Dynamin is functionally coupled to insulin granule exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.     

Cortical granule exocytosis is coupled with membrane retrieval in the egg of Brachydanio

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.     

Mast cell tryptase does not alter matrix metalloproteinase expression in human dermal fibroblasts: further evidence that proteolytically-active tryptase is a potent fibrogenic factor.

There is compelling in vitro and in vivo evidence to implicate mast cells in the development of fibrosis. However, an important question remains as to the mechanisms by which mast cells mediate fibrosis. Recent evidence from our laboratory (Gruber et al., 1997, J. Immunol. , 158:2310-2317) has revealed that tryptase, the unique and abundant serine protease of human mast cells, is capable of activating fibroblasts by stimulating chemotaxis, proliferation, and procollagen mRNA synthesis. Regulation of matrix metalloproteinase (MMP) expression is another key step in connective tissue remodeling. Therefore, the effect of tryptase on fibroblast MMP expression was investigated. Proteolytically active tryptase did not alter the cellular mRNA levels for fibroblast MMP-1, MMP-2, MMP-3, and MMP-9 as detected by RNase protection assays. Moreover, tryptase did not alter the basal levels of MMP-1, MMP-2, MMP-3, MMP-9, or the tissue inhibitor of MMP-1 (TIMP-1) in fibroblast conditioned media as detected by specific enzyme-linked immunosorbent assay (ELISA). These results indicate that tryptase does not increase MMP expression in normal dermal fibroblasts. Moreover, these data strengthen the potential role of this unique serine protease as a potent fibrogenic factor.     

Cortical granule exocytosis in sea urchin eggs is inhibited by drugs that alter intracellular calcium stores

In sea urchin eggs fertilization is accompanied by cortical granule exocytosis, a secretory event thought to be initiated by release of intracellularly sequestered calcium. We have examined the effect of two drugs on this process: chlortetracycline (CTC), a known chelator of intracellular calcium, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an antagonist of intracellular calcium release in both skeletal and smooth muscle. Preincubation of eggs for 10 min with either CTC or TMB-8 blocked sperm entry, inhibited the burst of 45Ca2+ efflux normally seen postinsemination, and prevented fertilization envelope elevation. Half-maximal inhibition occurred with 200 microM CTC and 60 microM TMB-8. Electron microscopy confirmed that cortical granule exocytosis had been blocked, although inhibition was not due to a direct effect on exocytosis. CTC and TMB-8 had no effect on Ca2+-stimulated granule fusion in isolated egg cortices. Rather, these drugs block the early events in egg activation: sperm incorporation and triggering of exocytosis. These two effects appear to be independent since addition of either drug just before insemination permits sperm entry but inhibits calcium release and cortical granule exocytosis.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

A role for serglycin proteoglycan in granular retention and processing of mast cell secretory granule components

In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.     

Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase

In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.     

ERK activation is only one role of PKC in TCR-independent cytotoxic T cell granule exocytosis

Cytotoxic T cells (CTLs) kill target cells by releasing lytic agents via regulated exocytosis. Three signals are known to be required for exocytosis: an increase in intracellular Ca²⁺, activation of protein kinase C (PKC) and activation of extracellular signal regulated signal kinase (ERK). ERK activation required for exocytosis depends on activity of PKC. The simplest possibility is that the sole effect of PKC required for exocytosis is ERK activation. Testing this requires dissociating ERK and PKC activation. We did this using TCR-independent stimulation of TALL-104 human leukemic CTLs. When cells are stimulated with thapsigargin and PMA, agents that increase intracellular Ca²⁺ and activate PKC, respectively, PKC-dependent ERK activation is required for lytic granule exocytosis. Expressing a constitutively active mutant MAP kinase kinase activates ERK independent of PKC. However, activating ERK without PKC does not support lytic granule exocytosis, indicating that there are multiple effects of PKC required for granule exocytosis.     

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.     

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.     

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in ~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted ~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquine-sensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.     

