Suppressive effects of carotenoids on the antigen-induced degranulation in RBL-2H3 rat basophilic leukemia cells.

In this study, the anti-degranulation effects of fifteen carotenoids were evaluated using RBL-2H3 rat basophilic leukemia cell line as a mast cell model. Nine carotenoids, fucoxanthin, zeaxanthin, ß-carotene, astaxanthin, 3-hydroxyechinenone, fucoxanthinol, lycopene, ß-cryptoxanthin, and siphonaxanthin significantly suppressed antigen-induced mast cell degranulation. Under the same conditions, the cellular carotenoid contents were quantified using high performance liquid chromatography-photodiode array (HPLC-PDA). There was no correlation between the cellular carotenoid contents and their anti-degranulation activities. These results indicate that the differences in the anti-degranulation activities of carotenoids were not related to their uptake by the cells.

Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation.

Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

Upregulation of heme oxygenase-1 by degranulation in rat basophilic leukemia cells.

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.

The role of phosphatidylinositol 4-kinase type IIalpha in degranulation of RBL-2H3 cells.

OBJECTIVE AND DESIGN: We have studied the role of phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) in activation of rat basophilic leukemia (RBL-2H3) cells. MATERIALS AND METHODS: Antigen-mediated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase and beta-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIalpha-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIalpha (K151A)-YFP. RESULTS: Neither PI4KIIalpha-YFP nor PI4KIIalpha (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIalpha-YFP showed significantly enhanced beta-hexosaminidase secretion but not an increase in [Ca(2+)](i) after antigen stimulation. The cells with PI4KIIalpha (K151A)-YFP showed no change in the [Ca(2+)](i) increase nor degranulation. The promotion of secretion by PI4KIIalpha-YFP was not observed using co-stimulation with Ca(2+) ionophore and the protein kinase C activator, phorbol myristate acetate. CONCLUSIONS: These results suggest that PI4KIIalpha plays a role in the exocytotic process downstream of Ca(2+) signaling in antigen-mediated mast cell activation.

Activation of the store-operated calcium current ICRAC can be dissociated from regulated exocytosis in rat basophilic leukaemia (RBL-1) cells.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Polyvalent cations as permeant probes of MIC and TRPM7 pores.

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite.

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 +/- 0.04 vs. 0.05 +/- 0.01 microm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.

Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase.

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.

Comparison of the activation of the Ca2+ release-activated Ca2+ current ICRAC to InsP3 in Jurkat T-lymphocytes, pulmonary artery endothelia and RBL-1 cells.

In many electrically non-excitable cells, Ca2+ entry is mediated predominantly by the store-operated Ca2+ influx pathway. The best-characterised store-operated Ca2+ current is the Ca2+ release-activated Ca2+ current (ICRAC). It is generally believed that high concentrations of intracellular Ca2+ buffer are required to measure ICRAC, due to Ca2+-dependent inactivation of the channels. Recently, we have recorded robust ICRAC in rat basophilic leukaemia (RBL-1) cells at physiological levels of Ca2+ buffering when stores were depleted by inhibition of the sarcoplasmic/ endoplasmic reticulum Ca2+-activated adenosine triphosphatase (SERCA) pumps. However, the second messenger inositol 1,4,5-trisphosphate (InsP3) was not able to evoke the current under such conditions, despite inducing substantial Ca2+ release. We have therefore suggested that a threshold exists within the Ca2+ stores which has to be overcome for macroscopic ICRAC to activate. To establish whether this is a specific feature of ICRAC in RBL-1 cells or whether it is a more general phenomenon, we investigated whether a threshold is also seen in other cell-types used to study store-operated Ca2+ entry. In Jurkat-T lymphocytes, ICRAC is activated weakly by InsP3 in the presence of low concentrations of Ca2+ buffer, whereas the current is large when SERCA pumps are blocked simultaneously, as in RBL-1 cells. Although the electrophysiological properties of ICRAC in the Jurkat cell are very similar to those of RBL-1 cells, the Na+ conductance in the absence of external divalent cations is quite different. Unexpectedly, we failed consistently to record any store-operated Ca2+ current in macrovascular pulmonary artery endothelia whereas robust ICRAC was seen under the same conditions in RBL-1 cells. Our results show that ICRAC has a similar profile of activation in the presence of physiological levels of Ca2+ buffering for Jurkat T-lymphocytes and RBL-1 cells, indicating that the threshold mechanism may be a general feature of ICRAC activation. Because ICRAC in pulmonary artery endothelia is, at best, very small, additional Ca2+ influx pathways may also contribute to agonist-induced Ca2+ entry.

