Diphosphoinositide Research Articles

Polyamines stimulate the phosphorylation of phosphatidylinositol in rat mast cell granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P] ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of polyamines, spermine and spermidine, to the granules caused an increase of DPI synthesis, which can be catalyzed by PI kinase. This effect of polyamines in the DPI synthesis was in a dose-dependent manner and maximal effects were observed at 1 mM spermine and 10 mM spermidine, respectively.

Changes in polyphosphoinositide metabolism during mediator release from stimulated rat mast cells

The metabolism of the polyphosphoinositides, diphosphoinositide (DPI) and triphosphoinositide (TPI), was studied during mediator release from rat mast cells. Serosal mast cells were purified by density gradient centrifugation and prelabeled with 32PO 4. Incorporation of 32PO 4 into DPI and TPI was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO 4 incorporation into DPI and TPI was increased by Concanavalin A (ConA) or compound 48/80. The concentration of ConA causing a half-maximal increase in DPI labeling was less than that required for a comparable change in histamine release. The increases in DPI labeling and histamine release in response to ConA were enhanced by phosphatidylserine. The addition of α-methylmannoside to mast cells after challenge with ConA rapidly halted DPI and TPI labeling. The results of these studies indicate that changes in the metabolism of polyphosphoinositides may be an intrinsic part of the biochemical mechanisms that control mediator release from mast cells.

Phorbol Myristate Acetate Stimulates Formation of Diphosphoinositide in Rat Mast Cell Granules

Rat mast cell granules were obtained by homogenization of highly purified rat mast cells and isolated in a Percoll gradient. Diphosphoinositide (DPI) synthesis in rat mast cell granules was assayed by measuring the incorporation of 32P from [gamma 32P]ATP into DPI in the absence of exogenous phosphatidylinositol (PI). Lipids were isolated with methanol/chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The addition of phorbol myristate acetate (PMA) to the granules caused an increase of 32P incorporation from [gamma 32P]ATP in the DPI fraction, which can be catalyzed by PI kinase. This effect of PMA in the DPI synthesis was dose dependent and maximal effects were observed at 10 ng/ml.

ラットｍａｓｔ ｃｅｌｌか粒ｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ ｋｉｎａｓｅに及ぼすａｄｅｎｉｎｅ ｎｕｃｌｅｏｔｉｄｅと抗アレルギー薬の影響

Rat mast cell granules were obtained by homogenation of highly purified rat mast cells and isolated in a Percoll gradient. PI kinase in rat mast cell granules were assayed by measuring the incorporation of 32P from [γ32P] ATP into diphosphoinositide (DPI) in the absence of exogenous PI. Lipids were isolated with methanol chloroform/HCl and were separated by thin-layer chromatography on oxalic acid impregnated silica gel plates. DPI areas were identified by staining with iodine, scraped and measured for 32P radioactivity. The enzyme was inhibited by 50-500μM adenosine, 5-500μM ADP and AMP. Among several anti-allergic drugs investigated, 10-1000μM theophylline and 1-100μM azelastine inhibited the enzyme, but 1-100μM disodium cromoglycate and ketotifen had little effect.

Studies of phosphorylation in rat mast cells (supplement 2). Diphosphoinositide kinase in rat mast cell granules

Rat mast cells were purified by a Percoll gradient, and the granules were isolated from the sonicated mast cells on a Percoll gradient. The granules were shown to contain a diphosphoinositide kinase which catalyzes the formation of triphosphoinositide (TPI) from diphosphoinositide (DPI). The enzyme requires ATP and Mg2+ for the activity. TPI formation is almost completely dependent on MgCl2 or MnCl2, and maximal response is observed at 20 mM or 1 mM, respectively. The Km for ATP is 3 microM. TPI formation in the granules is dependent on the reaction time. The maximal response is seen at 23 degrees C. NaCl, KCl, Na2HPO4 and KH2PO4 have no effect on the activity. One hundred microM adenosine, AMP, ADP, and 10 microM cyclic AMP inhibit the kinase activity.

Studies of phosphorylation in rat mast cells (second report)--phosphatidylinositol kinase in rat mast cell granule membranes

Granules were isolated from sonicated purified rat mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase which catalyzes the formation of diphosphoinositide (DPI) from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity. DPI formation is almost completely dependent on MgCl2 or MnCl2, and maximal response was observed at 20 mM or 2 mM, respectively. The effects of the divalent cations are competitive. Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few min. The maximal response is seen at 28 degrees C.

