The diacylglycerol kinase inhibitor R59022 potentiates superoxide production but not secretion induced by fMet-Leu-Phe: effects of leupeptin and the protein kinase C inhibitor H-7

The addition of low concentrations of the chemotactic factor fMet-Leu-Phe to rabbit neutrophils in the absence of cytochalasin B produces very little superoxide. This level of superoxide can be greatly increased in neutrophils pretreated for 30 min with 10 microM of the diacyl-glycerol kinase inhibitor R59022. This potentiation occurs also in the presence of cytochalasin B. In addition, while the small level of superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7), the increase by R59022 is completely abolished by this compound. In addition, this increase can be potentiated further by leupeptin. Unlike superoxide generation, the release of lysozyme or N-acetyl-beta-glucosaminidase produced by fMet-Leu-Phe is not stimulated by R59022. The results presented here suggest that stimulation of the oxidative burst requires the generation and the maintenance of a sufficient amount of diacylglycerol and/or the rearrangement of the cytoskeleton such as the inhibition of actin polymerization. Furthermore, the membrane-associated form of protein kinase C is the one responsible for the activation of the oxidative burst. The relationship between protein kinase C activation and the stimulated oxidative burst and the physiological role of chemotactic factors in the functions of the neutrophils are discussed.     

Actin polymerization in human eosinophils,unlike human neutrophils,depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca²⁺]_i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca²⁺-depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes. © 1996 Wiley-Liss, Inc.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Diacylglycerol kinase inhibitors R59022 and dioctanoylethylene glycol potentiate the respiratory burst of neutrophils by raising cytosolic Ca2+

The diacylglycerol kinase inhibitors, R59022 and dioctanoylethylene glycol (diC8-eg), potentiate stimulation of the respiratory burst by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. However, in contrast to the potentiation observed in intact cells, neither R59022 nor diC8-eg enhanced the effect of fMLP on O2 consumption in electropermeabilized neutrophils, under conditions where cytosolic [Ca2+] was held constant using EGTA. In unstimulated, intact cells treatment with the diacylglycerol kinase inhibitors elicited an increase in cytosolic Ca2+ ([Ca2+]i). The results suggest that enhancement of the respiratory burst by diC8-eg and R59022 is mediated by a rise in [Ca2+]i, rather than by inhibition of diacylglycerol kinase.     

The role of the cytosolic free Ca2+ transient for fMet-Leu-Phe induced actin polymerization in human neutrophils

We have addressed the important question as to if and how the cytosolic free Ca2+ concentration, [Ca2+]i, is involved in fMet-Leu-Phe induced actin polymerization in human neutrophils. Stimulation of human neutrophils with the chemotactic peptide (10(-7) M), known to result in a prompt rise of the [Ca2+]i to above 500 nM, also induced a rapid decrease of monomeric actin, G-actin, content (to 35% of basal) and increase of filamentous actin, F-actin, content (to 320% of basal). A reduction of the fMet-Leu-Phe induced [Ca2+]i transient to about 250 nM, resulted in a less pronounced decrease of G-actin content (to 80% of basal) and increase of F-actin content (to 235% of basal). A total abolishment of the chemotactic peptide induced [Ca2+]i rise, still led to a decrease of the G-actin content (to 85% of basal) and increase of F-actin (to 200% of basal). These results indicate that the [Ca2+]i rise is not an absolute requirement, but has a modulating role for the fMet-Leu-Phe induced actin polymerization. Another possible intracellular candidate for fMet-Leu-Phe induced actin polymerization is protein kinase C. However, direct activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) only resulted in a minor increase of F-actin content. The recent hypothesis that a metabolite of the polyphosphoinositide cycle, independently of [Ca2+]i and protein kinase C, is responsible for actin polymerization agrees well with these results and by the fact that preexposure to pertussis toxin totally abolished a subsequent increase of F-actin content induced by fMet-Leu-Phe.     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Spatial control of actin polymerization during neutrophil chemotaxis

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.     

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, “classical chemoattractants” such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and “pure chemoattractants” such as TGF-β1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the “non-classical” group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca²⁺]_i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca²⁺-independent G-protein-coupled pathway for post-receptor signal transduction triggered by “pure chemoattractants.” The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds. © 1996 Wiley-Liss, Inc.     

