Radioactivity In Phosphatidylinositol Research Articles

Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP 2) via phospholipase C- γ1 (PLC- γ1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC- γ1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC- γ1 translocation to the particulate fraction. Thus, although activation of PLC- γ1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC- γ1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC- γ1 induced by PDGF and slowed the translocation of PLC- γ1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP 2 in cells labeled with myo[ 3H]inositol. The increased synthesis of PtdInsP 2 could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC- γ1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC- γ1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC- γ1, and on PtdInsP 2 synthesis cannot be attributed to inhibition of PKC. © 1997 Elsevier Science B.V. All rights reserved.

Effect of Epidermal Growth Factor on Phosphatidylinositol 3-kinase Activity in Rabbit Corneal Epithelial Cells

We investigated the effect of epidermal growth factor (EGF) on phosphatidylinositol 3-kinase (PI 3-kinase) activity in SV-40 transformed rabbit corneal epithelial cells (RCEC) and in normal corneal epithelial cells grown in primary culture. The enzyme products, 3-phosphorylated phosphoinositides, were identified by TLC and by deacylation followed by separation on HPLC. Addition of 30 ng ml −1EGF to 32P-labeled SV-40 transformed or primary epithelial cell cultures, increased the radioactivity in phosphatidylinositol 3,4,5-trisphosphate (PIP 3) by about 96% over that of control incubations. The growth factor also significantly increased the radioactivity in phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2] and phosphatidic acid which was quite similar in the two cell types. The EGF-mediated increase in PIP 3formation in RCEC was dose- and time-dependent. Treatment of RCEC lysate with anti-PI 3-kinase antibody immunoprecipitated proteins that phosphorylated PI, PI(4)P and PI(4,5)P 2to corresponding 3-phosphorylated phosphoinositides. Incubation of RCEC with EGF resulted in a dose- and time-dependent increase in PI 3-kinase activity associated with the immunoprecipitate. Nonionic detergent, Nonidet P-40, strongly inhibited the PI 3-kinase activity in the immunoprecipitate, but it stimulated the PI 4-kinase activity in the cell lysate. Wortmannin, a fungal metabolite, also exerted a dose-dependent inhibitory effect on the activity of PI 3-kinase. Analysis of the PI 3-kinase immunoprecipitate by Western blot did not show any increase in concentration of the 85-kDa subunit of PI 3-kinase in EGF-treated cells. However, probing the immunoblot with anti-phosphotyrosine antibody showed a large increase (145%) in phosphorylation of the 85-kDa subunit of the enzyme. It can be concluded from these data that PIP 3and other 3-phosphoinositides exist in RCEC, and that EGF stimulates the production of these lipids by activation of PI 3-kinase, probably by increased tyrosine phosphorylation of the 85-kDa subunit of the enzyme.

Phosphorylation in vivo of chick brain microtubule-associated phospholipids.

Microtubules were prepared by temperature-dependent cycles of assembly/disassembly from chick brain labeled in vivo with 32Pi and the distribution of labeled phospholipids extracted from cold-insoluble and soluble microtubular protein fractions was analyzed by thin-layer and paper chromatography. While 32P-labeling was associated with all of the phospholipids identified after 2-D TLC, it was found that all of the relatively high radioactivity associated with phosphatidylserine (PS) was in fact associated with a minor co-migrating component which was subsequently identified as phosphatidylinositol(PI) by three independent separation procedures. It was estimated that the relative specific radioactivity in PI was several-fold higher than that associated with other microtubule-associated phospholipids. Additional experiments, in which the protein components of once-cycled microtubules were fractionated by gel permeation chromatography, provided evidence that the 36S component containing ring-like tubulin oligomers (36S) appears to be selectively associated with phospholipid components that were specifically enriched in 32P-PI. The possible significance of these findings is discussed in relation to the effects of phospholipids on microtubule dynamics and to the function of microtubules in their interactions with membranes.

Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.1 mM) for 30 min to RCEC, prelabeled with 32Pi, decreased the radioactivity in phosphatidylinositol 4-phosphate and PIP2 by 15 and 27%, respectively, and concomitantly increased the radioactivity in phosphatidylinositol and phosphatidic acid by 14 and 38%, respectively. When the concentration of CCh was increased to 1 mM, the changes in radioactivity were even more pronounced. Addition of CCh (0.1 mM) to the cells, prelabeled with myo[3H]inositol, increased the accumulation of [3H]inositol 1,4,5-trisphosphate ([3H]InsP3) by 115%, indicating stimulation of PLC-mediated PIP2 hydrolysis. Similar increases were also observed in [3H]InsP1 and [3H]InsP2. The effects of CCh on inositol phosphate accumulation were time- and dose-dependent, and were inhibited by atropine (10 microM), suggesting that the observed effects of CCh were mediated by activation of muscarinic cholinergic receptors. The effects of CCh were antagonized more potently by 4-diphenylacetoxy N-methyl-piperidine than by pirenzepine, indicating that the muscarinic receptors involved in PLC activation are probably of M3 type. By Western immunoblotting analysis with various anti-PLC antibodies, the RCEC were shown to contain PLC gamma 1 and PLC delta 1 in the soluble fraction and PLC beta 1 in the microsomal fraction. Addition of isoproterenol to RCEC, increased cAMP both in a time- and dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Organic Solvents Activate Human Platelets Through the Inositol Lipid-linked Signal Transduction System

It has recently been proposed that occupational exposure to organic solvents in vivo may lead to platelet activation and this has been substantiated by exposure of platelets to solvents in vitro. The present work was undertaken to study the effects of organic solvents on the platelet inositol lipid signal transduction system. Human platelets that had been prelabelled with [(32)P] P, were exposed to a saturated atmosphere of the organic solvents toluene, xylene or hexane. Extracts were analyzed for metabolites of the polyphosphoinositide cycle and ATP. All solvents studied induced a decrease in radioactivity in phosphatidylinositol 4,5-bisphosphate together with an increase in radioactivities in phosphatidylinositol 4-phosphate and phosphatidic acid. This is compatible with solvent-induced activation of the cells through the inositol lipid pathway. In cells exposed to toluene or xylene we could detect an increased level in inositol trisphosphates at 3 min of exposure. The solvent-induced changes in metabolic ATP could not explain the solvent-induced effects on the inositol lipid metabolism. It is concluded that the organic solvents toluene, xylene and hexane can activate human platelets through the inositol lipid-linked transmembrane signal system.

Elongation, desaturation, and esterification of essential fatty acids by fetal rat brain in vivo.

Tracer amounts of either [1-14C]linolenic (18:3n-3, LNA), or [1-14C]linoleic (18:2n-6, LA) acids were intracranially injected into 19- to 20-day-old rat fetuses, and the time course of the in vivo formation and esterification of their long chain polyenoic metabolites was determined for up to 20 h. A rapid disappearance of free LNA and LA, with apparent half-lives of 60 and 40 min, respectively, was noticed. One hour after LNA injection, 32.3% and 14.3% of the total brain radioactivity was found in the neutral glyceride (NG) and phospholipid (PL) fractions, respectively. After 20 h, PL radioactivity attained a level of 75%. Phosphatidylcholine (PC), diacylglycerol (DG), and triacylglycerol (TG) constituted 40%, 23%, and 9% of the total brain label at 1 h, and 35%, 10% and 14% at 20 h, respectively. Ethanolamine-containing PL (including plasmalogen) radioactivity accounted for about 10% up to 6 h and increased nearly 3-fold at 20 h, primarily due to an increase in the amount of labeled docosapentaenoic and docosahexaenoic acids (DHA), the elongation-desaturation products of LNA. A similar pattern of incorporation into NG and PL fraction was observed after the administration of [1-14C]LA. After 1 h, PC, DG, and TG species constituted 23%, 10%, and 23% of the total brain radioactivity, whereas after 20 h it accounted for 44%, 6%, and 10%, respectively. Although radioactivity in the ethanolamine PL also increased substantially from 4% at 1 h to 29% at 20 h, the main labeled fatty acid in this fraction was LA. Labeled arachidonic acid (AA) constituted 42.7% of the total radioactivity in phosphatidylinositol (PI) at 20 h. At this time, it comprised 12.5% and 14% of the total radioactivity in PC and ethanolamine PL, respectively, suggesting a high degree of esterification selectivity. Comparison of the total amounts of LA and LNA and their corresponding labeled AA and DHA metabolites in brain and liver after 3 and 6 h indicated that the contribution of liver metabolism to the elongation-desaturation under these conditions was negligible. One hour after intracerebral injection of [3H]DHA (22:6n-3) or [3H]AA (20:4n-6), 29.2% and 12% of total radioactivity, respectively, was found in the ethanolamine PL while 20% and 40% was incorporated in PC, respectively. PI labeling by [3H]AA was 6- to 8-fold higher than that seen in the presence of DHA. A high percent of radioactivity (26.9% and 18.2%) was found in DG and TG species.(ABSTRACT TRUNCATED AT 400 WORDS)

Metipranolol-induced changes in membrane phospholipids of isolated mast cells

Metipranolol significantly decreased32P incorporation into membrane phosphatidylcholine but increased the radioactivity in phosphatidylinositol of nonstimulated and 48/80-stimulated rat mast cells. In stimulated cells metipranolol significantly decreased3H-arachidonic acid liberation from total membrane phospholipids as well as thromboxane B2 formation. The effect of metipranolol on stimulated rat mast cells is most probably a result of its direct effect on membrane phospholipid and arachidonic acid turnover.

