Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Complete primary structure of a human plasma membrane Ca2+ pump

cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.     

Delayed activation of the plasma membrane calcium pump by a sudden increase in Ca2+: fast pumps reside in fast cells

There are four genes encoding isoforms of the plasma membrane Ca(2+) pump (PMCA). PMCA variability is increased by the presence of two splicing sites. Functional differences between the variants of PMCA have been described, but little is known about the adaptive advantages of this great diversity of pumps. In this paper we studied how the different isoforms respond to a sudden increase in Ca(2+) concentration. We found that different PMCAs are activated by Ca(2+) at different rates, PMCA 3f and 2a being the fastest, and 4b the slowest. The rate of activation by Ca(2+) depends both on the rate of calmodulin binding and the magnitude of the activation by calmodulin. We found that 2a is located in heart and the stereocilia of inner ear hair cells, 3f in skeletal muscle and 4b was identified in Jurkat cells. Both cardiac and skeletal muscle, and stereocilia recover very rapidly after a cytoplasmic Ca(2+)peak, while in Jurkat cells the recovery takes up to a minute. In stereocilia, 2a is the only method for export of Ca(2+), making the analysis of them unusually straightforward. This indicates that these rates of PMCA activation by Ca(2+) are correlated with the speed of Ca(2+) concentration decay after a Ca2 spike in the cells in which these variants of PMCA are expressed. The results suggest that the type of PMCA expressed will correspond with the speed of Ca(2+) signals in the cell.     

The Neurospora plasma membrane Ca2+ pump

Plasma membrane vesicles isolated from the eukaryotic microorganism Neurospora crassa by the concanavalin A method catalyze Mg2+-ATP dependent 45Ca2+ accumulation. Since the ATP-responsive vesicles are functionally inverted, the Ca2+ transport system presumably operates as a Ca2+ exit pump in the intact cell. The mechanism of the Ca2+ pump system involves two components: 1) an electrogenic, proton-translocating ATPase (EC 3.6.1.3), which utilizes the chemical energy of ATP hydrolysis to generate a transmembrane electrical potential and pH gradient, and 2) a Ca2+/H+ antiporter, which utilizes the transmembrane pH gradient to energize the active transport of Ca2+. Evidence for this mechanism is presented and the possible implications of these findings for the mechanisms of Ca2+ pumps in other cells are discussed.     

Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells

We examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on Ca2+ extrusion from cultured vascular smooth muscle cells (VSMCs) incubated in the absence of added extracellular Na+ (Na+o). Previously, strong experimental evidence was presented that the Na+o-independent Ca2+ extrusion from VSMCs is effected by the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., and Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Brief (2 min) pretreatment of VSMCs with 30-300 nM PMA suppressed the intracellular Ca2+ transient induced with 1 microM ionomycin to about 60% of the control, whereas it accelerated the concomitant Na+o-independent 45Ca2+ extrusion by up to 20%. When the Ca2+ transient was induced with 0.1 microM angiotensin II, the PMA pretreatment markedly suppressed it and reduced also the rate of 45Ca2+ efflux from cells slightly. These effects of PMA were mimicked by 1-oleoyl-2-acetylglycerol, another protein kinase C activator, but were abolished by prior treatment of cells with staurosporine, an inhibitor of protein kinase C, or prior long incubation of cells with PMA. Analysis of the effect of PMA on [Ca2+]i dependence of the rate of Na+o-independent 45Ca2+ efflux revealed that PMA increased the maximum Ca2+ efflux rate without a significant change in the affinity for Ca2+. These results strongly suggest that the plasma membrane Ca2+ pump in VSMCs can be stimulated by PMA and that protein kinase C is involved in regulation of [Ca2+]i in intact VSMCs.     

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.     

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca²⁺ pump is aCa²⁺-Mg²⁺-ATPase that expels Ca²⁺from cells to help them maintain low concentrations of cytosolic Ca²⁺. There are no known extracellularly acting PMCa²⁺ pump inhibitors, as digoxin and ouabain are forNa⁺ pump. In analogy with digoxin, we define caloxins asextracellular PM Ca²⁺ pump inhibitors and describe caloxin2A1. Caloxin 2A1 is a peptide obtained by screening a random peptidephage display library for binding to the second extracellular domain(residues 401-413) sequence of PM Ca²⁺ pump isoform1b. Caloxin 2A1 inhibits Ca²⁺-Mg²⁺-ATPase inhuman erythrocyte leaky ghosts, but it does not affect basalMg²⁺-ATPase or Na⁺-K⁺-ATPase in theghosts or Ca²⁺-Mg²⁺-ATPase in the skeletalmuscle sarcoplasmic reticulum. Caloxin 2A1 also inhibitsCa²⁺-dependent formation of the 140-kDa acid-stableacylphosphate, which is a partial reaction of this enzyme. Consistentwith inhibition of the PM Ca²⁺ pump in vascularendothelium, caloxin 2A1 produces an endothelium-dependent relaxationthat is reversed byN^G-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca²⁺ pump inhibitor selectedfor binding to an extracellular domain.

