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.     

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.     

LTB4 induced activation signals and responses in neutrophils are short-lived compared to formylpeptide

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.     

Relationship of actin polymerization and depolymerization to light scattering in human neutrophils: dependence on receptor occupancy and intracellular Ca++

When exposed to the N-formylated chemoattractant peptides, neutrophils undergo a transient ruffling followed by a polarization that involves a redistribution of F-actin (Fechheimer, M., and S. H. Zigmond, 1983, Cell Motil., 3:349-361). The cells also undergo a biphasic right angle light scatter response whose first phase is maximal 10-15 s after exposure to the stimulus, and whose second phase is longer in duration and maximal only after 1 min or more (Yuli, I., and R. Snyderman, 1984, J. Clin. Invest. 73:1408-1417). We now report that the first phase is accompanied by a transient polymerization of actin (monitored by cytometric analysis of phallacidin staining according to the method of Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271) and the second phase is accompanied by a more sustained polymerization of actin. Based on correlated measurements of ligand binding (Sklar, L. A., D. A. Finney, Z. G. Oades, A. J. Jesaitis, R. G. Painter, and C. G. Cochrane, 1984, J. Biol. Chem., 259:5661-5669) and intracellular Ca++ elevation (under conditions where we use the fluorescent Ca++ chelator Quin 2 to modulate intracellular Ca++ levels), we conclude that this first phase requires less than 100 receptors/cell (out of 50,000) and does not require the release of intracellular stores of Ca++. In contrast, the sustained polymerization requires both the occupancy of thousands of receptors (an estimated 10% of the receptors per minute) and may be somewhat sensitive to the availability of intracellular Ca++. When ligand binding is interrupted, F-actin rapidly depolymerizes with a half-time of no greater than approximately 15 s, and the transient light scatter response decays toward its initial value in parallel. Partial disaggregation of the cells follows the recovery of these responses. Based on these observations, we suggest that transient actin polymerization and transient cell ruffling give rise to transient aggregation as long as degranulation is limited.     

Three states for the formyl peptide receptor on intact cells

Three distinct states of the formyl peptide receptor have been described. These are: 1) the ternary complex of ligand, receptor, and G protein (LRG); 2) the rapidly dissociating occupied receptor (ligand-receptor complex (LR]; and 3) a desensitized slowly dissociating guanine nucleotide-insensitive receptor (desensitized ligand-receptor complex ("LRX"]. During cell activation there is a rapid interconversion among receptor states from a rapidly dissociating form (t 1/2 approximately 10 s) to a slowly dissociating form (t 1/2 greater than or equal to 2 min). Neither the dynamics of the states nor their interconversion is influenced by ribosylation of G protein in the presence of pertussis toxin. In contrast to ribosylation, treatment of cells with either 2-deoxyglucose or fluoride ion, both of which lead to a loss of adenine and guanine nucleotides, causes a time-dependent change in ligand dissociability. After short periods of treatment (5-15 min) rapid dissociation is observed; after longer times (30-60 min), slow dissociation is once again detected. When intact cells are first ribosylated and then energy-depleted, only a rapidly dissociating receptor is detected. These results are discussed in terms of a model with the following elements: 1) intact cell dynamics during cell activation are dominated by an energy-dependent interconversion from LR to LRX; 2) under activation conditions, LRG appears and disappears too rapidly to be detected; 3) in cells depleted of energy and guanine nucleotide, LRG is stabilized; 4) in cells both ribosylated and depleted of energy, LR is stabilized.     

Diffusion and partitioning of a pesticide, lindane, into phosphatidylcholine bilayers. A new fluorescence quenching method to study chlorinated hydrocarbon-membrane interactions.

Chlorinated hydrocarbons, such as the pesticide lindane (gamma-hexachlorocyclohexane), quench the fluorescence of carbazole. The observed quenching is a result of the molecular contacts which occur upon diffusional collisions. Because the amount of quenching depends upon the collisional frequency between carbazole and pesticide, this phenomenon provides a measure of both the diffusional rate of lindane and its local concentration. The carbazole fluorophore is localized within phosphatidylcholine bilayers by cosonicating the lipid with a newly synthesized phospholipid, beta-(11-(9-carbazole)-undecanoyl)-L-alpha-phosphatidylcholine. Using this probe in dimyristoyl-L-alpha-phosphatidylcholine vesicles, and the above mentioned quenching phenomena, we determined the lindane diffusion rate within the bilayer to be 5.7.10-7 cm2/s at 37 degrees C. Measurement of the apparent quenching constant at various dimyristoyl phosphatidylcholine concentrations yielded a lipid-water partition coefficient for lindane of 9500, which is in agreement with the value of 8980 obtained by our equilibrium dialysis experiments. Vesicles of dimyristoyl-L-alpha-phosphatidylcholine become saturated with lindane at a pesticide to lipid molar ratio of approx. 0.28. These results demonstrate the possibility of using the quenching of carbazole fluorescence to investigate the transport and partitioning of pesticides within biological membranes. This ability should prove useful in studies of the interactions of chlorinated hydrocarbons with cell membranes.     

Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity

Many plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non-freezing temperatures at the onset of winter. CA is associated with gene expression that requires transient calcium influx into the cytosol. Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold-acclimatize. Conversely, chemical agents causing increased calcium influx induce cold acclimatization-specific (cas) gene expression in alfalfa at 25 degrees C. How low temperature triggers calcium influx is, however, unknown. We report here that induction of a CA-specific gene (cas30), calcium influx and freezing tolerance at 4 degrees C are all prevented by cell membrane fluidization, but, conversely, are induced at 25 degrees C by membrane rigidification. cas30 expression and calcium influx at 4 degrees C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25 degrees C by the actin microfilament destabilizer cytochalasin D (CD). JK blocked the membrane rigidifier-induced, but not the calcium channel agonist-induced cas30 expression at 25 degrees C. These findings indicate that cytoskeleton re-organization is an integral component in low-temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidification and calcium influx in CA.     

G protein threshold behavior in the human neutrophil oxidant response: measurement of G proteins available for signaling in responding and nonresponding subpopulations

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.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

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.