A new model for the mechanism of stimulus-secretion coupling

By combining ultrastructural techniques with a biochemical approach to study the mechanism of mast cell stimulus-secretion coupling and by using purified secretory granules to confirm those early biochemical events which originate from within the secretory granule, a new model for the mechanism of secretory granule exocytosis has emerged. This model not only provides the mechanism by which an activated granule can achieve fusion with the plasma membrane, but it also provides the rationale for the linking of the various early biochemical events to the process of granule activation and thus to exocytosis. Although we still do not understand how the 'activating signal', which results from the stimulation of cell surface receptors, can be conveyed to the granule to cause its activation, we are certain that this 'signal' must cause an influx of water into the matrix of the target granule. This influx of water is what initiates the granule activation process. The major intragranular events which are triggered by this water influx include: (i) de novo membrane assembly; (ii) protein proteolysis; (iii) release of arachidonic acid from matrix-bound phospholipid by phospholipase A2; (iv) initiation of the arachidonic acid cascade and the synthesis of eicosanoids; (v) rapid phospholipid turnover; and (vi) the discharge of matrix materials into the cytoplasm of the activated cell via the fusion of de novo generated vesicles with the perigranular membrane. The ejection of some matrix contents which may include histamine, Ca2+, calmodulin, protease, the products of the arachidonic acid cascade and the products of phospholipid turnover into the cytosole, may serve to turn on the various metabolic machineries needed to initiate a cellular recovery phase.     

Phospholipid storage in the secretory granule of the mast cell

A spontaneous membrane assembly process has been postulated to account for the rapid perigranular membrane enlargement which occurs during mast cell secretory granule activation. This process requires the presence of a phospholipid store in the quiescent granule. By using purified granules with intact membranes we have determined the total phospholipid content of the average quiescent granule. The results suggest that the average quiescent granule contains sufficient phospholipid to sustain at least a trebling of its perigranular membrane surface area during activation. As much as two-thirds of the total cellular phospholipid is found in the granules, and since a large portion of this phospholipid is extruded into the extracellular space along with the granule matrix during exocytosis, it is implied that this phospholipid can serve as the substrate for the formation of the lipid-derived mediators of inflammation.     

Compound versus multigranular exocytosis in peritoneal mast cells

We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.     

Mechanism of secretory granule exocytosis: can granule enlargement precede pore formation?

Secretory granules have been observed to swell during the process of exocytosis. Swelling is an indication of osmotic stress. The probable role of osmotic pressure in facilitating membrane fusion makes it necessary to determine whether granule membrane 'swelling' can occur prior to its fusion with the plasma membrane (pore formation) in the process of exocytosis. By subjecting adjacent thin and semi-thin sections of an activated granule to ultrastructural examination for membrane enlargement, and to metachromatic staining for verification of pore formation it is concluded that the perigranular membrane can indeed enlarge prior to pore formation. However, the degree of membrane enlargement can far exceed the limit of 2-3% stretching allowed under normal osmotic stress for a membrane bilayer. Such an extensive membrane enlargement, which takes place in the mechanism of exocytosis, cannot be achieved without being accompanied by the insertion of additional membrane.     

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.     

Formation and exocytosis of secretory granules in the endocrine cells of normal and transplanted pancreas: an electromicroscopic study

The mechanism of secretory granule formation and exocytosis in the endocrine cells of normal and transplanted rat pancreas was studied using electron microscopy. On the one hand, formation of secretory granules starts with the dilatation of the 2 ends or the vesicularization of the middle parts of rough endoplasmatic reticulum (RER). On the other hand, prohormone ribosomes condense into the vesicles of the GOLGI apparatus. This probably indicates that the GOLGI complex is not the only source of formation of secretory granules. Exocytosis occurs with the formation of an electron dense streak between the perigranular membrane and the apical cell membrane. This is followed by the rupture of the streak at this midpoint allowing the granule to extrude into the space between the cell membrane and the parenchymal basal membrane. This fusion-rupture-extrusion mechanism repeats itself at the parenchymal and capillary basal membranes and also at the endothelium until it gets into the capillary lumen, showing that hormones of pancreatic endocrine cells may be actively transported into circulation as intact secretory granules. There is no significant morphological difference between the mechanism of secretory granule formation in normal and transplanted pancreatic tissue.     

