Effect of chronic and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on the function and structure of cultured chromaffin cells

Cultured bovine adrenal chromaffin cells were treated chronically with various concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the culture medium for 2–8 days or acutely for 10–15 min. Culture of cells with MPTP for periods of 2–8 days resulted in a marked loss of total cellular catecholamines and a parallel reduction in secretory response, but not the ratio of stimulated to unstimulated secretion. By the eighth day in culture, at the highest MPTP concentration (1000 μM), cell catecholamine content and secretion were only about 10% that of untreated cells. The proportion of total cellular catecholamines secreted was not altered by MPTP, suggesting that the secretory process was unaffected by the drug. The loss of secretory output was not prevented by inhibitors of monoamine oxidase or catecholamine uptake, drugs known to prevent MPTP-induced damage to central dopaminergic neurons. The subcellular organelles of MPTP-treated cells appeared relatively normal except for extensive depletion of the vesicle contents, in agreement with the biochemical data. The severity of the depletion appeared to be lessened in cells treated with monoamine oxidase inhibitors.Short term exposure to MPTP at concentrations less than 100 μM had little effect on secretion induced by carbachol. Higher concentrations of MPTP increased unstimulated release and reduced stimulated release. Pretreatment of the cells with MPTP resulted in a lasting reduction in their subsequent secretory responsiveness. MPTP alone, at concentrations greater than 100 μM induced catecholamine release that was unaffected by pretreatment of the cells with monoamine oxidase inhibitors or the catecholamine uptake inhibitor desipramine. MPTP-induced secretion by intact cells was calcium-dependent, while the small increase by permeabilized cells was not.     

Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro.

Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest that magnesium may decrease the secretory response by displacing the cellular calcium which is utilized in stimulus-secretion coupling.     

Molecular machinery and mechanism of cell secretion

Secretion occurs in all living cells and involves the delivery of intracellular products to the cell exterior. Secretory products are packaged and stored in membranous sacs or vesicles within the cell. When the cell needs to secrete these products, the secretory vesicles containing them dock and fuse at plasma membrane-associated supramolecular structures, called porosomes, to release their contents. Specialized cells for neurotransmission, enzyme secretion, or hormone release use a highly regulated secretory process. Similar to other fundamental cellular processes, cell secretion is precisely regulated. During secretion, swelling of secretory vesicles results in a build-up of intravesicular pressure, allowing expulsion of vesicular contents. The extent of vesicle swelling dictates the amount of vesicular contents expelled. The discovery of the porosome as the universal secretory machinery, its isolation, its structure and dynamics at nanometer resolution and in real time, and its biochemical composition and functional reconstitution into artificial lipid membrane have been determined. The molecular mechanism of secretory vesicle swelling and the fusion of opposing bilayers, that is, the fusion of secretory vesicle membrane at the base of the porosome membrane, have also been resolved. These findings reveal, for the first time, the universal molecular machinery and mechanism of secretion in cells.     

Secretory cargo composition affects polarized secretion in MDCK epithelial cells

Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a “sorting escort” (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as “sorting escorts” to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells. David V. Cohn—Deceased.     

Catecholamine Secretion by Chemically Skinned Cultured Chromaffm Cells

The secretory system of intact chromaffin cells is not accessible to direct chemical manipulation because of the selective permeability of the plasmalemma. We have devised a simple procedure for chemically "skinning" (permeabilizing) cultured adrenal medullary chromaffin cells by brief exposure to the detergent saponin. This procedure disrupts the continuity of the plasmalemma, thus allowing us to bypass those aspects of the secretory process controlled by the cell membrane and giving direct access to exogenous substances to the cellular secretory machinery. We report here that the skinned cells retain a fully competent secretory mechanism dependent only on exogenous calcium and MgATP. Saponin treatment had no significant effect on the total catecholamine content of the cells. Secretion could be initiated by either MgATP or calcium as long as the other was present in the medium. Catecholamine and dopamine-beta-hydroxylase release by the skinned cells was dependent on the calcium concentration of the medium. The ratio of secreted catecholamine and enzyme was similar to that of the cells, indicating that secretion occurred by an exocytotic mechanism. About half the total cellular content of the cytoplasmic enzyme lactic dehydrogenase was released during the permeabilization process and subsequent incubations, indicating plasmalemma permeability to molecules as large as protein. Calcium-induced secretion was unaffected by several drugs known to affect catecholamines and granule function. Saponin treatment of chromaffin cells in culture appears to be a simple means for allowing access to exogenous substances to the cells' secretory machinery. Therefore, it offers the opportunity to use chemical treatments, and perhaps specific antibodies to cellular components, to determine the role of these elements in the secretory process. These techniques should also be applicable to other cells known to secrete by an exocytotic mechanism.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.     

