Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum.

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10(5) greater than that remaining in the medium. The apparent Keq is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external 35SO42- exchanges with internal, unlabeled SO42- at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1 is characterized by the rapid release of 35SO42- when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t 1/2, 0.38 min). 35SO42- in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of 35SO42- induced by azide at pH 8.4 or unlabeled sulfate (t 1/2, 32 to 49 min). Early in the 35SO42- accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated 35SO42-. The kinetics of 35SO42- accumulation is consistent with the following sequential process: medium----pool 1----pool 2. Monensin (33 microns) accelerates the transfer of 35SO42- from pool 1 to pool 2. Valinomycin (0.2 microM) and tetraphenylboron- (1 mM) retard the transfer of 35SO42- from pool 1 to pool 2. At the concentrations used, neither of the ionophores nor tetraphenylboron- affect total 35SO42- uptake. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.     

Internalization pathway of C3b receptors in human neutrophils and its transmodulation by chemoattractant receptors stimulation.

On the surface of phagocytes, C3b receptors (CR1) bind C3b-coated particles and promote their ingestion after activation by appropriate stimuli such as lymphokines or the chemoattractant formyl methionyl leucyl phenylalanine (fMLP) and fibronectin. The aims of the present study were 1) to define at the electron microscopic level the nature of the process responsible for CR1 internalization and 2) to dissect the mechanism by which a physiological activator (fMLP) stimulates this process. CR1 was visualized either by the immunogold technique or by quantitative electron microscopic autoradiography using a monoclonal anti-CR1 antibody. Both techniques revealed that after anti-CR1 binding, CR1 cluster on the neutrophil surface in a time-, temperature-, and antibody-dependent fashion, but do not concentrate in coated pits. CR1 internalization requires receptor cross-linking (does not occur in the presence of Fab fragments of anti-CR1) and intact microfilaments. It results in the association of the internalized material with large flattened vacuoles, organized in stacks. Together with the surface localization of CR1 close to cytoplasmic projections (ruffles), these observations suggest that uptake of CR1 occurs through a macropinocytotic process. Eventually, CR1 concentrate in lysosomal structures. fMLP markedly stimulates this pattern of CR1 internalization without affecting their clustering or their lack of association with coated pits. Stimulation by fMLP is inhibited by pertussis toxin, unaffected by preventing receptor-triggered cytosolic free calcium [Ca2+]i elevations, and mimicked by phorbol myristate acetate. Taken together our data demonstrate 1) that, in neutrophils, CR1 is internalized via a coated pit independent macropinocytotic process, dependent on intact microfilaments and receptor cross-linking; 2) that, in the same cells, fMLP is internalized via the classical coated pits pathway; and 3) that fMLP amplifies CR1 uptake possibly via protein kinase C stimulation.     

Neutrophil C3bi receptors: formation of membrane clusters during cell triggering requires intracellular granules

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.     

Regulation of distinct pools of protein kinase C δ in beta cells

Previous studies from our laboratory have demonstrated the presence of several isoforms of protein kinase C (PKC), Ca²⁺-independent and Ca²⁺-dependent, in both whole islets and tumor-derived beta cells. In the basal state, a major proportion of the isoform was found in the crude membrane fraction with smaller amounts found in both the cytosolic and cytoskeletal fractions. Whole islets showed a similar distribution of the isoform. These studies were done to analyze the effects of insulin secretagogues on the distribution of PKC δ to different cellular pools in isolated insulinoma beta cells. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), produced a transient association of PKC δ with the beta cell cytoskeleton along with sustained decreases in cytosolic enzyme and transient increases in membrane enzyme. Neither glucose nor carbachol could acutely affect the subcellular distribution of PKC δ. Oleic acid decreased the amount of the enzyme associated with the cytoskeleton and led to a sustained decrease of cytosolic enzyme and a transient increase in membrane enzyme. Oleic acid was also able to prevent the increase in cytoskeletal enzyme induced by PMA. Both oleic acid and PMA potentiated glucose-induced insulin release but oleic acid, in contrast to PMA, was unable to initiate insulin release in the presence of substimulatory concentrations of glucose. These data demonstrate that different activators of PKC may have different effects on localization of the enzyme within the cells and suggest that there are at least three apparently distinct pools of PKC δ within the beta cell which may be important in insulin secretion or other aspects of beta cell function. © 1996 Wiley-Liss, Inc.     

