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By taking advantage of combinations of the many rich properties of photons, new forms of optical microscopy can now be used to visualize features of samples beyond thickness and density variations. We are now within reach of viewing the motions, orientations, binding kinetics and specific transient associations of previously 'submicroscopic' cellular structures and single molecules.

It is well-established that the binding of N-formyl peptides to the N-formyl peptide receptor on neutrophils can be described by a kinetic scheme that involves two ligand-bound receptor states, both a low affinity ligand-receptor complex and a high affinity ligand-receptor complex, and that the rate constants describing ligand-receptor binding and receptor affinity state interconversion are ligand-specific. Here we examine whether differences due to these rate constants, i.e. differences in the numbers and lifetimes of particular receptor states, are correlated with neutrophil responses, namely actin polymerization and oxidant production. We find that an additional receptor state, one not discerned from kinetic binding assays, is required to account for these responses. This receptor state is interpreted as the number of low affinity bound receptors that are capable of activating G proteins; in other words, the accumulation of these active receptors correlates with the extent of both responses. Furthermore, this analysis allows for the quantification of a parameter that measures the relative strength of a ligand to bias the receptor into the active conformation. A model with this additional receptor state is sufficient to describe response data when two ligands (agonist/agonist or agonist/antagonist pairs) are added simultaneously, suggesting that cells respond to the accumulation of active receptors regardless of the identity of the ligand(s).

Apoptosis, or programmed cell death, has been proposed as a biomarker for environmental contaminant effects. In this work, we test the hypothesis that in vitro assays of apoptosis are sensitive indicators of immunological effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophils. Apoptosis, necrosis, and viability as well as the related indicators F-actin levels, and active thiol state were measured in purified human neutrophils after treatment with contaminants. Effective concentrations observed were 0.3μM (60μg/L) mercury, 750μg/L Aroclor 1254, and 50μM (14,500μg/L) hexachlorocylcohexanes. Concentrations of contaminants that induced apoptosis also decreased cellular F-actin levels. Active thiols were altered by mercury, but not organochlorines. Comparison of these data with levels of contaminants reported to be threats to human health indicate neutrophil apoptosis is a sensitive indicator of mercury toxicity.

Polybrominated diphenyl ether (PBDE) flame-retardants have been identified as an emergent contaminants issue in many parts of the world. In vitro analyses were conducted to test the hypothesis that selected PBDEs congeners affect viability, apoptosis, and necrosis of thymocytes from laboratory-reared lake trout (Salvelinus namaycush). At current environmental levels (< 1 mg/L), effects of the tested PBDEs on thymocytes were negligible. However, at 100 mg/L, major effects were seen for congener brominated diphenyl ether 47 (BDE-47) and minor effects were seen for congener BDE-99.

Threshold behavior is an important aspect of signal transduction pathways that allows for responses to be turned on or off. Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G proteins available for signaling; progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to nonresponding cells. To quantify the threshold level of G proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, we used a plasma membrane associated G protein quantification assay in conjunction with a sorting flow cytometer and measured differences in the average number of G proteins available for signaling per cell in both the responding and the nonresponding subpopulations after pertussis toxin treatment. Although there appeared to be a threshold separating responding cells and nonresponding cells for a given sample, no discrete threshold was measured across multiple treatment conditions. A mathematical model of the early steps in signaling suggests that cell-to-cell variability in signal parameters, such as numbers of signal components and values of kinetic rate constants, obscures the measurement of a discrete threshold and leads to an apparent decrease in the threshold level of G proteins available for signaling as the total G proteins are decreased.

