Ionic control of the size of the vesicle matrix of beige mouse mast cells

Isolated matrices of the giant secretory vesicles of mast cells of the beige mouse were reliably produced by the osmotic lysis of isolated vesicles. These matrices maintained their form, and their sizes were easily measured using Nomarski optics. The size of the matrix depended on the ionic composition of the bathing solution. The physiologically relevant ions, histamine and serotonin, contracted the matrix. Multivalent cations condensed the matrix relative to univalents. Ag+, acid pH (below 5), and basic pH (above 9) expanded the matrix. In the presence of 10 mM histamine, lowering the pH from 9 to 5 contracted the matrix more than can be attributed to the pH-dependent matrix contraction in zero histamine. The nontitratable organic cation, dimethonium, contracts the matrix with little effect of pH in the range of 5-9. These results suggest that histamine acts as a matrix contractor in the divalent form. The dose-response (contraction) relation for histamine was gradual from micromolar to 316 mM (millimolar) histamine. Experiments with mixtures of histamine and sodium show antagonistic effects on the matrix but are inconsistent with either a model where ions compete for identical sites or a parallel model where ions interact with separate independent sites. In vigorous histamine washoff experiments, the half time for vesicle expansion in 10(-4) M pH buffer was approximately 4 s; in isotonic NaCl solution, it was 0.5 s. When 1 M histamine was presented to closely apposed matrices, fusion resulted. The matrix material returned to its initial shape after being mechanically deformed with a glass probe. These results suggest that the matrix size is controlled by its ion exchange properties. The matrix expansion can quantitatively account for the vesicular size increase observed upon exocytosis (as a postfusional event) and the osmotic nonideality of intact vesicles. The mechanical expansion is probably significant in the widening of the exocytotic pore and the dispersal of the vesicular contents.     

Morphological and functional characterization of beige mouse adrenomedullary secretory vesicles

We tested whether the giant secretory granules observed in the mast cells of the naturally occurring mutant beige mouse (BM) (C57BL/6N-bg) were also present in the adrenal chromaffin cells. The presence of large chromaffin granules (CG) would be a valuable tool for the study of exocytosis in neuronal tissues. Conversely, the observation of large vesicles within chromaffin cells that are different from CG could indicate that CG are of a different origin than granules of mast cells. Ultrastructural analysis demonstrated the presence of large lysososmal-like vesicles in the BM, and also a discrete increase in the number of CG with diameters larger than 240 nm but not of giant CG. In addition, amperometric measurements of single-event exocytosis, using carbon fiber microelectrodes, showed no differences between the quantal size of secretory events from BM and wildtype or bovine chromaffin cells. Minor but significant differences were found between the kinetics of exocytosis in BM cells andwild-type mouse cells. We conclude that CG, but not the abnormal-sized vesicles found in BM chromaffin cells contribute to the catecholamine secretion and that abnormal secretory granules are not present in adrenergic cell lineage.     

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.     

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [³⁵S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.

The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins.     

The molecular mechanism of nondegranulative release of biogenic amines.

According to current teaching biogenic amines are released by exocytosis, i.e. by evacuation of amine storing vesicles or granules into the extracellular space. The release of transmitter amines is quantal, i.e. occurs in packs of transmitter molecules. These packs are assumed to be identical with vesicle contents, in other words, the smallest releasable quantum equals the amine content of one vesicle. However, there are experimental observations which do not fit in with this version of an exocytotic release theory. Observed quantitative discrepancies could be explained if the release mechanism allowed a fractional release of transmitter amine from several vesicles instead of the total evacuation of a few. The lack of adequate knowledge about the mechanisms of storage of biogenic amines within the vesicles has up til now rendered it difficult to envisage the machinery behind a fractional release of the amine content of a vesicle. In extensive in-vitro studies we have found that the matrices of amine storing granules (i.e. from mast cells, chromaffin cells and nerve terminals) show the properties of weak cation exchanger materials, carboxyl groups serving as amine binding ionic sites. When exposed to cations like sodium and potassium ions, the amines are released from their storage sites according to kinetics characteristic of weak cation exchangers. In vivo, amine release from cat adrenals on splanchnic nerve stimulation also occurs according to ion exchange kinetics. Histamine release from mast cells is considered to occur as the result of degranulation, i.e. the expulsion of histamine storing granules to the extracellular space, a typical example of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles

To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.     

