Cellular arachidonate-releasing function of novel classes of secretory phospholipase A2s (groups III and XII)

Here we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A(2)s (sPLA(2)s), groups III and XII. Human group III sPLA(2) promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA(2) domain alone was sufficient for its in vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique N- or C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA(2) was constitutively expressed in two human cell lines, in which other sPLA(2)s exhibited different stimulus inducibility. Human group XII sPLA(2) had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA(2) exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA(2)s, general properties of cellular actions of a full set of mammalian sPLA(2)s in regulating AA metabolism are discussed.     

Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets

Human platelet membrane vesicles that accumulated Ca2+ in the presence of ATP were isolated on an isoosmotic KCl-Percoll gradient. ATP-dependent Ca2+ uptake was stimulated by oxalate and phosphate to steady-state levels of greater than 100 nmol/mg protein, and the accumulated Ca2+ could be largely released by ionophore A23187. Inositol 1,4,5-trisphosphate, in a dose-dependent manner (0.5-5.0 microM), caused the rapid release (less than 5 s) of 40-70% of the total A23187-releasable store of accumulated Ca2+. The membrane vesicles that release accumulated Ca2+ in response to inositol 1,4,5-trisphosphate were enriched in enzymes characteristically found in smooth endoplasmic reticulum. These results support the hypothesis that inositol 1,4,5-trisphosphate, produced by the hydrolysis of phosphatidylinositol 1,4-bisphosphate in response to stimulation of cell surface receptors, is a second messenger mediating the release of Ca2+ from intracellular storage sites.     

Critical role of mitochondrial damage in determining outcome of macrophage infection with Mycobacterium tuberculosis

Human macrophages (Mphi) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mphi death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca(2+) concentration ([Ca(2+)](m)) determine whether Mphi undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca(2+)](m) and examined their physiological consequences. Adding calcium ionophore A23187 to Mphi inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca(2+)](m), blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mphi infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mphi, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.     

Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.     

Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.     

Release of adenylate cyclase stimulating, GTP-binding protein, Gs from human erythrocyte membranes under the effect of fluoride-ion and guanine nucleotides

Human erythrocyte membranes were incubated in the presence of sodium fluoride or guanylylimidodiphosphate (GppNHp), a nonhydrolysable GTP analog. After centrifugation at 100000 g the activity of the adenylate cyclase-stimulating GTP-binding protein, Gs, was detected in the supernatant fraction. The release of the Gs activity from the membranes closely resembles Gs activation by GppNHp. The Gs activity release from the GppNHp-induced membranes is characterized by a lag period. The nucleotide concentration causing a half-maximal solubilization is about 9.10(-7) M. Approximately 50% of the Gs activity released from sodium fluoride-treated human erythrocyte membranes was associated with the cytoskeletal fraction extracted by a low ionic strength solution. The data obtained suggest that Gs exists in the membrane at lease in two compartmentalized states and is solubilized from both states during its activation.     

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.     

ALIX Is Recruited Temporarily into HIV-1 Budding Sites at the End of Gag Assembly

Polymerization of Gag on the inner leaflet of the plasma membrane drives the assembly of Human Immunodeficiency Virus 1 (HIV-1). Gag recruits components of the endosomal sorting complexes required for transport (ESCRT) to facilitate membrane fission and virion release. ESCRT assembly is initiated by recruitment of ALIX and TSG101/ESCRT-I, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. In contrast to previous models, we show that ALIX is recruited transiently at the end of Gag assembly, and that most ALIX molecules are recycled into the cytosol as the virus buds, although a subset remains within the virion. Our experiments imply that ALIX is recruited to the neck of the assembling virion and is mostly recycled after virion release.     

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.     

Effect ofClostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane

Summary Human diploid embryonic lung fibroblasts were cultivated in Eagle's Minimum Essential Medium and labeled with³H-uridine. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane. The assay method described is simple, sensitive and rapid and allows quantitative estimation of changes in membrane permeability before any morphological damage is observed microscopically. Crude commercial preparations of phospholipase C (E.C. 3.1.4.3.) (40 g/ml) were highly active on the cell membrane but most of the membrane damaging activity was found to be due to contaminating theta-toxin. However, also highly purified phospholipase C caused a membrane damage as measured by release of isotope through the plasma membrane. The release could be increased by including an optimal concentration of calcium ions in the incubation buffer, by treating the cells in a hypotonic medium and by simultaneous treatment with sublytic concentrations of Triton X-100. To our knowledge this is the first report of membrane damage on a live, intact, metabolizing human diploid cell caused by a highly purified phospholipase C. The results are in agreement with a dynamic membrane structure with the polar groups of a part of the phospholipids accessible at the membrane surface.     

The Role of Cellular Factors in Promoting HIV Budding

Human immunodeficiency virus type 1 (HIV-1) becomes enveloped while budding through the plasma membrane, and the release of nascent virions requires a membrane fission event that separates the viral envelope from the cell surface. To facilitate this crucial step in its life cycle, HIV-1 exploits a complex cellular membrane remodeling and fission machinery known as the endosomal sorting complex required for transport (ESCRT) pathway. HIV-1 Gag directly interacts with early-acting components of this pathway, which ultimately triggers the assembly of the ESCRT-III membrane fission complex at viral budding sites. Surprisingly, HIV-1 requires only a subset of ESCRT-III components, indicating that the membrane fission reaction that occurs during HIV-1 budding differs in crucial aspects from topologically related cellular abscission events.     

Effect of bilirubin on toxicity induced by trifluoperazine, dibucaine and praziquantel to erythrocytes

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 micromol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37 degrees C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p<0.05), and in elution of membrane cholesterol (73%, p<0.01) and phospholipids (123%, p<0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (-36%, p<0.01), as well as that of phospholipids by TFP (-28%, p<0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.     

