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PubMed1989

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release.

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.

Arachidonic Acids↗

PubMed1989

Platelet-activating factor-induced phosphoinositide metabolism in differentiated U-937 cells in culture.

Human monocytic leukemic U-937 cells, when differentiated with dimethylsulfoxide to macrophage-like state, express receptors for platelet-activating factor (PAF). In the differentiated U-937 cells, PAF induced hydrolysis of phosphoinositides and synthesis of inositol phosphates. PAF-induced production of inositol phosphates was rapid, concentration-dependent and was inhibited by a receptor antagonist CV3988, indicating that it was mediated via a specific receptor. In fura-2-loaded, differentiated U-937 cells, PAF induced immediate and concentration-dependent calcium mobilization [( Ca++]i) that was inhibited by CV3988, but not by calcium channel blockers. Addition of an increasing concentration of calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, to the medium inhibited a large fraction (approximately 75%) of PAF receptor-induced [Ca++]i mobilization thus suggesting the majority of [Ca++]i mobilization was originated from extracellular milieu and a small portion (approximately 25%) was originated from intracellular sources. The inositol phosphate production induced by PAF, however, was independent from the extracellular calcium and was not inhibited by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Neither [Ca++]i mobilization or phosphoinositide metabolism in U-937 cells was sensitive to treatment of pertussis toxin, but both types of effects were sensitive to treatment by an inhibitor of phospholipase C, manoalide. These results suggest that in differentiated U-937 cells PAF receptor is coupled through a pertussis toxin-insensitive guanine nucleotide binding protein to a phosphoinositide specific phospholipase C. Inositol-trisphosphate, and possibly diacylglycerol, could be the intracellular messengers for PAF receptor in U-937 cells.

Calcium↗

PubMed1989

[Phosphoinositide turnover and Ca2+ mobilization during phagocytosis in cultured chick retinal pigment epithelial cells].

Ca2+ mobilization and phosphoinositide turnover were examined during phagocytosis of latex particles in cultured chick retinal pigment epithelial cells (RPE cells). Ca2+ influx into cells and Ca2+ efflux from the cells were enhanced by about 1.5-fold and 3-fold compared to control cells, respectively. A high content of Ca2+ in RPE cells has been reported. Therefore, the Ca2+ efflux observed here may be a reflection of Ca2+ release into cytosol from the intracellular storage site(s). In [3H] inositol-prelabeled RPE cells, addition of latex particles elicited decreases in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol with the concomitant formation of inositol phosphates including inositol trisphosphate, indicating the hydrolysis of phosphoinositides by phospholipase C. These results suggest that phosphoinositide turnover may be closely coupled with Ca2+ mobilization during phagocytosis in RPE cells.

Animals↗

PubMed1989

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism.

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.

Arachidonic Acid↗

PubMed1987

Histamine-stimulated phosphoinositide hydrolysis in developing rat brain.

Histamine stimulation of phosphoinositide breakdown has been studied in brain cortex from developing rats by measuring the accumulation of [3H]inositol monophosphate in lithium-treated slices prelabeled with [3H]inositol. Histamine stimulated phosphoinositide hydrolysis as early as day 3 after birth. The maximal response increased about 3-fold up to day 15 and remained essentially at the same level until adulthood. This developmental pattern contrasted with the 8-fold increase in [3H]mepyramine binding to histamine H1 receptors which takes place from the first to the end of the fourth week of life. Nonlinear regression analysis of concentration-effect curves for histamine generated at 6, 10, and 15 days of age revealed the presence of two components in the response, whereas a single component was found in adults. A high potency component showed EC50 values increasing from 6.4 microM at day 6 to 26.5 microM at day 15, and its contribution to the maximal response augmented from 65.8% to 78.9% during the same period. In 6-day-old animals, the H1 receptor agonists N alpha-methylhistamine and N alpha,N alpha-dimethylhistamine produced single-component responses with maximal effects similar to that estimated for the high potency component for histamine, and their relative potencies were consistent with an H1 receptor-mediated response. Inhibition constants for mepyramine, calculated from the parallel displacement of the concentration-response curves for N alpha-methylhistamine and the high potency component for histamine (6.3 nM and 10.8 nM, respectively) closely agreed with the reported affinity of rat brain H1 receptors for this compound. The low potency component of the response (EC50 = 175 microM at day 6 and EC50 = 260 microM at day 15) did not involve histamine-H2, serotonin 5-HT2, alpha 1-adrenergic, or muscarinic receptors. Pretreatment of 6-day-old cortical slices with the irreversible antagonist phenoxybenzamine, at concentrations that completely abolish [3H]mepyramine binding, left about 25% of the response to 1 mM histamine unaffected. This portion of the response, which was, moreover, insensitive to 1 microM mepyramine, was of a magnitude similar to that of the low potency component. Thus, at early stages of rat brain development, histamine induces phosphoinositide hydrolysis in part through a mechanism different from activation of H1 receptors. The loss of histamine potency and efficacy at H1 receptors throughout development is discussed and the natural occurrence of H1 receptor subsensitivity suggested.