Expression differences in mitochondrial and secretory chaperonin 60 (Cpn60) in pancreatic acinar cells

In pancreatic acinar cells, chaperonin Cpn60 is present in all the cellular compartments involved in protein secretion as well as in mitochondria. To better understand the role Cpn60 plays in pancreatic secretion, we have evaluated its changes under experimental conditions known to alter pancreatic secretion. Quantitative protein A-gold immunocytochemistry was used to reveal Cpn60 in pancreatic acinar cells. Cpn60 immunolabelings in cellular compartments involved in secretion were found to decrease in acute pancreatitis as well as upon stimulation of secretion and in starvation conditions. A major increase in Cpn60 was recorded in diabetic condition. This was normalized by insulin treatment. Although in certain situations changes in secretory enzymes and in Cpn60 correlate well, in others, nonparallel secretion seemed to take place. In contrast, expression of mitochondrial Cpn60 in acinar cells appeared to remain stable in all conditions except starvation, where its levels decreased. Expression of Cpn60 in the secretory pathway and in mitochondria thus appears to behave differently, and Cpn60 in the secretory pathway must be important for quality control and integrity of secretion.     

Association of lipids with mucins may take place prior to secretion: studies with primary hamster tracheal epithelial cells in culture

Confluent cultures of hamster tracheal surface epithelial (HTSE) cells are highly enriched with secretory cells and secrete mucins. Ultrastructural studies of cellular localization of these mucins show that mucins are found not only inside secretory granules but also on the apical surface of secretory cells during active secretion, and secreted mucins are highly associated with lipids. In the present communication, we analyzed lipids associated with both cellular and secreted mucins following metabolic radiolabeling of these cultured cells with [3H]palmitic acid. We found that profiles of lipids associated with both cellular and secreted mucins are almost identical not only qualitatively but also quantitatively. It is concluded that the lipid association with mucins seems to take place before secretion. The origin of the cell surface-bound mucins is discussed.     

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.     

Cutting edge: selective requirement for the Wiskott-Aldrich syndrome protein in cytokine, but not chemokine, secretion by CD4+ T cells

The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4(+) T cells. Murine WASp-deficient CD4(+) T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4(+) T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4(+) T cell activity to be further fine-tuned to serve specialized effector functions.     

The story of cell secretion: events leading to the discovery of the 'porosome' - the universal secretory machinery in cells

Cell secretion has come of age, and a century old quest has been elegantly solved. We have come a long way since earlier observations of what appeared to be 'fibrillar regions' at the cell plasma membrane, and electrophysiological studies suggesting the presence of 'fusion pores' at the cell plasma membrane where secretion occurs. Finally, the fusion pore or 'porosome' has been discovered, and its morphology and dynamics determined at nm resolution and in real time in live secretory cells. The porosome has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membrane. The discovery of the porosome as the universal secretory machinery in cells and the discovery of the molecular mechanism of vesicular content expulsion during cell secretion have finally enabled a clear understanding of this important cellular process. This review outlines the fascinating and exciting journey leading to the discovery of the porosome, ultimately solving one of the most difficult, significant, and fundamental cellular process -cell secretion.     

Secretion machinery at the cell plasma membrane

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Three critical activities occur at the cell plasma membrane to ensure secretion. Membrane-bound secretory vesicles dock, fuse, and expel their contents to the outside via specialized and permanent plasma membrane structures, called porosomes or fusion pores. In recent years, significant progress has been made in our understanding of these three key cellular activities required for cell secretion. The molecular machinery and mechanism involving them is summarized in this article.     

Thiophosphorylation prevents catecholamine secretion by chemically skinned chromaffin cells

Skinned cells treated with the adenosine triphosphate analog, adenosine-5'-0-(3-thiotriphosphate) showed calcium-dependent thiophosphorylation of cellular constituents. Catecholamine secretion was inhibited when the analog was used as the substrate to promote secretion. The attenuation of secretion was proportional to the percentage of the analog in mixtures with adenosine triphosphate. Moreover, cells treated with the analog were subsequently unable to secrete when presented with MgATP, their normal substrate, indicating that the secretory systems was locked in the thiophosphorylated state. We hypothesize that phosphorylation is the calcium-dependent step required to prime the secretory system for secretion while dephosphorylation is the event required for exocytosis.     