ML-7 inhibits exocytosis of superoxide-producing intracellular compartments in human neutrophils stimulated with phorbol myristate acetate in a myosin light chain kinase-independent manner

ML-7, (5-iodonaphthalene-1-sulfonyl) homopiperazine, is commonly employed as a myosin light chain kinase (MLCK) inhibitor. In the present study, we demonstrated that ML-7 affects the superoxide (O(2)(-))-producing system of human neutrophils in an MLCK-independent manner. Human neutrophils were stimulated with phorbol myristate acetate (PMA), which does not activate MLCK. ML-7 inhibited extracellular release, but not intracellular production of O(2)(-) in the stimulated cells. Fluorescence microscopy revealed the generation of O(2)(-) at intracellular compartments in the stimulated cells exposed to ML-7. At the electron microscopic level, the reaction product of NADPH oxidase activity was found in intracellular compartments. ML-7 strongly inhibited the association of the oxidant-producing intracellular compartments with the plasma membrane. Furthermore, the upregulation of alkaline phosphatase activity, a marker enzyme of the oxidant-producing intracellular compartments, was also inhibited by ML-7. These findings indicate that ML-7 inhibits the fusion of the oxidant-producing intracellular compartments to the plasma membrane resulting in the inhibition of the extracellular release of O(2)(-) in PMA-stimulated human neutrophils in an MLCK-independent manner.     

Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes.

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.     

Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells

Human cultured mast cells (HCMC) release histamine and sulfidoleukotrienes (LTs) upon IgE-FcepsilonRI-mediated mast cell activation. We analyzed the Ca2+ and PKC signaling in HCMC and compared it to that in rodent mast cells. In HCMC, after IgE-mediated stimulation, an elevation of [Ca2+]i and PKC translocation to the membrane fraction was observed. As concerns Ca2+ signaling, 1) IgE-mediated histamine and LTs release was abolished after Ca2+ depletion, and the reconstitution of Ca2+ recovered the release of histamine and LTs. As regards PKC signaling, 1) staurosporine inhibited IgE-mediated mediator release. 2) PKC-downregulated mast cells did not release histamine and LTs. A23187 and PMA synergistically potentiated the activation of extracellular-regulated kinase and synergistically induced histamine and LTs release. These results demonstrated that HCMC might be useful for analysis of the signal transduction pathway for mediator release, such as histamine and LTs.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.     

Pancreatic hormones are expressed on the surfaces of human and rat islet cells through exocytotic sites

Human and rat insulin cells show insulin immunoreactivity, and glucagon cells show glucagon immunoreactivity on their membrane surfaces, respectively. The reaction occurs in the form of small dots on the islet cell surface and colocalizes with the chromogranin family of secretory granule markers. Electron microscopy reveals the labeling to occur at sites of exocytotic granule release, involving the surfaces of extruded granule cores. The surfaces of islet cells were labeled both by polyclonal and monoclonal antibodies, excluding that receptor-interacting, anti-idiotypic hormone antibodies were responsible for the staining. Human insulin cells were surface-labeled by monoclonal antibodies recognizing the mature secretory products, insulin and C-peptide but not with monoclonal antibodies specific for proinsulin. Thus, routing of unprocessed preproinsulin to the cell surface may not account for these results. It is concluded that the staining reflects interactions between the appropriate antibodies and exocytotic sites of hormone release.     

Neutrophils promote the release of alpha-6-fucosyltransferase from blood platelets through the action of cathepsin G and elastase

Human blood platelets release alpha-6-fucosyltransferase during coagulation of blood or after stimulation with thrombin or other agonists that cause platelet activation (Antoniewicz et al., FEBS Lett. 244 (1989) 388-390). However, in the absence of neutrophils the thrombin-stimulated platelets release only a small fraction of alpha-6-fucosyltransferase activity (Ko?cielak et al., Acta Biochim. Polon. 42 (1995) 35-40). We show that the effect of neutrophils is reproduced by cathepsin G or (less efficiently) by elastase, the two enzymes that are released by neutrophils during coagulation of blood. We have also localized alpha-6-fucosyltransferase to membrane and alpha-granule fractions of platelets that had been disrupted by nitrogen cavitation. It is concluded that thrombin-activated neutrophils release cathepsin G and elastase that promote degranulation of platelets and hence the secretion of alpha-6-fucosyltransferase.     

Hydrophobically modified biomineralized polysaccharide alginate membrane for sustained smart drug delivery

Hydrophobically modified biomineralized polysaccharide alginate membrane with smart drug release property using sodium palmitate as the hydrophobic component was prepared via a one-step method. The formation of CaHPO(4) in the membrane was clearly identified through scanning electron microscopy (SEM), energy dispersive X-ray spectrometer (EDS), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Indomethacin release profiles of the modified alginate membrane were found to be pH- and thermo-responsive. The drug release of modified alginate membrane was around 60% within 12 h, while that of the alginate membrane was higher than 90%. These results indicate that the hydrophobic and biomineralized polysaccharide components can hinder the permeation of the encapsulated drug and reduce the drug release effectively. The resulting membrane can be used as "smart" materials for sustained dual-responsive drug delivery.     

Stimulated platelets release amyloid beta-protein precursor

Human platelets can be stimulated by thrombin or ionomycin to secrete soluble truncated amyloid beta-protein precursor and particulate membrane fragments which contain C-terminal and N-terminal immunoreactive amyloid beta-protein precursor. This suggests a possible circulating source of beta-protein in serum which may play a role in the formation of amyloid deposits. The release of soluble amyloid beta-protein precursor could be involved in normal platelet physiology.