Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils.

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.

Direct determination of the stoichiometry of charge transfer complexes.

The stoichiometry of the charge transfer complex between N-acetyl-L-tryptophan and 1-methylnicotinamide chloride has been determined to be precisely 1:1 by direct measurement of the molecular weight of the complex. The result is of interest both in terms of a general method for determining the stoichiometry of charge transfer complexes, and in terms of the probable stoichiometry of specific charge transfer complexes between 1-methylnicotinamide chloride and the exposed tryptophyl side chains of certain proteins. In the latter case, the result provides experimental proof for the assignment of extinction coefficients of specified magnitudes to the homomorphic model complexes which serve as the basis for the interpretation of results with proteins.

[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.

Activation of human neutrophils by C3a and C5A. Comparison of the effects on shape changes, chemotaxis, secretion, and respiratory burst.

The effects of anaphylatoxin C3a on human neutrophils were studied in comparison with C5a. Both peptides induced a transient shape change response and a respiratory burst. In both cases C3a was 50- to 100-times less potent than C5a. A marked chemotactic response with bimodal concentration dependence was obtained with C5a, but no neutrophil chemotaxis was observed with C3a. Repeated stimulation led to homologous desensitization of shape changes and respiratory burst but no cross-desensitization, indicating that the two anaphylatoxins act through separate receptors. The lack of chemotactic activity suggests that C3a is not involved in neutrophil recruitment into infected or inflamed tissues.

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils.

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.

Shape oscillations: a fundamental response of human neutrophils stimulated by chemotactic peptides?

Neutrophils undergo periodic cytoskeletal rearrangements that lead to cycles of shape change, ultimately resulting in cell translocation. Repeated stimulation of resting neutrophils with subsaturating chemoattractant doses induced transient sinusoidal oscillations in neutrophil filamentous actin content at the second and subsequent stimulations. Oscillation frequencies increased with increasing concentration of the first stimulus. In contrast, neutrophils pretreated with the phosphatidylinositol 3-kinase inhibitor (17-hydroxy)wortmannin displayed shape oscillations with the first stimulation, and the frequencies were independent of agonist type and dose. We demonstrate that oscillations in filamentous actin, which may be critical for neutrophil motility, can be induced in untreated cells by natural peptide chemoattractants.

Real-time models of morphogenetic processes.

Morphological transformations are often describable by simple series kinetic models, A----B, A----B----C, etc., which allow assessment of the rates of interconversion of the distinguishable shapes or forms present and their probabilities of occurrence at various points in time, thus providing a means for kinetic comparisons with biochemical measurements of the molecular-level reactions that cause the transformations. When changes in cell morphology are followed turbidimetrically, the real-time progress curves can be simulated by fitting the data to a form of Beer's law for scattering by mixtures in which the species concentrations change with time in accordance with the chosen kinetic scheme. Because many even relatively large cells are mostly water, classical light scattering theory can be used to interpret the turbidimetric data in terms of simple geometrical models of average cell size and shape suggested by microscopic examination. Two examples are briefly considered, the stimulus-induced changes in blood platelet shape and apparent size and their correlation with cytosolic-free calcium, and apparent swimming motion exhibited by neutrophils in suspension.

Kinetic models of cell shape changes and other optically-detected responses to stimulation.

Certain cellular responses to stimulation can be described as a continuous series of virtually infinite but real transient states generated by reactions occurring at the molecular level within the cell. While any particular state can in principle be isolated and examined by using appropriate methods to stop or freeze the reaction, adjacent or nearby states will generally be indistinguishable from one another by kinetic means. In favorable cases, however, the progress curves can be fitted to comparatively very simple kinetic models involving a limited number of steps, which accurately describe the real-time response within the limitations of the experimental setup. The simple series models have their origins in the continuous myriad-state or "microscopic" series description, and the observable or "macroscopic" kinetic rate constants are statistically related to the rate constants describing the transitions between the real states of the ongoing response. This indicates that different aspects of stimulus-response coupling, e.g., shape changes, alterations in cytosolic calcium levels and so forth, can be compared in a self-consistent fashion by modeling the individual responses in terms of simple parallel series models.

Thermophilic aminopeptidase. IV. Cooperative effects in ANS binding by the thermophilic aminopeptidase I from B. stearothermophilus.

Aminopeptidase I is a membrane-bound metalloenzyme isolated from B. stearothermophilus which is thermostable and requires Co2+ for activity. The Co, Zn, and metal-free enzyme were titrated with ANS, and cooperative binding was noted with the active Co enzyme but not with the Zn (5% active) or apo (inactive) enzymes. There are a number of sites for ANS on each of the three enzyme forms and the agreement between the association constants of the Zn enzyme (identical and independent sites) and the non-cooperative sites of the Co enzyme suggest that these sites are intrinsically similar. However, binding to the first of these sites in the Co enzyme triggers a cooperative binding of a second molecule of ANS, and this cooperative binding is related to a concomitant decrease in enzymatic activity. The correspondence can be shown by comparison of the inhibition constant for the hydrolysis of Gly-Leu-Tyr (Ki-1=13,300 cm3/mmol) and the association constant for the cooperating site (12,500 cm3/mmol). The significance of these observations is discussed in terms of the nature of the binding sites and the possible consequences of the interactions on the regulation of aminopeptidase I activity.

