Search

Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.

The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2-EGFP. In serum-free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2-EGFP to the plasma membrane. Serum-induced translocation was blocked by transfection of short-form TRPV2 (s-TRPV2) lacking a pore-forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met-Leu-Phe (fMLP) also induced translocation of TRPV2-EGFP to the plasma membrane. The fMLP-induced translocation was blocked by an inhibitor of PI 3-kinase, LY294002, and pertussis toxin. Whole-cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s-TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s-TRPV2 or siRNA. Finally, fMLP-induced migration of macrophage was blocked by ruthenium red or transfection of s-TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP-induced calcium entry., (Copyright 2006 Wiley-Liss, Inc.)

Movements of D. discoideum vegetative amoebae responding to pteridine chemoattractants, folate acid and pterin, were recorded. A vector analysis of these images was performed to partition the speed and orientation components of these motility patterns. This study demonstrates that in addition to orientation (chemotaxis), stimulated speed (chemokinesis) is an important component of the directed migration of these amoebae. Furthermore, the primary difference in their response to folate versus pterin is in speed rather than orientation. The data support a model of directed migration of these cells in which there are (1) separate signal translation pathways consequent from folate versus pterin reception and (2) specific pathways leading to increase in orientation versus speed., ((c) 2005 Wiley-Liss, Inc)

It has been assumed that the natural chemotactic signal that attracts human polymorphonuclear leukocytes (PMNs) over long distances to sites of infection is in the form of a standing spatial gradient of chemoattractant. We have questioned this assumption on the grounds, first, that standing spatial gradients may not be stable over long distances for long periods of time and, second, that in the one animal cell chemotaxis system in which the natural chemotactic signal has been described in space and time, aggregation of Dicytostelium discoideum, the signal is in the form of an outwardly relayed, nondissipating wave of attractant. Here, it is demonstrated that PMNs alter their behavior in each of the four phases of a wave of PMN chemoattractant, fashioned after the Dictyostelium wave, in a manner similar to Dictyostelium. These results demonstrate that PMNs have all of the machinery to respond to a natural wave of attractant, providing support to the hypothesis that the natural signal that attracts PMNs over large distances to sites of infection in the human body may also be in the form of a wave., (Copyright 2003 Wiley-Liss, Inc.)

The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) were investigated with a phospho-specific antibody that recognized cofilin only when this protein was phosphorylated on ser-3. Unlike earlier studies that monitored changes in (32)P-labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 microM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide-specific phospholipase C (PI-PLC) (1.0 microM U73122). Products of PI-PLC, sn-1,2-diglyceride and inositol (1,4,5)-trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ca(2+), respectively. Treatment of neutrophils with agents that selectively activate PKC [4beta-phorbol 12-myristate 13-acetate (PMA) ] or cellular Ca(2+) (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolylmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 microM) and W-7 (50 microM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less-active analogs of these inhibitors (15 microM promethazine, 50 microM W-5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC-dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP-stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot)., (Copyright 2003 Wiley-Liss, Inc.)

Chronic myeloid leukemia (CML), a hematopoietic stem cell disorder, is characterized by the presence of Philadelphia chromosome (Ph1). Earlier studies have shown that various functions, such as chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), were defective in polymorphonuclear leukocytes (PMNL) from CML patients. These functions depend on actin microfilaments (MF). Further studies showed that fMLP-induced actin polymerization was lower in CML PMNL. To see if this defect is specific to stimulation by fMLP alone or is a global phenomenon involving other chemoattractant receptors, chemotaxis and actin polymerization were studied in response to fMLP, an analog of fMLP, formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenyalanine-O-methionine (FACC8), platelet-activating factor (PAF), and leukotriene B4 (LTB4). These compounds bind to different chemoattractant receptors. Chemotaxis and actin polymerization in response to all four chemoattractants were significantly lower in CML PMNL compared with PMNL from normal subjects and were differentially affected for the different chemoattractants. These results suggest a global phenomenon involving all four chemoattractant-stimulated pathways. This lower amount of F-actin may be responsible for the defective chemotaxis seen in these cells.