The organochlorine insecticide lindane is a known activator of neutrophil responses. In this work we delineated the biochemical pathways by which lindane stimulates neutrophil oxidant production. Plasma membrane GTPase activity was not stimulated by lindane, ruling out a role for lindane-induced activation of G-proteins or G-protein coupled receptors, whereas inhibition of phospholipase C inhibited lindane-induced oxidant production. Together these data pointed to phospholipase C as the direct target of lindane activation. Type I phosphoinositide 3-kinase was not significantly activated by lindane and an inhibitor of phosphoinositide 3-kinases inhibited oxidant production by only 40%. Thus, Type I phosphoinositide 3-kinase played a minor role, if any, in lindane-induced oxidant production. Lindane stimulated an increase in phosphatidylinositol phosphate suggesting a Type II or III phosphotidylinositol 3-kinase or phosphatidylinositol 4-kinase activity was also stimulated.

The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming. In unprimed cells G(ialpha2) and its effector, phospholipase C(beta2), were segregated in different membrane compartments; priming caused G(ialpha2) to move to the compartment in which phospholipase C(beta2) resided. Thus, an important component of the mechanism of priming may involve regulation of the location of G-proteins and effector molecules in plasma membrane compartments where their abilities to couple may be enhanced.

The immunotoxicity of chemical combinations commonly encountered by the lake trout (Salvelinus namaycush) immune system was the focus of this study. It was hypothesised that combinations of an environmental contaminant (mercuric chloride or Aroclor 1254) and an immunomodulatory agent (bacterial endotoxin or cortisol) might interact to produce a greater toxicity than that of the environmental contaminant alone at concentrations typically encountered in piscine blood and other tissues. Thus lake trout thymocytes were isolated and treated with mercuric chloride or Aroclor 1254 in the presence and absence of cortisol or lipopolysaccharide. Incubations were performed for 6 or 20 h at 4 degrees C or 10 degrees C. Lipopolysaccharide did not affect the toxicity of either contaminant. In contrast, cortisol enhanced the toxicity of both environmental contaminants. Hence, stressors that lead to increased cortisol production, but not lipopolysaccharide directly, may increase the toxicity of mercury and Aroclor 1254 to lake trout thymocytes.

Background: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands., Methods: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine., Results: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical., Conclusions: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated., (Copyright 2001 Wiley-Liss, Inc.)

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.e. signaling) and inactive conformations of the receptor, but do not include the dynamics of G-protein activation or receptor desensitization. Yet understanding how these dynamic events effect response behavior is crucial to determining ligand efficacy. We developed a mathematical model for G-protein coupled receptor signaling that includes G-protein activation and receptor desensitization, and used it to predict how activation and desensitization would change if either the conformational selectivity (the effect of ligand binding on the distribution of active and inactive receptor states) or the desensitization rate constant was ligand-specific. In addition, the model was used to explore the implications of measuring responses far downstream from G-protein activation. By comparing the experimental data from the beta(2)-adrenergic, micro-opioid, D(1)dopamine, and neutrophil N -formyl peptide receptors with the predictions of our model, we found that the conformational selectivity is the predominant factor in determining the amounts of activation and desensitization caused by a particular ligand., (Copyright 1999 Academic Press.)

Stimulation of neutrophils with LTB(4) or PAF results in the production of a rapidly oscillating actin polymerization/depolymerization response. Treatment of neutrophils with inhibitors of PKC prior to stimulation with ligand resulted in a masking of the F-actin oscillations. Because myosin has been shown to be a substrate for neutrophil PKC, this protein was investigated as a potential downstream mediator of F-actin oscillations. Stimulation of neutrophils with LTB(4) resulted in myosin light chain being serine phosphorylated in a PKC-dependent manner. This phosphorylation was shown to occur in a manner that is kinetically distinct from the myosin phosphorylation induced by FMLP, a potent activator of actin polymerization that alone does not induce F-actin oscillations. Additionally, disruption of intracellular actin-myosin interactions resulted in inhibition of LTB(4)- as well as PAF-induced F-actin oscillations. These data suggest that PKC and downstream phosphorylation of myosin as well as actin-myosin interaction may play roles in mediating the production of neutrophil F-actin oscillations., (Copyright 1999 Academic Press.)