Cloricromene inhibits leukotriene formation by human polymorphonuclear leucocytes by suppressing arachidonate release from membrane phospholipids

Cloricromene, an antithrombotic agent known to inhibit the release of arachidonic acid (AA) in stimulated human platelets, was tested for its effects on arachidonate release and metabolism in human polymorphonuclear leucocytes (PMNs). Cloricromene dose-dependently suppressed the release of leukotriene B 4 (LTB 4), as assessed by radioimmunoassay, from both isolated PMNs and human whole blood stimulated with the calcium ionophore A23187 or with serum-treated zymosan (STZ). The inhibitory effect was higher when the concentration of the stimulating agent was weaker. Cloricromene also inhibited dose-dependently the liberation of LTB 4, LTC 4, LTD 4 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid as assessed by HPLC in the supernatant ofA23187-stimulated PMNs. Finally, the drug was able to suppress the release of [ 3H]AA from purified human PMNs prelabelled with the radioactive fatty add and stimulated with either A23187 or with STZ. The A23187-induced decrease in the radioactivity of phosphatidylinositol, the phospholipid class mainly involved in AA release in stimulated PMNs, was also inhibited by Cloricromene. Cloricromene suppresses leukotriene formation in human PMNs by reducing AA release from membrane phospholipids, possibly through interference with phospholipase A 2 activation; this activity may contribute to the leucocyte-inhibitory effects reported previously for Cloricromene.

Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells.

The effects of phorbol esters on phospholipase C (PLC) activity towards phosphoinositides and phosphatidylcholine (PC) in bovine corneal epithelial cells were examined. The cells were labeled with 32Pi, myo[3H]inositol or methyl[14C]choline, and PLC stimulated by incubation of the cells with Ca2+ ionophore, ionomycin. The PLC activity was assessed by monitoring the loss of radioactivity from the labeled phospholipids or the accumulation of their radioactive metabolites. The data from this study can be summarized as follows: Addition of 20 microM ionomycin to the prelabeled cells resulted in a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and somewhat slower hydrolysis of phosphatidylinositol (PI) and phosphatidylcholine (PC) with concomitant several-fold increase in phosphatidic acid (PA). The effects of the ionophore were time- and dose-dependent. Incubation of the cells with phorbol 12,13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) caused increased radioactivity in PC and PA, whereas the radioactivity in PI and PIP2 remained unchanged. The effects of PDBu were inhibited by staurosporine and H-7, and inactive derivatives of phorbol esters failed to exert any effect on phospholipid metabolism. Pretreatment of the corneal epithelial cells with PDBu or PMA abolished the ionomycin-induced hydrolysis of phosphoinositides and PC. The data suggest that activation of protein kinase C by phorbol esters in corneal epithelial cells results in inhibition of PLC activity towards phosphoinositides and PC through a mechanism probably involving phosphorylation of the enzyme.

Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells.

We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 cells were responsive to TRH. In cells prelabeled in situ and then permeabilized, TRH stimulated an increase in 3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity initially present in lipids. This increase in 3H-labeled IPs was 6.3 times the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior to stimulation. When prelabeled cells were exchanged with unlabeled Ins after permeabilization there was only a 10-16% decrease in 3H-labeled IP accumulation stimulated by TRH even though 3H-labeled lipids decreased to 52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In cells labeled by base exchange after permeabilization TRH stimulated a very small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol 4,5-bisphosphate. These data show that in permeabilized GH3 cells base exchange can occur in the absence of de novo PI synthesis and that lipids that are preferentially labeled by base exchange comprise a pool that is less responsive to TRH than total Ins lipids.

