scholarly journals Agonist-stimulated inositol phosphate metabolism in avian erythrocytes

1990 ◽  
Vol 269 (1) ◽  
pp. 65-72 ◽  
Author(s):  
L R Stephens ◽  
C P Berrie ◽  
R F Irvine
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Intact Cells ◽  
Inositol Phosphate Metabolism ◽  
Avian Erythrocytes ◽  
32P Incorporation ◽  
The Individual ◽  
Chicken Erythrocytes ◽  
Purinergic Stimulation

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5′-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.

scholarly journals The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection

1989 ◽  
Vol 264 (2) ◽  
pp. 323-333 ◽  
Author(s):  
T Radenberg ◽  
P Scholz ◽  
G Bergmann ◽  
G W Mayr
Keyword(s):  
Mammalian Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Detection System ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Phosphate Metabolism ◽  
Qualitative And Quantitative ◽  
Inositol Phosphate Metabolism ◽  
Avian Erythrocytes

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.

Synthesis of inositol phosphate ligands of plant hormone–receptor complexes: pathways of inositol hexakisphosphate turnover

2012 ◽  
Vol 444 (3) ◽  
pp. 601-609 ◽  
Author(s):  
David E. Hanke ◽  
Paroo N. Parmar ◽  
Samuel E. K. Caddick ◽  
Porntip Green ◽  
Charles A. Brearley
Keyword(s):  
Hormone Receptor ◽  
Plant Hormone ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Leaf Tissue ◽  
Phosphate Metabolism ◽  
Inositol Hexakisphosphate ◽  
Receptor Complexes ◽  
Inositol Phosphate Metabolism ◽  
Phosphate Ligands

Reduction of phytate is a major goal of plant breeding programs to improve the nutritional quality of crops. Remarkably, except for the storage organs of crops such as barley, maize and soybean, we know little of the stereoisomeric composition of inositol phosphates in plant tissues. To investigate the metabolic origins of higher inositol phosphates in photosynthetic tissues, we have radiolabelled leaf tissue of Solanum tuberosum with myo-[2-3H]inositol, undertaken a detailed analysis of inositol phosphate stereoisomerism and permeabilized mesophyll protoplasts in media containing inositol phosphates. We describe the inositol phosphate composition of leaf tissue and identify pathways of inositol phosphate metabolism that we reveal to be common to other kingdoms. Our results identify the metabolic origins of a number of higher inositol phosphates including ones that are precursors of cofactors, or cofactors of plant hormone–receptor complexes. The present study affords alternative explanations of the effects of disruption of inositol phosphate metabolism reported in other species, and identifies different inositol phosphates from that described in photosynthetic tissue of the monocot Spirodela polyrhiza. We define the pathways of inositol hexakisphosphate turnover and shed light on the occurrence of a number of inositol phosphates identified in animals, for which metabolic origins have not been defined.

scholarly journals Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

2012 ◽  
Vol 448 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Yixing Zhou ◽  
Shilan Wu ◽  
Huanchen Wang ◽  
Yoichi Hayakawa ◽  
Gary S. Bird ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Mass Action ◽  
Surface Receptors ◽  
Entry Pathway ◽  
Inositol Phosphate Metabolism ◽  
Metallothionein Promoter ◽  
Inositol Tetrakisphosphate ◽  
A Cell ◽  
Signalling Cascade

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117–28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

scholarly journals Epidermal-growth-factor-induced formation of inositol phosphates in human A431 cells. Differences from the effect of bradykinin

1988 ◽  
Vol 252 (3) ◽  
pp. 857-863 ◽  
Author(s):  
B C Tilly ◽  
P A van Paridon ◽  
I Verlaan ◽  
S W de Laat ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Cell System ◽  
Ionophore A23187 ◽  
A431 Cells ◽  
Inositol Phosphate Metabolism ◽  
Absolute Requirement ◽  
Epidermal Growth