Influence of the antiallergic drug oxatomide and derivatives on membrane structures: relation with inhibition of calcium influx in rat basophilic leukemia cells.

Oxatomide is an H1 antihistaminic drug that also inhibits mediator release from mast cells. From previous studies, it appeared that inhibition of the influx of extracellular calcium is the major cause of this inhibition of exocytosis. Here, we explored the role of drug-membrane interactions in the inhibition of mediator release. We investigated the effects on phase transition and fluidity of artificial membranes. All compounds studied distorted the phase transition in L-alpha-dipalmitoylphosphatidylcholine liposomes, which correlated with the drug-induced increase in membrane fluidity measured by fluorescence anisotropy of the bilayer interacting probe 1-[4-(trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. Erythrocytes were used to study membrane effects on a cellular level. The hypotonic-induced haemolysis of erythrocytes was inhibited by the drugs. Compounds which increased membrane fluidity of liposomes to a greater extent were also more active in decreasing haemolysis. Drug-induced disturbance of the membranes is related to their effect on the activity of store-operated Ca2+ channels. The activity of these channels in rat basophilic leukemia cells, assayed as 45Ca2+ influx, was most effectively inhibited by oxatomide derivatives, thereby inducing a more rigid membrane structure. Small changes in molecular structure affect the activity of the drugs and these structure-activity relations are discussed.

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells.

The role of ATP in both the activation of store-operated Ca2+ current ICRAC and in Ca2+-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, ICRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4, 5-trisphosphate (InsP3) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of ICRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca2+-dependent vesicular fusion was triggered by dialysing cells with 10 microM Ca2+ and guanosine-5'-O-(3-thiotriphosphate (GTP[gamma-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of ICRAC or for the capacitance increases elicited by high concentrations of intracellular Ca2+.

Ca2+-dependent capacitance increases in rat basophilic leukemia cells following activation of store-operated Ca2+ entry and dialysis with high-Ca2+-containing intracellular solution.

Ca2+-dependent vesicular fusion was studied in single whole-cell patch-clamped rat basophilic leukemia (RBL) cells using the capacitance technique. Dialysis of the cells with 10 microM free Ca2+ and 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) resulted in prominent capacitance increases. However, dialysis with either Ca2+ (225 nM to 10 microM) or GTP[gamma-S] alone failed to induce a capacitance change. Under conditions of weak Ca2+ buffering (0.1 mM EGTA), activation of Ca2+-release-activated Ca2+ (CRAC) channels by dialysis with inositol 1,4,5-trisphosphate (InsP3) failed to induce a capacitance increase even in the presence of GTP[gamma-S]. However, when Ca2+ATPases were inhibited by thapsigargin, InsP3 and GTP[gamma-S] led to a pronounced elevation in membrane capacitance. This increase was dependent on a rise in intracellular Ca2+ because it was abolished when cells were dialysed with a high level of EGTA (10 mM) in the recording pipette. The increase was also dependent on Ca2+ influx because it was effectively suppressed when external Ca2+ was removed. Our results demonstrate that ICRAC represents an important source of Ca2+ for triggering a secretory response.

The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells.

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

Increased degranulation and phospholipase A2, C, and D activity in RBL cells stimulated through FcepsilonR1 is due to spreading and not simply adhesion.

Rat basophilic leukemia cells will adhere to and spread out on fibronectin coated surfaces in an integrin dependent manner. Adhesion and spreading on fibronectin leads to increased degranulation, inositol phosphate production, phospholipase D activation, and increased production of prostaglandin D2 and leukotriene C4 when the cells are activated through the high affinity IgE receptor. Rat basophilic leukemia cells will also adhere to surfaces coated with anti-rat class I antibodies, poly-L-lysine, and a lectin purified from Tetragonolobus purpureas. In all cases, antigen activated cells, which were adherent, displayed increased signaling, degranulation and eicosanoid production as compared to cells which were non-adherent. Cells which adhere to either anti-rat class I antibodies or poly-L-lysine also spread even though this is not mediated through integrins. In contrast, adhesion to the lectin from Tetragonolobus did not cause any appreciable spreading unless the cells were also triggered through the IgE receptor. Cells were also able to bind to fibronectin immobilized on polystyrene beads which mimics adhesion but does not allow spreading. However, these cells exhibited no increased signaling, degranulation, or eicosanoid production. Furthermore, rat basophilic leukemia cells can be modified by incubating them in the presence of biotinylated-phosphatidylserine which becomes incorporated into the membrane. These modified cells will adhere to streptavidin coated plates while unmodified cells will not. However, these modified cells do not spread, even after activation with antigen, and they show no increased degranulation or production of eicosanoids. These results indicate that adhesion itself is not sufficient for upregulation of the cells in response to antigen and that spreading of the cells may be the critical component.

