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ABSTRACT: Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis.
Biochemical Journal 07/1989; 260(3):915-22. · 4.90 Impact Factor
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ABSTRACT: Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.
Journal of Neurochemistry 07/1989; 52(6):1751-8. · 4.06 Impact Factor
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ABSTRACT: Chromogranin A (CgA) and related acidic proteins are widely distributed in the organism. They are also present in entero-endocrine cells and in other members of the paraneuron family. Therefore, CgA has been claimed as an universal marker of this cellular community. To yield precise data about the distribution of CgA in entero-endocrine cells, all segments of the gastro-intestinal tract of five mammalian species (man, cattle, pig, cat, guinea-pig) were investigated immunohistochemically for CgA. In serial semithin plastic sections, all CgA-immunoreactive endocrine cells were identified for resident amines or peptides. CgA could be found in ten hormonally identified endocrine cell types and in two or three other endocrine cell types. Entero-endocrine cells containing amines (histamine, serotonin) regularly exhibited CgA-immunoreactivities. In contrast, peptide-containing endocrine cells were largely heterogeneous: Their CgA-immunoreactivities varies among the species, among the gastro-intestinal segments, and even among the members of the same cell population. Hence, seen histochemically, CgA is no universal marker for entero-endocrine cells. Seen biochemically, the observed heterogeneities of CgA-immunoreactivities theoretically can be attributed to various factors (species-specificities of CgA, subclasses of chromogranins, processing of CgA or its pro-protein). Most probably, these heterogeneities are caused by species- or cell-specific differences in the extent of processing of CgA. In addition, some findings point to certain interrelations between the processing or storage of CgA and resident peptides in the secretion granules of enteroendocrine cells.
Histochemistry 02/1989; 92(4):265-75.
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ABSTRACT: Chromogranin A (CgA) and related acidic proteins are widely distributed in the organism. They are also present in entero-endocrine cells and in other members of the paraneuron family. Therefore, CgA has been claimed as an universal marker of this cellular community. To yield precise data about the distribution of CgA in entero-endocrine cells, all segments of the gastro-intestinal tract of five mammalian species (man, cattle, pig, cat, guinea-pig) were investigated immunohistochemically for CgA. In serial semithin plastic sections, all CgA-immunoreactive endocrine cells were identified for resident amines or peptides. CgA could be found in ten hormonally identified endocrine cell types and in two or three other endocrine cell types. Entero-endocrine cells containing amines (histamine, serotonin) regularly exhibited CgA-immunoreactivities. In contrast, peptide-containing endocrine cells were largely heterogeneous: Their CgA-immunoreactivities varied among the species, among the gastro-intestinal segments, and even among the members of the same cell population. Hence, seen histochemically, CgA is no universal marker for entero-endocrine cells. Seen biochemically, the observed heterogeneities of CgA-immunoreactivities theoretically can be attributed to various factors (species-specificities of CgA, subclasses of chromogranins, processing of CgA or its proprotein). Most probably, these heterogeneities are caused by species- or cell-specific differences in the extent of processing of CgA. In addition, some findings point to certain interrelations between the processing or storage of CgA and resisdent peptides in the secretion granules of entero-endocrine cells.
Histochemie 01/1989; 92(4):265-275. · 2.59 Impact Factor
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ABSTRACT: Chromaffin cells of the adrenal medulla synthesize, store and secrete catecholamines. These cells contain numerous electron-dense secretory granules which discharge their contents into the extracellular space by exocytosis. The subplasmalemmal area of the chromaffin cell is characterized by the presence of a highly organized cytoskeletal network. F-Actin seems to be exclusively localized in this area and together with specific actin-binding proteins forms a dense viscoelastic gel; fodrin, vinculin and caldesmon, three actin cross-linking proteins, and gelsolin, an actin-severing protein, are found in this subplasmalemmal region. Since fodrin-, caldesmon- and alpha-actinin-binding sites exist on secretory granule membranes, actin filaments can also link secretory granules. Chromaffin granules can be entrapped in this subplasmalemmal lattice and thus the cytoskeleton acts as a barrier preventing exocytosis. When cells are stimulated, molecular rearrangements of the subplasmalemmal cytoskeleton take place: F-actin depolymerizes and fodrin reorganizes into patches. In addition, introduction of monospecific antifodrin immunoglobulins into digitonin-permeabilized cells blocks exocytosis, demonstrating the crucial role of this actin-binding protein. In bacterial toxin-permeabilized chromaffin cells, experiments using actin-perturbing agents such as cytochalasin D and DNAase I suggest that exocytosis is in part controlled by the cytoskeleton. The intracellular signal governing the cytoskeletal reorganization (associated with exocytosis) is calcium. Calcium inhibits some and activates other actin-binding proteins and consequently causes dissolution of the subplasmalemmal cytoskeleton. This dissolution of cytoskeletal filaments should result in granule detachment and permit granules free access to exocytotic sites on the plasma membrane.
