The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.     

Elevated glucose alters A23187-induced release of arachidonic acid from porcine aortic endothelial cells by enhancing reacylation.

Cultured porcine aortic endothelial cells were conditioned in normal (5.2 mM) and elevated (15.6 mM) glucose, prelabeled with [14C]arachidonic acid and stimulated with ionophore A23187. Elevated glucose cultures released less radiolabeled products and less [14C]arachidonic acid. Analysis of cellular lipids revealed that elevated glucose reduced net loss of radiolabel from diacylphosphatidylethanolamine, did not affect early phosphatidylinositol hydrolysis, and increased net loss from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine. Uptake of radiolabel upon stimulation was examined to measure the role of reacylation on the diminished net release of radiolabel in elevated glucose cultures. Enhanced acylation of [3H]arachidonic acid into cellular lipids, especially PI, was observed in stimulated and resting cultures with elevated glucose. Further, pretreatment of the cultures with an acyltransferase inhibitor, thimerosal, prior to A23187 stimulation in radiolabeled cultures, abolished the effects of glucose on eicosanoid and arachidonic acid release. Differences in the ionophore-induced net loss of radiolabel from diacylphosphatidylethanolamine and phosphatidylinositol of the two glucose treatments were diminished by thimerosal exposure, while net loss of radiolabel from diacylphosphatidylcholine and alkenylacylphosphatidylethanolamine were unaffected. The data indicate that elevated glucose alters deacylation and enhances reacylation of arachidonic acid into endothelial cells and particularly into phosphatidylinositol. Enhanced reacylation may explain some of the altered lipid pathways that have been observed in experiments that elevate glucose concentrations or involve diabetes.     

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.     

Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation

Abstract: Awake adult male rats were infused intravenously with [³H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline-treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k ^⋆_brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [³H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A₂ and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse-mediated changes in brain phospholipid metabolism in vivo.     

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.     

Stimulation of deacylation in Madin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetradecanoylphorbol-13-acetate-treated cells

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.     

Low molecular weight GTP-binding proteins in human neutrophil granule membranes

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.     

Distribution of arachidonic acid in choline- and ethanolamine-containing phosphoglycerides in subfractionated human neutrophils

Human neutrophils were fractionated on Percoll gradients and the various subcellular fractions were analyzed for phospholipid and fatty acid composition. The results showed that plasma membranes and azurophilic granules were enriched with ethanolamine-(PE) relative to choline-(PC) containing phosphoglycerides. A remarkable degree of uniformity existed throughout the gradient with respect to the subclass composition of the subcellular PC and PE components. In each fraction 50-60% of the PC was diacyl, 40-45% was 1-O-alkyl-2-acyl (ether linked), and 2-5% was 1-O-alk-1'-enyl-2-acyl (plasmalogenic). For PE, 20-25% was diacyl, 7-12% ether linked, and 64-76% plasmalogenic. When neutrophils were incubated for 15 min with [1-14C]arachidonic acid and subfractionated most of the PC-associated label was intracellularly localized. A similar result was observed in PE, however, when the cells were allowed to stand for 2 h in fatty acid-free buffer following the 15 min of labeling and then subfractionated there was a sizable migration of [14C]arachidonate into plasma membrane PE. In all cases the diacyl subclass was labeled most heavily after 15 min but after an additional 2 h of incubation in fatty acid-free buffer there was a direct transfer of label to the ether- and plasmalogenic-linked PC and PE subclasses. It was also found that arachidonoyl-coenzyme A 1-acyl-lysophosphatide acyltransferase activity was inherent in all three major membrane types but was enriched in the endoplasmic reticulum/secondary granule fraction. Arachidonate consistently accounted for roughly 5% of the PC and 17% of the PE fatty chain composition in each subcellular fraction. These findings demonstrate that, despite the uniform arachidonate and PC and PE subclass composition within the various neutrophil subcellular fractions, the bulk of the PC- and PE-associated arachidonate is localized in intracellular membranes.     

The Inositol Lipids of Paramecium tetraurelia and Preliminary Characterizations of Phosphoinositide Kinase Activity in the Ciliary Membrane

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphati-dylinositol-bis-phosphate (PI-P₂) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [γ-³²P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P₂. Kinase activity was activated by millimolar [Mg²⁺] and inhibited by millimolar [Ca²⁺]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated     

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[14C]eicosatrienoate was two-to threefold that of 5,11,14-[14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.     

