scholarly journals Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations

1989 ◽  
Vol 258 (1) ◽  
pp. 23-32 ◽  
Author(s):  
I H Batty ◽  
A J Letcher ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Inositol Phosphates ◽  
Periodate Oxidation ◽  
Chemical Degradation ◽  
Inositol Polyphosphate ◽  
Tissue Slices ◽  
Inositol 1 ◽  
Agonist Stimulation ◽  
Cortex Slices ◽  
Hydrolysis Of

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.

scholarly journals Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation

1989 ◽  
Vol 260 (1) ◽  
pp. 237-241 ◽  
Author(s):  
I H Batty ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Muscarinic Receptor ◽  
Metabolic Flux ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Inositol Polyphosphate ◽  
Receptor Stimulation ◽  
Cortex Slices ◽  
Rapid Kinetics

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.

Oxytocin and vasopressin stimulate inositol phosphate production in human gestational myometrium and decidua cells

1986 ◽  
Vol 6 (7) ◽  
pp. 613-619 ◽  
Author(s):  
Michael P. Schrey ◽  
Alison M. Read ◽  
Philip J. Steer
Keyword(s):  
Arachidonic Acid ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Diacylglycerol Lipase ◽  
Inositol 1 ◽  
Inositol Phosphate Production ◽  
Acid Accumulation ◽  
Free Arachidonic Acid ◽  
Hydrolysis Of ◽  
Related Increase

The involvement of phosphoinositide hydrolysis in the action of oxytocin and vasopressin on the uterus was investigated in gestational myometrium and decidua cells by measuring the production of inositol phosphates. Both peptides stimulated a dose related increase in all three inositol phosphates in myometrium. This may be related to the control of sarcoplasmic Ca++ levels in the myometrium. Oxytocin and vasopressin also stimulated inositol 1-phosphate (IP) production in decidua cells. The hydrolysis of phosphatidylinositol by decidua homogenates exhibited a precursor-product relationship for diacylglycerol and arachidonic acid accumulation. Hence both peptides may mobilise free arachidonic acid, for prostaglandin biosynthesis, from decidua cell phosphoinositides by the sequential action of phospholipase C and diacylglycerol lipase.

Phosphoranylation de polyols: Voie d'accès aux phosphates d'intérêt biologique. I. Cas du myo-inositol

1988 ◽  
Vol 66 (12) ◽  
pp. 2965-2974 ◽  
Author(s):  
Brigitte Duthu ◽  
Douraid Houalla ◽  
Robert Wolf
Keyword(s):  
Acid Hydrolysis ◽  
Inositol Phosphates ◽  
Original Method ◽  
Inositol 1 ◽  
Reaction Proceeds ◽  
Hydrolysis Of

An original method for the phosphorylation of an unprotected myo-inositol is described, which yields several myo-inositol phosphates at the same time. The reaction proceeds via a partial or complete phosphoranylation of the cyclitol by means of the aminobicyclophosphane 8, followed by oxidation of the resulting bicyclophosphoranes bearing a P—H bond and acid hydrolysis of the neutral phosphates thus formed. In the case of the tris-phosphoranylation we identified, among the HPLC fractions, the myo-inositol-1,2-(cycl)phosphate 22, the myo-inositol-1-phosphate 23, and the myo-inositol-2-phosphate 24.

scholarly journals Lithium stimulates accumulation of second-messenger inositol 1,4,5-trisphosphate and other inositol phosphates in mouse pancreatic minilobules without inositol supplementation

1994 ◽  
Vol 304 (1) ◽  
pp. 251-258 ◽  
Author(s):  
J F Dixon ◽  
L E Hokin
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase C ◽  
Feedback Inhibition ◽  
Inositol Phosphates ◽  
Plasma Concentrations ◽  
Second Messenger ◽  
Inositol Polyphosphates ◽  
Peripheral Tissues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cortex Slices

