Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells.

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event.

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.

Chemotactic factor-induced membrane potential changes in rabbit neutrophils monitored by the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide.

Rabbit neutrophil leucocytes take up the cationic, fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide (DiS-C3-(5)). Treatment with valinomycin and K+ then produces characteristic changes in suspension fluorescence that indicate that the dye enters the cells in a potential-dependent fashion and that the resting membrane potential lies between -66 and -86 mV. The peptide, N-fMet-Leu-Phe, a potent chemoattractant for neutrophils, added to stained cell suspensions, induces fluorescence intensity changes. These occur over an 8-10 min period. The time course of this response is profoundly affected by the omission of Ca2+ from the medium. When this ion is present (1.26 mM) a small, transient increase in intensity is observed, superimposed on a sustained decrease. On the other hand, in the absence of added Ca2+ a large, transient increase is observed. The ED50 for this is 1.1 x 10(-10) M. These changes are not elicited by N-fMet-Phe (10(-9) M) and are inhibited by the antagonist Boc-Leu-Phe-Leu-Phe. However, a component of zymosan-activated rabbit plasma, which is complement-derived, induces identical fluorescence changes that are not inhibited by the antagonist, confirming that neutrophil activation by complement operates through an independent receptor. The fluorescence responses to the chemotactic peptide and the activated-plasma component may be interpreted in terms of changes in neutrophil membrane potential brought about by alterations in cell ionic permeability at an early stage during activation. The transient increase corresponds to a depolarisation that may be associated with a change in Na+ permeability, while the sustained decrease corresponds to a membrane hyperpolarisation.

The relationship between mitogen-induced membrane potential changes and intracellular free calcium in human T-lymphocytes.

We have investigated the effects of mitogenic lectins on human T-lymphocytes, isolated from peripheral blood, and cells from the T-cell clone, HPB-ALL, using the fluorescent dyes, bis-thiobarbiturate tri-methineoxonol (bisoxonol) and quin2 to sense changes in membrane potential and intracellular free [Ca2+], respectively. The resting potential of both cell types is close to the K+ equilibrium potential. Changes from the resting level occur when mitogenic concentrations of either concanavalin A or phytohaemagglutinin are added. T-lymphocytes undergo a decrease in emission, maximal at 1 to 2 min, corresponding to a small membrane hyperpolarization. This is followed by a depolarization to approximately the resting level. HPB-ALL cells, on the other hand, respond to the mitogens by a sustained increase in fluorescence, denoting a depolarization, that is maximal at 4 to 5 min and 7 to 9 min, respectively. The Ca2+-dependence of these phenomena indicates that the membrane potential response, in both cell types, is the resultant of two opposing effects: a Ca2+-sensitive ion movement tending to hyperpolarize the cells and a Ca2+-insensitive effect that generates a depolarization. Our results suggest that Ca2+-activated K+ channels are responsible for the first effect and that an inward Na+ movement accounts for the depolarization signal in T-lymphocytes. In HPB-ALL cells only part of the depolarization is Na+-dependent. Although the effects elicited by phytohaemagglutinin occur more slowly than those produced by concanavalin A, similar membrane potential and [Ca2+]i changes occur.

Cell-surface changes during mitogenic stimulation of lymphocytes assessed by the binding of wheat-germ agglutinin and other plant lectins.

Lymphocytes from murine lymph node, cultured in the presence of an optimally mitogenic dose of phytohaemagglutinin, were stained with fluoresceinated lectins and analysed by flow cytometry. A marked increase in the ability of lymphocytes to bind wheat-germ agglutinin was observed that is particularly pronounced for the blast cells, reaching a maximum at about 40 h, when they are 5.5-times brighter than cells at zero time. The corresponding intensification of the small cells is 2-fold. Much smaller increases in binding accompanying blast transformation were observed when fluoresceinated concanavalin A or Lens culinaris haemagglutinin were used. Polyacrylamide gel electrophoresis of plasma membranes followed by treatment of the gels with radioactively labelled lectins and autoradiography also showed a very distinct increase in the binding of wheat-germ agglutinin to membranes from mitogen-stimulated porcine lymphocytes. Less marked changes in the binding of concanavalin A, Lens culinaris heamagglutinin and Ricinus communis agglutinin 120 were also noted. The apparent multiplicity of glycoproteins that bind each lectin, suggests that in each case the sites are heterogeneous. We conclude that lymphocytes stimulated by the T-cell mitogen phytohaemagglutinin expose new glycoprotein receptors for wheat-germ agglutinin that are most abundant on blast cells at 40 h. Attempts to characterize the receptor biochemically suggest that the carbohydrate moiety recognised by wheat-germ agglutinin is present on a glycoprotein of approx. 120 kDa molecular mass and also possibly on glycoproteins of 170-190 kDa.