Morphological study of rat mast cells stimulated with compound 48/80 at different temperatures

Changes of mast cells stimulated with compound 48/80 were morphologically investigated at different temperatures. Peritoneal mast cells of male rats were stimulated in vitro at 4 or 17° C. At 17° C, mast cells stimulated for 10 s gave decreased fluorescent reactions for phalloidin. At 30 s stimulation, they showed typical exocytosis initiated by fusions of peripherally located secretory granules to the plasma membrane. In contrast, mast cells stimulated at 4° C exhibited neither decrease of phalloidin reactions nor typical excytosis even after 30 s. It was inferred that the fusions were mediated by cytoplasmic elements, probably the actin filaments previously suggested to prevent release of secretory granules. Furthermore, the space between the perigranular membrane and granular contents was enlarged in some mast cells stimulated at 4° C. The morphological changes suggested that equivocal events occurred also in the cytoplasm of these cells. The mast cells showed no typical exocytosis at 4° C.     

Requirement for metalloendoprotease in exocytosis: evidence in mast cells and adrenal chromaffin cells

Exocytosis is initiated by the receptor-mediated influx of calcium that results in fusion of the secretory vesicle with the plasma membrane. We examined the possibility that calcium-dependent exocytosis in mast cells and adrenal chromaffin cells requires metalloendoprotease activity. Metalloendoprotease inhibitors and dipeptide substrates block exocytosis in these cells with the same specificity and dose dependency as that with which they interact with metalloendoproteases. Metalloendoprotease activity is identified in these cells with fluorogenic synthetic substrates, which also blocked exocytosis. Metalloendoprotease activity is highest in the plasma membrane of chromaffin cells. The metalloendoprotease appears to be required in exocytosis at a step dependent on or after calcium entry, since exocytosis initiated by direct calcium introduction in both mast cells and chromaffin cells is blocked by metalloendoprotease inhibitors.     

CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-¹⁴C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.     

Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.     

Phospholipases stimulate secretion in RBL mast cells

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.     

MATURATION OF RAT MAST CELLS : An Electron Microscope Study

Electron microscope study of rat mast cell maturation corroborates certain interpretations of features of mast cell differentiation based on light microscope studies. In addition, the ultrastructural variation observed in the granules of differentiating mast cells suggests that granule formation begins with the elaboration of dense granules about 70 mµ in diameter inside Golgi vacuoles. These progranules appear to aggregate inside a membrane and fuse to form dense cords 70 to 100 mµ in diameter. These dense cords are embedded in a finely granular material possibly added to the developing granule by direct continuity between perigranular membranes and cisternae of rough endoplasmic reticulum. The dense cords and finely granular material then appear to be replaced by a mass of strands about 30 mµ in diameter, thought to be a reorganization product of the two formerly separate components. A process interpreted as compaction of the strands completes the formation of the dense, homogeneous granules observed in mature rat mast cells. The similarity between mast cell granule formation and the elaboration of other granules is considered, with special reference to rabbit polymorphonuclear leukocyte azurophil granules. The relationships between the ultrastructural, histochemical, and radioautographic characteristics of mast cell granule formation are considered, and the significance of the perigranular membrane is discussed.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.     

Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions

We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.     

Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis.

We have studied the admittance of patch-clamped mast cells during exocytosis and found that they are adequately described by a four parameter equivalent circuit. On the basis of these measurements, we show that, contrary to current belief, when using a phase sensitive detector, small capacitance changes due to exocytosis or endocytosis should be studied by measuring current 90 degrees out of phase, relative to the component that corresponds to changes in series resistance. We have extended the theory on phase-detectors to include the errors in the estimation of step changes of membrane capacitance. We show that the measured capacitance of a secretory granule can be up to 80% smaller than its true value, during the course of a typical mast cell degranulation. We also describe a software-based phase-detector that simplifies capacitance measurements.     

Mast cell degranulation and its inhibition by an anti-allergic agent tranilast. An electron microscopic study

Degranulation of IgE-sensitized rat mast cells by antigen was studied quantitatively in vitro and in vivo by electron microscopy. The inhibition of this degranulation by an anti-allergic drug, N-(3,4-dimethoxycinnamoyl)anthranilic acid (Tranilast), was also examined both in vitro and in vivo. In the in vitro study using peritoneal mast cells, alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane were observed and were accompanied by histamine release. Scanning electron microscopy disclosed the extrusion of smooth, round bodies from pores formed on the cell surface. In the in vivo study of passive cutaneous anaphylaxis (PCA), the characteristic features of mast cell degranulation were obvious 5 min after the injection of antigen; leakage of dye increased progressively from 5 to 30 min but was not found at 6 h. From quantitative analysis of the substructure of mast cells, it was demonstrated that degranulation of IgE-sensitized mast cell induced by antigen was achieved by sequential exocytosis both in vitro and in vivo. Tranilast inhibited these changes to a remarkable extent and it was concluded that the inhibition of mast cell degranulation by this drug might play an important role in anti-allergic treatment.