Functional asymmetry of the auditory centers in the guinea pig

Antibodies to triphosphoinositide (TPI, I-phosphatidylinositol-3,4-bisphosphate) were raised in rabbits immunized with a hapten suspension of TPI, lecithin and cholesterol (1:4:30 by wt) in 0.1% methylated BSA. The hapten suspension was administered i.v. every two days for three weeks with each rabbit receiving a total of 3 mg TPI. Immunodiffusion with the partially purified immunoglobulin fraction and the comparison of complement-isofixation curves demonstrate complete cross-reactivity between TPI and diphosphoinositide (DPI) and very little specificity towards phosphatidylinositol (PI) and cardiolipin.

O2E1024) - Studies of diphosphoinositide(DPI) and phosphatidylinositol (PI) kinase in rat mast cell granules.

O2E1024) - Studies of diphosphoinositide(DPI) and phosphatidylinositol (PI) kinase in rat mast cell granules.

Gonadotropin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.

Fertilization increases the polyphosphoinositide content of sea urchin eggs.

Fertilization of the sea urchin egg stimulates a wave of exocytosis of cortical vesicles, but the mechanism by which fertilization regulates this secretion is not fully understood. We describe here experiments which suggest that polyphosphoinositide metabolism could be a factor in this regulation. We find that the cortical vesicle exocytosis in the egg of Strongylocentrotus purpuratus is preceded by a 40% increase in its content of triphosphoinositide (TPI) and a 22% increase of diphosphoinositide (DPI).

Effects of corticotropin-(1-24)-tetracosapeptide on polyphosphoinositide metabolism and protein phosphorylation in rabbit iris subcellular fractions.

Effects of the neuropeptide corticotropin-(1-24)-tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 X g supernatant fraction [30-50% (NH4)2SO4 precipitate; ASP 30-50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The 32P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [gamma-32P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP 30-50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30-60 s), and it was dependent on the presence of Mg2+ in the incubation medium; in general it was inhibited by high concentrations (greater than 0.2 mM) of Ca2+. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50-100 microM of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 microM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP 30-50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K).(ABSTRACT TRUNCATED AT 250 WORDS)

Defective phospholipid metabolism in the retinular cell membrane of [formula omitted] (no receptor potential) visual transduction mutants of [formula omitted

The phosphorylation of photoreceptor phospholipids in the three alleles of Drosophila visual mutants ( norpA : no receptor potential A gene) was studied. In the normal strain, the γ- 32P of ATP was transferred mainly to phosphatidic acid (PA) and diphosphoinositide (DPI), while, in the mutants, we found that the phosphorylation of PA was drastically reduced, but that of DPI was not. The radioactivity incorporation into PA closely parallels with the degree of the mutant genes' expressivity among the three alleles of norpA tested. Therefore, the abnormality found in the phosphorylation of diglycerol to PA may be closely related to the primary mutant defect in the phototransduction mechanism.

POSSIBLE ROLES OF POLYPHOSPHOINOSITIDES (PPI) IN THE INFORMATION TRANSDUCTION VIA BIOLOGICAL MEMBRANES

The polyphosphoinositides (PPI), diphosphoinositide (DPI) and triphosphoinositide (TPI), are quantitatively minor components of various eukaryotic tissues. They are of special biochemical interst because of their extraordinarily high affinity for Ca++ and the rapid turnover rate of their monoesterified phosphate groups. In addition, their metabolic turnover is accelerated with appropriate stimulations to the tissues. In this paper, we reviewed representative studies os PPI and discussed possible roles of these lipids in the stimulus-response coupling via biological membranes.Several results from the studies on the PPI in nervous tissues suggested a strong correlation between PPI and the, K+ -permeability of excitable membranes. It was also mentioned that PPI might play essential roles in ACTH-activated memory in the limbic system, ACTH-activated steroidgenesis in the adrenal cortex, ····etc.In contrast with the phosphatidylinositol (PI)-breakdown or the phospholipid-methylation that is suggested to contribute to an earlier step in the information transduction process via cellular membranes, PPI-metabolism may have important roles at a later step in the transduction process and contribute to various cellular responses.

Effect of hyperphenylalaninemia on polyphosphoinositides content of rat brain.

Hyperphnylalaninemia (experimental PKU) induced in developing rats by treatment with p-chlorophenylalanine (PCPA) plus phenylalanine (PHE) causes a significant reduction in the triphosphoinositide (TPI) and diphosphoinositide (DPI) content of brain. Since TPI and DPI play an important role in excitable nervous membranes, the functional abnormality in experimental and perhaps in genetic PKU may be related to the reduction in TPI and DPI content.