Cell motility and changes in cell shape are largely powered by actin polymerization and depolymerization. Eight to ten second periodic changes in human polymorphonuclear neutrophil (PMN) shape were detected by video-image analysis of PMN crawling on a surface and by right angle light scattering (RALS) in suspended PMN. However, sustained RALS oscillations in suspended PMN requires pre-treatment with an inhibitor of phosphatidylinositol 3-kinase or an activator of protein kinase C. Here, we show that cross-linking of the beta(2) (CD18) or beta(3) (CD61), but not beta(1) (CD 29) integrins in the presence of a low dose of formyl-Methionyl-Leucyl-Phenylalanine (fMLP) enables similar 8-s periodic RALS oscillations in suspended PMN in response to stimulation with two consecutive doses of chemoattractants. This effect did not appear to be due to increased surface expression of CD18 or CD61. RALS oscillations occurred in phase with 8-s oscillations in the stable F-actin pool and peaks in F-actin correlated with predominance of cells exhibiting a nascent lamella. Thus, simulation of surface attachment by CD18 and CD61 cross-linking after exposure to fMLP in suspended cells supports shape oscillations that are the result of actin-driven cyclic extension/retraction of nascent lamellae at the same frequency as the shape changes previously observed in crawling PMN., (Copyright 1999 Wiley-Liss, Inc.)

Hepatocyte growth factor (HGF) and its receptor c-met are present in several human tissues but their expression in mesothelial cells has not been examined. In this study, we have investigated the expression of HGF and c-met in normal human mesothelial cells and 11 human malignant mesothelioma cell lines. Using RT-PCR and Western blotting we found that HGF is produced by 3/11 mesothelioma cell lines whereas c-met is expressed in 11/11 mesothelioma cell lines. In addition, c-met expression was also found in 6/6 cell samples obtained from pleural fluids of patients with mesothelioma. In contrast, neither normal cultured mesothelial cells nor mesothelial cells obtained directly from patients without mesothelioma expressed HGF nor c-met. We have also analysed the biological function of HGF and c-met in mesothelioma cell lines. Recombinant human (rh) HGF stimulated both directional (chemotactic) and random (chemokinetic) motility in all mesothelioma cell lines tested. Furthermore, mesothelioma serum free conditioned medium containing HGF stimulated mesothelioma cell migration. This effect could be blocked in the presence of neutralizing anti-HGF monoclonal antibodies (MAbs) in the assay. Addition of HGF to mesothelioma cells cultured on collagen type IV was associated with induction of bipolar shape and protrusion of prominent pseudopodia. We have also found that rhHGF was mitogenic for mesothelioma cells. Our findings suggest that expression of HGF/c-met is involved not only in mesothelioma progression but also in its growth and migration and that c-met expression found in mesothelioma cells taken directly from patients may be of diagnostic importance.

Oral fibroblasts stimulated invasion of oral-carcinoma cells into the collagen matrix. The mechanisms of the fibroblast-induced stimulation of invasiveness was further investigated by examining cell motility and proteolytic activity of tumor cells, using mainly an adenoid-cystic-carcinoma cell line (ACCS) and normal fibroblasts from gingival tissues. Conditioned medium from the fibroblasts grown in serum-free medium was fractionated on a Superdex 200 pg column, and Peak 1 eluted at 200 to 300 kDa and Peak 2 eluted at 50 to 100 kDa were found to contain different specific activity. Treatment of ACCS cells with Peak 1 resulted in an increase in the production of proteolytic enzymes. Peak 2 stimulated both chemotaxis and chemokinesis of ACCS cells. A chemotactic factor was purified from the heparin-unbound fraction of Peak 2 by anion exchange and hydrophobic chromatography, and was named "fibroblast-derived motility factor (FDMF)". At 1 microg/ml, FDMF stimulated chemotaxis of ACCS cells by 4-fold compared with unstimulated controls. Characterization of the physicochemical properties of FDMF suggested that it might be different from any known motility factors. Exposure of ACCS cells to FDMF resulted in reduced amounts of actin stress fiber in the cytoplasm and induction of tyrosine phosphorylation of several cellular proteins detectable 30 to 60 min after treatment. These FDMF-induced changes were blocked by pre-treatment either with genistein or with pertussis toxin. These findings suggest that FDMF may be a novel protein which stimulates cell motility via a signaling pathway mediated by a pertussis-toxin-sensitive G protein and tyrosine phosphorylation.