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses. The model is divided into two parts, ligand/receptor/G-protein dynamics and the actin polymerization mechanism. Fast (receptor precoupled to G-protein) and slow (free receptor) signaling pathways involving ligand/receptor/G-protein interactions produce an activated signaling molecule. The actin polymerization mechanisms utilizes an actin binding protein which complexes with actin monomer and inhibits polymerization in an unstimulated cell. During stimulation, the activated signaling molecule enhances the dissociation of monomer/binding protein complexes, allowing the actin polymerization response to occur. The fast and slow signaling pathways are predicted to have different roles in controlling the time course of this actin polymerization. Additionally, precoupled receptors are predicted to have a larger ligand association rate constant than non-precoupled (free) receptors. Model simulations agree with many of the experimentally observed characteristics of both the stimulated F-actin response and ligand/receptor binding kinetics for both the fluorescent peptide ligand CHO-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine-fluorescein (CHO-NLFNTK-fl) and the non-fluorescent peptide ligand CHO-methionyl-leucyl-phenylalanine (CHO-MLF).

Observing the qualitative characteristics of response behavior as key variables in the signal transduction cascade are changed can provide insight into the fundamental roles of these interactions in producing cellular responses. Using flow cytometric assays and pertussis toxin (PT) treatment of human neutrophils, we have shown that actin polymerization stimulated with the chemoattractants N-formyl-Met-Leu-Phe, leukotriene B4, and interleukin-8 exhibits threshold behavior in terms of G-protein number. Partial PT treatment resulted in both responding and nonresponding populations of cells upon stimulation. As PT treatment was increased, the responding population of cells continued to respond maximally, while the number of cells responding decreased. We also showed that N-formyl peptide-stimulated oxidant production exhibits threshold behavior in terms of G-protein number, and the threshold for oxidant production is significantly greater than that for actin polymerization. The threshold behavior observed with PT treatment contrasted with the graded response behavior seen when cells were stimulated with different doses of ligand. For actin polymerization, only one population of cells was observed at submaximal ligand concentrations, and as ligand concentration was decreased the whole population responded submaximally. For oxidant production, as ligand concentration was decreased there were two populations of cells, but the responding cells responded submaximally. A mathematical model incorporating receptor/ligand binding and G-protein activation was developed to account for these differences in response behavior. Our results predict that an early signal transduction event in addition to, and not initiated by G-protein activation, is necessary to account for actin polymerization and oxidant production in neutrophils.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses have been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step. Advantages and limitations of each protocol are discussed and data is presented demonstrating some of the properties of the protocols.

Polymorphonuclear leukocytes (PMN) play a central role in the host response to injury and infection. These terminally differentiated phagocytes have a limited life span, after which they undergo spontaneous apoptosis. PMN life span can be significantly prolonged by several naturally occurring cytokines, and PMN are now known to be capable of cytokine production in response to various antigenic stimuli. These facts suggest the possibility that PMN possess an autocrine/paracrine mechanism for the control of their own survival. The present study was undertaken to test this hypothesis. Supernatants from PMN that had been incubated with Candida albicans for 18 h significantly decreased the number of fresh PMN demonstrating features of apoptosis and increased the percentage of viable PMN during in vitro culture. This was demonstrated by monitoring morphologic features of apoptosis with fluorescence microscopy and DNA endonuclease activity with agarose gel electrophoresis. Significant levels of tumor necrosis factor (TNF) were detectable in the supernatants of PMN that had been stimulated with C. albicans, as determined using a TNF-sensitive cell line. Neutralization of TNF biologic activity with a specific monoclonal antibody partially abrogated the supernatant-mediated prolongation of PMN survival. The present study demonstrates that PMN possess a mechanism for the modulation of their own survival, which in part may be through the production of TNF.