Phospholipid phosphorylation in erythrocyte of spontaneously hypertensive rats.

The rapid turnover of phosphoinositides within membranes suggests that these lipids play an important role in membrane function. Since various abnormalities have been described in the erythrocyte membrane of the spontaneously hypertensive rat (SHR) we have studied the turnover of phosphoinositides in the erythrocyte of SHR and age-matched normotensive Wistar-Kyoto rat (WKY). This was achieved by measuring the incorporation of 32P into inositol lipids after incubation of 1) intact erythrocytes with [32P]orthophosphate and 2) isolated ghost membranes with [gamma-32P]ATP. In both series of experiments more than 99% of the radioactivity incorporated into lipids was into the polyphosphoinositides diphosphoinositide (DPI) and triphosphoinositide (TPI). In both intact erythrocytes and ghost membranes, the levels of 32P incorporated into DPI and TPI were significantly different in SHR than in WKY. Further analysis of factors known to influence the labeling of DPI and TPI indicated that this could be ascribed to decreased activities of phosphatidylinositol kinase and/or DPI kinase, with respect to ATP as substrate. Moreover comparison of data obtained in intact cells with those obtained with ghost membranes suggests that within the SHR erythrocyte, membrane-cytosol interactions may occur that could also be responsible for the alteration of phosphoinositide labeling observed in hypertensive animals. Since phosphoinositides have been reported to be involved in the Ca2+-gating system of membrane, our findings could be associated with the abnormal Ca2+ binding and transport recently described in SHR erythrocyte.

Immunochemical studies of phospholipids: Production of antibodies to triphosphoinositide

Antibodies to triphosphoinositide (TPI, I-phosphatidylinositol-3,4-bisphosphate) were raised in rabbits immunized with a hapten suspension of TPI, lecithin and cholesterol (1:4:30 by wt) in 0.1% methylated BSA. The hapten suspension was administered i.v. every two days for three weeks with each rabbit receiving a total of 3 mg TPI. Immunodiffusion with the partially purified immunoglobulin fraction and the comparison of complement-isofixation curves demonstrate complete cross-reactivity between TPI and diphosphoinositide (DPI) and very little specificity towards phosphatidylinositol (PI) and cardiolipin.

Incorporation of 32PO4 into phospholipids of blood platelets.

Human or rabbit platelets in an artificial medium without phosphate were incubated with carrier-free [32P] orthophosphate for I h, washed and resuspended in Tyrode-albumin solution containing unlabelled phosphate. Aliquots of platelet suspension were subjected to lipid extraction at various times and the extracted phospholipids were separated by thin layer chromatography. The specific radioactivities of triphosphoinositide (TPI) and diphosphoinositide (DPI) were highest during the labelling period and then declined rapidly after the platelets were resuspended in the medium containing unlabelled phosphate. The percentage of total phospholipid 32P in TPI and DPI decreased from over 95% at 1 h to less than 50% at 12 h with either rabbit or human platelets. The specific radioactivity of monophosphoinositide (MPI) reached its greatest value 6-8 h after labelling. In the in vitro incubation studies, phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE) were negligibly labelled in the initial hours but at 12 h (rabbit or human platelets) PC contained more than 30% of the radioactivity. PE and PS contained less than 10% of the total phospholipid-bound radioactivity at the end of the incubation period. The pattern of incorporation of [32P]orthophosphate into the inositol phospholipids, PC, PS and PE that had been found in the in vitro studies was confirmed by an in vivo study in which rabbit platelets labelled with 32P in vitro were infused into rabbits and harvested after 35 h. These findings indicate that the phosphates of all the phospholipids studied in these experiments turn over in vitro and in vivo but the phosphates of the inositol phospholipids turn over most rapidly.

Phosphoinositides and other phospholipids in sympathetic ganglia and nerve trunks of rats. Effects of neuronal activity and inositol analogs ( - and -hexachlorocyclohexane (lindane)) on ( 32 P)-labelling, synaptic transmission and axonal conduction.