Altered surface distribution of both C3b receptors and Fc receptors on neutrophils induced by anti-C3b receptor or aggregated IgG

Human neutrophils to which monospecific Fab' or F(ab')2 anti-C3b receptor had been bound at 0 degrees C were incubated for timed intervals at temperatures ranging from 0 degrees C to 37 degrees C, after which the cells were labeled with TRITC -conjugated second antibody. Neutrophils bearing Fab' anti-C3b receptor and incubated for up to 30 min at 37 degrees C, and cells bearing F(ab')2 anti-C3b receptor and incubated at 0 degrees C, exhibited diffusely distributed punctate clusters of receptors. Neutrophils bearing the bivalent anti-receptor and incubated at 30 degrees C or 37 degrees C for 5 min had redistributed C3b receptors into caps and patches that were associated with subplasmalemmal accumulations of myosin. The redistribution of cross-linked C3b receptors was inhibited by pretreatment of the neutrophils with either cytochalasin D or chlorpromazine. On approximately one-half of the cells demonstrating capped C3b receptors there was a corresponding redistribution of Fc receptors, as demonstrated by subsequent binding of FITC-aggregated IgG (FITC agg-IgG). In contrast, capping of C3b receptors did not alter the diffuse distribution of HLA-A on these cells. Cross-linking of Fc receptors on neutrophils by FITC agg-IgG also induced temperature-dependent capping of these receptors that was inhibited by cytochalasin D and chlorpromazine. In approximately one-half of the cells demonstrating capped Fc receptors, subsequent labeling of C3b receptors revealed a similar redistribution of these receptors. Thus, the neutrophil responds to cross-linking of either C3b receptors or Fc receptors by a cytoskeletal-dependent rearrangement of both receptors that causes their overlapping topographic distribution, demonstrating a form of cooperative interaction between these two types of receptors that are involved in the phagocytic reactions of these cells.     

Evidence for linkage between HLA antigens and receptors for complement components C3b and C3d in human-mouse hybrids

A linkage between HLA-A and HLA-B antigens and receptors for complement components C3b and C3d has been demonstrated by means of human-mouse somatic cell hybridization. In 26 hybrid clones formed between the human lymphoblastoid cell lines RPMI 6410 and Raji and the murine fibroblast line 1T22, HLA-A and HLA-B antigens and receptors for C3b and C3d segregated conjointly. No segregation of the C3b and C3d receptors from each other or from HLA antigens was observed. The results suggest that the genes for the C3b and C3d receptors are located on the same chromosome that encodes the major histocompatibility complex of the human.     

Fc and C3bi receptors and the differentiation antigen BH2-Ag are randomly distributed in the plasma membrane of locomoting neutrophils

Reports from several laboratories suggest that neutrophils arrested during locomotion preferentially bind immune complexes at the front of the cell. Such asymmetry of binding has been interpreted as indicating an active modulation of phagocytic receptors to the anterior of the cell. To investigate this further, we have used digital analysis of fluorescence images to determine the binding patterns of mAbs directed against the Fc receptors, the receptors for the C3bi fragment of C3, and a neutrophil-specific antigen. We found that all three proteins are distributed nearly identically along the length of migrating neutrophils, and their distribution very closely parallels the anterior to posterior distribution of the plasma membrane. The use of mAbs offered an important advantage in that the binding of antireceptor antibodies, unlike the binding of ligands, should be independent of potential changes in the affinity of the receptors. We conclude that the anterior distribution of the phagocytic receptors in the plasma membrane of locomoting neutrophils parallels the overall increase in membrane area at the front of a migrating cell and that specific translocation of phagocytic receptors does not occur.     

Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils.

Cell adherence plays a central role in many host defense mechanisms. Human peripheral blood neutrophils possess cell surface receptors that contribute to cell adherence or detachment. Receptors specific for the C3bi cleavage fragment of the third component of complement (CR3) promote adhesion, whereas histamine receptors promote detachment. In the present study, we tested the ability of histamine to down-regulate the physiological effects of CR3 receptors. Histamine decreased the binding of 51Cr-labeled neutrophils to complement-coated surfaces (C3-coated surfaces) in a dose-dependent fashion. Scanning electron microscopic and optical microscopic observations of neutrophils on C3-coated surfaces revealed polarized or spherical cell morphologies in the absence or presence of histamine, respectively. Histamine inhibited the ability of CR3 to cluster on plasma membranes of neutrophils adherent to C3-coated surfaces as shown by fluorescence microscopy. In addition, histamine diminished but did not abolish the FMLP-stimulated increase in plasma membrane CR3 expression as measured by fluorometry. Histamine did not inhibit the release of marker proteins from specific or gelatinase containing granules by neutrophils in suspension. Histamine also diminished the FMLP-stimulated production of respiratory burst oxidants from cells in suspension or cells allowed to adhere to fibrinogen substrates. We suggest that histamine may modulate selective changes in neutrophil function by diminishing adherence and preventing changes in cell shape following cell activation.     