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor- mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

Actin polymerization modifies stimulus-oxidase coupling in rat neutrophils

Oxidase activity in rat neutrophils was monitored by oxygen consumption rate and luminol-dependent chemiluminescence. Two agents which inhibit actin polymerization, cytochalasin B and dihydrocytochalasin B, produced a marked enhancement (up to 10-fold) of oxidase activation induced by two Ca2+-dependent stimuli, chemotactic peptide and ionophore A23187. In contrast, activation by the calcium-independent stimulus, phorbol myristate acetate, was unaffected by these agents. Other agents that interact with the cytoskeleton, phalloidin and colchicine have no effect on activation by any stimulus tested. The effect of cytochalasin B, when added after stimulation by chemotactic peptide, was transient with t0.5 approx. 10 s. Similarly, the degree of actin polymerization following stimulation by chemotactic peptide was transient, decaying with a t0.5 of approx. 10 s. The half-maximal concentration of cytochalasin B for inhibition of actin polymerization was similar to that for enhancement of oxidase activation. It was concluded, therefore, that the intracellular Ca2+ rise in rat neutrophils that accompanies stimulation by chemotactic peptide affects actin polymerization in a manner that modifies oxidase activation.     

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca²⁺ ([Ca²⁺]_i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β- -glucuronide. We found that the transient rise in [Ca²⁺]_i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca²⁺ transient is prevented by the Ca²⁺ chelator BAPTA, degranulation can still be induced by a stimulated Ca²⁺ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca²⁺ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca²⁺]_i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.     

Neutrophil chemotaxis by pathogen-associated molecular patterns--formylated peptides are crucial but not the sole neutrophil attractants produced by Staphylococcus aureus

The chemotactic migration of phagocytes to sites of infection, guided by gradients of microbial molecules, plays a key role in the first line of host defence. Bacteria are distinguished from eukaryotes by initiation of protein synthesis with formyl methionine. Synthetic formylated peptides (FPs) have been shown to be chemotactic for phagocytes, leading to the concept of FPs as pathogen-associated molecular patterns (PAMPs). However, it remains unclear whether FPs are major chemoattractants released by bacteria and whether further chemoattractants are produced. A Staphylococcus aureus mutant whose formyltransferase gene was inactivated (Deltafmt) produced no FPs and the in vitro and in vivo ability of Deltafmt culture supernatants to recruit neutrophils was considerably reduced compared with those of the parental strain. However, some chemotactic activity was retained, indicating that bacteria produce also unknown, non-FP chemoattractants. The activity of these novel PAMPs was sensitive to pertussis toxin but insensitive to the formyl peptide receptor inhibitor CHIPS. Deltafmt culture supernatants caused reduced calcium ion fluxes and reduced CD11b upregulation in neutrophils compared with wild-type supernatants. These data demonstrate an important role of FPs in innate immunity against bacterial infections and indicate that host chemotaxis receptors recognize a larger set of bacterial molecules than previously thought.     

Signaling to migration in neutrophils: importance of localized pathways

Neutrophils, a major type of blood leukocytes, are indispensable for host defense of bacterial infections. Directed migration in a gradient of chemotactic stimuli enables these cells to rapidly find the site of infection and destroy the invading pathogens.

Chemotactic factors bind to seven-transmembrane-domain receptors and activate heterotrimeric G-proteins. Downstream of these proteins a complex interrelated signaling network is activated in human neutrophils. Stimulation of phospholipase Cβ results in activation of protein kinase C isoforms and increases in cytosolic calcium. Activation of the enzyme phosphoinositide 3-kinase results in increased production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidyl 3,4-bisphosphate. In addition, small GTP-binding proteins of the Rho family, the mitogen-activated protein kinase cascade, tyrosine kinases and protein phosphatases are activated. The enzyme phosphoinositide 3-kinase and the small cytosolic GTP-binding proteins Rho and Rac emerge as key regulators of neutrophil migration. A steep internal gradient of phosphatidylinositol 3,4,5-trisphosphate, with a high concentration in the leading lamellae, is thought to regulate polarized actin polymerization and formation of protrusions, together with Rac which may be more directly involved in initiating actin reorganization. Rho may regulate localized myosin activation, tail retraction, cell body traction and dynamics of adhesion.

