scholarly journals Secretagogue-induced phosphoinositide metabolism in human leucocytes

1984 ◽  
Vol 222 (2) ◽  
pp. 307-314 ◽  
Author(s):  
R W Dougherty ◽  
P P Godfrey ◽  
P C Hoyle ◽  
J W Putney ◽  
R J Freer
Keyword(s):  
Phospholipase C ◽  
Lysosomal Enzyme ◽  
Time Course ◽  
Inositol Phosphates ◽  
Biological Response ◽  
Enzyme Secretion ◽  
Receptor Interaction ◽  
Polymorphonuclear Leucocytes ◽  
Phosphoinositide Metabolism ◽  
Binding Condition

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.

Inositol phosphate production in response to [Arg8]vasopressin, endothelin 1, and prostaglandin F2α in rat aorta and mesenteric arteries

1992 ◽  
Vol 70 (10) ◽  
pp. 1408-1416 ◽  
Author(s):  
Angèle Parent ◽  
Paul V. Nguyen ◽  
Xiao Ping Yang ◽  
Ernesto L. Schiffrin
Keyword(s):  
Phospholipase C ◽  
Blood Vessels ◽  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Prostaglandin F2α ◽  
Rat Aorta ◽  
Mesenteric Arteries ◽  
Phosphoinositide Metabolism ◽  
Endothelin 1

Vascular tissues such as rat aorta and mesenteric arteries are extensively used experimentally for the study of cardiovascular diseases. To further our understanding of the signal transduction mechanisms involved in responses to several potent vasoconstrictors, such as [Arg8]vasopressin (AVP), endothelin 1 (ET-1), and prostaglandin F2α (PGF2α), we have investigated the time course for production of inositol monophosphate (InsP1), bisphosphate (InsP2), and trisphosphate (InsP3) in response to these agonists as well as their relative potency for phosphatidylinositol hydrolysis. Time-course studies of production of the different inositol phosphates in response to AVP and PGF2α showed an early increase after 15–30 s of stimulation. Thereafter InsP3 declined towards baseline, with a secondary increase towards steady state after 5–10 min. Rapid turnover of InsP3 was reflected by accumulation of InsP1 and InsP2 in the presence of LiCl (20 mM) to inhibit monophosphatases. After 15–30 min of stimulation, there was accumulation of the Ins(1,3,4)P3 isomer. All three agonists induced greater accumulation of InsP2 in mesenteric arteries than in thoracic aorta, suggesting that turnover of Ins(1,4,5)P3 may be faster in the former than in the latter. The accumulation of total inositol phosphates induced by maximum concentrations of ET-1 was greater than in response to AVP or PGF2α. Dose–response curves showed that the rank order of potency for stimulation of production of inositol phosphates was AVP > ET-1 > PGF2α, similar to the sensitivity of blood vessels to these agents. Comparison of responses to ET-1 and ET-3 showed that the receptors stimulated by endothelins were of the isopeptide selective ETA subtype. In conclusion, all three agonists (AVP, ET-1, PGF2α) stimulate phospholipase C activity in rat aorta and in mesenteric arteries, although with different potencies. This study demonstrates that intact blood vessels allow a detailed investigation of inositol phosphate responses to different agonists of interest in cardiovascular research.Key words: phosphoinositide metabolism, phospholipase C, inositol trisphosphate, vasoconstrictors, blood vessels.

IMPAIRMENT OF PLATELET PHOSPHOINOSITIDE METABOLISM IN PRIMARY HYPERTENSION

1987 ◽  
Author(s):  
S Koutouzov ◽  
A Remmal ◽  
P Marche ◽  
P Meyer
Keyword(s):  
Phospholipase C ◽  
Time Course ◽  
Blood Platelets ◽  
Second Messengers ◽  
Calcium Handling ◽  
Phosphoinositide Metabolism ◽  
Experimental Conditions ◽  
Spontaneously Hypertensive ◽  
Serotonin Secretion ◽  
Wistar Kyoto

Blood platelets from hypertensive patients and spontaneously hypertensive rats (SHR) display multiple abnormalities when compared with cells from normotensive controls. The major features of the modified platelet profile are an enhanced rate of adhesion/aggregation in response to many stimuli, a greater sensitivity for thrombin and adrenaline to produce increases in cytoplasmic free Ca2+, and an exaggerated release reaction. Furthermore, the resting levels of cytosolic free Ca2+ ions are specifically and constantly increased. Since phosphoinositides are involved in the stimulus-response coupling mediated by intracellular Ca2+ mobilization, the metabolism of these lipids was investigated in platelets of SHR and compared with those of normotensive Wistar-Kyoto rats (WKY). Following 32P-labelling of quiescent platelets, labeled lipids were analyzed both in platelets at rest and after thrombin stimulation. In resting platelets, the 32P associated with each of the phosphoinositides and phosphatidic acid (PA) was similar in SHR and WKY indicating that both the pool size of the various lipids and their basal turnover did not differ between the two strains. By contrast, within the first seconds after thrombin stimulation (10-60 sec), the dose-response and time-course curves of agonist-induced increase in 32P-PA were markedly shifted to the left and reached higher equilibrium levels in SHR. Since thrombin-induced 32P-PA formation is held as the most sensitive index of phospholipase C activity, our results indicate that this enzyme displays hyperreactivity in SHR (vs WKY). It is therefore likely that in SHR, the enhanced physiological responses (serotonin secretion, aggregation) that we observed under the same experimental conditions may be related to an increased formation of Phospholipase C products (inosi-toltriphosphate and diacylglycerol) which are the two second messengers responsible for internal Ca2+ mobilization and activation of protein kinase C, respectively. Therefore, these data suggest that the hypersensitivity of Phospholipase C may be involved in the overall alteration of cell calcium handling and hence in the SHR platelet responses.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