In human A431 epidermoid carcinoma cells, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids and raises cytoplasmic free [Ca2+]. In this paper, we investigate the action of EGF on inositol phosphate metabolism, and we compare it with the previously described effects of bradykinin on the same cell system [Tilly, van Paridon, Verlaan, Wirtz, de Laat & Moolenaar (1987) Biochem. J. 244, 129-135]. In cells prelabelled with [3H]inositol, EGF slowly but persistently (for at least 30 min) stimulates the formation of [3H]inositol phosphates, whereas bradykinin causes an immediate but transient release of inositol phosphates, which lasts for only a few minutes. The EGF effect is additive to bradykinin stimulation and does not require extracellular Ca2+. In contrast, inositol phosphate formation induced by Ca2+-ionophore A23187 has an absolute requirement for external Ca2+. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate completely abolishes the response to EGF and to sub-optimal doses of bradykinin, suggesting a negative-feedback function of protein kinase C. Pretreatment of the cells with pertussis toxin has no effect on inositol phosphate formation induced by either EGF or bradykinin. Unlike bradykinin, EGF stimulates very little accumulation of inositol 1,4,5-trisphosphate, with only a small and rather variable release of Ca2+ from intracellular stores. EGF rapidly but transiently increases inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate, but the effects are much smaller than those of bradykinin. In addition, EGF increases both inositol mono- and bis-phosphate. At 10 min after EGF addition, inositol monophosphate, unlike the other inositol phosphates, is still increasing. It is concluded that the EGF-dependent pattern of stimulation is different from that observed in bradykinin-stimulated A431 cells, suggesting that there are separate mechanisms of inositol-lipid hydrolysis involved.

scholarly journals Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1972 ◽  
Vol 128 (3) ◽  
pp. 531-541 ◽  
Author(s):  
Janet M. Stein ◽  
C. N. Hales
Keyword(s):  
Phosphatidic Acid ◽  
Specific Radioactivity ◽  
Phospholipid Composition ◽  
Fat Cells ◽  
Fat Pad ◽  
Fat Cell ◽  
32P Incorporation ◽  
The Individual ◽  
Adrenergic Blocking ◽  
Stimulation Of

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [32P]Pi into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [32P]Pi into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [32P]Pi into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.

Elevation of inositol tetrakisphosphate parallels inhibition of Ca(2+)-dependent Cl- secretion in T84 cells

1993 ◽  
Vol 264 (3) ◽  
pp. C671-C676 ◽  
Author(s):  
U. Kachintorn ◽  
M. Vajanaphanich ◽  
K. E. Barrett ◽  
A. E. Traynor-Kaplan
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Chloride Secretion ◽  
Epithelial Cell Line ◽  
T84 Cells ◽  
Cell Monolayers ◽  
Calcium Dependent ◽  
Inositol Phosphate Metabolism ◽  
Inositol Tetrakisphosphate ◽  
Calcium Elevation

Carbachol and histamine both stimulate calcium-dependent chloride secretion in the colonic epithelial cell line, T84. However, pretreatment of cell monolayers with carbachol blocks subsequent chloride secretion induced by thapsigargin but not the calcium elevation stimulated by this agent, whereas histamine pretreatment blocks neither thapsigargin-induced chloride secretion nor calcium elevation. To examine whether inositol phosphate metabolism might account for this difference, we measured levels of radiolabeled inositol phosphates: Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins-(1,3,4,6)P4, Ins(3,4,5,6)P4, InsP5, and InsP6 after cell stimulation. Although both carbachol and histamine increase Ins (1,4,5)P3 at 5 s, there is a greater and more persistent increase in the levels of Ins(1,3,4)P3 and InsP4 at later time points after carbachol than histamine, which corresponded to the suppression of the chloride secretory response.

scholarly journals Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
H. Ian BATTY ◽  
A. Richard CURRIE ◽  
C. Peter DOWNES
Keyword(s):  
Phospholipase C ◽  
Time Course ◽  
Inositol Phosphates ◽  
Specific Radioactivity ◽  
Lipid Transport ◽  
Intact Cells ◽  
Whole Cells ◽  
Metabolic Compartmentation ◽  
Astrocytoma Cells ◽  
Inositol Phospholipids

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A ‘chase’ protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5ʹ-[γ-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.

scholarly journals Cholera-toxin and corticotropin modulation of inositol phosphate accumulation induced by vasopressin and angiotensin II in rat glomerulosa cells

1988 ◽  
Vol 253 (3) ◽  
pp. 765-775 ◽  
Author(s):  
G Guillon ◽  
N Gallo-Payet ◽  
M N Balestre ◽  
C Lombard
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adp Ribosylation ◽  
Glomerulosa Cells