Journal of Experimental Biology 10/1988; 139:253-66. · 3.00 Impact Factor
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ABSTRACT: Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.
European Journal of Cell Biology 07/1988; 46(2):316-26. · 2.81 Impact Factor
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ABSTRACT: Chromogranin A (CGA) is the major protein of the secretory granule from chromaffin cells and also is found in a variety of endocrine cells. Although the sequence of this acidic glycoprotein has been elucidated recently, its biological function is unknown. Here we have purified CGA from chromaffin granules; the final preparation contained the 74-kDa native CGA together with two degradation products--three bands near 60 kDa and a single band of 43 kDa. This preparation was found to inhibit (a maximum inhibition of 60% at 1 microM) the nicotine-induced, but not the high K+-evoked, catecholamine secretion from bovine chromaffin cells maintained in primary culture. Spontaneous release was also affected in the nanomolar CGA protein concentration range. The observation that the inhibitory effect is strictly dependent on a preincubation step together with the modification of the CGA protein profile during this preincubation step suggests that the degradation peptide(s) rather than the 74-kDa native CGA--the approximately equal to 60-kDa bands or the 43-kDa singlet band--is actually involved in secretory cell activity. This was demonstrated by using trypsin-generated peptides that were inhibitory without the preincubation period. The finding that unprocessed CGA is not active on chromaffin cell secretion suggests that this molecule is a precursor of a peptide(s) that is able to regulate catecholamine secretion. Thus, the present data suggest that a CGA-derived peptide(s) could exert a feedback control on chromaffin cell secretory activity--a mechanism that might be of importance during stress situations.
Proceedings of the National Academy of Sciences 04/1988; 85(5):1712-6. · 9.68 Impact Factor
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ABSTRACT: Cultured chromaffin cells can be permeabilized with digitonin; the cell interior is then accessible to the cytoplasm, and addition of calcium provokes release of catecholamines. Increasing the incubation time between the permeabilization step and calcium-induced stimulation resulted in a progressive inhibition of secretion reaching 60% after 20 min. Cytosoluble proteins which leak from detergent-permeabilized cells were collected, dialyzed, and concentrated. When these proteins were added back to permeabilized cells which were unable to secrete, catecholamine release was fully restored, suggesting that certain proteins necessary for exocytosis had been dialyzed from these cells. One of the released proteins was characterized as calmodulin. However, addition of calmodulin alone was ineffective in maintaining or restoring secretory activity in digitonin-permeabilized cells, excluding calmodulin as the sole factor responsible for the loss of release. Protein kinase C was also identified as one of the leaked proteins. This enzyme is known to be retained in cells in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). However, under TPA-dependent conditions, there was also a loss of secretory activity. The present paper shows that among the proteins leaked from digitonin-permeabilized cells, there are specific proteins crucial to the exocytotic mechanism.
Journal of Biological Chemistry 01/1988; 262(34):16671-6. · 4.77 Impact Factor
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ABSTRACT: An ultrastructural examination of cultured bovine chromaffin cells permeabilized with Staphylococcus aureus alpha-toxin or digitonin revealed differences in the preservation of cell morphology. The toxin-treated cells closely resembled control cultured cells whereas digitonin-treated cells showed gradations in cytoplasmic densities suggesting extraction, some swelling of the endoplasmic reticulum and, occasionally, discontinuities in the plasma membrane and free granules in the extracellular medium. In both cell models, there was a swelling of the mitochondria. Horseradish peroxidase labelling of permeabilized cells marked the cytoplasm of digitonin-treated cells but only the surface of toxin-treated cells, demonstrating that larger lesions were caused by digitonin. In stimulated cells, the decrease in volumetric density of chromaffin granules correlated well with catecholamine release. The sites of secretory activity could be demonstrated in toxin-treated cells using horseradish peroxidase as a surface marker. Although both cell systems secrete catecholamines in response to calcium stimulation, their calcium requirements and the kinetics of release were different. In alpha-toxin-treated cells, 100 microM free calcium induced maximal catecholamine release. In digitonin-treated cells, 20 microM evoked maximal release but secretion was blocked at 100 microM. Catecholamine release terminated in digitonin-treated cells within 10 min but continued in alpha-toxin-treated cells for at least 60 min. In addition, the maximal release observed in toxin-treated cells (50%) was always greater than that observed in digitonin-permeabilized cells (20%). The results suggest that both exocytosis and granule translocation are operational in alpha-toxin-treated cells, but that the translocation step or the docking of granules at the plasma membrane may be impaired in digitonin-treated cells.
