scholarly journals Glucose-induced accumulation of inositol trisphosphates in isolated pancreatic islets. Predominance of the 1,3,4-isomer

1986 ◽  
Vol 237 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Turk ◽  
B A Wolf ◽  
M L McDaniel
Keyword(s):  
Pancreatic Islets ◽  
Anion Exchange ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Isolated Pancreatic Islets ◽  
Stimulus Secretion Coupling

Anion-exchange h.p.l.c. analysis of [3H]inositol phosphates derived from glucose-stimulated isolated pancreatic islets that had been prelabelled with myo-[3H]inositol revealed that the predominant inositol trisphosphate was the 1,3,4-isomer [Ins(1,3,4)P3]. The 1,4,5-isomer [Ins(1,4,5)P3] was also detectable, as was a more polar inositol phosphate with the chromatographic properties of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Glucose-induced accumulation of Ins(1,3,4)P3 was augmented by Li+ and occurred after maximal accumulation of Ins(1,4,5)P3. These findings suggest a possible role for Ins(1,3,4)P3 or its probable precursor Ins(1,3,4,5)P4 in stimulus-secretion coupling in pancreatic islets.

Effect of cholecystokinin on the accumulation of inositol phosphates in isolated pancreatic islets

1989 ◽  
Vol 257 (6) ◽  
pp. G865-G870
Author(s):  
J. Florholmen ◽  
D. Malm ◽  
B. Vonen ◽  
P. G. Burhol
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Maximal Effect ◽  
Isolated Pancreatic Islets ◽  
Chromatography Analysis ◽  
Inositol Tetrakisphosphate ◽  
Per Se ◽  
Hydrolysis Of

Sulfated cholecystokinin octapeptide (CCK-8S) potentiated glucose-induced secretion in isolated pancreatic islets with a maximal effect at 12 mM glucose, whereas no effect was observed at 3.3 and 25 mM glucose. This effect of CCK-8S was maximal at 10(-7) M. Anion-exchange fast-protein liquid chromatography analysis of [3H]inositol phosphates derived from islets prelabeled with myo-[3H]inositol showed that glucose induced accumulation of the 1,4,5-isomer of inositol trisphosphate and of inositol tetrakisphosphate. At 3.3 mM glucose, CCK-8S stimulated accumulation of inositol trisphosphate and inositol tetrakisphosphate to levels induced by 25 mM glucose alone. The net effect of CCK-8S on the accumulation of the inositol phosphates was maximal at 12 mM glucose and decreased at higher glucose concentrations. At 12 mM glucose the accumulation of inositol phosphates increased gradually up to 10(-7) M CCK-8S. This study indicates that CCK-8S potentiates glucose-induced insulin secretion through a mechanism involving the hydrolysis of polyphosphoinositides and the generation of inositol phosphates. However, activation of the inositol cycle per se did not seem to induce insulin secretion.

scholarly journals Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate

1984 ◽  
Vol 219 (2) ◽  
pp. 655-659 ◽  
Author(s):  
R P Rubin ◽  
P P Godfrey ◽  
D A Chapman ◽  
J W Putney
Keyword(s):  
Phospholipase C ◽  
Exocrine Pancreas ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Receptor Activation ◽  
Early Step ◽  
Pancreatic Acini ◽  
Rat Exocrine Pancreas ◽  
Stimulus Secretion Coupling

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.

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 A rapid separation method for inositol phosphates and their isomers

1987 ◽  
Vol 245 (3) ◽  
pp. 655-660 ◽  
Author(s):  
K A Wreggett ◽  
R F Irvine
Keyword(s):  
Anion Exchange ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Separation Method ◽  
Ammonium Formate ◽  
Rapid Separation ◽  
Large Numbers

A technique is described using ACCELL QMA anion-exchange SEP-PAKs (Waters Associates) with ammonium formate-based solutions, whereby a sample can be processed within minutes to yield resolution of inositol phosphates. Isomers of inositol trisphosphate can then be separated by using this technique in combination with a rapid (5-6 min) isocratic h.p.l.c. procedure. The use of QMA SEP-PAKs offers a degree of reproducibility comparable with that of h.p.l.c. while maintaining the capacity for automation, allowing large numbers of samples to be processed rapidly.

scholarly journals Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol

1988 ◽  
Vol 251 (1) ◽  
pp. 279-284 ◽  
Author(s):  
M L Rand ◽  
J D Vickers ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Thromboxane A2 ◽  
Second Messenger ◽  
Inhibitory Effect ◽  
Thromboxane B2 ◽  
Rabbit Platelets ◽  
Platelet Functions ◽  
Stimulation Of

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.

scholarly journals Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration

1985 ◽  
Vol 229 (2) ◽  
pp. 361-367 ◽  
Author(s):  
F Di Virgilio ◽  
L M Vicentini ◽  
S Treves ◽  
G Riz ◽  
T Pozzan
Keyword(s):  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Human Neutrophils ◽  
Chemotactic Peptide

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.