Abstract— Paired vagus nerves, phrenic nerves or superior cervical sympathetic ganglia from adult white rats were incubated for 4 h at 37°C in a bicarbonate‐buffered physiological solution containing glucose and 32P1. At the end of incubation triphosphoinositide (TPI) contained more 32P than any other lipid in the vagus nerves and was second only to phosphatidylcholine (PC) in the phrenic nerves. In the sympathetic ganglia phosphatidylinositol (PI) contained more 32P than did TPI, but both had less than PC. Conducted nerve impulses, initiated by electrical stimulation during the final 3 h of incubation, caused a highly significant increase in the [32P]‐labelling of PI in ganglia (as previously reported) probably decreased the labelling of TPI in the vagus nerves, and decreased the labelling of phosphatidylethanolamine (PE) in the ganglia. Addition to the incubation medium of §‐ or γ‐hexachlorocyclohexane (analogs of inositol) reversibly blocked transmission through the sympathetic ganglia at concentrations less than 0·1 mM. The §‐isomer also blocked conduction along axons at similar concentrations; only the γ‐isomer (lindane) exerted a selective effect on synaptic transmission. In the ganglia, the §‐isomer increased the [32P]‐labelling of PI and diphosphoinositide (DPI) relative to that of PC. The γ‐isomer did not affect the relative labelling of PI in the ganglia, whereas it decreased that of TPI, but only at relatively high concentrations. Thus, various affects of the hexachlorocyclohexanes were not explicable by assuming that they acted as analog inhibitors of inositol metabolism. In the ganglia, the hexachlorocyclohexanes reduced the effect of neuronal activity on the labelling of PI in proportion to the extent by which they blocked transmission. This metabolic effect was therefore presumed to be secondary to a ganglionic blocking action.

Biosynthesis of triphosphoinositide in rat kidney cortex

An enzyme, diphosphoinositide (DPI) kinase, that catalyzes the phosphorylation of DPI by γ −32P-ATP to form labeled triphosphoinositide (TPI) has been found to be predominantly localized in the plasma membrane fraction from rat kidney cortex. The isolated plasma membrane fraction when compared with the homogenates showed a 15-fold increase in DPI kinase activity. The enzyme required added Mg 2+ for full activity and was inhibited by Cu 2+ or Hg 2+. The apparent K m for DPI was calculated to be 1.43 × 10 −4 m and for ATP, 2.5 × 10 −3 m at 15 m m MgCl 2. An excess amount of either substrate (DPI or ATP) caused an inhibition of the enzyme activity. The inhibitory effect of excess ATP could be overcome by an increase of Mg 2+ concentration in the assay medium. The enzyme activity was considerably decreased by EDTA (Tris salt) at concentrations above 2 m m in the presence of 15 m m MgCl 2. It was inhibited by sulfhydryl reagents, such as N-ethylmaleimide (1 m m) and p-chloromercuribenzoic acid (0.2 m m). In addition, reduced glutathione (1 m m) and cysteine (1 m m) exerted an inhibitory effect. NaCl (100 m m) and KCl (100 m m) reduced the formation of TPI, while NH 4Cl (50 m m) and LiCl (50 m m) were stimulatory. Both reaction products, ADP (1–3 m m) and TPI (0.2–1 m m), lowered the kinase activity. In the presence of detergents (nonionic or cationic), the enzyme activity was impaired.

A simple procedure for the study of inositol phosphatides in cat brain slices.

A procedure is described for the separation of monophosphoinositide (MPI), diphosphoinositide (DPI), and triphosphoinositide (TPI) from small samples of tissue. The method is readily applicable to experiments with tissue slices. It is based upon the finding that MPI is readily extracted into chloroform–methanol (2:1), and DPI and TPI are extracted when 1 part of concentrated hydrochloric acid is added to 300 parts of the solvent. The phosphoinositides are then separated chromatographically on formaldehyde-treated paper.In experiments in which slices of cat brain were incubated in the presence of inorganic32P, the phosphorus compounds of high specific radioactivity remaining in the tissue residue after extraction of lipids and nucleic acids, and referred to previously as "inositide P" or "residue organic P", were found to be chiefly protein-bound DPI and TPI, with small amounts of protein-bound phosphatidyl serine and phosphatidyl ethanolamine.Increasing the concentration of K+ion in the medium from 5.9 mM to 105 mM caused a decrease in the incorporation of inorganic32P into the "lipid extract", MPI, DPI, and TPI of cat brain slices. Addition of chlorpromazine (0.1 mM), or acetylcholine (10 mM) with eserine sulfate (0.5 mM), caused an increase in the incorporation of inorganic32P into the "lipid extract" and MPI, but with no corresponding increase in the incorporation into DPI and TPI.