Neuroscience 01/1988; 23(3):1143-55. · 3.38 Impact Factor
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ABSTRACT: A sensitive and selective radioimmunoassay for chromogranin A (Chrg A) has been developed to quantitate content, release, and biosynthesis of this secretory protein in neuroendocrine tissues. An antiserum raised against Chrg A from bovine adrenal medulla was found to detect predominantly only the Mr 70-75 kilodalton Chrg A in its native form, allowing the use of this antiserum as a quantitatively specific probe for Chrg A in cell-free extracts of the adrenal medulla and chromaffin cells. Chrg A comprises about 10% of the total protein of the chromaffin cell. It is released in parallel with Met-enkephalin and catecholamines from the bovine chromaffin cell in primary culture in response to nicotine and nicotinic cholinergic agonists. From 14 to 22% of total Chrg A is released from the cell during a 15-min exposure to a maximally stimulatory dose of nicotine (10-100 microM). Chrg A release on nicotinic stimulation is blocked by D-600 and hexamethonium to the same extent as Met-enkephalin and catecholamine release. The parallel time course and percent release of Chrg A and Met-enkephalin indicate that these secretory polypeptides are contained in, and released from, functionally identical cellular compartments. Chrg A and Met-enkephalin pentapeptide sequences are present in the chromaffin cell at a ratio of about 2:1, although Chrg A is far more abundant on a mass basis. Chrg A and Met-enkephalin biosynthesis appear to be differentially regulated within the chromaffin cell, since chronic treatment of cells with nicotine and forskolin causes an elevation of Met-enkephalin pentapeptide without a concomitant elevation of intracellular levels of Chrg A.
Journal of Neurochemistry 08/1987; 49(1):65-74. · 4.06 Impact Factor
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ABSTRACT: Chromaffin cells purified from bovine adrenal medulla and maintained in primary culture were used to study the effects of hyperosmolarity on the nicotine- and high potassium-induced secretory response. A similar study was also performed on cells permeabilized with digitonin and with alpha-toxin from Staphylococcus aureus. Hyperosmolarity does not affect the spontaneous release of catecholamines from either intact cells or permeabilized cells. The nicotine-induced secretion and high potassium-induced secretion from intact cells are inhibited by hypertonic solutions; a 100% inhibition of net release was observed at 660 mOsm (sucrose as osmotic agent). Veratridine- and the cation ionophore X537-A-induced release were both depressed under hyperosmotic conditions. Hyperosmolarity was shown to have reversible effects on the secretory response of intact cells. Finally, hyperosmolarity has intracellular effects on catecholamine release evoked by calcium from both detergent- and alpha-toxin-permeabilized cells. Our data show that hyperosmolarity has multiple effects on the cell membrane and the protein constituents associated with it, but has also a significant effect on intracellular reactions concerned with exocytosis.
Biochimica et Biophysica Acta 02/1987; 927(1):18-25. · 4.66 Impact Factor
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ABSTRACT: Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.
Journal of Neurochemistry 02/1987; 48(1):233-5. · 4.06 Impact Factor
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Annals of the New York Academy of Sciences 02/1987; 493:435-47. · 3.15 Impact Factor
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ABSTRACT: Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx. Once calcium enters the cell, calcium acts at pharmacologically distinct sites to elicit secretion and enhanced biosynthesis of Metenkephalin. This is demonstrated by the calcium-independent stimulation of enkephalin secretion by 1 mM barium, in contrast to the dependence on extracellular calcium of barium-stimulated biosynthesis of this peptide. The synthesis and secretion of VIP is also coupled to acetylcholine and elevated potassium stimulation by calcium influx. Treatment with barium demonstrates that calcium acts at distinct sites to stimulate secretion and biosynthesis of this peptide; however induction of VIP by barium and veratridine shows greater sensitivity to the calcium channel blocker methoxyverapamil (D600) than does the induction of Met-enkephalin by these agents. These differences in D600 sensitivity may be due to differences in calcium metabolism or voltage-dependent calcium channels in enkephalin-producing and VIP-inducible subpopulations of chromaffin cells. Chromogranin A levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels, although it is secreted in parallel with enkephalins and catecholamines from chromaffin cells in response to secretagogues. We suggest that peptide hormones such as VIP and enkephalins are regulated by calcium-dependent stimulus-secretion-synthesis coupling in the chromaffin cell. Cyclic AMP is a positive regulator of enkephalin and VIP biosynthesis, but does not affect acute release of these peptides. The cAMP/protein kinase A system may be a distal mediator of peptide biosynthesis stimulated by secretagogues. Alternatively, cAMP may be involved in early developmental establishment of phenotype or long-term regulation of peptide biosynthesis by other hormones or neurotransmitters. Chromogranin A may represent a class of intravesicular, soluble proteins that are expressed constitutively by the chromaffin cell in the presence or absence of positive regulators of other systems. The biosynthesis of chromogranin A may be coupled to the production or assembly of the secretory vesicle itself.