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.

Expression of c-Met, the receptor for hepatocyte growth factor (HGF), and the biological roles of HGF were examined in cultured human glioma cells. All of the 5 glioma cell lines examined expressed c-Met protein as well as the c-met gene. Expression of the c-met gene was also confirmed in a glioblastoma tissue. Three cell lines (MGM-3, U251, KG-1-C) demonstrated chemotactic response to HGF in a dose-dependent manner. The response was not only chemotactic but also chemokinetic as judged by a checkerboard analysis. The amounts of c-Met mRNA and protein were abundant in the cell lines which showed a migratory response to HGF. Moreover, c-Met protein expression was highest in U251 with the highest migratory response to HGF. Among the cell lines, KG-1-C produced notable amounts of HGF protein as well as of c-Met, suggesting that HGF may act in an autocrine fashion in this case. HGF did not act as an apparent growth factor in the glioma cell lines examined. Furthermore, HGF stimulated the production of metalloproteinase, probably gelatinase A, in U251 cells.

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-beta 1 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 Ca(2+)-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.

Prostate tumor cells preferentially metastasize to bony sites and lymph nodes at a frequency in excess of that which would be predicted by random tumor cell dissemination. In order to determine whether chemoattractants in these organs promote organ-specific metastasis, we utilized human cell lines derived from and/or related to these organs as sources of potential chemoattractants. Secretory proteins derived from the cell lines MG-63 (osteosarcoma), SK-ES-1 (Ewing's sarcoma), and KG-1 (leukemia) stimulated chemomigration of the TSU-pr1 prostate tumor cells in a dose-dependent manner in Boyden chambers. In addition, secretory proteins from a human prostatic stromal cell line (hPS) and from the TSU-Pr1 prostate tumor cell line were also able to stimulate chemomigration of the TSU-pr1 cells through Boyden chambers. Since lymph nodes and bony sites represent organs of hematopoietic/lymphoid proliferation and activation, we undertook identification of specific cytokines present at these sites which may promote the chemomigration of prostate tumor cells. In this context, the cytokines interleukin-1 alpha, interleukin-2, interleukin-6, tumor necrosis factor-beta, transforming growth factor-beta, interferon alpha 2-a, and granulocyte-macrophage colony-stimulating factor did not stimulate chemomigration of the TSU-pr1 prostate tumor cell line. In contrast, the cytokine epidermal growth factor (EGF) stimulated chemomigration of the TSU-pr1 prostate tumor cells through the Boyden chambers in a dose-dependent manner. Western blot analysis of secretory proteins from the cell lines KG-1, SK-ES-1, MG-63, hPS, and TSU-pr1 identified EGF-immunoreactive proteins in all cases. In addition, EGF immunoreactivity was localized to the stroma of the human prostate, the osteogenic stroma of pelvic medullary bone, and the stroma within the capsule and trabeculae of pelvic lymph nodes. Hence, these results demonstrate that the cytokine EGF promotes the chemomigration of the TSU-pr1 prostate tumor cell line, and that EGF within the stroma of pelvic lymph nodes and medullary bone may act as a chemoattractant for prostate tumor cells, thereby facilitating the preferential formation of metastatic foci within these organs.