     

The liver plasma membrane Ca2+ pump: hormonal sensitivity

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.     

Adherence of human neutrophils changes Ca signaling during activation with opsonized particles

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) upon activation of human neutrophils by opsonized particles (serum-treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo-1 loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo-1 loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura-2 loaded neutrophils adherent to serum-coated coverslips. Our study shows that the opsonized particle-induced change in [Ca²⁺]_i in neutrophils is altered during adherence of the cells to a serum-coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotaxis.     

The peripheral Ca pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Modulation of plasma membrane Ca2+ pump by membrane potential in cultured vascular smooth muscle cells

We examined the effect of membrane potential (Em) on the activity of the plasma membrane Ca2+ pump in cultured rat aortic smooth muscle cells (VSMCs). Inside-negative K+ diffusion potential higher or lower than the resting Em (-46 mV) was artificially imposed on VSMCs with various concentrations of extracellular K+ (K+o) and 1 microM valinomycin. We found that the recovery phase of the intracellular Ca2+ transient elicited with 1 microM ionomycin was accelerated by depolarizing Em, whereas it was retarded by hyperpolarizing Em. The rate of extracellular Na+ (Na+o)-independent 45Ca2+ efflux from VSMCs stimulated with 1 microM ionomycin increased almost linearly with a change in Em from -98 to -3 mV. This effect of Em was abolished by extracellularly added LaCl3 or a combination of high pH (pH 8.8) and high Mg2+ (20 mM), conditions that presumably inhibit the plasma membrane Ca2+ pump (Furukawa, K.-I., Tawada, Y., & Shigekawa, M. (1988) J. Biol. Chem. 263, 8058-8065). Intracellular contents of Na+ and K+ and intracellular pH, on the other hand, were not influenced by the change in Em under the conditions used. These results indicate that alteration in Em can modulate the intracellular Ca2+ concentration in intact VSMCs by changing the rate of Ca2+ extrusion by the plasma membrane Ca2+ pump. The data strongly suggest that the plasma membrane Ca2+ pump in VSMCs is electrogenic.     

Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

Background  

Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Delayed activation of calcium pump during transient increases in cellular Ca2+ concentration and K+ conductance in hyperpolarizing human red cells

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium

In vascular endothelial cells, depletion of intracellularCa²⁺ stores elicited capacitativeCa²⁺ entry (CCE) that resulted inbiphasic changes of intracellular Ca²⁺ concentration([Ca²⁺]_i)with a rapid initial peak of[Ca²⁺]_ifollowed by a gradual decrease to a sustained plateau level. Weinvestigated the rates of Ca²⁺entry, removal, and sequestration during activation of CCE and theirrespective contributions to the biphasic changes of[Ca²⁺]_i.Ca²⁺ buffering by mitochondria,removal byNa⁺/Ca²⁺exchange, and a fixed electrical driving force forCa²⁺ (voltage-clamp experiments)had little effect on the CCE signal. The rates of entry ofMn²⁺ andBa²⁺, used as unidirectionalsubstitutes for Ca²⁺ entry throughthe CCE pathway, were constant and did not follow the concomitantchanges of[Ca²⁺]_i.Pharmacological inhibition of the plasma membraneCa²⁺ pump, however, abolished thesecondary decay phase of the CCE transient. The disparity between thebiphasic changes of[Ca²⁺]_iand the constant rate of Ca²⁺entry during CCE was the result of a delayed,Ca²⁺-dependent activation of thepump. These results suggest an important modulatory role of the plasmamembrane Ca²⁺ pump in the netcellular gain of Ca²⁺ during CCE.

     

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.     

Intracellular organelle motility and membrane fusion processes in human neutrophils upon cell activation

Release and subcellular fractionation experiments indicate that fusion of a novel tertiary granule with the plasma membrane is concomitant with human neutrophil activation. Phorbol 12-myristate 13-acetate (PMA) induced a respiratory burst in human neutrophils as well as a high release of gelatinase, a marker of the tertiary granule. Preincubation of neutrophils with cytochalasin E induced a partially activated or 'primed' state, in which cells were unable to generate superoxide anion, but showed a reduced latency period for this activity. Fusion of tertiary granules with the cell surface also occurred during priming, although to a lesser extent than in PMA stimulation. The rapid tertiary granule degranulation, preceding that of specifics and azurophilics, seems to play an important role in the functionality and secretory properties of human neutrophils.