Sequential expression of cell surface C3bi receptors during neutrophil locomotion

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein- and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (10(-7) M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule-like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules----lamellipodia and cell body----uropod----trailing filopodia during chemotaxis.     

Evidence for multiple intracellular calcium pools in GH4C1 cells: investigations using thapsigargin.

The actions of thapsigargin (Tg), a plant sesquiterpene lactone, on Ca2+ homeostasis were investigated in digitonin-permeabilized GH4C1 rat pituitary cells. Tg (1 microM) caused a rapid and sustained increase in ambient Ca2+ concentration [( Ca2+]) and inhibited the rise in [Ca2+] induced by subsequent addition of TRH (100 nM), inositol 1,4,5-trisphosphate (IP3, 10 microM), or the nonhydrolyzable GTP analogue guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S, 10 microM). However, neither IP3 nor GTP gamma S pretreatment, which themselves release sequestered Ca2+, prevented the Ca2+ accumulation induced by Tg. Pretreatment with heparin (100 micrograms/ml, 10 min), an IP3 receptor antagonist, did not affect Ca2+ accumulation induced by Tg, although it abolished the rise in [Ca2+] induced by IP3. The ability of Tg to increase [Ca2+] was dependent on added ATP. We conclude that, in GH4C1 cells, Tg acts, in part, on TRH-, IP3- and GTP gamma S-sensitive Ca2+ pools; however, Tg also acts on an ATP-dependent pool of intracellular Ca2+ which is not sensitive to TRH, IP3 or GTP gamma S, indicating a complexity of intracellular Ca2+ pools not previously appreciated in these cells.     

Detection of a neoantigen on human C3bi and C3d by monoclonal antibody

A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.     

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-beta-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with alpha-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.     

Differential interaction of the C3b/C4b receptor and MHC class I with the cytoskeleton of human neutrophils

As measured by fluorescence microscopy and radioligand binding, C3b/C4b receptors (CR1) became attached to the detergent-insoluble cytoskeleton of human neutrophils when receptors were cross-linked by affinity-purified polyclonal F(ab')2 anti-CR1, dimeric C3b, or Fab monoclonal anti-CR1 followed by F(ab')2 goat anti-mouse F(ab')2. CR1 on neutrophils bearing monovalent anti-CR1 was not attached to the cytoskeleton. In contrast, cross-linked CR1 on erythrocytes and cross-linked MHC Class I on neutrophils were not cytoskeleton associated. A possible role for filamentous actin (F-actin) in the binding of cross-linked CR1 to neutrophil cytoskeleton was suggested by three observations. When neutrophils were differentially extracted with either Low Salt-detergent buffer or High Salt-detergent buffer, stained with FITC-phalloidin, and examined by fluorescent flow cytometry, the residual cytoskeletons generated with the former buffer were shown to contain polymerized F-actin, whereas cytoskeletons generated with the latter buffer were found to be depleted of F-actin. In parallel experiments, High Salt-detergent buffer was also found to release cross-linked CR1 from neutrophils. Second, depolymerization of F-actin by DNAse I released half of the cytoskeletal-associated cross-linked CR1. Third, immunoadsorbed neutrophil CR1, but not MHC Class I or erythrocyte CR1, specifically bound soluble 125I-actin. In addition, Fc receptor and CR3, other phagocytic membrane proteins of neutrophils, specifically bound 125I-actin. These data demonstrate that CR1 cross-linked on neutrophils becomes associated with detergent-insoluble cytoskeleton and that this interaction is mediated either directly or indirectly by actin.     