Aging↗

PubMed1988

Phosphoinositide content of erythrocyte membranes in cystic fibrosis.

Phosphoinositide content was measured in erythrocyte membranes from 11 patients with cystic fibrosis (CF) and from 12 control subjects to determine whether altered levels of phosphatidylinositol-4-phosphate (Ptdlns4P) or phosphatidylinositol-4,5-bisphosphate (Ptdlns(4,5)P2) are responsible for the decrease in Ca2+-adenosine triphosphatase (Ca2+-ATPase) activity in this disorder. Isolated membranes were extracted with an acidified chloroform-methanol solvent system. The recovered lipids were separated by one-dimensional thin-layer chromatography and quantified with a colorimetric assay for phosphorus. The results are expressed in molar percent, moles of phosphoinositide times 100 divided by the total number of moles of phospholipid per membrane. The means +/- SEM of Ptdlns(4,5)P2, Ptdlns4P, and phosphatidylinositol (Ptdlns) in CF membranes (1.07 +/- 0.18, 1.02 +/- 0.22, and 2.32 +/- 0.36 molar percent, respectively) were indistinguishable from controls (0.91 +/- 0.14, 0.85 +/- 0.12, and 2.21 +/- 0.32 molar percent, respectively) (P greater than 0.20 for all three pairs). The accuracy of quantitative recovery throughout the procedure was determined by adding a radioactive internal standard, L-3-phosphatidyl[2-3H]inositol to 10 membrane preparations. Although quantitative recoveries, as determined by percent radioactivity recovered, varied from 54% to 92%, mean Ptdlns(4,5)P2, Ptdlns4P, and Ptdlns levels appropriately corrected from tracer loss were still indistinguishable between the two groups. We conclude that absolute phosphoinositide levels are not altered in cystic fibrosis erythrocyte membranes and that the differences in Ca2+-ATPase activity cannot be explained on this basis.

Cystic Fibrosis↗

PubMed1987

Identification of polyphosphoinositide-specific phospholipase C and its resolution from phosphoinositide-specific phospholipase C from human platelet extract.

Sonicated platelet extract, obtained after high-speed centrifugation, was fractionated by phosphocellulose chromatography, and assayed for phosphodiesteric cleavage of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2) using (i) single bilayer vesicles from total platelet membrane lipids and (ii) single bilayer vesicles from individual pure lipids. When total platelet lipids were used as the substrate, two clear-cut, but incompletely resolved, peaks of phosphoinositide-specific phospholipase C (PI-PLC) were detected; one of these activities hydrolysed PIP alone, whereas the second peak hydrolysed all three phosphoinositides. When pure phosphoinositides were used as the substrate, three distinct peaks of PI-PLC were observed; two of these peak activities correspond to the peaks when assays were conducted with total platelet membrane lipids. The third peak of activity hydrolysed PIP2 and PIP, but not PI. These results clearly show that there exist PIP/PIP2-specific PLC enzymes that do not act on PI, and escaped detection thus far because of the general practice of using PI as the substrate to monitor PI-PLC activities. I propose that these PIP/PIP2-specific PLC enzymes are the real candidates that respond to agonist occupation of receptors on the cell surface to generate the two putative second messengers inositol triphosphate and diacylglycerol.

Blood Platelets↗

PubMed1988

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine.

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.

Adenine↗

PubMed1988

Insulin and oxytocin effects on phosphoinositide metabolism in adipocytes.