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.

scholarly journals Automated isocratic high-performance liquid chromatography of inositol phosphate isomers

1989 ◽  
Vol 262 (3) ◽  
pp. 997-1000 ◽  
Author(s):  
K A Wreggett ◽  
R F Irvine
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Anion Exchange ◽  
High Performance ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Direct Analysis ◽  
Isocratic Elution ◽  
Anion Exchange Resins ◽  
Exchange Resins

Isomers of inositol phosphates from biological samples can be analysed by anion-exchange h.p.l.c., by using isocratic elution with phosphate buffers. The method involves the preliminary processing of the extracted samples with conventional soft-gel anion-exchange resins, including the commonly used Dowex resins, followed by direct analysis with h.p.l.c. of a portion of relevant fractions. Run times (up to 20 min) and collected fraction numbers (up to 24) are minimal, so that if the method is used in conjunction with automated h.p.l.c. injection a high throughput of samples is achieved.

scholarly journals The inositol phosphates in WRK1 rat mammary tumour cells

1992 ◽  
Vol 286 (2) ◽  
pp. 459-468 ◽  
Author(s):  
N S Wong ◽  
C J Barker ◽  
A J Morris ◽  
A Craxton ◽  
C J Kirk ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Periodate Oxidation ◽  
Tumour Cells ◽  
Mammary Tumour ◽  
Inositol Polyphosphate ◽  
Inositol Hexakisphosphate ◽  
Site Specific

1. A detailed structural survey has been made of the inositol phosphates of unstimulated and vasopressin-stimulated WRK-1 rat mammary tumour cells. Inositol phosphate peaks were separated by h.p.l.c., and structural assignments were made for more than 20 compounds by combinations of: (a) co-chromatography with labelled standards; (b) site-specific enzymic dephosphorylation; (c) complete and partial periodate oxidation, followed by h.p.l.c. of polyols and their stereospecific oxidation by dehydrogenases; and (d) ammoniacal hydrolysis. 2. The ‘inositol monophosphates’ fraction from unstimulated cells included an uncharacterized peak, probably containing some glycerophosphoinositol, and Ins(1:2-cyclic)P. Stimulation provoked accumulation of both Ins1P and Ins3P, of Ins2P, and of Ins5P and/or the enantiomers Ins4P and Ins6P. The proportions of Ins1P and Ins3P were determined by partial periodate oxidation and enantiomeric identification of the resulting glucitols. 3. Three inositol bisphosphate peaks were detected in unstimulated cells: Ins(1,4)P2 [this was distinguished chemically from its enantiomer Ins(3,6)P2], Ins(3,4)P2 and/or Ins(1,6)P2, and Ins(4,5)P2 and/or Ins(5,6)P2. On stimulation, Ins(1,4)P2 and Ins(3,4)P2 [and/or Ins(1,6)P2] levels increased, and Ins(1:2-cyclic,4)P2 and Ins(1,3)P2 were also formed. 4. Three inositol trisphosphate peaks were obtained from unstimulated cells: all increased during stimulation. These were Ins(1,3,4)P3 [with some Ins(1:2-cyclic,4,5)P3], Ins(1,4,5)P3 and Ins(3,4,5)P3 [and/or Ins(1,5,6)P3]. During stimulation, another compound, probably Ins(1,4,6)P3, appeared in the ‘Ins(1,4,5)P3 peak’. The ‘Ins(3,4,5)P3 peak’ contained a second trisphosphate, probably Ins(2,4,5)P3. 5. Three inositol tetrakisphosphates, namely Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, were present in unstimulated cells, and all accumulated during stimulation. 6. Ins(1,3,4,5,6)P5, which is the most abundant inositol polyphosphate in these cells, a less abundant inositol pentakisphosphate and inositol hexakisphosphate were all unresponsive to stimulation.

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