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R F Irvine

Publications and source records attributed to R F Irvine.

At least 55 records · Page 3Linked to original sources
PubMed1994

Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets.

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

Animals↗
PubMed1994

Rapid turnover of phosphatidylinositol 3-phosphate in the green alga Chlamydomonas eugametos: signs of a phosphatidylinositide 3-kinase signalling pathway in lower plants?

When Chlamydomonas eugametos gametes were incubated in carrier-free [32P]P1, the label was rapidly incorporated into PtdInsP and PtdInsP2 and, after reaching a maximum within minutes, was chased out by recirculating unlabelled P1 in the cell. This pulse-chase labelling pattern reflects their rapid turnover. In contrast, 32P incorporation into the structural lipids was slow and continued for hours. Of the radioactivity in the PtdInsP spot, 15% was in PtdIns3P and the rest in PtdIns4P, and of that in the PtdInsP2 spot, 1% was in PtdIns(3,4)P2 and the rest in PtdIns(4,5)P2, confirming the findings by Irvine, Letcher, Stephens and Musgrave [(1992) Biochem. J. 281, 269-266]. When cells were labelled with carrier-free [32P]P1, both PtdInsP isomers incorporated label in a pulse-chase-type pattern, demonstrating for the first time in a plant or animal system that D-3 poly-phosphoinositides turn over rapidly in non-stimulated cells, with kinetics similar to those shown by the D-4 isomers. In animal systems such lipids are already established as signalling molecules, and the data suggest that a similar role must be sought for them in lower plants such as Chlamydomonas.

Animals↗
PubMed1994

The nuclear phosphoinositide cycle--does it play a role in nuclear Ca2+ homoeostasis?

The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions.

Animals↗
PubMed1993

Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate.

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures), and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a 'safe' binding site for iron during its transport through the cytosol or cellular organelles.

Ferric Compounds↗
PubMed1993

Evidence for receptor-specific regulation of the metabolism of the second messenger, inositol trisphosphate, in human endothelial cells.

Primary cultures (1-2 weeks-old, no passage) of endothelial cells from the human umbilical vein were pre-labelled with [3H]inositol, stimulated for varying times (15 seconds to 5 minutes) in the presence of LiCl with either thrombin or histamine, and the extracted radiolabelled isomers of inositol phosphates were analysed by anion-exchange chromatography. The doses of each receptor-agonist were chosen to stimulate a large accumulation of inositol phosphates at approximately the same rate. At all times of stimulation the relative accumulation of inositol trisphosphate isomers and inositol tetrakisphosphate was different upon stimulation with these agonists, particularly at earlier times. These results argue for the receptor-specific regulation of the inositol trisphosphate 3-kinase. The significance of this observation is discussed with respect not only to the current ideas on the control of calcium homoeostasis by inositol phosphates, but also in consideration of their role as second messengers controlling different end-points of vascular function.

Cells, Cultured↗
PubMed1993

Monoclonal antibodies to phosphatidylinositol 4-phosphate 5-kinase: distribution and intracellular localization of the C isoform.

We have raised a panel of monoclonal antibodies to PtdIns4P 5-kinase C purified from bovine brain [Divecha, Brooksbank and Irvine (1992) Biochem. J. 288, 637-642]. This panel includes antibodies which specifically recognize PtdIns4P 5-kinase C both in a native catalytically active condition, and/or when presented on Western blots. Some of the former antibodies will also inhibit PtdIns4P 5-kinase C activity. We have used the blotting antibodies to study the bovine tissue distribution of PtdIns4P 5-kinase C and its distribution in mammalian species. We have also studied its localization in Jurkat cells and found it to be predominantly bound to membranes, with only a minority localized to the cytoskeleton. Neither PtdIns4P 5-kinase activity nor PtdIns4P 5-kinase C, as detected by Western blotting, were increased in the cytoskeleton after stimulation of Jurkat cells with OKT3. These antibodies should prove to be extremely useful tools with which to study the regulation of PtdIns4P 5-kinase C.

Animals↗
PubMed1993

Nuclear diacylglycerol is increased during cell proliferation in vivo.