Effects of divalent cations,temperature, osmotic pressure gradient,and vesicle curvature on phosphatidylserine vesicle fusion

Summary Fusion of phosphatidylserine vesicles induced by divalent cations, temperature and osmotic pressure gradients across the membrane was studied with respect to variations in vesicle size. Vesicle fusion was followed by two different methods: 1) the Tb/DPA fusion assay, whereby the fluorescent intensity upon mixing of the internal aqueous contents of fused lipid vesicles was monitored, and 2) measurement of the changes in turbidity of the vesicle suspension due to vesicle fusion. It was found that the threshold concentration of divalent cations necessary to induce vesicle fusion depended on the size of vesicles; as the diameter of the vesicle increased, the threshold value increased and the extent of fusion became less. For the osmotic pressure-induced vesicle fusion, the larger the diameter of vesicles, the smaller was the osmotic pressure gradient required to induce membrane fusion. Divalent cations, temperature increase and vesicle membrane expansion by osmotic pressure gradient all resulted in increase in surface energy (tension) of the membrane. The degree of membrane fusion correlated with the corresponding surface energy changes of vesicle membranes due to the above fusion-inducing agents. The increase in surface energy of 9.5 dyn/cm from the reference state corresponded to the threshold point of phosphatidylserine membrane fusion. An attempt was made to explain the factors influencing fusion phenomena on the basis of a single unifying theory.     

Orientation of Membrane Vesicles from Escherichia coli as Detected by Freeze-Cleave Electron Microscopy

The application of freeze-cleave electron microscopy to whole cells of Escherichia coli revealed that the particles exposed on the resulting two inner membrane faces are asymmetrically distributed. This method can therefore be used to determine the orientation of membrane vesicles from E. coli. Membrane vesicles freshly prepared in potassium phosphate buffer (K(+)-vesicles) by osmotic lysis of spheroplasts consisted almost entirely of right-side-out vesicles. Their size suggested that each cell gives rise to one vesicle. When the membrane vesicles were subjected to one cycle of freezing and thawing, the number of inside-out vesicles rose to about 25%. However, due to the small size of most of the inside-out vesicles, these contribute only 2 to 3% of the total membrane surface area of the preparation. The inside-out vesicles appear to arise from infoldings of the membrane of right-side-out vesicles. They also accumulate within the latter, thus producing multivesicular membrane sacs. Na(+)-vesicles (vesicles prepared in sodium phosphate buffer) subjected to freezing and thawing appeared to lose structural rigidity more than did K(+)-vesicles. In contrast to the membrane vesicles prepared by the osmotic lysis of spheroplasts, those obtained by breaking intact cells by a single passage through a French pressure cell were uniformly very small (only 40 to 110 nm in diameter); approximately 60 to 80% were inside-out. To reconcile the polarity of the membrane vesicles with the enzymic activities of such preparations, we propose that "dislocation" of membrane proteins occurs during osmotic lysis of spheroplasts.     

Compartmentalization of monoaminergic synaptic vesicles in the storage and release of neurotransmitter

Monoaminergic nerves are characterized by the presence of a population of small synaptic vesicles (40-60 nm in diameter) containing a few large vesicles (80-90 nm in diameter). Thus, although both types of vesicles contain monoamines, the small vesicles must be considered as the organoid responsible for the storage and release of the neurotransmitter, whereas the large ones possibly are involved in the modulation of the process. The small vesicles are electron-lucent or have an osmiophilic electron-dense core that is always linked to the vesicle membrane. Considering morphological and histochemical evidence under different experimental conditions, we proposed the existence of two compartments in the small vesicles: the core and the matrix, corresponding respectively to the electron-dense core and the electron-lucent space between the core and the vesicle membrane in osmium tetroxide fixations. The sizes of both compartments are inversely related, i.e., the smaller the core, the larger the matrix and vice versa. The core even disappears, giving way to a small electron-lucent vesicle made exclusively by the matrix. Thus, the matrix is a constant component of the vesicle, whereas the core is a transient one. Each compartment has a different pool of amine: a loosely bound, easily releasable pool in the matrix and a tightly bound, more resistant pool in the core. These two pools subserve, respectively, a tonic or phasic release of the neurotransmitter, correlated with a tonic or phasic stimulation of the receptor. The core may be considered as a storage or reserve pool. Experimental evidence from our laboratory supports the concept that different mechanisms are operative in both compartments in the release of the neurotransmitter. For instance, a Ca2(+)-independent release would be primarily concerned with the neurotransmitter contained in the matrix, and a Ca2(+)-dependent efflux would be primarily related with the neurotransmitter stored in the core. However, it still must be established that a simple relationship exists between each kind of stimulus and each vesicle compartment, rather than both compartments being integrated in a dynamic functional unit.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

Temporal separation of vesicle release from vesicle fusion during exocytosis

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.     

PC12 Cells that Lack Synaptotagmin I Exhibit Loss of a Subpool of Small Dense Core Vesicles

Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca²⁺ sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.     