[Stimuli sensitive changes in electrical surface properties of soft membranes: from a synthesized polymer to a biological system].

The electrical surface properties of biological cells have been studied, which provided us with the fundamental knowledge about the cell surface. The change in shape or biological functions of cells may affect the surface properties and can be detected by electrokinetic measurements. Biological cell surfaces are covered with polysaccharide chains, some are charged and some are not. Some polysaccharides produce a hydrogel matrixes under a proper condition. We thus consider it reasonable that cell surface is approximated by a hydrogel surface. Electrophoretic mobility measurements are useful for studying the surface properties of biological cells suspended as colloidal particles in an electrolyte solution. The electro-osmotic velocity measurements on the other hand are advantageous to the study of the surface properties of slab-shaped biological systems such as membranes. This work was started with a hydrogel, as a model material. As a hydrogel, poly(N-isopropylacrylamide) poly(NIPAAm), abbreviated as hereafter, was chosen, because this hydrogel changes its volume depending on temperature. The dependence of the electrophoretic mobility of latex particles covered with poly(NIPAAm) hydrogel layer or of the electro-osmotic mobility on poly(NIPAAm) plate upon temperature and ionic strength of the dispersing medium was well explained with an electrophoretic mobility formula for "soft particles" developed by Ohshima. The electrokinetic measurements and the explanation of data with an electrophoretic mobility formula for "soft particles" give us information about the surface charge density and the "softness" of soft surfaces. On the basis of the findings with hydrogels, we have discussed the relationship between the changes in shape or function of the biological cells and the change in physicochemical surface properties using these measurements. To study the change in physicochemical properties of the cell surface caused by apoptosis, we have measured the electrophoretic mobilities of intact and apoptotic human promyelocytic leukemia cell lines, HL-60RG cells. We have also studied the differences observed in surface properties of malignant lymphosarcoma cell line, RAW117-P, and its variant, RAW117-H10, with a high metastatic property to the liver. In both cases, the cell surfaces became softer by the changes of biological functions. We have applied electrophoresis and electro-osmosis measurements to the study of the electrokinetic surface properties of rat basophilic leukemia cells, RBL cells. It was also found that the surface of Human umbilical vein endothelial cells, HUVEC, is considerably soft as compared with those of other biological cells we have studied before.

Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study.

Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled 35S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation).

ATP gamma S induces actin and myosin rearrangement during histamine secretion in a rat basophilic leukemia cell line (RBL-2H3).

Rat basophilic leukemia cells (RBL-2H3) undergo morphological and cytoskeletal changes during antigen (DNP-BSA) or calcium ionophore-induced secretion of allergic mediators from intact or permeabilized cells. We describe the novel finding that the phosphatase-resistant ATP analogue, ATP gamma S, mimics antigen-induced serotonin secretion and cytoskeletal rearrangements in permeabilized cells. Confocal microscopy of unstimulated cells shows that myosin and F-actin are concentrated at the plasma membrane. Upon addition of ATP gamma S, F-actin becomes rearranged into membrane ruffles and also associates with myosin in a cytoplasmic meshwork, concentrated perinuclearly. F-actin and myosin ultimately become colocalized into parallel microfilament bundles located on the basolateral membrane. During this period the cell height decreases whilst the cell area increases more than twofold. Gel electrophoresis shows that the cytoskeletal proportion of actin remains unchanged, indicating that the rearrangements occur within the total F-actin pool. The distribution of microtubules and intermediate filaments is unchanged in the presence of ATP gamma S. These results suggest that overcoming a phosphatase may be sufficient to induce secretion in RBL-2H3 cells, and that this secretion may be regulated by F-actin and myosin rearrangements.

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.