Molecular species synthesized by phosphatidylinositol synthases from potato tuber, pea leaf and soya bean

5–9 different molecular species of phosphatidylinositol (PI) were separated, combining HPLC and GLC analyses, from microsomes extracted from potato tuber, pea leaf and germinated soya beans. In microsomes from the three plant tissues, 16:0/18:2-PI was the most abundant molecular species. The second most abundant species was 16:0/18:3-PI in pea leaf and potato tuber, whereas 18:0/18:2-PI was more abundant than 16:0/18:3-PI in soya bean microsomes. Labelling PI molecular species from [ 14 C] myo -inositol, in the presence of EDTA to avoid any exchange reaction, showed that only two molecular species (16:0/18:2-PI and 16:0/18:3-PI) were prominently produced by PI synthases from the three tissues. The molecular species 18:0/18:2-PI and 18:2/18:3-PI were slightly labelled (less than 10% of total PI radioactivity) in soya bean and pea leaf, respectively. A good correspondence could be observed between the quantitative distributions of PI molecular species and the relative labellings of the same species in the three tissues. Those results suggest that de novo synthesis of PI molecular species is not followed by subsequent alterations (acyl exchanges or desaturations) of these molecules.

Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils.

Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.

Importance of the presence of the N-terminal tripeptide of substance P for the stimulation of phosphatidylinositol metabolism in rat parotid gland: A possible activation of phospholipases C and D

In this study, we have compared the effects of Substance P (SP) and an SP deprived of the N-terminal tripeptide, SP(4–11), on phosphoinositide metabolism by measuring phosphoinositide breakdown, inositol phosphate production and inositol incorporation into phosphoinositides. This work shows that SP and SP(4–11) have similar effects on phosphatidylinositol-4.5 bisphosphate (PIP 2) metabolism. In fact, SP(4–11), like SP, induces a rapid PIP 2 breakdown. On the contrary, SP and SP(4–11) have different effects on phosphatidylinositol (PI) metabolism since SP induces a decrease of radioactivity in PI, whereas SP(4–11) does not. Both peptides stimulate [ 3H]-inositol mono-, bis- and trisphosphate (respectively IP 1, IP 2, IP 3) production in a time and dose-dependent manner. The kinetic of IP 3 production is directly correlated with the one of PIP 2 breakdown. The time course of IP 1 production after SP(4–11) shows a time delay, while the one after SP does not. Since SP evokes an IP 1 production without any delay and a large decrease of radioactivity in PI (which cannot account for the small amount measured in IP 1 accumulation) we suggest that SP could activate a PI specific phospholipase C (leading to a PI breakdown) and a phospholipase D. These activations would require the complete structure of SP while the classical PIP 2 specific phospholipase C activation (which induces PIP 2 breakdown) would only require the carboxamide part of the peptide. So the complete structure of SP would be necessary to have a complete response (stimulation of PIP 2 and PI metabolism).

Arginine vasopressin stimulates phosphoinositide turnover in an enriched rat Leydig cell preparation

An enriched rat Leydig cell preparation was preincubated with [ 14C]arachidonic acid. Stimulation of the cells with arginine vasopressin (AVP) (1 μM) for 2 min caused a significant increase in labelled phosphatidic acid and a significant fall in radioactivity in phosphatidylinositol and phosphatidylinositol 4-monophosphate + phosphatidylinositol 4,5-bisphosphate. Preincubation with dibutyryl cyclic AMP had no effect on the AVP-induced phospholipid turnover. Leydig cells were preincubated with myo-[2- 3H]inositol for 22 h and then with 10 mM LiCl for 10 min. Exposure to AVP (1 μM) induced a rise in labelled inositol phosphates. The response was inhibited when the cells were preincubated with the phorbol ester, 4β-phorbol 12-myristate 13-acetate (0.16 μM) for 10 min. These results provide evidence for an AVP-induced phospholipase C stimulation in rat Leydig cells and suggest a protein kinase C-dependent feedback inhibition of the stimulation. Other agonists that might have a regulatory function in the testis were tested for possible effects on phosphoinositide metabolism. Of prostaglandin E 2 (10 μm), angiotensin II (0.1 μM), and bradykinin (0.9 μM), only the latter induced a significant increase in the labelled inositol phosphates. This suggests that Leydig cells possess a bradykinin receptor which can activate phospholipase C.