Background: The present study was undertaken to determine if tyrosine phosphorylation signal transduction pathways, which are known to be activated in polymorphonuclear leukocytes (PMN) by lipopolysaccharide (LPS), play a role in priming of PMN oxidative burst and protection of PMN from apoptosis by LPS, and to determine if an interface between these two signaling pathways exists., Methods: PMN were combined with or without 10-fold serial dilutions (0.1 ng-1 microgram/ml) of LPS and incubated at 37 degrees C/5% CO2. After 24 h PMN apoptosis was assessed using fluorescence microscopy and DNA agarose gel electrophoresis. Additional PMN were pretreated with the tyrosine kinase inhibitors genistein and herbamycin A before addition of LPS. Tyrosine phosphorylation was detected by immunoblotting. Oxidant production was quantitated by following the oxidation of a chromophore to its fluorescent product., Results: LPS delayed the onset of apoptosis and prolonged the survival of PMN in a dose-dependent fashion. Both tyrosine kinase inhibitors blocked the protective effect of LPS on PMN apoptosis; however, only genistein blocked the priming effect of LPS on PMN oxidative burst., Conclusions: Tyrosine phosphorylation signal transduction pathways are central to protection of PMN from apoptosis by LPS. Although tyrosine phosphorylation pathways also play a role in priming of the oxidative burst in PMN, our data suggest that there is not an interface between these important signaling pathways.

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life-threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult. This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70-90% of PMN displaying features of apoptosis by 4 h after UV exposure. Bacterial lipopolysaccharide and granulocyte-macrophage colony-stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV-accelerated apoptosis. The UV-accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.

With the aid of high time resolution kinetic data extracted from a flow cytometer, we determined that there are two N-formyl peptide receptor states for human neutrophils at 4 degrees C: a low affinity and a high affinity state. Competitive binding of FMLP, FNLP, and t-BOC with FNLPNTL-FL revealed different kinetic rate constants for two distinct reactions that control the lifetime of the low affinity ligand-receptor complex. For these ligands, the rate constant for dissociation of ligand from the low affinity receptor state (the first reaction) ranges in order of magnitude from 10(-2) to 1 s-1, and the conversion rate constant from the low affinity receptor state to the high affinity receptor state (the second reaction) ranges from 10(-4) to 10(-2) s-1. The antagonist t-BOC differed most significantly from the three agonists by having an association rate constant for the low affinity receptor on the order of 10(5) M-1 s-1; the value for all three agonists was on the order of 10(7) M-1 s-1. Characterization of the receptor conversion at 4 degrees C revealed that it is irreversible (or very slow) and independent of Gi protein and that neither receptor state is a form of receptor precoupled to Gi protein. The affinity conversion and the dissociation characteristics of each receptor state determine the duration of the signaling complex and may contribute to differences in ligand efficacy.

High resolution kinetic data of the binding of fluorescent peptide to the N-formyl peptide receptor of neutrophils at 37 degrees C has allowed for the development of a ligand binding model that predicts statistically larger binding rate constants than those previously reported for intact neutrophils. The new model accounts for ligand association and dissociation, receptor up-regulation, ligand-receptor complex internalization, a change in receptor affinity, and the quenching of internalized fluorescent ligand. We determined that receptor up-regulation is both agonist- and temperature-induced and is inhibited by both phenylarsine oxide and pertussis toxin treatment. Model fits of ligand association to pertussis toxin-treated cells show that while receptor up-regulation was inhibited, rate constants for ligand binding, receptor affinity conversion, and internalization of ligand-receptor complexes were unaffected. Results suggest Gi-protein-mediated receptor up-regulation and Gi-protein-independent receptor affinity conversion. Simulation of ligand infusion using our model gives insight into the quantitative and dynamic relationship between the low affinity ligand-receptor complex and the actin polymerization response.