Previous studies showed that lithium, beginning at therapeutic plasma concentrations in the treatment of manic depression, increased the accumulation of second-messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in cerebral cortex slices of guinea pig and rhesus monkey [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385; Dixon, Lee, Los and Hokin (1992) J. Neurochem. 59, 2332-2335; Dixon, Los and Hokin (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 8358-8362]. These studies have now been extended to a peripheral tissue, mouse pancreatic minilobules. In the presence of carbachol, concentrations of lithium from 1 to 20 mM sharply and progressively increased the accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate, followed by a decrease. Assay of these inositol polyphosphates by either the prelabelling technique or mass assay gave similar results. Atropine quenching of cholinergically stimulated pancreatic minilobules led to a rapid disappearance of Ins(1,4,5)P3. This disappearance was impeded by lithium. This suggested that the lithium-induced elevation in Ins(1,4,5)P3 was due to inhibition of the 5-phosphatase and, on the basis of the markedly elevated concentrations of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and inositol 1,4-bisphosphate in the presence of lithium, probably by feedback inhibition by these latter two compounds. An additional mechanism, i.e. a stimulatory effect of lithium on phospholipase C, cannot, however, be ruled out. The other reaction product of phospholipase C, inositol cyclic 1:2,4,5-trisphosphate, also increased in the presence of lithium. This may also be due to inhibition of the 5-phosphatase, which is the exclusive mechanism for removal of this compound. The effects of lithium on the accumulation of other inositol phosphates paralleled that of Ins(1,4,5)P3, with the exception of inositol 3,4-bisphosphate, which decreased. This was presumably due to the inhibition of Ins(1,3,4)P3 1-phosphatase by lithium. Unlike mouse cerebral cortex slices [Lee, Dixon, Reichman, Moummi, Los and Hokin (1992) Biochem. J. 282, 377-385], inositol supplementation was not required to demonstrate lithium-stimulated Ins(1,4,5)P3 accumulation in mouse pancreatic minilobules. This indicates that inositol depletion sufficient to impair lithium-stimulated Ins(1,4,5)P3 accumulation does not occur in mouse pancreatic minilobules, even though an elevation of cytidine diphosphodiacylglycerol occurred, indicating some inositol depletion due to lithium. Elevation of Ins(1,4,5)P3 by lithium may be a general phenomenon in the central nervous system and peripheral tissues under non-rate-limiting concentrations of inositol.

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.

scholarly journals Differences between muscarinic-receptor- and Ca2+-induced inositol polyphosphate isomer accumulation in rat cerebral-cortex slices

1990 ◽  
Vol 267 (3) ◽  
pp. 835-838 ◽  
Author(s):  
J G Baird ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Muscarinic Receptor ◽  
Degradation Products ◽  
The Other ◽  
Rat Cerebral Cortex ◽  
Inositol Polyphosphate ◽  
Receptor Stimulation ◽  
Other Hand ◽  
Cortex Slices ◽  
Polyphosphate Metabolism

Muscarinic-receptor stimulation or depolarization by elevated K+ leads to increased accumulation of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4 and several degradation products of these polyphosphates separated by h.p.l.c. On the other hand, agents such as ionomycin and maitotoxin, which increase intracellular Ca2+ directly, produce a small accumulation of [3H]Ins(1,4,5)P3 and markedly increase [3H]Ins(1,4)P2, but [3H]Ins(1,3,4,5)P4, [3H]Ins(1,3,4)P3 and [3H]Ins(1,3)P2 are virtually unaffected. Ca2(+)-dependent [3H]inositol polyphosphate metabolism may involve different pools of lipids and/or phosphoinositidases.

scholarly journals Uptake of monosaccharides by guinea-pig cerebral-cortex slices

1969 ◽  
Vol 112 (3) ◽  
pp. 367-371 ◽  
Author(s):  
P. Joanny ◽  
J. Corriol ◽  
H. Hillman
Keyword(s):  
Cerebral Cortex ◽  
Guinea Pig ◽  
Diffusion Kinetics ◽  
Cerebral Tissue ◽  
Tissue Slices ◽  
Saturation Kinetics ◽  
Cortex Slices ◽  
Kinetics Of

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.

scholarly journals Evidence for lithium-sensitive inositol 4,5-bisphosphate accumulation in muscarinic cholinoceptor-stimulated cerebral-cortex slices

1992 ◽  
Vol 287 (2) ◽  
pp. 437-442 ◽  
Author(s):  
S Jenkinson ◽  
R A Challiss ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase D ◽  
Inositol Phosphate ◽  
Intact Cell ◽  
Inositol Phosphate Metabolism ◽  
Kinase C ◽  
Cortex Slices ◽  
A Minor ◽  
Hydrolysis Of ◽  
Stimulation Of

Stimulation of [3H]inositol-prelabelled rat cerebral-cortex slices with carbachol results in the accumulation of four [3H]inositol bisphosphate isomeric species, Ins(1,3)P2, Ins(1,4)P2, Ins(3,4)P2 and Ins(4,5)P2. Although the last isomer ran as a minor peak on h.p.l.c., its accumulation was dramatically enhanced in the presence of Li+ (1 mM), such that at 30 min it represented almost 35% of the total bisphosphate fraction. The accumulation of Ins(4,5)P2 appeared to be very sensitive to Li+ (EC50 = 94 +/- 3 microM), strongly implicating a Li(+)-sensitive metabolism. Evidence for this is provided from the rapid but Li(+)-sensitive decay of Ins(4,5)P2 when muscarinic-receptor stimulation is antagonized by atropine at a time when accumulations have reached a new steady state. Manipulation of phospholipase D by activators and inhibitors of protein kinase C did not suggest a role for phospholipase D hydrolysis of PtdInsP2 in the formation of Ins(4,5)P2. Attempts to reveal Ins(4,5)P2 metabolism, or indeed its synthesis from Ins(1,4,5)P3, were not successful with broken cell preparations and strongly suggest discrete compartmentation of inositol phosphate metabolism in the intact cell.

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