Micropipets are used either to deliver a given concentration of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) to a local region of a human neutrophil or to create a membrane tether. Pseudopods, which have a cylindrical shape and grow at a constant rate, are formed in either case. After reaching a maximum extension, they retract, even in the presence of chemoattractant. As a pseudopod grows, cell granules begin to penetrate the pseudopod region to a "boundary" that defines a distance to the pseudopod's leading edge that is almost constant. The exclusion of granules from this domain indicates that it is filled with a dense network. The formation of this network involves the plasma membrane because pseudopod growth ceases when a membrane tether is pulled away from the leading edge. The rate of pseudopod growth depends on fMLP concentration just as the number of occupied N-formyl peptide receptors depends on this concentration. The experimental data are explained by assuming that F-actin network is formed next to the plasma membrane. The newly formed network displaces the membrane and the dominant process in the network region then becomes F-actin depolymerization. The rate of pseudopod growth is determined by the rate of the process leading to network formation. This process is apparently an enzymatic type of reaction. It has a positive enthalpy change and, therefore, is endothermic.

Nitric oxide (NO) has been shown to be both an intercellular and intracellular messenger. We propose here that exogenous NO induces chemotactic locomotion of human neutrophils. Indeed, when human neutrophils were placed in a gradient of a nitric oxide donor (S-nitroso-N-acetylpenicillamine; SNAP), a directed locomotion was induced, as evidenced by experiments of chemotaxis under agarose. Degraded SNAP (i.e., SNAP solution which had previously released NO) did not induce directed locomotion. Moreover, oxyhemoglobin, a scavenger of free NO, suppressed the chemotactic effect of SNAP, whereas LY-83583, a soluble guanylate cyclase inhibitor, inhibited the SNAP-mediated chemotaxis in a dose-response manner. Other unrelated NO donors, SIN-1 and S-nitroso-cysteine--a natural S-nitroso-compound, also induced a directed locomotion of neutrophils. Taken together, these in vitro experiments indicate that exogenous NO could mediate the chemotaxis of neutrophils and thus suggest that NO could contribute to neutrophil recruitment in vivo.

Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as egg-derived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half-maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5-27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor metastasis is the primary cause of death for cancer patients. The metastatic cascade requires successful tumor cell invasion into and through vascular and parenchymal barriers. We have shown that autocrine motility factor (AMF, autotaxin) and the insulin-like growth factors (IGFs) induce tumor-cell migration. Since granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to prime neutrophils for chemotaxis, we have therefore studied the influence of GM-CSF upon tumor cells and report that GM-CSF stimulates migration of these cells in a dose-dependent fashion. The ED50 for A2058 human melanoma cell line chemotaxis to GM-CSF is approx. 60 pM. The motile response to GM-CSF was additive to that of IGF-I and AMF, both of which are potent attractants for tumor cells. Pre-treatment of cells for 2 hr with non-toxic concentrations of pertussis toxin (PT) or amiloride resulted in a 50% inhibition of chemotaxis to GM-CSF. Therefore, GM-CSF, through PT- and amiloride-sensitive signal pathways, is a potent attractant for melanoma cells, the response to which is additive to that of other attractants. The presence of the GM-CSF receptor in A2058 melanoma cells was indicated by Northern-blot analysis which identified message transcripts of 2.1 and 3.0 kb. These data emphasize the versatility of the melanoma cell migration response to an array of cytokines, including GM-CSF.

We compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F-actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine-labeled G-actin under polymerizing conditions. Endogenous F-actin was stabilized and stained by lysis of cells into fluorescein-labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F-actin concentration. Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophores went up; (2) if peptide was diluted shortly before lysis, the endogenous F-actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F-actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F-actin. The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin-actin complex (which stably caps barbed ends), then washed before the rhodamine G-actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991]. The fact that the distribution of exogenous actin polymerization paralleled the endogenous F-actin suggests that the number of free barbed ends per F-actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cell.