Calcium fluxes and calcium buffering in human neutrophils.

Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.

Stochastic response of human blood platelets to stimulation of shape changes and secretion.

Stopped-flow turbidimetric data indicate that platelets stimulated with low levels of thrombin undergo a shape transformation from disc to "sphere" to smaller spiny sphere that is indistinguishable from the shape change induced by ADP through different membrane receptor sites and a dissimilar receptor trigger mechanism. Under conditions where neither secretion nor aggregation occur, the extinction coefficients for total scattering by each of the three platelet forms are independent of the stimulus applied, and both reaction mechanisms can be described as stochastic (Poisson) processes in which the rate constant for the formation of the transient species is equal to the rate constant for its disappearance. This observation is independent of the shape assignment, and as the concentration of thrombin is increased and various storage organelles secrete increasing amounts of their contents into the external medium, the stochastic pattern persists. Progressively larger decreases in the extinction coefficients of the intermediate and final platelet forms, over and above those that reflect shape alterations alone, accompany or parallel the reaction induced by the higher thrombin concentrations. The excess turbidity decrease observed when full secretion occurs can be wholly accounted for by a decrease in platelet volume equal in magnitude to the fraction of the total platelet volume occupied by alpha granules. Platelet activation, as reported by the whole body light scattering of either shape changes alone or shape changes plus parallel (but not necessarily also stochastic) alpha granule secretion, thus manifests itself as a random series of transient events conceivably with its origins in the superposition of a set of more elementary stochastic processes that could include microtubule depolymerization, actin polymerization, and possibly diffusion. Although the real nature of the control mechanism remains obscure, certain properties of pooled stochastic processes suggest that a reciprocal connection between microtubule fragmentation and the assembly of actin-containing pseudopodal structures and contractile elements--processes that may exhibit reciprocal requirements for calcium--might provide a hypothetical basis for a rate-limiting step.

Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility.

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37 degrees C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility.

Flexibility in the specificity site of serine proteases.

The statistical availability of tryptophan and tyrosine residues with one ring face fully exposed to solvent was examined for two serine proteases and their derivatives by investigating the formation of charge transfer (CT) complexes between the aromatic donor residues of the protein and the acceptor 1-methylnicotinamide chloride. The availability of the ring face of one of the two exposed tryptophan residues in trypsin has been previously shown to be pH dependent and to parallel the acid side of the pH-activity profile of the enzyme. The present results indicate that, in diisopropylphosphoryl-trypsin (DIP-trypsin), this residue [which was identified as Trp-215 in native trypsin (chymotrypsin numbering)] is locked in a relatively rigid, pH-independent conformation with one ring face rotated out toward the solvent. In the zymogen and DIP-zymogen, the ring face is essentially unavailable. Chymotrypsin, like trypsin, has a pH-depent tryptophan residue available for complexation with the CT acceptor, but unlike trypsin, the pH dependence is apparently associated with dimerization of the enzyme. These and other data suggest this residue is the same as in the homologous trypsin structure, i.e., Trp 215, and that the ring face is mostly buried in the zymogen. Comparison of the crystal structure models of chymotrypsin and chymotrypsinogen shows that, as the specificity pocket opens up from its collapsed structure upon zymogen activation, the ring face of Trp-215 moves out and rotates relative to the surface of the enzyme in such a fashion as to become more accessible to solvent. These observations are in accord with the present CT results and provide additional support for the assignment of changes in Trp-215 availability to parallel changes in the conformation of the specificity pocket of these serine proteases. The present investigation also shows that, although a tryptophan ring face is partly exposed in DIP-chymotrypsin, its statistical availability more closely resembles that of the zymogen than the native enzyme. The reverse appears to be true for DIP-trypsin, which suggests the possibility that the specificity pocket in DIP-chymotrypsin may be partially collapsed while the catalytic residues are frozen in the conformation of the acyl-enzyme.

Charge-transfer studies of the availability of aromatic side chains of proteins in guanidine hydrochloride.