黑胸散白蚁腹腺的形态学与细微构造

对黑胸散白蚁(Reticulitermes chinensis Snyder)腹腺整体装片和切片进行了描述.腹腺分前、后两部分.“腹腺前部”有两类分泌细胞和一个中央腔.两类分泌细胞中,一类是圆形,个体较大;另一类细胞突起很长,具扁平的核.复盖于腹腺前部的体壁上有许多小管和一些感器,表明腹腺前部的分泌细胞产物可能是经体壁上的小管或者先贮存于中央腔中,再经体壁小管逸出体外.“腹腺后部”由大的椭圆形分泌细胞组成.根据腹腺前部紧贴于第Ⅴ腹节表皮层,而腹腺后部是可动的,并且复盖于腹腺后部的体壁上无排出小管,作者认为这些细胞的分泌物可能是释放入血淋巴中.腹腺前部及腹腺后部分泌细胞的分泌物可能有不同的机能,有待于进一步研究.     

Studies of tracheal secretion using serous cell cultures and monoclonal antibodies

The glycoconjugate composition of tracheal secretions varies with physiological and pathophysiological parameters. Believing that these differences might be explained by metabolic or regulatory modifications of particular cell types, we have developed strategies for biochemical analysis at the cellular level. We have produced monoclonal antibodies whose determinants are restricted to a single secretory cell type (serous, mucous, or goblet cell granules, or ciliated cell glycocalyx). By enzyme immunoassay (ELISA), we have characterized four of the antibodies biochemically, and have also used the antibodies as quantitative molecular probes to detect release of antigen from mixed cell explants. Four of the antigens are carried by carbohydrate moieties of high molecular weight glycoproteins. Western blot analysis shows their molecular weight in reducing gels (SDS-PAGE) to exceed 200 kD. When used in parallel with pulse-chase labeling studies, the antibodies are both more sensitive and specific (than bound radioactivity) in detecting gland or goblet cell secretion in response to autonomic drugs or proteases. We have also isolated and cultured serous gland cells for physiological and biochemical studies. These cells express serous cell phenotype as reflected by ultrastructure, histochemistry, and lysozyme activity. Biochemical analysis of their secretory products reveals glycoconjugate components which are heterogeneous with respect to both molecular weight and charge. Radiolabeled secretory products eluting in the void volume of Sepharose C1 4B were completely degraded by chondroitinase ABC. This indicates that the major glycoconjugate produced by serous cell is a proteoglycan resembling chondroitin sulfate.     

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.     

Glucocorticoids stimulate the production of preprocalcitonin-derived secretory peptides by a rat medullary thyroid carcinoma cell line

A rat medullary thyroid carcinoma cell line, CA-77, has been established as a model system for investigating calcitonin biosynthesis and secretion. Growth of this cell line in serum-free defined medium provided suitable conditions for studying steroid hormone effects on the production of calcitonin and related peptides. After exposure for 5 days to a variety of steroids, only dexamethasone and corticosterone increased cellular content of calcitonin and a second secretory peptide (CCAP) derived from the same mRNA translation product as calcitonin. Glucocorticoids had no effect on cellular somatostatin, another secretory product of these cells. Increasing doses of dexamethasone progressively elevated cellular calcitonin and CCAP, with a maximal effect at 10(-8) M; 10(-9) M and lower doses were ineffective. On a molar basis, corticosterone was approximately 50-fold less potent than the synthetic glucocorticoid. An increase in cellular calcitonin content was observed only after 48 h of glucocorticoid treatment; a maximum increase (13-fold) occurred after 7 days. Glucocorticoids also increased basal calcitonin secretion. Similar effects were observed for cellular and secreted CCAP. Withdrawal of dexamethasone after 4 days of treatment lowered cellular calcitonin toward the level of control cultures. Dexamethasone pretreatment potentiated the acute secretory response to calcium for both calcitonin and CCAP, while no such enhancement was noted for calcium stimulation of somatostatin secretion. We conclude that the glucocorticoids specifically stimulate the production and secretion of calcitonin and CCAP, two secretory peptides derived from preprocalcitonin.     

Distinct linkage between post-translational processing and differential secretion of progastrin derivatives in endocrine cells

Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C-terminal fragments derived from progastrin differ in characteristic manners in levels and patterns of carboxyamidation and tyrosine sulfation in accordance with the secretory pathway taken. Point mutations introduced into a sorting motif disrupt these patterns, suggesting that differences in post-translational modifications are attributable to differential intracellular sorting of precursors. The results suggest a two-step sorting mechanism for progastrin leading to differential secretion of processed fragments via different secretory pathways.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.