Highly purified nuclei were prepared from livers and kidneys of rats undergoing compensatory hepatic or renal growth, the former being predominantly by cellular proliferation, and the latter mostly by cellular enlargement. In liver, an increase in nuclear diacylglycerol (DAG) concentration occurred between 16 and 30 h, peaking at around 20 h. At the peak of nuclear DAG production a specific translocation of protein kinase C to the nucleus could be detected; no such changes occurred in kidney. There was no detectable change in whole-cell DAG levels in liver, and the increase in DAG was only measurable in nuclei freed of their nuclear membrane. Overall, these results suggest that there is a stimulation of intranuclear DAG production, possibly through the activation of an inositide cycle [Divecha, Banfić and Irvine (1991) EMBO J. 10, 3207-3214] during cell proliferation in vivo.

Animals↗
PubMed1993

Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus.

The presence of phosphoinositide-mobilizing enzymes has been investigated in purified rat liver nuclei by radiolabelling and by probing with antibodies. A Ca(2+)-activated phosphoinositidase C (PIC) is present and was shown immunologically to be the beta 1 isoform. No gamma- or delta-PIC was found. However, only 5% of the total beta 1-PIC isoform is nuclear, with the majority being cytosolic. G alpha q and G alpha 11, the suggested physiological activators of beta 1-PIC, were not present. A PtdIns4P 5-kinase is also present, which immunologically is shown to be the C isoform. All of these nuclear inositide enzymes still remained after the removal of the nuclear envelope with Triton X-100, consistent with the concept of an intranuclear inositide cycle [Divecha, Banfic and Irvine (1991) EMBO. J. 10, 3207-3214].

Animals↗
PubMed1992

Purification and characterization of phosphatidylinositol 4-phosphate 5-kinases.

We detail the purification and characterization of three distinct isoforms of PtdIns4P 5-kinase present in bovine brain. One of these, PtdIns4P 5-kinase C, was purified to apparent homogeneity, and SDS/PAGE analysis demonstrated a single polypeptide and molecular mass 53 KDa. These three isoforms were shown to differ in their kinetic properties, and immunological characterization with an antibody raised to PtdIns4P 5-kinase C demonstrated that this isoform was unrelated to the other two. Furthermore, PtdIns4P 5-kinase C was shown to be the bovine brain homologue of the Type II PtdIns4P 5-kinase previously purified from human erythrocytes [Bazenet, Ruano, Brockman & Anderson (1990) J. Biol. Chem. 265, 18012-18022].

Animals↗
PubMed1992

Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance.

1. Ins(1,3,4,5)P4 binding sites were studied in cerebellar and hepatic microsomes from rat, and in bovine adrenal-cortical microsomes. 2. At pH 7.0, all three tissues showed specific binding, with Ins(1,3,4,5)P4 being the most potent competing ligand of those tested [which included Ins(1,4,5)P3, Ins(1,3,4,5,6)P5 and InsP6] and Scatchard analysis suggested two sites; a site with high affinity and high specificity [Kd (1-6) x 10(-9) M] and a site with low affinity and low specificity [Kd (2-6) x 10(-7) M]. 3. At pH 5.5, cerebellar and bovine adrenal microsomes showed similar binding properties: two affinities with a similar specificity for Ins(1,3,4,5)P4 as at pH 7.0. 4. However, when assayed in a low-ionic strength acetate-based buffer at pH 5.0, cerebellar microsomes retain specific Ins(1,3,4,5)P4 binding sites, whereas bovine adrenal and hepatic microsomal binding sites lose much of their specificity, as InsP6 and Ins(1,3,4,5,6)P5 are equally as potent as Ins(1,3,4,5)P4. 5. Pi (25 mM), which is frequently included in Ins(1,3,4,5)P4 binding assays, had a small inhibitory effect on binding of cerebellar and adrenal microsomes at pH 5.5, but a large effect at pH 7.0, so that a considerable decrease occurs in the amount of specific binding at pH 5.5 compared with that at pH 7.0, if Pi is omitted from the binding assay. 6. Cerebellar and adrenal microsomes were used in a ligand-displacement mass assay (conducted under near-physiological conditions, at pH 7.0) on extracts of cerebral-cortex slices stimulated with agonists, and both preparations faithfully detected the increases in Ins(1,3,4,5)P4 that occurred, implying that Ins(1,3,4,5)P4 is the principal ligand on these binding sites in intact cells. 7. Apparent contradictions in the literature with regard to Ins(1,3,4,5)P4 binding sites in neuronal and peripheral tissues can be largely accounted for by the data, and the properties of the binding sites detected at physiological pH are consistent with the possibility that they are putative receptors for the proposed second-messenger role for Ins(1,3,4,5)P4.