Neutrophils stimulated with a low concentration of chemotactic peptide N-formyl-met-leu-phe respond with a brief pulse of actin polymerization and a persistent change in morphology from round to polarized. The recovery of the actin polymerization response is unlikely to be due to usual receptor desensitization mechanisms. We hypothesized that cell polarization and redistribution of signaling components effect the depolymerization phase of the actin response. In this report we show that the overall actin depolymerization is equivalent to the reduction in the volume occupied by F-actin while its concentration at the front of the polarized cell remains undiminished. To test whether the confinement of F-actin to a small volume can be caused by receptor redistribution, we observed receptors and F-actin in the same cell and found that their localization was different. To explain our findings, we propose a model based on the affinity of a signaling component, other than the receptor, for the areas of the cell rich in F-actin.

The aim of this study was to determine if exposure of cells to oxidants and metabolic inhibition, conditions which are present during ischemia-reperfusion, act synergistically to produce cytoskeletal disruption. Adherent bovine pulmonary artery endothelial cells were subjected to metabolic inhibition by incubating the cells in glucose-free buffer containing 650 nM oligomycin for 2 hr. Cells were rescued from metabolic inhibition by washing the cells with buffer containing 5.5 mM glucose and were simultaneously exposed to 0, 25, 100, or 5000 microM H2O2. At various time points during recovery from metabolic inhibition the microfilaments and microtubules were stained for microscopic evaluation. Intracellular ATP levels were determined by the luciferin/luciferase assay. Cells that were not metabolically inhibited showed minimal microfilament disruption at lower doses of H2O2. Cells that were subjected to metabolic inhibition but not exposed to H2O2 showed microfilament disruption after 2 hr of metabolic inhibition, but normal microfilament architecture was seen in over 95% of the cells by 1 hr after recovery from metabolic inhibition. Cells that were metabolically inhibited and then exposed to doses of H2O2 as low as 25 microM showed marked microfilament disruption at 1 and 2 hr after the metabolic inhibition was relieved. The microtubules were distorted, but did not depolymerize except when exposed to concentrations of H2O2 > or = 5000 microM. Metabolic inhibition appeared to selectively potentiate the effect of subsequent oxidant exposure and the potentiation largely affected microfilament architecture with secondary effects on microtubule morphology and endothelial cell shape.

We previously showed that activation of polymorphonuclear leukocytes by leukotriene B4 (LTB4) and platelet-activating factor produces a rapidly oscillating actin polymerization/depolymerization response. In this study, we show that 1) oscillations are not due to the stimulated cyclic release of autocoids that could bind to cell surface receptors and activate subsequent cycles; 2) oscillations are not related to oscillations of ligand binding; and 3) the particular kinetic pattern is a property of the receptor, not of the binding constants of the ligand. The major conclusion of these studies is that the oscillations are a property of the intrinsic signaling pathways triggered by these chemoattractants. We also questioned whether increased actin nucleation activity was induced by LTB4 and found that, although LTB4 induced a transient actin nucleation response, there was not a direct correlation between oscillations of the actin polymerization/depolymerization and the actin nucleation activity. This suggests that processes other than actin nucleation, such as release of monomeric actin from monomer sequestering proteins and regulation of depolymerization, are likely to be involved.

The rate of binding of a ligand to receptors on the cell surface can be diffusion limited. We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport. We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient. A moderately transport-limited kinetics is not readily recognizable from the shape of the binding curve and may lead to erroneous estimates of the rate coefficients. We apply our results to practically important cases: a cell suspension in a stirred volume of liquid and a confluent cell colony under a laminar stream. Using typical numbers characterizing the ligand-receptor interactions, we show that stirring and perfusion can be important factors determining the reaction rates. With the confluent colony, the early reaction kinetics requires a different treatment, and we provide it for the case of low receptor occupancy. We show that, even with a fast perfusion, a cell monolayer can transiently generate a zone of depletion of the ligand, and that would affect the early stages of the reaction. Our results are expressed in a simple analytical form and can be used for the design and interpretation of experimental data.