Vasopressin (VP) and angiotensin II (AT II) stimulate the production of inositol phosphates (IP) in rat glomerulosa cells. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), but not VP or AT II, stimulates IP production in a myo-[3H]inositol-prelabelled glomerulosa-cell membrane preparation. In combination with GTP[S], these hormones potentiate the response to GTP[S], indicating the existence of a G-protein involved in the coupling of the VP and AT II receptor with the phospholipase C. ADP-ribosylation with pertussis toxin (IAP) revealed the specific labelling of a single molecule of 41 kDa. No significant inhibition of VP- or AT II-stimulated IP accumulation was detected in intact cells when the whole 41 kDa molecule was endogenously ADP-ribosylated by IAP treatment. On the contrary, when glomerulosa cells were infected with cholera toxin (CT), both the VP- and AT II-stimulated IP accumulations were inhibited in a dose-dependent manner. Yet these effects were partial even at high concentrations of CT, and could not be related to the ADP-ribosylation of ‘alpha s’ molecules. Similarly, when the cells were infected with 1 microgram of CT/ml, the specific binding of VP and AT II decreased by 50-60%. Such results may signify that the treatment primarily affects the densities of the hormone receptors. When glomerulosa cells were incubated for 15 h in the presence of 10 nM-corticotropin (ACTH), a condition in which the intracellular concentration of cyclic AMP was increased 3-fold, the maximum IP response to 0.1 microM-VP or -AT II was decreased by 50%. When similar experiments were carried out only after a 15 min incubation period with the same concentration of ACTH, the increase in cyclic AMP was more pronounced, but no inhibition of hormone-induced IP accumulation was observed. Altogether, these results may suggest that CT exerts its action on the VP- or AT II-sensitive phospholipase C systems via a prolonged increase in intracellular cyclic AMP.

scholarly journals The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+

1985 ◽  
Vol 231 (2) ◽  
pp. 269-278 ◽  
Author(s):  
R V Farese ◽  
J S Davis ◽  
D E Barnes ◽  
M L Standaert ◽  
J S Babischkin ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
De Novo ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Phospholipid Content ◽  
Phospholipid Synthesis ◽  
Rat Adipose Tissue ◽  
Phosphatidylinositol 4 ◽  
Protein Kinase C Activation

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated ‘second messenger’ which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular ‘messenger’ for controlling metabolic processes during insulin action.

scholarly journals Phosphoinositide hydrolysis by guanosine 5′-[γ-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes

1988 ◽  
Vol 252 (2) ◽  
pp. 583-593 ◽  
Author(s):  
T K Harden ◽  
P T Hawkins ◽  
L Stephens ◽  
J L Boyer ◽  
C P Downes
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Specific Radioactivity ◽  
Major Product ◽  
Erythrocyte Membranes ◽  
Low Concentrations ◽  
Phosphate Response ◽  
Phosphatidylinositol 4 ◽  
Rate Of Accumulation

Phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] of turkey erythrocytes were labelled by using either [32P]Pi or [3H]inositol. Although there was little basal release of inositol phosphates from membranes purified from labelled cells, in the presence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) the rate of accumulation of inositol bis-, tris- and tetrakis-phosphate (InsP2, InsP3 and InsP4) was increased 20-50-fold. The enhanced rate of accumulation of 3H-labelled inositol phosphates was linear for up to 20 min; owing to decreases in 32P specific radioactivity of phosphoinositides during incubation of membranes with unlabelled ATP, the accumulation of 32P-labelled inositol phosphates was linear for only 5 min. In the absence of ATP and a nucleotide-regenerating system, no InsP4 was formed, and the overall inositol phosphate response to GTP[S] was decreased. Analyses of phosphoinositides during incubation with ATP indicated that interconversions of PtdIns to PtdIns4P and PtdIns4P to PtdIns(4,5)P2 occurred to maintain PtdIns(4,5)P2 concentrations; GTP[S]-induced inositol phosphate formation was accompanied by a corresponding decrease in 32P- and 3H-labelled PtdIns, PtdIns4P and PtdIns(4,5)P2. In the absence of ATP, only GTP[S]-induced decreases in PtdIns(4,5)P2 occurred. Since inositol monophosphate was not formed under any condition, PtdIns is not a substrate for the phospholipase C. The production of InsP2 was decreased markedly, but not blocked, under conditions where Ins(1,4,5)P3 5-phosphomonoesterase activity in the preparation was inhibited. Thus the predominant substrate of the GTP[S]-activated phospholipase C of turkey erythrocyte membranes is PtdIns(4,5)P2. Ins(1,4,5)P3 was the major product of this reaction; only a small amount of Ins(1:2-cyclic, 4,5)P3 was released. The effects of ATP on inositol phosphate formation apparently involve the contributions of two phenomena. First, the P2-receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) greatly increased inositol phosphate formation and decreased [3H]PtdIns4P and [3H]PtdIns(4,5)P2 in the presence of a low (0.1 microM) concentration of GTP[S]. ATP over the concentration range 0-100 microM produced effects in the presence of 0.1 microM-GTP[S] essentially identical with those observed with 2MeSATP, suggesting that the effects of low concentrations of ATP are also explained by a stimulation of P2-receptors. Higher concentrations of ATP also increase inositol phosphate formation, apparently by supporting the synthesis of substrate phospholipids.(ABSTRACT TRUNCATED AT 400 WORDS)

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