Tumor cell locomotion is an integral part of the metastatic process. We present a new autocrine motility factor (AMF) derived from the serum-free conditioned medium of the Dunning R-3327 rat prostate adenocarcinoma AT2.1 tumor cell subline AT2.1-AMF, prepared by concentration of components less than or equal to 30 kDa- in size and washed free of low-molecular-weight growth factors, stimulated motility of AT2.1 cells in modified Boyden chamber migration assays. This stimulated migration was dose-dependent, and by checkerboard analysis was both chemotactic and chemokinetic. AT2.1-AMF activity was labile to heat, acid, base, reduction, oxidation, and proteases. Lyophilization and treatment with 6M urea caused a mild decrease (less than 20%) in migration-stimulating capability. Tumor-cell specificity was demonstrated for AMF of AT2.1 and AT3.1 Dunning sublines, and the A2058 human melanoma cell lines. AT2.1 cell migration to AT2.1-AMF was inhibited by 2 hr pre-treatment with cholera toxin (0.1 microgram/ml) or forskolin (100 microM), but not altered by 2 hr pre-treatment with pertussis toxin (1.0 microgram/ml). This indicates that guanine nucleotide binding protein-mediated regulation of cAMP is involved in modulating the AT2.1 cell response to its AMF. The AT2.1-AMF belongs to a related family of tumor autocrine motility factors and represents a new model for understanding the role of tumor-cell migration in the metastatic process of human prostate cancer.

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.

Definition of the kinetics of ligand-activated actin polymerization in the neutrophil is important for ultimately understanding the mechanisms utilized for regulation of actin polymerization in this non-muscle cell. To better define the kinetics of formyl peptide (fMLP)-induced actin polymerization in neutrophils we determined F-actin content at 5 second intervals after activation of human neutrophils with a range (10(-11)-10(-9) M) of fMLP concentrations. The state of actin polymerization was monitored by quantifying F-actin content with NBD phallacidin binding in both flow cytometric and extraction assays. Results demonstrate three successive kinetic periods of fMLP-induced actin polymerization in neutrophils, a lag period, a 5 second period when rate of polymerization is maximal, and a period of declining rate of actin polymerization as F-actin content approaches a maximum. The duration of the lag period, the maximum rate of polymerization, and the maximum extent of polymerization all depend upon the fMLP concentration. The lag period varies from 0 to 12 seconds and is followed in 5-10 seconds by a 5 second burst of actin polymerization when the rate is as great as 9% increase in F-actin content per second. After the 5 second burst of polymerization, the rate of polymerization rapidly declines. The study defines three distinct kinetic periods of fMLP-induced actin polymerization during which important rate-limiting biochemical events occur. The mechanistic and motile implications of kinetic periods are discussed.

The addition of low concentrations (less than 10(-7) M) of the calcium ionophore A23187 to rabbit neutrophils releases the intracellular pool of calcium previously shown in radioactive steady-state and chlortetracycline fluorescence studies to be mobilized by chemotactic factors. A23187 at these concentrations elicits no functional responses from these cells. However, A23187, added before chemotactic factors such as fMet-Leu-Phe and leukotriene B4, inhibits the ability of the latter stimuli to induce, in the presence of cytochalasin B, an exocytotic release of the neutrophil's cytoplasmic granules. These results imply that the chemotactic-factor-induced release of intracellular calcium is a necessary event for the optimal activation of the neutrophils. Phorbol ester-induced neutrophil degranulation on the other hand is unaffected by exposure to A23187, thereby completely dissociating its mechanism of action from rises in cytoplasmic free calcium.

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA-5E (visceral endodermlike) cells produced little of this factor. However, PYS-2 (parietal endodermlike) and Dif 5 endoderm cells were found to produce significant amounts of endoderm-derived mitoattractant (EDM) activity. The activity secreted by the Dif 5 cells was partially purified using gel filtration chromatography using chemotaxis and mitogenic assays as markers for biological activity. The partially purified activity competes with [125I]iodo-platelet-derived growth factor (PDGF) for binding to target cells, and the biological activity is neutralized with anti-PDGF IgG, suggesting shared domains in the two molecules. However, the factor appears to be different from PDGF, based on its thermal stability, molecular weight, and charge. The differentiated endoderm cells including retinoic acid (RA)-treated F9, Dif 5, PSA-5E, and PYS-2 cells also exhibit specific [125I]iodo-PDGF binding, and the PSA-5E cells respond to PDGF as a chemoattractant. Conceivably, such a PDGF-like factor may contribute to the regulation of cell growth and migration during the early stages of embryogenesis.