The effects of hormones on phosphoinositide metabolism were examined in rat adipocytes prelabeled with 32Pi or [3H]inositol. Oxytocin and vasopressin produced large decreases in labeled polyphosphoinositides and increases in phosphatidic acid and inositol phosphates, whereas insulin was without effect, although it stimulated lipogenesis from glucose. Likewise, insulin did not elevate 1,2-diacylglycerol measured chemically by high pressure liquid or thin-layer chromatography in fat cells or pads. It also did not increase the radioactivity in 1,2-diacylglycerol in ghosts prepared from fat cells previously labeled with [3H]arachidonic acid, although oxytocin and vasopressin increased this. It is therefore concluded that insulin does not stimulate the breakdown of polyphosphoinositides to yield 1,2-diacylglycerol and inositol phosphates in adipocytes and that the insulin-like actions of oxytocin must be due to other changes. Insulin induced small, but significant and equal increases (40% at 30 min) in the incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in adipocytes. The effects were not dependent upon glucose and were not evident before 15 min. Oxytocin also produced large increases in the labeling of the three phosphoinositides. Insulin stimulated the incorporation of [3H]glycerol into the three phosphoinositides and also phosphatidic acid, phosphatidylserine, and phosphatidylethanolamine by 50-100% in cells incubated without glucose. No changes in the labeling of glycerol 3-phosphate, lysophosphatidic acid, phosphatidylcholine, and triacylglycerol were detected, and there was a small increase (30%) in 1,2-diacylglycerol labeling. It is concluded that insulin increases the synthesis of phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylethanolamine, and phosphatidylserine in fat cells partly by stimulating a reaction(s) located between glycerol 3-phosphate and phosphatidic acid in the biosynthetic pathway.

Adipose Tissue↗

PubMed1988

Role of phosphoinositide-derived second messengers in mediating anti-IgM-induced growth arrest of WEHI-231 B lymphoma cells.

Anti-IgM irreversibly inhibits the growth of WEHI-231 B lymphoma cells and induces phosphoinositide hydrolysis--producing diacylglycerol, which activates protein kinase C, inositol 1,4,5-trisphosphate, which induces the release of calcium from intracellular storage sites into the cytoplasm, and other inositol polyphosphates. The roles of two of the possible second messengers, cytoplasmic free calcium and diacylglycerol, in mediating the action of anti-IgM on WEHI-231 cells were assessed by elevating [Ca2+]i with ionomycin and by activating protein kinase C with phorbol 12,13-dibutyrate (PdBu). The combination of 250 nM ionomycin and 4 to 7 nM PdBu was found to cause growth arrest and cell volume decrease responses in WEHI-231 cells which were similar to those caused by anti-IgM, although clearly slower. Both anti-IgM and the combination of mimicking reagents induced growth arrest of WEHI-231 cells in the G1 phase of the cell cycle. In both cases, this growth arrest was mitigated by addition of bacterial LPS. Moreover, 250 nM ionomycin plus 4 to 7 nM PdBu did not inhibit the growth of two other murine B lymphoma cell lines, each of which did exhibit increased phosphoinositide hydrolysis but not growth arrest in response to anti-Ig. Taken together, these results suggest that ionomycin and PdBu, at the concentrations used, did not inhibit WEHI-231 growth by general toxicity, but rather by mimicking the effects of the natural second messengers generated from Ag receptor cross-linking. Thus, the phosphoinositide-derived second messengers Ca2+i and diacylglycerol are capable of playing important roles in mediating the action of anti-IgM on WEHI-231 B lymphoma cells. However, the response of WEHI-231 cells to anti-IgM could not be fully reproduced with ionomycin and phorbol diester. These results suggest that another second messenger induced by anti-IgM may also play an important role in mediating the growth arrest of these cells.

Animals↗

PubMed1988

Homologous desensitization of thrombin-induced phosphoinositide breakdown in hamster lung fibroblasts.

Activation of phosphoinositide breakdown is thought to be an important signaling pathway involved in the mitogenic effects of alpha-thrombin in Chinese hamster lung fibroblasts. We have previously shown that the initial strong stimulation of inositol phosphate formation induced by thrombin in quiescent hamster cells (CCL39 line) is rapidly attenuated. We now report that this desensitization of phospholipase C to thrombin 1) is independent of protein kinase C activation, because thrombin-induced desensitization normally occurs in cells that have been depleted in protein kinase C by a prolonged treatment with a phorbol ester, and 2) is even independent of phosphoinositide hydrolysis because the desensitization still occurs, although at a lesser degree, at 4 degrees C, in the absence of any phospholipase C activity. Furthermore, phospholipase C desensitization to thrombin is homologous. It does not affect the response to thrombin-free serum or the direct activation by A1F-4 of the GTP-binding protein (G-protein) coupled to phospholipase C. We therefore conclude that the desensitization of phospholipase C to thrombin does not result from an impairment of the G-protein-phospholipase C complex, or from a depletion in phosphoinositides, but rather from a modification of thrombin receptors leading to their uncoupling from G-protein. This modification is slowly reversible because, upon thrombin removal, a prolonged incubation (approximately 2 h) restores responsiveness of the cells to thrombin. Although the desensitization seems to depend on thrombin receptor occupancy, it cannot be accounted for by an internalization of the occupied receptors, because it is not blocked at 4 degrees C. The exact mechanism underlying this homologous desensitization of thrombin receptors remains to be elucidated.