Many chemoattractant-activated responses in neutrophils show transient kinetics, suggesting that rapid desensitization occurs during the time course of the response. We found that desensitization of the actin polymerization response to N-formyl peptides is, in a large part, due to inhibition by adenosine released from cells to the medium and depletion or a chemical inactivation of the agonist. To reduce the influence of these factors, we stimulated neutrophils in a very diluted suspension, sometimes with continuous replacement of the medium. The actin polymerization response to a high agonist concentration was greatly enhanced and prolonged under these conditions, often without any tendency to subside within 10 min at 25 degrees C. It has previously been shown that the N-formyl peptide receptor converts from a rapidly dissociating to a slowly dissociating and presumably inactive form during activation. Under the conditions of low cell concentration, the conversion to a slowly dissociating receptor still occurred. Thus the prolonged response was not due to prolonged presence of rapidly dissociating receptors. We conclude either that a low number of rapidly dissociating receptors, which we failed to see, is sufficient to maintain actin polymerization or that slowly dissociating receptors can support the actin response. In contrast to responses stimulated by high agonist concentrations, the responses to low concentrations of the agonists were transient. The results of other authors indicate that low concentrations of N-formyl peptides do not desensitize the receptors. Other mechanisms, which are specific for the actin polymerization response, must be involved in response termination to low concentrations of N-formyl peptides. Activation at low cell density will be a useful approach for studying other processes (Ca2+ elevation, oxidant production, etc.) and chemoattractants (leukotriene B4, interleukin 8, etc.) for which an understanding of the kinetics due to desensitization of the components of the receptor-mediated activation pathway is desired.

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) approximately 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.

Oxidants derived from the partial reduction of oxygen are thought to play a significant role in neuronal injury. We present here a cellular model of neuronal injury mediated by hydrogen peroxide (H2O2) using the PC 12 rat pheochromocytoma cell line. The organization of microtubules and microfilaments within neurites of PC 12 cells differentiated by exposure to nerve growth factor was examined after H2O2 injury using fluorescence microscopy. Concentrations of H2O2 as low as 100 microM produced an initial periodic pattern of microtubule depolymerization over 3-4 h which later progressed to complete depolymerization. Neuritic microspikes containing actin filaments were relatively more resistant to injury by H2O2 than microtubules. Blebbing of PC 12 cell bodies and neurites also was seen after H2O2 injury and the blebs appeared to contain microtubules. The destructive changes affecting neuritic structure preceded but were not essential for PC 12 cell lysis. Exposure of the cells to the Ca2+ ionophore, ionomycin (25 microM) also produced the same pattern of microtubule depolymerization in PC 12 neurites as was seen after H2O2 injury suggesting that H2O2 may mediate its destructive effect on the neurites via elevation of intracellular Ca2+.

Depletion of cellular ATP is associated with profound effects on the cytoskeleton, particularly disruption of microfilaments. We examined this process in bovine pulmonary artery endothelial cells by inducing differential reductions of cellular ATP using mitochondrial inhibition and variable amounts of glucose. Reduction of cellular ATP to levels < 40% of control produced discrete stages in the visible disruption of microfilaments. Using the deoxyribonuclease I assay, a reversible 11% decrease in monomeric (G) actin occurred in conjunction with microfilament disruption. Polyacrylamide gel electrophoretic (PAGE) analysis of the detergent-insoluble cytoskeleton did not reveal any differences in actin content between normal or ATP-depleted cells. Image analysis of adherent endothelial cells that had been fixed and stained with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin revealed that an increase of F-actin of approximately 20% occurred in cells depleted of ATP. If the cells were lysed with detergent before fixation, the increase in F-actin was lost. PAGE analysis and electron microscopy of detergent-soluble material from the cells obtained by ultracentrifugation directly demonstrated the presence of a labile pool of F-actin within the cells, which increased with ATP loss. These observations suggest that ATP may play an important role in the organization and remodeling of microfilaments within cells.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.

Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.

