness

1991 ◽  
Vol 278 (1) ◽  
pp. 49-56 ◽  
Author(s):  
W S Zawalich ◽  
K C Zawalich ◽  
S Ganesan ◽  
R Calle ◽  
H Rasmussen
Keyword(s):  
Insulin Release ◽  
Phorbol Ester ◽  
Inositol Phosphate ◽  
Secretory Response ◽  
Second Phase ◽  
Significant Impairment ◽  
Inositol Phosphate Accumulation ◽  
Pi Hydrolysis ◽  
Quantitative Immunoblotting ◽  
Insulin Secretory Response

Collagenase-isolated rat islets were labelled for 2 h in myo-[2-3H]inositol solution supplemented with 2.75 mM-glucose. The phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 microM) was also present in some experiments. After labelling, islets were washed and then perifused in 2.75 mM-glucose to establish basal [3H]inositol-efflux and insulin-secretory rates. Subsequently, the responses of these islets to stimulation with various agonists were assessed. Inositol phosphate accumulation was measured at the termination of the perifusion. In separate experiments, the cellular location of protein kinase C (PKC) after PMA pretreatment was measured by quantitative immunoblotting of membrane and cytosolic fractions. The following observations were made. (1) Labelling in 0.1-1 microM-PMA had no deleterious effect on total [3H]inositol incorporation during the 2 h labelling period. However, islets labelled for 2 h in 1 microM-PMA were unable to respond, in terms of increases in insulin release, to a 1 microM-PMA stimulus during the subsequent perifusion. (2) As compared with the responses of control islets labelled in 2.75 mM-glucose alone, islets labelled in the additional presence of 1 microM-PMA displayed a significant impairment in phosphoinositide (PI) hydrolysis, but an enhancement of both first-and second-phase insulin secretion, in response to subsequent 20 mM-glucose stimulation. (3) Decreasing extracellular Ca2+ level to 0.1 mM and including the Ca(2+)-channel antagonist nitrendipine (0.5 microM) along with 1 microM-PMA during the [3H]inositol-labelling period did not alter the response of the islets to the subsequent addition of 20 mM-glucose. Glucose-induced PI hydrolysis was still inhibited and 20 mM-glucose-induced insulin release was still enhanced. (4) A markedly amplified and sustained insulin-secretory response to 200 microM-tolbutamide in the presence of 2.75 mM-glucose was also obtained from 1 microM-PMA-pretreated islets. This contrasts sharply with the small and transient response to tolbutamide noted in control islets. (5) When present only during the perifusion phase of the experiments, nitrendipine (0.5 microM) abolished the amplified insulin-secretory responses to both 20 mM-glucose and 200 microM-tolbutamide noted in PMA-pretreated islets. (6) Prior labelling in 1 microM-PMA dramatically amplified the insulinotropic effect of 25 mM-K+ or 5 microM-A23187 stimulation. The amplified insulin-secretory response to K+, but not to A23187, was abolished by inclusion of nitrendipine during the perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration

1989 ◽  
Vol 259 (3) ◽  
pp. 743-749 ◽  
Author(s):  
W S Zawalich ◽  
K C Zawalich ◽  
H Rasmussen
Keyword(s):  
Insulin Release ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Secretory Response ◽  
Second Phase ◽  
Rat Islets ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Pi Hydrolysis ◽  
Insulin Secretory Response

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.

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.

scholarly journals Beryllium competitively inhibits brain myo-inositol monophosphatase, but unlike lithium does not enhance agonist-induced inositol phosphate accumulation

1993 ◽  
Vol 291 (2) ◽  
pp. 369-374 ◽  
Author(s):  
W S Faraci ◽  
S H Zorn ◽  
A V Bakker ◽  
E Jackson ◽  
K Pratt
Keyword(s):  
Rat Brain ◽  
Inositol Phosphate ◽  
Bipolar Depression ◽  
Neuroblastoma Cell ◽  
Brain Slices ◽  
Human Neuroblastoma Cell ◽  
Inositol Monophosphatase ◽  
Human Neuroblastoma ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

Despite limiting side-effects, lithium is the drug of choice for the treatment of bipolar depression. Its action may be due, in part, to its ability to dampen phosphatidylinositol turnover by inhibiting myo-inositol monophosphatase. Beryllium has been identified as a potent inhibitor of partially purified myo-inositol monophosphatase isolated from rat brain (Ki = 150 nM), bovine brain (Ki = 35 nM), and from the human neuroblastoma cell line SK-N-SH (Ki = 85 nM). It is over three orders of magnitude more potent than LiCl (Ki = 0.5-1.2 mM). Kinetic analysis reveals that beryllium is a competitive inhibitor of myo-inositol monophosphatase, in contrast with lithium which is an uncompetitive inhibitor. Inhibition of exogenous [3H]inositol phosphate hydrolysis by beryllium (IC50 = 250-300 nM) was observed to the same maximal extent as that seen with lithium in permeabilized SK-N-SH cells, reflecting inhibition of cellular myo-inositol monophosphatase. However, in contrast with that observed with lithium, agonist-induced accumulation of inositol phosphate was not observed with beryllium in permeabilized and non-permeabilized SK-N-SH cells and in rat brain slices. Similar results were obtained in permeabilized SK-N-SH cells when GTP-gamma-S was used as an alternative stimulator of inositol phosphate accumulation. The disparity in the actions of beryllium and lithium suggest that either (1) selective inhibition of myo-inositol monophosphatase does not completely explain the action of lithium on the phosphatidylinositol cycle, or (2) that uncompetitive inhibition of myo-inositol monophosphatase is a necessary requirement to observe functional lithium mimetic activity.

Mechanisms underlying AlCl3 inhibition of agonist-stimulated inositol phosphate accumulation

1994 ◽  
Vol 47 (8) ◽  
pp. 1417-1425 ◽  
Author(s):  
Timothy J. Shafer ◽  
Amy C. Nostrandt ◽  
Hugh A. Tilson ◽  
William R. Mundy
Keyword(s):  
Inositol Phosphate ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation

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.

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