Size measurements on isolated rat heart cells using Coulter analysis and light scatter flow cytometry.

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells. Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.

ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.

The role and mechanism of the GTP-binding protein GE in the control of regulated exocytosis.

Recent advances in the understanding of the terminal stages of the pathway of regulated secretion (exocytosis) have depended in large part on the use of permeabilized secretory cells in which the cytosol is directly accessible to manipulation from the outside. As well as Ca2+, a role for GTP, and hence a GTP-binding protein (GE), is plainly apparent in the exocytotic mechanism of some cells. In metabolically depleted and permeabilized mast cells, the combination of Ca2+ and a guanine nucleotide are a sufficient stimulus for the release of the total cell content of histamine and hexosaminidase. ATP is not required and so a phosphorylation reaction does not comprise a necessary step in the terminal pathway. Phosphorylating nucleotides, however, can modulate the exocytotic reaction in a manner that suggests that the reverse reaction, namely protein dephosphorylation, might be an enabling step. Support for this idea is derived from experiments in which the cells are conditioned to become responsive to Ca2+ alone; in these circumstances the dependence of secretion on the presence of a guanine nucleotide can be reimposed by okadaic acid, an inhibitor of protein phosphatases.

Thirty years of stimulus-secretion coupling: from Ca(2+) toGTP in the regulation of exocytosis.

Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognised as only one of a number of intracellular activators. In cells of haematopoietic origin (including mast cells), the key activator is one or more GTPases. Cells of this class, stimulated with GTPgammaS can undergo exocytosis in the effective absence of Ca(2+). A number of GTP-binding proteins that mediate exocytosis (G(E)) have been proposed but the best evidence supports roles for members of the Rho family of monomeric GTPases and for betagamma-subunits derived from G(i3). While preactivated Rac and Cdc42 can induce secretion from permeabilised mast cells in the absence of a guanine nucleotide betagamma-subunits only act to enhance the secretion induced by other GTP-binding proteins (likely to be members of the Rho family of monomeric GTPases). Further work is required to identify downstream effectors activated by these GTP-binding proteins and to show how they interact with the SNAP and SNARE isoforms known to be present in these cells.

Characterisation of the ATP4- receptor that mediates permeabilisation of rat mast cells.

ATP (as the tetrabasic acid, ATP4-) applied externally to rat mast cells causes the formation of lesions which permit influx and efflux of low molecular weight, normally impermeant aqueous solutes. To monitor membrane permeabilisation we have used two fluorescent dyes, ethidium which stains the nucleus, and TMA-DPH which stains the cytosolic surfaces of intracellular membranes following entry into the cells Permeabilisation by ATP is not affected by the metabolic status of the cells, and is maintained at temperatures as low as 8 degrees C. We have tested the ability of 30 structural analogues of ATP to effect mast cell permeabilisation. The analogues include those having substituents in the 2- and 8-positions of the purine ring, structural and optical isomers of the ribose sugar, and variations in the triphosphate chain. The pattern of selectivity displayed by the rat mast cell ATP4- receptor is distinct from those characteristic of the P1 purinoceptor for adenosine and the P2X and P2Y purinoceptors for adenine nucleotides.

ATP inhibits onset of exocytosis in permeabilised mast cells.

ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role for protein phosphorylation in the late stages of the activation pathway. We have measured the time-course of exocytosis from permeabilised cells triggered to release hexosaminidase following addition of Ca2+ to cells equilibrated for 2 min with GTP-gamma-S. If ATP is included at the time of permeabilization, then exocytosis commences after a delay, the duration of which depends on the square root of the product [Ca2+][GTP-gamma-S], and which may extend to beyond 3 min. When ATP is excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector combination. These results suggest that the achievement of a new steady-state, induced by Ca2+ and GTP-gamma-S, and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.

The dual effector system for exocytosis in mast cells: obligatory requirement for both Ca2+ and GTP.

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.

Late events in regulated exocytosis.