Endothelin is a potent vasoconstrictor peptide which has recently been localized in the gastrointestinal tract. We have investigated the transmembrane signaling properties of endothelin in isolated smooth muscle cells of the rabbit rectosigmoid. Endothelin induced a dose dependent contraction of smooth muscle cells in a range of 10(-10) to 10(-6)M. In normal buffer, contraction peaked at 30 sec and was sustained for up to 8 min. Incubation in 0Ca/2mM EGTA abolished the sustained contraction induced by endothelin, but had no effect on the initial transient contraction. Preincubation of saponin treated cells with G protein antisera had no effect on control cell length. Preincubation of saponin treated isolated smooth muscle cells with specific G protein antisera (rabbit antisera) for Go alpha or Gs for 60 minutes did not inhibit contraction induced by endothelin. Preincubation with an antiserum to Gi3 alpha inhibited the initial transient contraction induced by endothelin and preincubation with an antiserum to Gi1-2 alpha inhibited the sustained phase of the endothelin induced contraction. Our data indicate that: 1) Endothelin induces a direct sustained contraction of smooth cells from the rectosigmoid; 2) The transmembrane signalling of endothelin is through two specific GTP binding components that are Gi alpha, one for the initial transient contraction, and the other for the sustained phase of the contraction.

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Interleukin 2-activated peripheral blood lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to lyse tumor cells in a nonspecific fashion. Although the mechanisms involved in this cell-mediated lysis are unknown, previous work has shown that T-lymphocyte-mediated cytolysis is associated with massive fluctuations in the intracellular calcium concentration within target cells. Studies were undertaken to determine whether intracellular calcium concentration changes were associated with LAK-mediated cytotoxicity and to determine the effects of calcium channel blockade on in vitro cell-mediated cytotoxicity. Natural killer (NK) and LAK cell cytotoxicity in vitro were measured against head and neck squamous cell carcinoma (UMSCC-11a and UMSCC-38) and Daudi cell lines. Assays were performed in parallel with flow cytometry to measure changes in intracellular calcium concentration within the target cells. Compared with NK cells, LAK cells showed enhanced cytotoxicity against the UMSCC-11a and Daudi lines but not the UMSCC-38 cell line. Both NK and LAK cell cytotoxicity against all the target cell lines directly paralleled significant increases in calcium concentration in the target cells. The addition of verapamil hydrochloride inhibited the rise in intracellular calcium concentration in the sensitive target cells and significantly inhibited both NK and LAK cell cytotoxicity in all the cell lines. The NK cell activity was more sensitive than LAK cell activity to verapamil inhibition. These data suggest that changes in the target cell calcium concentration are early and important events in both NK and LAK cell cytotoxicity in vitro.

Pertussis toxin (PT) inhibits polymorphonuclear leukocyte (PMN) function by ADP-ribosylating and inactivating guanine nucleotide binding proteins (G-proteins) that transduce activation by chemoattractants such as N-formyl peptides (FP). Studies of PMN activation during the time course of PT treatment yielded these results. 1) Responses were differentiated based on their sensitivity to PT treatment. Suboptimal PT treatment that resulted in 50% inhibition of the FP-induced actin-associated right angle light scatter response resulted in greater than 90% inhibition of oxidant production. Exhaustive PT treatment was required to completely inhibit the right angle light scatter response. This is consistent with previous observations that, relative to oxidant production, actin polymerization requires 100-fold fewer active N-formylpeptide receptors to elicit the response. This differential sensitivity to PT treatment has important implications for studies that use pertussis toxin to determine if the neutrophil G-protein is involved in the signaling of responses. If inhibition of oxidant production is used as the only indicator of the effectiveness of PT treatment, significant cytoskeletal changes may still be activated in these cells. Inhibition of actin polymerization is a much more rigorous indicator of complete G-protein inhibition by PT. 2) Analysis of FP-induced actin polymerization and cytosolic calcium elevation using flow cytometry, which measures individual cell responses, revealed that PT treatment resulted in the conversion of PMN from a responding to a non-responding population. In contrast, in control PMN, submaximal doses of FP caused submaximal stimulation of all the cells. The all-or-none effect of PT may result from heterogeneous insertion of the A-promoter of PT into the cell or it may result from a sharp threshold of coupled G-proteins required to transduce the responses.