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles

It is proposed that the role of calcium in calcium-induced exocytosis is to open Ca-activated K channels present in vesicle membranes. The opening of these channels coupled with anion transport across the vesicle membranes would result in an influx of K and anions, increasing the osmotic pressure of the vesicles. For those vesicles situated very close to the cell plasma membrane, this would lead to fusion with the membrane and exocytosis of the vesicle contents. This model can account for facilitation and other key properties of transmitter release. In addition, the model predicts that vesicles with a higher transmitter content, and hence higher initial osmotic pressure, would be preferentially discharged. The model also predicts that a faster response can be obtained for small vesicles than for large vesicles, providing a rationale as to why neurotransmitters, which must be released quickly, are packaged in small vesicles.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Catecholamine-rich cells and varicosities in bovine splenic nerve,vesicle contents and evidence for exocytosis

The bovine splenic nerve trunk contins mast cells, ganglion cells, small intensely flurescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structure could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300–800nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120–140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45–55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicle in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Interrogating the role of liposome size in mediating the dynamics of a chromophore in the acyl chain region of a phospholipid bilayer

We have examined the temperature-dependent reorientation dynamics of perylene imbedded in bilayers of 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), where the bilayers exist in the form of unilamellar vesicles. Previous work using 100-nm diameter DMPC vesicles has shown that the phase transition from the gel phase to the fluid phase can be detected using the reorientation dynamics of perylene. In this work we explore the vesicle size dependence of the perylene reorientation dynamics in DMPC vesicles. The size of the vesicles is determined by extrusion and the reorientation dynamics of perylene are measured as a function of vesicle size between 100-nm and 5-microm diameter. We find that, while the phase transition for DMPC is seen in smaller vesicles, perylene becomes insensitive to the phase transition for vesicles larger than ca. 800-nm diameter. We also find a discontinuous change in perylene reorientation dynamics with increasing vesicle size, and we consider this result in the context of the location of perylene within the bilayer.     

Regulation of releasable vesicle pool sizes by protein kinase A-dependent phosphorylation of SNAP-25

Protein kinase A (PKA) is a key regulator of neurosecretion, but the molecular targets remain elusive. We combined pharmacological manipulations of kinase and phosphatase activities with mutational studies on the exocytotic machinery driving fusion of catecholamine-containing vesicles from chromaffin cells. We found that constitutive PKA activity was necessary to maintain a large number of vesicles in the release-ready, so-called primed, state, whereas calcineurin (protein phosphatase 2B) activity antagonized this effect. Overexpression of the SNARE protein SNAP-25a mutated in a PKA phosphorylation site (Thr-138) eliminated the effect of PKA inhibitors on the vesicle priming process. Another, unidentified, PKA target regulated the relative size of two different primed vesicle pools that are distinguished by their release kinetics. Overexpression of the SNAP-25b isoform increased the size of both primed vesicle pools by a factor of two, and mutations in the conserved Thr-138 site had similar effects as in the a isoform.     

A theory of osmotic lysis of lipid vesicles

Osmotic lysis of vesicles is shown to begin when the membrane expansion due to osmotic pressure exceeds its critical value, delta S, at which a membrane ruptures to form a pore. The dependence of delta S on the vesicle radius and respective osmotic pressures are obtained. It is found that osmotic pressure necessary for small (100 A) vesicles to rupture should exceed 30 atm, for large (10 000 A) vesicles it being as small as 10(-3) atm. In the case of large (greater than or approximately 1000 A) vesicles the value of relative expansion of the membrane at which its rupture occurs in a reasonable time only depends slightly on the vesicle radius. For instance, for 10 000 A vesicles it amounts to 3%. The tension of membrane rupture is about 8 dyn/cm for large vesicles. Membrane tension, although it decreases considerably as a result of rupture and pore formation, does not vanish completely. It supports the residual intravesicular pressure causing the efflux of vesicle (cell) contents. Simultaneously, osmotic influx of water through the membrane occurs that results in either complete rupture of the membrane with the efflux of the whole of the contents, or its gradual washout in either of two, quasi-steady or pulse-wise regimes. In the first case a pore is steadily open, whereas in the second case it alternately opens and closes, ejecting about 5% of internal solution each time. Lysis kinetics is analyzed. Pulse-wise regime of lysis is shown to be the most likely one.     

Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion.

The detection of exocytotic fusion in patch-clamped secretory cells depends on measuring an increase in the cell membrane capacitance as new membrane is added to the plasma membrane. However, in the majority of secretory cells, secretory vesicles are too small (< 200 nm in diameter) to cause a detectable signal. We have found that incubations of normal mouse mast cells with the hydrophobic anion dipicrylamine (DPA), increases cell membrane capacitance by about three times. The large capacitive current induced by DPA was voltage-dependent, having a maximum value at -10 mV. The DPA-induced charge movement could be described by a single barrier model in which the DPA molecules move between two stable states in the bulk lipid matrix of the membrane. More importantly, the DPA treatment produced a sevenfold increase in the size of the capacitance steps observed upon the exocytotic fusion of single secretory granules. A similar amplification of DPA on the secretory vesicle capacitance was observed in a cell with larger (< or = 5 microns in diameter) or with smaller secretory granules (< 250 nm in diameter). Additionally, the increased granule membrane capacitance enlarged the transient capacitive discharge measured upon formation of a fusion pore in normal mast cell granules. Our results indicate that hydrophobic ions provide an important tool for high resolution studies of membrane capacitance.