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 A comparative study of endothelin- and platelet-activating-factor-mediated signal transduction and prostaglandin synthesis in rat Kupffer cells

1992 ◽  
Vol 281 (2) ◽  
pp. 485-492 ◽  
Author(s):  
C R Gandhi ◽  
K Stephenson ◽  
M S Olson
Keyword(s):  
Prostaglandin E2 ◽  
Phospholipase C ◽  
Kupffer Cells ◽  
Inositol Phosphates ◽  
Platelet Activating Factor ◽  
Phosphoinositide Metabolism ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Hydrolysis Of ◽  
Stimulation Of

Endothelin-3 (ET-3) stimulated phosphoinositide metabolism and synthesis of prostaglandins in cultured rat Kupffer cells. ET-3-induced hydrolysis of phosphoinositides was characterized by the production of various inositol phosphates and of glycerophosphoinositol. The mechanism of ET-3-stimulated metabolism of phosphoinositides and synthesis of prostaglandins appeared to be distinct from the effect of platelet-activating factor (PAF) on these processes described previously [Gandhi, Hanahan & Olson (1990) J. Biol. Chem. 265, 18234-18241]. On a molar basis ET-3 was significantly more potent than PAF in stimulating phosphoinositide metabolism, e.g. ET-3-induced hydrolysis of phosphoinositides occurred at 1 pM, whereas PAF was ineffective at concentrations less than 1 nM. Upon challenging Kupffer cells with both ET-3 and PAF, an additive stimulation of phosphoinositide metabolism was observed, suggesting that the actions of these factors may be exerted on separate phosphoinositide pools. Treatment of Kupffer cells with pertussis toxin resulted in an inhibition of ET-3-induced phospholipase C activation; in contrast, cholera toxin treatment caused potentiation of ET-3-stimulated phospholipase C activity. Both toxins, however, inhibited PAF-stimulated phospholipase C activity. The present results suggest that the stimulatory effects of ET-3 and PAF on the phosphodiesteric metabolism of phosphoinositides in Kupffer cells require different guanine-nucleotide-binding proteins. Furthermore, the effects of bacterial toxins on ET-3- and PAF-induced phosphoinositide metabolism were not mediated by cyclic AMP. ET-3-induced metabolism of phosphoinositides was inhibited completely in Kupffer cells pretreated with ET-3, suggesting homologous ligand-induced desensitization of the ET-3 receptors. In contrast, similar experiments using PAF showed only a partial desensitization of subsequent PAF-induced phosphoinositide metabolism. In contrast to the increased production of prostaglandins E2 and D2 observed upon stimulation of Kupffer cells with PAF, ET-3 stimulated the biosynthesis of prostaglandin E2 only. Consistent with their additive effects on phosphoinositide metabolism, PAF and ET-3 exhibited an additive stimulation of the synthesis of prostaglandin E2.

scholarly journals The role of phosphoinositide metabolism in signal transduction in secretory cells

1988 ◽  
Vol 139 (1) ◽  
pp. 135-150 ◽  
Author(s):  
J. W. Putney
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Regulatory Proteins ◽  
Secretory Cells ◽  
Phosphoinositide Metabolism ◽  
Surface Receptors ◽  
Hydrolysis Of ◽  
Stimulation Of ◽  
Plasma Membrane Phospholipid

Activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration, due to the release, or mobilization, of intracellular Ca2+ stores and to the entry of Ca2+ from the extracellular space. Stimulation of these same receptors also results in the phospholipase-C-catalysed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. Analogous to the adenylyl cyclase signalling system, receptor-mediated stimulation of phospholipase C also appears to occur through one or more intermediary guanine nucleotide-dependent regulatory proteins. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. There is strong evidence that Ins(1,4,5)P3 stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of Ins(1,4,5)P3 are terminated by its metabolism through two distinct pathways. Ins(1,4,5)P3 is dephosphorylated by a 5-phosphatase to Ins(1,4)P2; alternatively, Ins(1,4,5)P3 can also be phosphorylated to Ins(1,3,4,5)P4 by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; a recent proposal that Ins(1,4,5)P3 by emptying an intracellular Ca2+ pool, secondarily elicits Ca2+ entry will be considered. This review summarizes our current understanding of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

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