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.

In the first paper of a series (Gutowski, et al., 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).

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.

Leukotriene B4 (LTB4) was shown to be a potent stimulator of neutrophil actin polymerization as detected by right-angle light scatter and rhodamine-phalloidin staining of F-actin. When we compared the kinetics of this neutrophil cytoskeletal response to the chemoattractants formylpeptide and LTB4, we observed that the response to LTB4 was markedly shorter-lived. To understand the basis of this result, we re-examined the kinetics of superoxide generation, elastase release, intracellular calcium elevation, and phosphoinositide metabolism in neutrophils stimulated with LTB4 and N-formylhexapeptide. LTB4 was relatively inefficient in producing superoxide and in releasing elastase. Although both responses were initiated with similar rapidity, they turned off sooner with LTB4 as compared with N-formylhexapeptide stimulation. Intracellular calcium elevation, a signal that is necessary for superoxide generation and degranulation, was of similar magnitude but of shorter duration in response to LTB4 as compared with the N-formylhexapeptide. The LTB4-induced rise of phosphatidic acid also was not sustained as long as the N-formylhexapeptide-induced increase. Prior exposure of neutrophils to LTB4 did not inhibit subsequent stimulation of superoxide generation by N-formylhexapeptide. Thus, the inability of LTB4 to stimulate superoxide generation was not due to LTB4-induced global inhibitory signals. The deficiency in LTB4-induced superoxide and elastase responses may be related to short-lived LTB4-induced activation signals and/or the number of receptors contributing to these responses.

Chemotaxis by human phagocytes enables these cells to find sites of injury or inflammation in the host. This function is a complex orchestration of biochemical and morphologic events that begins with the occupancy of specific surface receptors for chemotactic agents. The response is propagated and amplified by a biochemical cascade of transduction reactions. This cascade is mediated by receptor interactions with guanyl nucleotide binding proteins, subsequent activation of phospholipase C, and a resulting increase in phospholipid turnover. These events are spatially and temporarily integrated as elevations in the concentrations of intracellular Ca++ and diacylglycerol combine to activate protein kinase C. At the macromolecular and subcellular level, integration of these functions with as yet undiscovered pathways results in stimulation of actin polymerization, shape change, degranulation, and membrane flow. Final integration of these events results in the morphologic process of directed migration of the cell along concentration gradients of chemotaxins. During this process, chemotactic receptors are continuously regulated and feed back information to the response system that adjusts the biochemistry and morphology accordingly. This article has briefly considered some of the relationships between these transduction events and the control of a specific chemotactic receptor system employing the N-formylated chemotactic peptides. It is hoped that knowledge of this regulation will aid the physician in understanding the origin of chemotactic disorders derived from defects in chemotactic receptor systems of the phagocytic cells.

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.

The response of human neutrophils to N-formyl peptides were studied under conditions where ligand binding was controlled by infusing a cell suspension with the peptide over a time period comparable to the normal half-time for binding. Receptor occupancy was measured in real time with a fluorescently labeled peptide using flow cytometry. This binding was approximated by a simple reversible model using typical on (7 X 10(8) M- min-1) and off (0.35/min) rate constants and the infusion rates (0.02-0.2 nM/min). Under conditions of stimulus infusion intracellular calcium elevation, superoxide generation, and right angle light scatter and F-actin formation were measured. As the infusion rate was decreased into the range of 10 pM/min, lowering the rate of increase of receptor occupancy to approximately 0.5% per min, the calcium and right angle light scatter responses elongated in time and decreased in magnitude. Superoxide generation decreased below infusion rates of approximately 100 pM/min (occupancy increasing at a rate in the range of 5% per min). This behavior could contribute to differences between chemotactic responses, which appear to require low rates of receptor occupancy over long periods, and bactericidal or inflammatory responses (free radical generation and degranulation), which require bursts of occupancy of several percent of the receptors.

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydro-carbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal gamma-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.