Regulation of intracellular pH in human neutrophils

The intracellular pH (pHi) of isolated human peripheral blood neutrophils was measured from the fluorescence of 6-carboxyfluorescein (6-CF) and from the equilibrium distribution of [14C]5,5-dimethyloxazolidine -2,4-dione (DMO). At an extracellular pH (pHo) of 7.40 in nominally CO2-free medium, the steady state pHi using either indicator was approximately 7.25. When pHo was suddenly raised from 7.40 to 8.40 in the nominal absence of CO2, pHi slowly rose by approximately 0.35 during the subsequent hour. A change of similar magnitude in the opposite direction occurred when pHo was reduced to 6.40. Both changes were reversible. Intrinsic intracellular buffering power, determined by using graded pulses of CO2 or NH4Cl, was approximately 50 mM/pH over the pHi range of 6.8-7.9. The course of pHi obtained from the distribution of DMO was followed during and after imposition of intracellular acid and alkaline loads. Intracellular acidification was brought about either by exposing cells to 18% CO2 or by prepulsing with 30 mM NH4Cl, while pHo was maintained at 7.40. In both instances, pHi (6.80 and 6.45, respectively) recovered toward the control value at rates of 0.029 and 0.134 pH/min. These rates were reduced by approximately 90% either by 1 mM amiloride or by replacement of extracellular Na with N-methyl-D-glucamine. Recovery was not affected by 1 mM SITS or by 40 mM alpha-cyano-4-hydroxycinnamate (CHC), which inhibits anion exchange in neutrophils. Therefore, recovery from acid loading is probably due to an exchange of internal H for external Na. Intracellular alkalinization was achieved by exposing the cells to 30 mM NH4Cl or by prepulsing with 18% CO2, both at a constant pHo 7.40. In both instances, pHi, which was 7.65 and 7.76, respectively, recovered to the control value. The recovery rates (0.033 and 0.077 pH/min, respectively) were reduced by 80-90% either by 40 mM CHC or by replacement of extracellular Cl with p-aminohippurate (PAH). SITS, amiloride, and ouabain (0.1 mM) were ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.     

Lipid monolayer-coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils

We have used epifluorescence and photobleaching techniques to study the lateral distribution and motion of fluorescein-conjugated Fab fragments of anti-C3b receptor antibody bound to human neutrophils when the cells rest on various solid supports (microscope slides or cover slips). Supports composed of quartz, glass, or alkylated glass induced cellular adhesion, spreading, and an extensive lateral redistribution of C3b receptors (but not HLA antigens). The neutrophil C3b receptors become patchy, and the patches apparently undergo nonrandom translational motion. Many patches are found on the upper surfaces of the cells removed from the region of cell membrane-glass contact. In contrast, neutrophils supported by lipid monolayer-coated glass do not adhere or spread, and the C3b receptor remains uniform and diffuses freely (D approximately equal to 2 X 10(-10) cm2/s).     

Human neutrophils and eosinophils have structurally distinct Fc gamma receptors

Human Fc gamma receptors were isolated from surface radioiodinated granulocytes and eosinophils by using repetitive affinity chromatography on human IgG-Sepharose columns. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated that cell preparations containing eosinophils possessed a 43,000 Mr Fc gamma-binding macromolecule. Nylon wool-filtered cells from patients with eosinophilia and cell cultures derived from normal donors provided highly purified eosinophil preparations that expressed only the 43,000 Mr Fc gamma receptor. Granulocyte populations yielded the 52,000 to 68,000 Mr Fc gamma receptor characteristic of neutrophils as well as the Fc gamma-binding macromolecules apparently derived from eosinophils. The 43,000 Mr Fc gamma receptor of the eosinophil and the 31,000 and 34,000 Mr fragments that appear to be derived from it were able to rebind selectively to human IgG1-Sepharose, Fc gamma 1-Sepharose, IgG3-Sepharose, and Fc gamma 3-Sepharose. In contrast, the 52,000 to 68,000 Mr Fc gamma receptor from neutrophils could rebind only to IgG1-Sepharose and Fc gamma 1-Sepharose. The results demonstrate that the Fc gamma receptor of human eosinophils is distinct in structure from the neutrophil Fc gamma receptor and that these Fc gamma receptors, at least in their solubilized states, differ in specificity for human IgG3.     

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.     

Discrete intracellular Ca2+ pools coupled to two distinct Ca2+ influx pathways in human platelets

Ca(2+) influx is an important event associated with platelet activation and regulated by the content of intracellular Ca(2+). Previous studies have suggested two different Ca(2+) pools and two Ca(2+) influx pathways exist in platelets. In the present study, we have investigated the regulation of thrombin- and thapsigargin-induced Ca(2+) entry into human platelets, using fluorescent indicators to monitor Ca(2+) mobilization and membrane potential. It was found that depletion of thapsigargin-sensitive Ca(2+) stores was coupled to Ca(2+) influx through a Ca(2+)-selective pathway. Additional release of Ca(2+) from the thapsigargin-insensitive pool by thrombin caused the opening of a nonselective cation channel.