Annals of the New York Academy of Sciences 02/1987; 493:308-23. · 3.15 Impact Factor
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ABSTRACT: Chromogranin A (CGA) is the major soluble protein within secretory vesicles of chromaffin cells. A polyclonal antiserum was raised against bovine CGA and characterized in two-dimensional immunoblots. Cellular and subcellular distribution of CGA in bovine pancreatic islet was investigated by immunocytochemistry. At the light microscopic level, CGA-like immunoreactivity was found in the same cells that react with antibodies against insulin, glucagon, and somatostatin. A minority of cells containing pancreatic polypeptide also showed faint immunostaining. At the ultrastructural level (protein A-gold technique), CGA-like immunoreactivity was confined exclusively to the secretory vesicles. Whereas the hormones were localized mainly in the central part of the secretory vesicles, CGA was present predominantly in the periphery. These findings indicate that a CGA-like protein is a regular constituent of the matrix of secretory vesicles in pancreatic endocrine cells.
Journal of Histochemistry and Cytochemistry 01/1987; 34(12):1673-82. · 2.72 Impact Factor
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ABSTRACT: Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.
Journal of Biological Chemistry 06/1986; 261(13):5777-83. · 4.77 Impact Factor
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ABSTRACT: Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.
The Journal of Cell Biology 03/1986; 102(2):636-46. · 10.26 Impact Factor
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ABSTRACT: Cytoskeleton proteins (actin, myosin, alpha-actinin, spectrin, tubulin, neurofilament subunits) and their regulatory proteins (calmodulin, gelsolin) have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localizations have been revealed by biochemical, immunocytochemical, and ultrastructural techniques. alpha-Actinin and spectrin are components of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules, but is present mainly in the cytosol and close to the cell surface. Trifluoperazine, a calmodulin antagonists, blocks stimulation-induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin-binding sites have also been found in chromaffin granule membranes and their calmodulin-binding proteins have been characterized. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions are discussed.
Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire 07/1985; 63(6):661-79.
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ABSTRACT: The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.
Journal of Neurochemistry 03/1985; 44(2):526-39. · 4.06 Impact Factor
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ABSTRACT: Bovine adrenal medullae were homogenized in the presence or in the absence of EGTA and different subcellular fractions were prepared by differential and density gradient centrifugations. In the presence of the chelating agent, 69% of the total calmodulin, measured by radioimmunoassay, was present in the cytosol; the rest was bound to different membrane-containing fractions (nuclei, microsomal, and crude granule fraction). When the chelating agent was omitted, 43% of the calmodulin was present in the cytosol, the remaining calmodulin being membrane-bound. Further resolution of the crude granule fraction by sucrose density centrifugation demonstrated that the distribution of calmodulin in the density gradient was similar to the distribution of chromaffin granules rather than to that of mitochondria, Golgi elements, and lysosomes. In this case, there was also more calmodulin bound to chromaffin granules when EGTA was omitted from the density gradient. Experiments with 125I-calmodulin indicated the presence of high-affinity binding sites (KD = 1.3 X 10(-8) M; Bmax = 30 pmol/mg protein) for calmodulin in chromaffin granule membranes. Further, photoaffinity crosslinking experiments with 125I-calmodulin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography indicated the presence of three calmodulin-binding polypeptide complexes (84,000; 41,000; and 38,000 daltons) in chromaffin granule membranes. These polypeptides were not labelled when either Ca2+ was omitted or an excess of nonradioactive calmodulin was present in the photolysis buffer, indicating the Ca2+ dependency and the specificity of the interaction. On the basis of the results described, it is suggested that the cellular levels of Ca2+ control the cellular distribution of calmodulin and its binding to specific chromaffin granule membrane proteins. Further, it is also suggested that the interactions between calmodulin and granule proteins might play a role in stimulus-secretion coupling.
Journal of Neurochemistry 11/1984; 43(4):1087-97. · 4.06 Impact Factor