Biological activities and metabolism of the lysophosphoinositides and glycerophosphoinositols

The lysophospholipids are integral components of the plasma membrane that have often been considered as side products of the phospholipase A2 (PLA2)-dependent production of arachidonic acid and the deacylation/reacylation processes involved in phospholipid homeostasis. Data indicating roles of these lipid derivatives in hormone responses and cell transformation have now led to a different view, and the understanding of their involvement in the modulation of cell function is building up. Here, we will summarise the current knowledge concerning the biological roles of the lysophosphoinositides and the glycerophosphoinositols (their fully deacylated counterparts) in the framework of their known effects, and those of the other lysophospholipids and glycerophospholipids.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.     

Intracellular location of T200 and Mo1 glycoproteins in human neutrophils

Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.     

Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation

We have studied the capacity of human neutrophils to release arachidonic acid from diacylglycerol, employing 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol and 1-[1-14C]stearoyl-2-arachidonoyl-sn-glycerol as exogenous substrates. We have found that arachidonic acid is removed from diacylglycerol by the sequential action of two enzymes. First, the sn-1 position is split by 1-diacylglycerol lipase activity, and then, arachidonic acid is released from the resulting 2-monoacylglycerol by a 2-monoacylglycerol lipase. The specific activity of the 2-monoacylglycerol lipase, using 2-[1-14C]arachidonoyl-sn-glycerol as exogenous substrate, was at least 9-fold higher than that of 1-diacylglycerol lipase, indicating that the action of the 1-diacylglycerol lipase is the rate-limiting step in arachidonic acid release from diacylglycerol. Postnuclear supernatants from A23187-treated cells showed a 2.5-fold increase in both lipase activities. The arachidonic acid-releasing diacylglycerol lipase system showed an optimum pH of 4.5 and was not inhibited by EGTA or stimulated by Ca2+, Mg2+, Mn2+, Zn2+, or Co2+. However, arachidonic acid release was inhibited by Hg2+, suggesting the involvement of sulfhydryl groups in catalytic activity. The subcellular distribution of both 1-diacylglycerol lipase and 2-monoacylglycerol lipase activities was examined in resting and A23187-treated human neutrophils by fractionation of postnuclear supernatants on continuous sucrose gradients. Both lipases were localized mainly in the membrane of gelatinase-containing granules, which were resolved from cytosol, plasma membrane, phosphasomes, and specific and azurophilic granules. When neutrophils were stimulated by the calcium ionophore A23187, a drastic shift of the 1-diacylglycerol lipase and 2-monoacylglycerol lipase toward the plasma membrane was detected. This shift was due to fusion of gelatinase-containing granules with the plasma membrane upon neutrophil stimulation. As a result of the membrane fusion process, the capacity to release arachidonic acid from diacylglycerol was increased. This translocation from the membrane of gelatinase-containing granules to the plasma membrane may play an important role in regulating the diacylglycerol level in stimulated human neutrophils.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'- nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium- dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.     

A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.     

Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation

A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation.     

Angiotensinase activity and angiotensin receptor binding in guinea pig aorta.

The angiotensinase (EC 3.4.99.3) activity of the subcellular fractions of guinea pig aorta has been studied in relation to their [14C]angiotensin binding capacity. The enzyme activity occurs in the following decreasing order: supernatant greater than plasma membrane fraction greater than 105 000 X g pellet greater than mitochondrial fraction. The specific binding of [14C]angiotensin to these fractions follows the same pattern. Pretreatment of the subcellular fractions at 47 degrees C for 20 min was performed in an attempt to differentiate binding of angiotensin to the pharmacological receptor from binding to the destroying enzymes. This procedure decreased the angiotensinase activity in the plasma membrane fraction only whereas the specific binding of [14C]angiotensin to this fraction was not significantly decreased, suggesting that the plasma membrane angiotensinase is a thermolabile enzyme.     

Isolation and partial characterization of glycolipids and a carbohydrate from the secondary granules and plasma membrane of polymorphonuclear leukocytes

Chloroform/methanol extracts of the secondary granule and plasma membrane fractions of polymorphonuclear leukocytes have been shown to contain both non-polar and polar carbohydrate-containing materials. The ratio of the polar to the non-polar material was much higher in the plasma membrane than the secondary granule fraction. The non-polar material contains at least two ceramide-like glycolipids and accounts for most of the broad band of periodic acid/Schiff-positive material which migrates at the dye front in sodium dodecyl sulfate electrophoretic gels of granule and plasma membrane extracts. The polar material appears to be a single substance containing no fatty acids or sialic acid and is composed of glucose, hexosamine and a carboxylic acid derivative of pentose. Expressed on a per mg of protein basis, the amount of carbohydrate associated with the polar material in the plasma membrane fraction was about five times that of the secondary granule fraction.