Aluminum↗

PubMed1988

Phosphoinositides and cicletanine.

In vascular smooth muscle cells, the activation of histaminergic receptors of the H1 subtype induces an increase in cytosolic free calcium. This has been shown to involve the metabolism of inositol-containing lipids. Since according to its pharmalogical characteristics, cicletanine could be considered as an H1 antagonist, it was of interest to check whether the drug behaved as such with regard to phosphoinositide metabolism. Phosphoinositide metabolism was studied by measuring the 32P labelling of phosphatidylinositol (PI) and of its phosphorylated derivatives in cultured smooth muscle cells isolated from guinea-pig aorta and incubated in the presence or absence of various histaminergic agonists and/or antagonists. The results clearly demonstrate that the increase in 32P-PI could be selectively induced by treatment of cells with histamine or 2-pyridylethylamine dihydrochloride (2-PEA) (an H1 agonist) but not with the H2 agonist dimaprit. In addition, histamine- or 2-PEA-induced 32P-PI increase was inhibited by pretreatment of cells with the H1 antagonist mepyramine, whereas the H2 antagonist cimetidine was without effect. These data demonstrate that the histamine-induced 32P-PI increase is mediated through H1 receptors. Under the same experimental conditions, cicletanine was as effective as mepyramine with an IC50 value of 10(-6) M. The development of tension in arterial smooth muscle is under the control of variations of calcium concentration in the intracellular space. Therefore, the blockade by cicletanine of histamine-enhanced phosphoinositide turnover may participate in the mechanism by which the drug exerts its antihypertensive action.

Angiotensin II↗

PubMed1988

Lysergic acid diethylamide and 2,5-dimethoxy-4-methylamphetamine are partial agonists at serotonin receptors linked to phosphoinositide hydrolysis.

Based on electrophysiological, radioligand binding, and behavioral studies in laboratory animals, it is generally believed that the psychotomimetic effects of the phenethylamine and indolealkylamine hallucinogens are mediated by central serotonin (5-HT) receptors, in particular the 5-HT-2 subtype. Agonist-stimulated phosphoinositide hydrolysis was utilized to determine the potency and efficacy of racemic 1-(2,5)-dimethoxy-4-methyl-phenyl)-2-aminopropane (DOM), and d-lysergic acid diethylamide (LSD) at the 5-HT-2 receptor in rat cerebral cortex and the 5-HT-1c receptor in rat choroid plexus. Both DOM and LSD stimulated phosphoinositide hydrolysis in cerebral cortex. These effects were blocked by the 5-HT-2 antagonists, ketanserin and spiperone, but not by antagonists of muscarinic, alpha-1 adrenergic or histaminergic receptors. The maximum responses of DOM and LSD, respectively, were 76% and 25% of the maximum response to 5-HT. However, LSD was 500 times more potent than was racemic DOM. Consistent with a partial agonist effect, LSD partially blocked the effect of 5-HT, with a maximal inhibition equivalent to the intrinsic activity of LSD alone. In choroid plexus, DOM and LSD stimulated phosphoinositide hydrolysis and both responses were blocked by mianserin and less effectively by spiperone. The maximum effect of DOM was 67% of that of 5-HT, whereas the maximum effect of LSD was only 34% of the maximum response of 5-HT. LSD was 50 times more potent than was racemic DOM. LSD partially antagonized the effect of 5-HT in the choroid plexus, consistent with a partial agonist effect at the 5-HT-1c receptor in this tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

DOM 2,5-Dimethoxy-4-Methylamphetamine↗

PubMed1988

Pharmacological characterization of serotonin-stimulated phosphoinositide turnover in brain regions of the immature rat.

The effects of serotonin (5-HT) and related agonists and antagonists on phosphoinositide turnover have been investigated in several brain regions of the immature rat. In the presence of LiCl, 5-HT caused a marked increase in total [3H]inositol phosphate levels in cortical (maximal effect + 420%, EC50 = 7 microM) and to a lesser extent in hippocampal and striatal slices prepared from the immature (8-day-old) rat; the cortical 5-HT-induced phosphoinositide response was tetrodotoxin resistant. The magnitude of the increase in the cortical phosphoinositide response caused by 5-HT was maximal at 1 day postnatal and progressively declined to reach 6% of this maximal response in the adult. After incubation of immature (8-day-old) rat cortical slices for 2.5 min with 5-HT (in the absence of LiCl), inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate levels increased about 2-fold. A variety of 5-HT2 or mixed 5-HT1/5-HT2 agonists stimulated total [3H]inositol phosphate formation in the immature rat cortex and hippocampus with a rank order of potency [alpha(+)-methyl-5-HT greater than quipazine greater than MK 212 greater than 5-HT] which resembles their potencies at the 5-HT2 binding site. In contrast, the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin, the 5-HT1B agonists 1-(m-trifluoromethylphenyl)piperazine and 1-(m-chlorophenyl)-piperazine and the 5-HT3 agonist 2-methyl-5-HT were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Phosphoinositide interconversion in thrombin-stimulated human platelets.