Adrenal Cortex↗
PubMed1992

Inositol polyphosphate metabolism and inositol lipids in a green alga, Chlamydomonas eugametos.

Swimming suspensions of Chlamydomonas eugametos were pelleted and homogenized, and the metabolism of inositol polyphosphates by cellular homogenates or supernatants was investigated. Ins(1,4,5)P3 was dephosphorylated under physiological conditions to yield a single InsP2, Ins(1,4]2. In the presence of ATP it was phosphorylated to give Ins(1,3,4,5)P3 as the only InsP4. The Ins(1,4,5)P3 3-kinase activity was predominantly soluble, was not detectably affected by calmodulin or Ca2+, and had a Km for Ins(1,4,5)P3 of 50 microM (two orders of magnitude higher than its mammalian counterpart). Ins(1,3,4,5)P4 was dephosphorylated by the cellular supernatants to Ins(1,3,4)P3 and Ins(1,4,5)P3, and could be phosphorylated to Ins(1,3,4,5,6)P4. No Ins(1,3,4)P3 6-kinase activity could be detected, and experiments with [3H]Ins(1,4,[32P]5)P3 revealed that Ins(1,3,4,5,6)P5 is formed from Ins(1,4,5)P3 with little loss of the 5-phosphate, i.e. the predominant route of synthesis is probably by a direct 6-phosphorylation of Ins(1,3,4,5)P4. Similar experiments with an (NH4)2SO4 fraction of turkey erythrocyte cytosol gave essentially the same result, i.e. direct phosphorylation of Ins(1,3,4,5)P4 in the 6 position is the predominant route of synthesis of InsP5 from that InsP4 in vitro. No InsP6 formation was detected in any of these experiments, but labelling of intact C. eugametos with [3H]inositol revealed that the cells do synthesize InsP6. The lipids of C. eugametos cells contain PtdIns, PtdIns(4)P and PtdIns(4,5)P2 [Irvine, Letcher, Lander, Drøbak, Dawson & Musgrave (1989) Plant Physiol. 64, 888-892]. Further examination of 32P-labelled lipids revealed that about 20% of the PtdInsP was the PtdIns(3)P isomer, and about 1% or less of the PtdInsP2 was the PtdIns(3,4)P2 isomer. The overall inositide metabolism of C. eugametos resembles that of a mammalian cell more closely than it does that of a plant cell or slime mould, and this suggests firstly that the known metabolism of inositol polyphosphates arose at an early time in eukaryotic evolution, and secondly that Chlamydomonas might prove a useful organism for genetic and comparative studies of inositide enzymology.

Animals↗
PubMed1992

Inositol lipids in cell signalling.

In the past year, major advances have been made in our understanding of the regulation of phosphoinositidase C, and of the action of the inositol trisphosphate receptor and how it may generate 'quantal' Ca2+ release. The functions of inositol tetrakisphosphate and of the 3-phosphorylated inositol lipids continue to generate controversy, but both may be well on the way towards some clarification. Finally, we may have to extend our concept of the inositide cycle to include an intranuclear signalling function.

Animals↗
PubMed1992

Phospholipids in the nucleus--metabolism and possible functions.

Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca2+ homoeostasis.

Animals↗
PubMed1992

Inositol phosphates and Ca2+ entry: toward a proliferation or a simplification?

Intracellular mobilization of Ca2+ by inositol trisphosphate (IP3) is only a temporary phenomenon; the more crucial process of stimulated entry of Ca2+ by inositol phosphates is still poorly understood, with apparently conflicting data and hypotheses arising from different tissues and experimental protocols. There is clear evidence that the intracellular Ca2+ stores themselves can control Ca2+ entry and that IP3 may exert a direct effect on Ca2+ entry over and above its function in emptying those stores. There is also clear evidence that inositol tetrakisphosphate (IP4) can control Ca2+ entry, but there is controversy over whether it is actually necessary. Thus at present the combined evidence suggests that there must be a multiplicity of mechanisms extant, with different mechanisms being emphasized in different tissues. Alternatively, there could be one common mechanism, discussed here, which may lead to apparently different emphases as a result of the various experimental protocols.

Animals↗