The impact of these different signaling pathways on reversible actin polymerization, development of polarity, reversible adhesion and migration, and the putative targets of these pathways in neutrophils, are reviewed in this article. Insight into mechanisms regulating migration of neutrophils could potentially lead to novel therapeutic strategies for counteracting chronic activation of neutrophils which leads to tissue damage.     

Actin polymerization induced by chemotactic peptide and concanavalin A in rat neutrophils

Changes in the state of actin in rat neutrophils were studied after chemotactic peptide and concanavalin A stimulation by using the DNase I inhibition assay. Actin polymerization occurred within seconds after stimulation with F-Met-Leu-Phe and concanavalin A. Pretreatment of cells with cytochalasin D prevented chemotactic peptide-induced actin polymerization. The addition of F-Met-Leu-Phe to lysed cells did not produce any change in actin state. These data offer strong evidence for receptor-induced actin polymerization and support the models implicating actin microfilament formation as a crucial event in cell activation. The observations on platelets, lymphocytes, neutrophils, and islets of Langerhans from different species suggest that actin polymerization might be a universal intracellular event accompanying cell surface receptor perturbation in eukaryotic cells.     

Correlation between chemotactic peptide-induced changes in chlorotetracycline fluorescence and F-actin content in human neutrophils: a role for membrane-associated calcium in the regulation of actin polymerization?

Several observations indicate that the triggering event for receptor-mediated actin polymerization takes place in or close to the plasma membrane. Stimulation of human neutrophils with the chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) causes rapid and transient changes in both chlorotetracycline (CTC) fluorescence and the cellular content of filamentous actin (F-actin), thus suggesting a regulatory role for membrane-bound calcium in actin polymerization. In the present study, tetracaine, a proposed antagonist to membrane-bound calcium, totally inhibited the rebinding of the membrane calcium released by fMet-Leu-Phe. This was accompanied by a magnified and sustained increase in the cellular content of F-actin. In agreement, N-ethylmaleimide, an inhibitor of motile functions, completely abolished the fMet-Leu-Phe-triggered changes in both CTC fluorescence and F-actin content and rapidly reversed the responses when added after the peptide. The tumor promoter phorbol-12-myristate-13-acetate, caused only small changes in CTC fluorescence and F-actin content, and reduced a subsequent fMet-Leu-Phe-induced CTC response and actin polymerization. Inhibition of the breakdown of phosphatidylinositol 4,5-bisphosphate, by calcium depletion, had no significant effects on the fMet-Leu-Phe-induced CTC response and alterations in F-actin content, whereas pretreatment with pertussis toxin totally inhibited both these responses. Consequently, the strong correlation between changes in CTC fluorescence and F-actin content, found in this study, suggests a triggering or modulating role of membrane-associated calcium on actin polymerization in human neutrophils.     

Identification of a common receptor for three types of rat cytokine-induced neutrophil chemoattractants (CINCs)

Rat cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/macrophage inflammatory protein-2 (MIP-2), members of the CXC chemokine family, are potent chemotactic factors for neutrophils. In order to identify the receptor for CINCs, rat CXC chemokine receptor 2 (CXCR2) was cloned and expressed in HEK293 cells. CINC-1, CINC-2 and CINC-3 induced calcium mobilizations dose-dependently in CXCR2-transfected cells, whereas formyl-methionyl-leucyl-phenylalanine (FMLP) did not. CINC-3 induced enhancement of cytoplasmic calcium concentration more potently than CINC-1 and CINC-2, and desensitized calcium transients induced by CINC-1 and CINC-2, which were essentially identical to those observed in rat neutrophils. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of three types of CINCs almost completely. The mutant CINC-3, whose amino-terminal amino acid sequence (SELR) was replaced to AAR, lost chemotactic activity of its own but inhibited that of CINC-1 and CINC-2 potently, and that of CINC-3 weakly. The results indicate that rat CXCR2 on neutrophils is the unique receptor for all three types of CINCs, and CINC-1/-2 and CINC-3 exert different biological activities through the common receptor.     

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.     

Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH

Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.