Stimulation of platelets and other secretory cells by agonists results in the degradation of phosphoinositides by phospholipase C. Kinetic studies suggest that hydrolysis of phosphatidylinositol 4,5-diphosphate (PI-4,5-P2) is an initial event in this process. Platelets contain much larger amounts of phosphatidylinositol (PI) than PI-4,5-P2, and approximately 50% of total phosphoinositides are degraded upon stimulation. We have investigated whether degradation of PI occurs by direct phospholipase C hydrolysis or by phosphorylation to PI-4,5-P2 followed by phospholipase C action on the latter compound. When platelets are incubated for 3 min with 32Pi prior to stimulation, the phosphoinositides are labeled to different specific activities. Under these nonequilibrium conditions, the time course of change in specific activity reflects turnover. The rise in specific activity of phosphatidylinositol 4-phosphate (PI-4-P) is similar in stimulated and unstimulated cells, indicating that there is little increase in the conversion of PI to PI-4-P during thrombin stimulation. In addition, the specific activity of the 4-phosphate in PI-4-P during thrombin stimulation is less than both the 5-phosphate of PI-4,5-P2 and the phosphate group of phosphatidic acid, indicating that the 4-phosphate moiety is not labeled to equilibrium with ATP. This finding is inconsistent with a rapid flux of PI via PI-4-P to PI-4,5-P2 during thrombin stimulation, in which case the 4-phosphate would be at maximum specific activity. We, therefore, conclude that the bulk of PI breakdown that occurs in thrombin-stimulated platelets occurs via direct phospholipase C hydrolysis of PI.

Blood Platelets↗

PubMed1985

Insulin and epidermal growth factor do not affect phosphoinositide metabolism in rat liver plasma membranes and hepatocytes.

Recent studies with viral oncogene tyrosine kinases have suggested that these kinases may phosphorylate phosphoinositides and diacylglycerol. Since the receptors for insulin and epidermal growth factor (EGF) also possess tyrosine kinase activity, we have investigated possible effects of insulin and EGF on phosphoinositide metabolism in rat liver plasma membranes and rat hepatocytes. In plasma membranes prepared from rats injected 18 h prior with [3H]myo-inositol or incubated with [gamma-32P]ATP, phosphatidylinositol-4-P and phosphatidylinositol-4,5-P2 were formed, but there were no effects of either insulin or EGF although these agents stimulated protein tyrosine phosphorylation. In hepatocytes incubated with [3H]myo-inositol, label was incorporated into phosphatidylinositol, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2, but there was no effect of insulin. Incubation of hepatocytes with [3H]myo-inositol plus insulin or EGF for 2 h also did not alter the formation of [3H]myo-inositol-1,4,5-P3 from [3H]phosphatidylinositol-4,5-P2 induced by vasopressin. These findings suggest that the tyrosine kinase activity of liver insulin and EGF receptors is not important in phosphoinositide formation.

Adenosine Triphosphate↗

PubMed1985

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells.

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.

Astrocytoma↗

PubMed1985

[Effects of aminoglycosides on phosphoinositide metabolism in renal proximal tubules. In vivo and in vitro studies].

Phosphoinositides play a major role in the interaction between aminoglycosides and renal epithelial cells. We therefore investigated both in vivo and in vitro, in isolated proximal tubules and in various parts of the rabbit kidney, the influence of neomycin, gentamicin and amikacin on the phosphoinositide metabolism. This was carried out by measuring the 32p incorporation into phosphatidylinositol 4,5-bisphosphates (PI-P2) and phosphatidylinositol 4-phosphates (PI-P). In aminoglycoside-treated animals, compared to untreated controls, the 32 p-PI-P2 and 32p-PI-P levels were decreased and increased respectively in proximal tubules and in cortical areas but these levels were unchanged in papilla and in medulla. Similar variations could be obtained after exogenous addition of aminoglycosides. PI-P2 is a key membrane component in the regulation of both the Ca2+ transport and the intracellular Ca2+ mobilization. Thus, the aminoglycoside-induced modifications of the phosphoinositide metabolism may be considered as a primary event in the drug toxicity which may result from alterations in the cell calcium homeostasis.

Amikacin↗