To understand the intracellular mechanisms that control exocytosis it is necessary to have access to the cell interior. This is achieved by plasma membrane permeabilisation or by application of patch-pipettes. These conditions permit control over the cytosol composition and also allow leakage of soluble factors that may have roles in the exocytotic mechanism. Different permeabilisation methods allow different extents of leakage and therefore provide complementary data. The exocytotic machinery itself remains intact and can be activated by providing Ca2+ and/or a guanine nucleotide. In some cells there is evidence for the participation of two guanine nucleotide-binding proteins (GP and GE), as well as a Ca(2+)-binding protein. In others Ca2+ is the only requirement. In a number of cell types, ATP is not required for the late steps in the secretory pathway.

Rat mast cells degranulate in response to microinjection of guanine nucleotide.

Investigation of regulated exocytosis has frequently required the use of permeabilised cell preparations. This has provided evidence that Ca2(+)-binding and guanine nucleotide-binding proteins can mediate secretion. Since the manner and extent of membrane permeabilisation affect the requirements for Ca2+ and guanine nucleotide, we have introduced such effectors directly into intact, rat peritoneal mast cells by microinjection. During this brief procedure (approximately 1 s) a glass needle forms a seal with the plasma membrane. Following injection and withdrawal of the needle the membrane reseals without apparent loss of cell contents. Using fluorescent dye, we estimate that the volume injected is approximately 5 fl and that the dilution of injected solutes is approximately 100-fold. Injection of the nucleotides inosine triphosphate, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta gamma-imido]triphosphate causes degranulation. The EC50 for GTP gamma S is approximately 10 microM (concentration in the needle) equivalent to an intracellular concentration of approximately 100 nM. However, the effect of GTP gamma S is dependent on the presence of Ca2+ in the external medium. This may be explained by a transitory influx of Ca2+ that occurs during impalement, since the seal between needle and membrane will not prevent the movement of small ions. Thus an increase in cytoplasmic Ca2+ appears to be necessary for secretion induced by GTP gamma S. Using metabolic inhibitors we have investigated the requirement for ATP. Under conditions where [ATP]i has fallen to 60 +/- 18 microM (S.E.M., n = 3) the mast cell agonist, compound 48/80, is unable to induce degranulation, yet injection of GTP gamma S still activates the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.

The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].

Monitoring exocytosis from single mast cells by fast voltammetry.

We have used fast differential ramp voltammetry with carbon-fibre electrodes to monitor exocytotic secretion in single rat mast cells. The oxidation peak and other aspects of the electrochemical profile of the substance released were similar to those of 5-hydroxytryptamine (5-HT) and the signals were increased by preloading the secretory granules with exogenous 5-HT. Metabolic blockade inhibited both visible degranulation and the electrochemical signal. For comparison, quinacrine, which is fluorescent and accumulates in secretory vesicles, was used as an alternative means of detecting secretion in single cells. The amplitude of the electrochemical signals observed during degranulation correlated well with the loss of quinacrine fluorescence. Both methods were used to record successive rounds of secretion in single mast cells in response to repeated applications of compound 48/80.

Wave-length dependencies of light scattering in normal and cold swollen rabbit corneas and their structural implications.

1. The studies described herein involve the use of light scattering measurements to characterize the ultrastructural arrangement of the constituent collagen fibrils in rabbit corneal stromas.2. Theoretical light scattering techniques for calculating the scattering to be expected from the structures revealed by electron micrographs are discussed, and comparison with the experimental light scattering tests the validity of these structures.3. The wave-length dependence of light transmission and of angular light scattering from normal corneas is in agreement with the short range ordering of collagen fibrils depicted in electron micrographs.4. The transmission measurements on oedematous rabbit corneas indicate that transmission decreases linearly with the ratio of thickness to normal thickness.5. The wave-length dependence of transmission through cold swollen corneas indicates that the increased scattering is caused by large inhomogeneities in the ultrastructure. Electron micrographs do, indeed, reveal the presence of such inhomogeneities in the form of large regions completely devoid of fibrils.

The effect of mitogenic lectins and monoclonal antibodies on intracellular free calcium concentration in human T-lymphocytes.

The effect of mitogenic lectins and the mitogenic antibody UCHT1 on the cytoplasmic free Ca2+ concentration ( [Ca2+]i) in human lymphocytes was investigated by using the fluorescent Ca2+-indicator quin2 . Phytohaemagglutinin, concanavalin A and UCHT1 increased [Ca2+]i in T-cells to a maximum level within 2 min. No T-cell response was seen with poke weed mitogen. None of the mitogens affected non-T-cell [Ca2+]i.