Further evidence that the CCK2 receptor is coupled to two transduction pathways using site-directed mutagenesis

A heterogeneity of CCK2 receptors has been reported which could correspond to different states of coupling to G proteins and/or association with different second messenger systems. To investigate these hypotheses, the wild-type CCK2 receptor and three mutants F347A, D100N and K333M/K334T/R335L, expected to modify the coupling of the G protein with the third intracellular loop of the receptor, were transfected into Cos-7 cells and their binding and signalling properties were evaluated using the natural ligand CCK8. Activation of wild-type as well as F347A, D100N or K333M/K334T/R335L CCK2 receptors by this ligand led to a similar arachidonic acid release which was blocked by pertussis toxin and the phospholipase A2 inhibitor, mepacrine. Nevertheless, in contrast to the wild-type CCK2 receptor, addition of CCK8 to cells transfected with the F347A or K333M/K334T/R335L mutants did not result in the production of inositol phosphates while the maximum increase in this second messenger formation was reduced by 30% with the D100N mutant. Taken together, these results suggest that the CCK2 receptor is coupled to two G proteins and that Phe347 and the cluster of basic residues K333/K334/R335 probably play a key role in Gq protein stimulation leading to inositol phosphate production but not in activation of the G protein coupled to phospholipase A2. These data bring additional support at the molecular level to the existence of different affinity states of CCK2 receptors suggested from the results of binding assays and behavioural studies.     

Norepinephrine-induced cardiac hypertrophy and fibrosis are not due to mast cell degranulation

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg·h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg·h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Freeze-fracture studies of chemotactic peptide-induced exocytosis in neutrophils: evidence for two patterns of secretory granule fusion

Rabbit neutrophils were fixed in 2% glutaraldehyde and then either quick-frozen and freeze-fractured or embedded and thin-sectioned. Cells exposed to 10(8) M N-formylmethionyl-leucylphenylalanine (FMLP) and 5 micrograms/ml cytochalasin B at 22 degrees C underwent a rapid, compound exocytosis. Within 10 sec after stimulation, narrow pores were seen joining individual peripheral granules with the plasma membrane. Sequential fusion of interior granules occurred between 20 and 60 sec and took on two different patterns. The first consisted of a linearly directed series of fusion events resulting in a radial array of tapering invaginations directed toward the cell center. The second pattern consisted of an undirected fusion of larger granules to form highly branched structures. These granules were often connected by narrow tubules and in some cases a series of fused granules would end in a small, vesiclelike tip. This suggested that sequential fusion may involve a set of smaller vesicles as well as the granule membranes themselves.     

Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.     

Elevated blood glucose after compound 48/80 treatment is not related to hepatic mast cell degranulation in rats

Blood glucose, hepatic glycogen, and the histological integrity of hepatic mast cells, were evaluated in anesthetized rats receiving iv injections of 0.125 mg/kg body weight compound 48/80 (a mast cell degranulator) and/or of 0.001 to 10.0 mg/kg body weight lodoxamide tromethamine (an inhibitor of mast cell degranulation). A nonglucogenic dose of lodoxamide, 0.001 mg/kg body weight, prevented dissipation of histochemically demonstrable fluorescence in mast cells (degranulation) without inhibiting compound 48/80-induced hyperglycemia and hepatic glycogenolysis. These results suggest that this glucotropic response is independent of compound 48/80-evoked release of mediators such as serotonin from mast cells.     

Further evidence that the pigment in the Dubin-Johnson syndrome is not melanin

The pigment in the Dubin-Johnson syndrome (DJS) is shown unequivocally not to be a typical melanin or closely related polymer. In electron spin resonance (ESR) studies of DJS pigment from a hepatoma, it is shown that, unlike true melanins, the pigment associated with the DJS syndrome has no free radical in the absence of light. Exposure to even low levels of visible light over a broad frequency range induces a free radical in the DJS pigment. Previous studies did not appreciate the sensitivity to light of this pigment and therefore erroneously concluded that the DJS pigment had a permanent free radical. The light induced ESR signal in DJS tissue has spectroscopic properties that differ significantly from any known melanins. The pigment is not extracted by lipophilic solvents and is centrifuged down at 50,000g, but not at 5,000g.