The ability of aromatic tryptophyl and tyrosyl side-chain donors to form charge-transfer (CT) complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride has been used to investigate the degree of exposure of these aromatic residues in denaturated proteins. The coplanar geometry of the CT complexes requires that virtually a full ring face of the donor be available for interaction with the acceptor, and the aromatic donor residues of lysozyme, trypsin, chymotrypsin, and the zymogens of the latter two enzymes do not appear to be wholly "exposed" in 6 M guanidine hydrochloride. Comparison of the CT proerties of the proteins with the corresponding properties of model complexes suggests that the incomplete exposure is due at least in part to statistical fluctuations in the continuously mobile, randomly coiled polypeptide chain which result in residues being alternately fully exposed and partly covered. Reduction and alkylation of the disulfide cross-links increase the apparent availability of the aromatic residues but the exposure is still less than that expected from a comparable mixture of tryptophan and tyrosine residues. Previous studies on the exposure of the aromatic residues of lysozyme and trypsin in aqueous salt solutions, when taken together with the present results, further suggest that there are two distinct kinds of surface environment possible on native proteins in solution. Some residues appear to be located in areas of the protein surface which are characterized by relatively fixed or stable local conformations, and have apparent CT association constants closely resembling these of comparable model complexes. Other residues may be located in a region where the protein conformation is flexible or continuously mobile, as evidenced by their smaller apparent association constants. It is probably significant that Trp-62 of lysozyme and Trp-215 of trypsin, both specificity site residues, appear to belong to the class of residues which can be considered as being in a flexible environment on the protein surface.

The use of n-methylnicotin amide chloride as a conformational probe for chicken egg-white lysozyme.

Chicken egg-white lysozyme forms a yellow complex with N-methylnicotinamide chloride. Titration studies utilizing the appearance of the yellow color as a measure of complex formation indicate that the weak complex (association constant k = 3.2 liter mole(-1)) involves a single class of binding sites on the lysozyme molecule. By analogy with similar titration studies on model compounds containing the indole moiety, the site for N-methylnicotinamide binding is probably the indole ring of a single, solvent-available tryptophan residue. The yellow color itself apparently arises from a charge transfer transition, with the indole ring system serving as the donor and N-methylnicotinamide as the acceptor. Complete resolution of the charge transfer spectrum of the lysozyme-N-methylnicotinamide complex was not achieved due to the very high absorbance of the protein near the short-wavelength absorption edge of the band. However, it is possible to consider the spectrum as the sum of two Gaussian bands whose positions and relative intensities agree remarkably well with the positions and relative intensities obtained by Gaussian fitting of the charge transfer spectra of several model complexes between substituted indoles and N-methylnicotinamide. The geometry for such complex formation requires that the ring faces of both donor and acceptor be more or less completely available for complexation. The possible use of N-methylnicotinamide as a molecular probe for tryptophan residues having at least one indole ring face freely available to the solvent is discussed.

Optimal efficiency of human platelet shape changes.

The light scattering extinction coefficients and kinetic rate constant(s) for the disc to sphere and the sphere to (smaller) 'spiny sphere' shape changes induced in platelets by saturating amounts of ADP are compared at 31, 37 and 41 degrees C. Platelets exhibit optimum efficiency at 37 degrees C, as judged by not only the rates of the shape changes but also by alterations in the overall sizes and shapes of the three platelet forms. Unstimulated (discoid) platelets appear to be flatter at 37 degrees C, the disc to sphere reaction appears to be impaired at other than 37 degrees C, and the pseudopodia which characterize the final spiny sphere may be more prominent at 41 degrees C.

Arachidonic acid and other unsaturated fatty acids alter membrane potential in PC12 and bovine adrenal chromaffin cells.

The action of arachidonic acid and other fatty acids on membrane potential in PC12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1-40 microM) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K(+)-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC12 and bovine chromaffin cells by modulating K+ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca2+ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca2+ influx and secretion. We propose a model where internally generated fatty acids act as a feedback to desensitize the stimulated cell via inhibition of receptor-dependent Ca2+ influx and induction of membrane hyperpolarization.

Corresponding oscillations in neutrophil shape and filamentous actin content.

Human neutrophils pretreated with 17-hydroxywortmannin responded to the chemotactic agonist formyl-Met-Leu-Phe with a transient doubling in filamentous actin content which was characterized by prominent oscillations. These oscillations closely matched transient oscillations in suspension turbidity measured in parallel. The experimental data could be simulated using A----B----C stochastic series kinetic models with an oscillating intermediate species (B), allowing quantitative comparison of the frequencies of the oscillations (0.092 +/- 0.006 and 0.094 +/- 0.004 Hz) and the overall reaction rate constants for actin mobilization and turbidity changes (0.11 +/- 0.02 and 0.14 +/- 0.03 s-1, respectively). The total cell volume remained constant, indicating that stimulus-induced extension of lamellipods reduces the body volume by an amount proportional to the mass displaced outward. Light scattering theory predicts that a decrease in body size decreases the turbidity and that fluctuations in body size due to lamellipod extension and retraction cycles like those exhibited by crawling neutrophils result in turbidity oscillations (lamellipods scatter very little by comparison to the cell body, and both aggregation and degranulation were absent). The experiments thus suggest that the cyclic variations in F-actin content are correlated with periodic fluctuations in lamellipod size. The available evidence appears to be consistent with the hypothesis that actin polymerization provides the main driving force for lamellipod extension and that depolymerization causes lamellipod retraction.