Evidence for the incorporation of [ 32P]orthophosphate into leaf inositol phospholipids

Leaf discs of brinjal, tomato, sugar cane and maize rapidly incorporated [ 32P]orthophosphate into total phospholipids. Analyses of the labelled lipid extracts by thin-layer chromatography, autoradiography and comparison with inositol phospholipid standards demonstrated the labelling of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate in addition to other phospholipids. The presence of polyphosphoinositides was further confirmed by deacylation of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and separation of the water-soluble products, glycerophosphoinositol phosphate and glycerophosphoinositol bisphosphate by formate exchange chromatography. Incorporation of [ 32P]orthophosphate into inositol phospholipids was time-dependent, with monoester phosphate groups attaining isotopic equilibrium within 90 min of incubation. After 2 h, incorporation of label into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate was about 15, 10 and 3%, respectively, of the total phospholipids. The ratio of radioactivity in phosphatidylinositol/ phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate was about 5:5:1 in brinjal leaves. However, this ratio may be an overestimate of the amounts of inositol phospholipids present, as other lysophospholipids may comigrate with standards.

Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium

Slices of rat brainstem that had been prelabeled by in vivo injection of [ 3H]inositol were stimulated with carbachol in the presence of lithium and changes measured in the radioactivity of inositol lipids and water-soluble inositol phosphates. For the latter, significant increases were seen for inositol mono- and bisphosphate but not inositol trisphosphate. Analysis of whole tisseu phosphoinositides revealed significantly reduced radioactivity in phosphatidylinositol and phosphatidylinositol 4-phosphate, whereas myelin showed decreases in those as well as phosphatidylinositol 4,5-bisphosphate. These effects were blocked by atropine. Stimulation of the tissue slices with elevated K + resulted in increased formation of inositol phosphate and decreased radioactivity in phosphatidylinositol. The effect was not blocked by atropine and in the presence of this agent, which reduced background reaction, all 3 phosphoinositides showed significant K +-induced loss of label. Elevated K + and carbachol thus function through different mechanisms in this system. Carbachol is believed to affect myelin phosphoinositides through direct interaction with muscarinic receptors which were recently shown to be present in this membrane.

Reduction of inositol triphosphate in retinal microvessels by glucose and restimulation by myo-inositol

The formation of inositol phospholipids (IPLs) and inositol phosphate esters (IPEs) in response to glucose was studied in isolated retinal microvessels from porcine eyes. Retinal microvessels incubated from 60 hr with myo-[3H]inositol were sequentially extracted to obtain IPLs and IPEs. [3H]Inositol-labelled IPLs were deacylated to produce the corresponding glycero derivatives. Both deacylation products and water-soluble IPEs were monitored by anion-exchange chromatography. In the presence of high glucose (30 mM) the labelling in inositol triphosphate (IP3) was reduced to 77% and was restimulated by adding myo-inositol (final concentration 0.4 mM) to 158% of the control under physiological conditions of glucose (5 mM) and myo-inositol (0.04 mM). With a fixed glucose concentration (5 mM), IPE accumulation was observed with increasing concentrations of exogenous myo-inositol. Under physiological conditions (glucose 5 mM, myo-inositol 0.04 mM) the distribution (percentage) of radioactivity in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) was 63:19:18. The myo-inositol concentration dependence of IPL formation was also demonstrated. A decrease in IP3 in response to high glucose without changing PIP2 but with a reduction in PI indicated that PI may act as a reservoir to replace a possible loss of PIP2. These findings suggest that availability of myo-inositol by retinal microvessels may be essential to maintain the normal signal transduction and cell proliferation associated with IPL turnover under high glucose concentration.

Phosphatidylinositol Cycle Metabolites in Samanea saman Pulvini

The major metabolites of the phosphatidylinositol cycle from extracts of [(32)PO(4)]- and [(3)H]-inositol-labeled Samanea saman pulvini were separated. The membrane localized phosphoinositides were separated by thin layer chromatography, identified by comparison with purified lipid standards, and quantitated based on incorporation of radiolabel. The ratio of radioactivity in phosphatidylinositol:phosphatidylinositol 4-phosphate:phosphatidylinositol 4,5-bisphosphate is about 32:8:1. The aqueous inositol phosphates were separated by anion exchange chromatography using conventional liquid chromatography and by high performance liquid chromatography (HPLC) and were identified by comparison with standards. Analysis by HPLC reveals that (32)P-labeled pulvini have inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate that co-migrate with red blood cell inositol phosphates, but (3)H-inositol-labeled pulvini appear to have a variant profile.

Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets

Human platelets were pulse-labelled with [ 32P]P i and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse-labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the γ-phosphoryl of ATP. Upon incubation of the platelets at 37°C, the specific 32P radioactivity of ATP (β- and γ-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [γ- 32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin-induced changes in [ 32P]PI differed markedly at the various times after the [ 32P]P i-pulse. Immediately after pulse-labelling, [ 32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [ 32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [ 32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10 11 cells upon thrombin stimulation of platelets.

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction.

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)