Biomedical subjects

L A Sklar

Publications and source records attributed to L A Sklar.

At least 19 recordsLinked to original sources

PubMed1992

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.

Antigens, CD↗

PubMed1992

Heparin enhances the interaction of infective Leishmania donovani promastigotes with mouse peritoneal macrophages. A fluorescence flow cytometric analysis.

Human visceral leishmaniasis results from the infection of macrophages by the protozoan parasite Leishmania donovani. Both forms of the parasite, the extracellular promastigote and the obligate intracellular amastigote, require cell surface molecules to ensure their recognition and uptake by the host cell, the macrophage. We have proposed previously that the heparin-binding protein on the surface of promastigotes is an adhesion molecule. The present report provides experimental evidence to support this hypothesis. Fluorescence flow cytometry using FITC-heparin was employed to study the heparin-binding protein of L. donovani promastigotes and amastigotes. We demonstrate the presence of the heparin-binding protein on the surface of amastigotes and document the heparin specificity of the binding protein for both forms of the parasite. Two-color fluorescence analysis was performed to compare R-PNA reactivity and FITC-heparin binding during the parasite's 7-day growth curve. Using this strategy we show that the expression of heparin binding activity coincides with the differentiation of the noninfective promastigote into the infective metacyclic from of the parasite. Macrophages that were challenged for 30 min with heparin-treated, FITC-labeled parasites became 2.82-fold more fluorescent than their counterparts which were exposed to non-heparin-treated FITC-labeled promastigotes. Finally, using Kolmogorov-Smirnov analysis we show that the adhesion of promastigotes to mouse peritoneal macrophages is significantly enhanced in the presence of 3.3 microM heparin. The experiments described in the present report provide evidence for the hypothesis that L. donovani's heparin-binding protein is a virulence factor that functions as an adhesion molecule in the parasite-macrophage interaction.

Animals↗

PubMed1992

Reversal of inhibitory pathways in neutrophils by protein kinase antagonists: a rational approach to the restoration of depressed cell function?

Neutrophil functions are sensitive to both stimulatory and inhibitory pathways. For example, the endogenous hormones histamine, prostaglandin E1, adenosine, and catecholamine were found to inhibit the oxidant responses of human neutrophils by formyl peptide to 6.2, 16.8, 11.4, and 15.4%, respectively, of the initial response. The inhibition of cell function is mimicked by dibutyryl cAMP and forskolin, consistent with a pathway involving cAMP and an A kinase. Because of likely roles of kinases in both stimulatory and inhibitory pathways, we evaluated the potential for regulating either pathway by kinase inhibitors. Preincubation of intact neutrophils with membrane-permeable but nonspecific protein kinase antagonists blocked the isoproterenol-mediated inhibition of superoxide generation. The isoquinoline sulfonamides H-7, H-8, and H-9 at 100 microM reversed inhibition to 60.1, 66.6, and 70.9%, respectively, of the response of control cells. H-9 also antagonized the inhibition of superoxide production induced by other agents that regulate intracellular cAMP (prostaglandin E1, histamine, adenosine, forskolin, and dibutyryl cAMP). A synthetic peptide used as a specific but impermeable protein kinase A antagonist restored superoxide production inhibited by isoproterenol and cAMP up to 70% in electroporated cells. A small number of proteins are targets of cAMP-dependent phosphorylation in electroporated cells, and phosphorylation is inhibited in the presence of the peptide inhibitor. Taken together, these data show that a peptide inhibitor and isoquinoline sulfonamides reverse the inhibition of the respiratory burst in neutrophils evoked by the inhibitory pathways. Drugs that reverse the effect of endogenous inhibitors of neutrophil activation (by restoring cell function) have important therapeutic implications in restoring cell functions in patients whose cell functions are depressed under physiological conditions.

Cell Membrane Permeability↗

PubMed1991

Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils.

The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1991

Differential amplification of antagonistic receptor pathways in neutrophils.

In human neutrophils approximately 500 ligand-occupied beta-adrenergic receptors almost completely inhibit the superoxide production generated by at least 50,000 formyl peptide receptors, suggesting a massive amplification of the inhibitory receptor signals. We estimated two stages of amplification. In the first stage, we quantitated the ligand-dependent GTPase activities. For the formyl peptide receptor, the number of phosphates released from GTP in the presence of the saturating ligand is relatively modest, i.e. approximately 1/min/receptor, even though there are approximately 200 Gn (Gi type II) proteins/formyl peptide receptor in neutrophil membranes. In contrast, the number of GTPs cleaved in the presence of a beta-adrenergic agonist is approximately 100/min/beta-adrenergic receptor, and there are about 700 Gs/beta-adrenergic receptor in membranes. Thus the signal of the beta-adrenergic receptor is already massively amplified at the G protein, whereas the signal of the formyl peptide receptor is likely to be amplified at subsequent steps. New kinetic evidence from intact cells and biochemical evidence from permeabilized cells is provided that the second messenger of the inhibitory pathway is cAMP. To estimate the amplification of this step, we determined the cAMP concentration necessary to maximally inhibit superoxide anion production of formyl peptide-stimulated electropermeabilized cells, and we compare these concentrations to previously determined values of cAMP production in neutrophils. We conclude that each receptor may generate up to 10,000 molecules of cAMP.

