Adhesion in cell migration.

Adhesive interactions play a central role in cell migration. The regulation of these interactions requires the coordination of a multiplicity of signals, both spatially and temporally. The role of the integrin family has received considerable recent attention. Progress has been made in the elucidation of the mechanisms by which growth factors and other motogenic factors stimulate migration. Major advances have also been made in understanding the mechanisms by which the formation and breakdown of adhesive complexes are regulated, including the participation of members of the rho family. Despite these advances, many important questions remain, and the field seems well positioned to answer them.

Signal transduction events and Fc gamma R engagement in human neutrophils stimulated with immune complexes.

Signal transduction events have been evaluated in human neutrophils stimulated with immune complexes consisting of polyclonal rabbit antibody complexed with BSA. Immune complexes induced dose-related O2- responses, but very small increases in intracellular calcium ([Ca2+]i) levels were observed, in contrast to FMLP-stimulated cells. Measurements employing [45Ca2+] demonstrated that calcium influx and efflux in cells stimulated with immune complexes was substantially less than fluxes found in FMLP-stimulated cells. With respect to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation under conditions in which the O2- responses to immune complexes or FMLP were similar, the Ins(1,4,5)P3 response to immune complexes was much smaller (by 65%) as compared to that induced by FMLP. Although pertussis toxin-treated cells showed a greatly diminished O2- response (by 89%) to FMLP, the response to immune complexes was largely resistant (only 26% reduction) to the inhibitory effects of this toxin. Antibodies to Fc gamma R indicated that engagement of Fc gamma RII and Fc gamma RIII, but not Fc gamma RI, receptors was related to the O2- response of neutrophils to immune complexes. O2- formation occurred in neutrophils incubated with Staphylococcus aureus cell walls bearing antibodies to Fc gamma RII or Fc gamma RIII. These data indicate that, in human neutrophils stimulated with immune complexes, signal transduction events involve engagement of Fc gamma RII and Fc gamma RIII. The O2- response is largely pertussis-toxin insensitive, is not associated with a significant increase in levels of [Ca2+]i, and is associated with relatively little formation of Ins(1,4,5)P3. This is in contrast to cells stimulated with FMLP in which O2- responses are largely pertussis toxin-sensitive and associated with large increases in [Ca2+]i as well as formation of Ins(1,4,5)P3. Signal transduction events involving Fc gamma R appear to be quite different from those events related to engagement of FMLP receptors.

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes.

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.

Ca2(+)-induced secretion by electropermeabilized human neutrophils. The roles of Ca2+, nucleotides and protein kinase C.

Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2(+)-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP beta S ATP gamma S, and App[NH]p were effective. GTP gamma S and GDP beta S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 degrees C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.

Exposure of human neutrophils to exogenous nucleotides causes elevation in intracellular calcium, transmembrane calcium fluxes, and an alteration of a cytosolic factor resulting in enhanced superoxide production in response to FMLP and arachidonic acid.

Exposure of human neutrophils to micromolar concentrations of both hydrolyzable and nonhydrolyzable purine nucleotides caused the generation of transient rises in intracellular calcium (Ca2+), Ca2+ fluxes across the membrane, and primed the cells for enhanced production of superoxide (O2-) when subsequently exposed to agonists such as FMLP and arachidonic acid. The neutrophils were most sensitive to adenosine triphosphate (ATP) and ATP-gamma-S, which produced Ca2+ transients and enhanced O2- production at concentrations as low as 1 to 5 mumol/L, with a doubling of O2- generation at 25 to 50 mumol/L. Adenosine diphosphate (ADP), guanosine triphosphate (GTP), and 5'-adenylylimidodiphosphate (AMP-PNP) required approximately 10-fold higher concentrations to cause similar effects. Adenosine did not cause Ca2+ fluxes or a Ca2+ transient and was inhibitory of O2- production. There was a strong correlation between a nucleotide's ability to generate a Ca2+ response and its ability to enhance O2- generation. Nitrogen cavitation and subcellular fractionation of the neutrophils after a brief exposure to ATP, ATP-gamma-S, and AMP-PNP revealed that the enhanced O2- generating capacity was stable and detectable in a cell-free assay system. By combining variously treated cytosolic and membrane fractions, it was found that the enhanced O2- production was attributable to a modification of a component(s) of the cytosol.

Effects of 5-fluorouracil on protein synthesis and secretion of the rat parotid gland.

100 mg/kg of FU were injected intraperitoneally once daily for three days. Animals were anaesthetized with 50 mg/kg of sodium pentobarbital before cannulation of the parotid duct. The total volume, amylase and protein content of the saliva were determined after stimulation with either 5 mg/kg pilocarpine or 5 mg/kg isoproterenol in FU-treated, pair-fed, and control animals. Saliva from FU-treated animals was significantly lower (p less than 0.05) in volume, amylase and protein content than that of both control groups. SDS, anionic and cationic gel electrophoresis of parotid saliva revealed no qualitative changes in the types of proteins secreted. FU reduced the total glandular amylase per unit DNA in both unstimulated and isoproterenol-stimulated parotids (p less than 0.05). Decreased protein synthesis may be the mechanism underlying the depleted secretory protein stores because the contents of isolated secretory granules from experimental glands contained less radiolabelled protein than those of either control group, and whole-gland homogenates had marked reductions in the activities of three lysosomal enzymes and in total RNA content. The secretory granules of experimental animals contained less labelled protein than those of controls, but experimental animals secreted a greater proportion of their total glandular radiolabelled secretory protein into saliva relative to amylase, suggesting that newly synthesized secretory proteins were preferentially secreted.

The effects of heavy metal cations and sulfhydryl reagents on degranulation from digitonin-permeabilized neutrophils.

Digitonin-permeabilized neutrophils were exposed to micromolar levels of a variety of heavy metal cations and sulfhydryl oxidants to gain insight into the potential biochemical mechanisms underlying neutrophil degranulation. The results from this study suggest that the oxidation of intracellular sulfhydryl groups may play a role in neutrophil signal transduction. Evidence to support this conclusion is based on the observation that cupric phenanthroline and Cu2+/cysteine, agents reported to induce disulfide bond formation, evoke significant granule enzyme release when presented to permeabilized neutrophils. The stimulatory actions of these compounds occur in the absence of Ca2+ and are blocked by the sulfhydryl reducing agent, dithiothreitol. In addition, we observed marked potentiation of Ca2+-induced secretion by potentially physiological levels of Ni2+. Although we are unaware of any Ni2+-requiring enzymes in eukaryotic cells that are likely to be pertinent to degranulation, the ability of this divalent metal cation to lower the Ca2+ requirements for granule secretion suggests that it may play an important regulatory role in Ca2+-dependent processes. Finally, we observed significant granule release when permeabilized neutrophils were exposed to the heavy metal cations, Hg2+ and Ag+. The apparent stimulatory actions of these metals were the result of lysis rather than degranulation. Thus, the ability of these metals to lyse intracellular organelles such as lysosomal granules may contribute to their toxicological properties.