We have found that exogenous leukotriene B4 modifies calcium homeostasis in rabbit neutrophils in a manner essentially analogous to that of the chemotactic peptide f-Met-Leu-Phe. Leukotriene B4 causes a rapid and dose-dependent increase in membrane permeability to calcium and a release of calcium from previously unexchangeable intracellular pool(s). The net result of these changes is to transiently elevate the intracellular level of exchangeable calcium. A stereoisomer of leukotriene B4 with greatly reduced secretory activity toward neutrophils (5S, 12S-di HETE) is essentially without effect on the rate of 45Ca uptake at concentrations equal to those that produce near maximal enhancement by leukotriene B4. Leukotriene B4, in addition to its effects on calcium metabolism, also increases the rate of 22Na influx into rabbit neutrophils. The relationships between the action of leukotriene B4 on calcium homeostasis and the neutrophil-directed activities of arachidonic acid and its lipoxygenase metabolites are discussed

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.

Membranes prepared from rabbit neutrophils exhibit GTPase activity which can be stimulated by the chemotactic factor fMet-Leu-Phe. The maximum contribution of the ATPase activities to the basal and the fMet-Leu-Phe-stimulated GTPase activities are less than 20% and 9%, respectively. The basal GTPase activity has a Vmax = 34.2 +/- 1.3 (pmol/mg protein, min) and a Km = 0.39 +/- 0.03 microM; and the fMet-Leu-Phe-stimulated has a Vmax = 52.3 +/- 2.5 (pmol/mg protein, min), and a Km = 0.29 +/- 0.02 microM. The GTPase activity can be stimulated by fMet-Leu-Phe and leukotriene B4. Unlike these two chemotactic factors, concanavalin A does not stimulate this GTPase activity. In addition, the rise in intracellular concentration of free calcium produced by concanavalin A is not inhibited by pertussis toxin treatment. Both the basal and stimulated GTPase activities are affected by pertussis toxin, cholera toxin and N-ethylmaleimide.

The relationship between the chemotactic-factor-elicited changes in the intracellular pH and the shape of human neutrophils was investigated using simultaneous measurements of both parameters. The results demonstrate first that fMet-Leu-Phe and leukotriene B4 elicit qualitatively similar pH and shape change responses from the neutrophils. A relationship between the chemoattractant-elicited decrease in cytoplasmic pH and the shape changes is indicated by several findings including: 1) the similarities in the time courses of the two responses, 2) the ability of propionic acid to induce a transient and pertussis-toxin-sensitive shape change response, and 3) the ability of the calcium ionophore A23187 to similarly induce both responses under conditions when the degranulation is minimized. On the other hand, several other results indicate that the drop in pH is not a sufficient condition for the chemotactic-factor-stimulated shape changes. These include: 1) the ability of pertussis toxin to inhibit the shape changes induced by propionic acid and by A23187 without affecting the drop in pH, and 2) the observation that the drop in pH induced by propionic acid persists significantly longer than the shape change. Increasing the cytoplasmic pH by adding ammonium chloride was also found to cause shape changes in the neutrophils. The response to the base differs in two important aspects from that caused by propionic acid: it is pertussis-toxin-insensitive, and it is long-lived. Chemotactic factors have been found to induce a shape change under conditions when the internal pH was artificially increased or decreased, indicating that it is not the absolute cytoplasmic pH that represents the internal signalling parameter. The results are discussed in terms of the activation of the cytoskeletal network of the neutrophils by chemotactic factors.

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of 45Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of 45Ca. Arachidonate also increases the uptake of 22Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to 45Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.

The preincubation of rabbit neutrophils with the chemotactic factor F-Met-Leu-Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Ca and 22Na was found upon preincubation with F-Met-Leu-Phe. In the presence of extracellular Ca2+, this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F-Met-Leu-Phe. In the absence of added extracellular Ca2+ an initial and rapid (less than 1 minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of the neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils.

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3'dipentyloxacarbocyanine [di-O-C5(3)], 3,3'dipentylindocarbocyanine [di-I-C5(3)], and 3,3'dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.