Cell Membrane↗

PubMed1991

Real-time analysis of the assembly of ligand, receptor, and G protein by quantitative fluorescence flow cytometry.

We describe a general approach for the quantitative analysis of the interaction among fluorescent peptide ligands (L), receptors (R), and G proteins (G) using fluorescence flow cytometry. The scheme depends upon the use of commercially available fluorescent microbeads as standards to calibrate the concentration of fluorescent peptides in solution and the receptor number on cells in suspension. We have characterized a family of fluoresceinated formyl peptides and analyzed both steady-state and dynamic aspects of ligand formyl peptide-receptor interactions in digitonin-permeabilized human neutrophils. Detailed receptor-binding studies were performed with the pentapeptide N-formyl-Met-Leu-Phe-Phe-Lys-fluorescein. Equilibrium studies showed that GTP [S] caused a loss of binding affinity of approximately two orders of magnitude, from approximately 0.04 nM (LRG) to approximately 3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from approximately 1 x 10(-3) s-1 (LRG) to approximately 1 x 10(-1) s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (approximately 3 x 10(7) s-1 M-1) were statistically indistinguishable and close to the diffusion limit. In the presence of guanine nucleotide (LR), the kinetic data were adequately fit by a single-step reversible-binding model. In the absence of guanine nucleotides, not all receptors have rapid access to G to form the LRG ternary complex. Mathematically, those R that have rapid access to G are either precoupled to R or the association of G with R is fast compared to the association of L with R. The physiological consequences of coupling heterogeneity are discussed.

Amino Acid Sequence↗

PubMed1991

Kinetics of N-formyl peptide receptor up-regulation during stimulation in human neutrophils.

The kinetics of receptor up-regulation was examined in isolated neutrophils and in whole blood by flow cytometry during cell activation. Stimulation of neutrophils prepared without exposure to LPS with chemoattractants induced fast up-regulation of N-formyl peptide receptors and C receptor type 3 (CR3). Biphasic N-formyl peptide binding curves were detected for saturating concentrations of N-formyl peptide at 37 degrees C. The bulk of the rapid binding during the first 30 to 60 s is attributed to already expressed binding sites whereas the slow binding over the next 3 to 4 min represents a time course of receptor up-regulation. Support for this interpretation comes from conditions under which the number of binding sites and the progress of the binding curves were affected. Cells treated with LPS, which caused expression of internal N-formyl peptide receptors, exhibited rapid, monophasic binding curves with increased total binding. In LPS-untreated, calcium-depleted cells, N-formyl peptide receptor up-regulation was inhibited and rapid, monophasic binding to a smaller total number of expressed sites was observed. Cytochalasin B enhanced the total number of available N-formyl peptide receptors in LPS-untreated but not LPS-treated cells. In both cases binding was rapid and monophasic suggesting that receptors were either fully or rapidly up-regulated. Although not studied in real-time, C receptor type 3 up-regulation was similar to N-formyl peptide receptor up-regulation in response to LPS, or stimulation by N-formyl peptide, C product C5a, leukotriene B4, and platelet-activating factor in isolated cells and in whole blood. After stimulation with formyl peptide, LPS, or C product 5a, the release of vitamin B12-binding protein paralleled up-regulation of receptors. These data indicate that untreated cells up-regulate N-formyl peptide receptors during cell response at a rate of approximately 10,000/min in a calcium-dependent manner whereas LPS-treated cells already express the bulk of their receptors. In cytochalasin B-treated, degranulating cells 30,000 to 50,000 receptors were up-regulated within a minute.

Calcium↗

PubMed1991

Conformational dynamics of the formyl peptide receptor: a prototype for studies of receptor dynamics and binding pocket structure.

We have used spectrofluorometric and flow cytometric techniques to examine the interactions of formyl peptide ligands (L) with their cell surface receptors (R). Kinetic studies suggest that L binds to R at a diffusion limited rate and that R undergoes rapid transitions involving three states (LR, LRG, the ternary complex of L and R with the G protein, and a desensitized receptor "LRX" which forms within seconds) prior to internalization. A spectroscopic analysis of the interaction between L and R show that the binding pocket of R is large enough to contain no more than 6 amino acids and that a fluorescein-labelled pentapeptide is quenched upon binding to R. We hypothesize that histidine 90 (putatively located in the extracellular loop connecting the second and third transmembrane domains) protonates L and quenches the probe. New technology will extend the analysis of structure and dynamics to low affinity peptide receptors of living biological systems. Such technology will have implications in the design of peptidomimetic ligand and drug molecules.

Animals↗

PubMed1990

Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils?

Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated [32P]PIP3 levels decayed toward initial values. However, recovery of [32P]PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of [32P] PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

Actins↗

PubMed1990

Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi.

Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.

Adenosine Diphosphate Ribose↗

PubMed1990

Neutrophil activation by inflammatory microcrystals of monosodium urate monohydrate utilizes pertussis toxin-insensitive and -sensitive pathways.

The activation of leukocytes by particulates is a critical event in certain inflammatory syndromes, including acute gout associated with microcrystals of monosodium urate monohydrate. In this study we have evaluated mechanisms of human neutrophil activation by urate crystals. Both N-formyl-nor-leu-leu-phe-nor-leu-tyr-lys and uncoated urate crystals (0.5 to 5 mg/ml) but not urate crystals coated with human low density lipoprotein (which suppresses crystal-induced neutrophil responsiveness), stimulated pertussis toxin (PT)-sensitive GTPase activity in purified preparations of human neutrophil membranes. Hydroxyapatite crystals (up to 5 mg/ml) were inactive. Pretreatment of neutrophil membranes with cholera toxin also inhibited crystal-induced and formylated peptide-induced GTPase activity. Pretreatment of whole neutrophils with PT resulted in nearly complete inhibition of formylated peptide-induced cytosolic calcium mobilization, release of superoxide and release of the azurophil granule constituent alpha-mannosidase. In contrast, identical pretreatment of whole neutrophils with PT only partially suppressed urate crystal-induced alpha-mannosidase and superoxide release and failed to inhibit crystal phagocytosis and increases in cytosolic free calcium. Mechanisms of neutrophil activation by monosodium urate crystals appear to be heterogeneous in comparison with activation by formylated peptides and there is no absolute requirement for PT-sensitive membrane G proteins in neutrophil responsiveness to urate crystals.

Cell Membrane↗

PubMed1990

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance.

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.

Amino Acid Sequence↗

PubMed1990

Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein.

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.

Acute-Phase Proteins↗

PubMed1990

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model.

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.

Cell Adhesion↗

PubMed1990

Kinetics and amplification in neutrophil activation and adaptation.

Neutrophil activation and adaptation are beginning to be understood in quantitative and mechanistic detail. Neutrophil responses to chemoattractants involve cell surface receptors, guanine nucleotide binding (G) proteins and intracellular second messengers which are generated within a few seconds of the presentation of the ligand. Novel real-time fluorescent methods have made it possible to examine the dynamics of assembly and disassembly of receptors and G proteins during cell activation and to model, by computer, the complex sequence of ligand-receptor events. Rapid amplification cascades of signals are observed in both stimulatory and inhibitory receptor pathways. The adaptation of neutrophils involves uncoupling between receptors and G proteins, both by sequestering receptors from the transduction sequence and by reducing the activatibility of the G proteins.

Adaptation, Physiological↗

PubMed1990

An HPLC procedure for separating polyphosphoinositides on hydroxylapatite.

We describe a method for separation of several phosphoinositides by high performance liquid chromatography (HPLC) for the purposes of identification, analysis, and possible purification of cell phospholipid extracts. The phosphoinositides were separated on an hydroxylapatite column using a solvent system consisting of tetrahydrofuran-ethanol-water with a gradient of triethylamine phosphate ranging from 1 to 100 mM. Increasing triethylamine phosphate concentrations over a series of isocratic steps resulted in the resolution of [3H]phosphoinositide standards (phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate) and their lyso-derivatives. The eluted peaks were collected and quantitated using scintillation counting, and the samples of the eluates were subjected to further analysis by thin-layer chromatography to verify their integrity and identity.

Chromatography, High Pressure Liquid↗

PubMed1989

Oscillating actin polymerization/depolymerization responses in human polymorphonuclear leukocytes.

Leukotriene B4 and platelet-activating factor induced a rapidly oscillating actin polymerization/depolymerization response in human polymorphonuclear leukocytes. N-Formylpeptides were deficient in the ability to induce these oscillations. Flow cytometric analysis of filamentous actin verified that all cells were synchronously responding in this cyclic manner. The hypothesis was tested that these oscillations were analogous to chemical oscillations, i.e. oscillations of intermediate species in chemical systems that are far from equilibrium (Epstein, I. R., Kustin, K., DeKepper, P., and Orban, M. (1983) Sci. Am. 248, 112). Actin polymerization/depolymerization cycles were terminated by adding receptor antagonist a few seconds after initiation of the response by agonists. Thus the oscillations did not represent chemical oscillations that hypothetically could result from a rapid jump of the intracellular milieu to a state far from equilibrium. Rather, continued occupancy of receptors and/or occupancy of new receptors was required to sustain the oscillations. This suggested that the oscillations resulted from regulated polymerization and depolymerization pathways. In simultaneous measurements of actin-associated right angle light scatter and intracellular calcium, no calcium oscillations were detected. Thus, cycles of actin polymerization/depolymerization were not regulated by calcium oscillations.

Actins↗

PubMed1989

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils.

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.

Autoradiography↗