Search PubMed⌕ Search

Biomedical subjects

D Aunis

Publications and source records attributed to D Aunis.

At least 145 records · Page 8Linked to original sources
PubMed1989

A reassessment of guanine nucleotide effects on catecholamine secretion from permeabilized adrenal chromaffin cells.

The role of guanine nucleotides in catecholamine secretion was investigated in alpha-toxin-permeabilized chromaffin cells. The stable GTP analogues, GTP-gamma-S (guanosine 5'-(gamma-thio)triphosphate) and GMP-PNP (guanosine 5'-(beta,gamma-imido)triphosphate), potentiated calcium-evoked catecholamine release in a dose-dependent manner. This effect was reversed by GDP-beta-S (guanosine 5'-(beta-thio)diphosphate) indicating that a GTP-binding protein plays a modulatory role in the calcium-dependent secretory process in chromaffin cells. Calcium and the phosphorylating nucleotide ATP were both necessary for secretion, even in the presence of GTP analogues, suggesting that the activation of a GTP-regulatory protein alone does not trigger exocytosis in these cells. TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C, was found to mimic the effects of the GTP analogues, inducing a dose-dependent potentiation of the calcium-evoked release in alpha-toxin-permeabilized cells. Treatment of the permeabilized cells with sphingosine, a potent inhibitor of protein kinase C, completely abolished the stimulatory effects of both TPA and GTP-gamma-S. Moreover, long term incubation of chromaffin cells with TPA, a treatment which depletes cells of protein kinase C activity, suppressed the stimulatory effects of GTP-gamma-S. Protein kinase C is activated when it becomes membrane-bound in the presence of calcium and diacylglycerol; here, GTP-gamma-S was found to enhance the calcium-induced translocation of protein kinase C to membranes in alpha-toxin-permeabilized cells. These results suggest that guanine nucleotides modulate secretion by activating protein kinase C-linked events in chromaffin cells. Furthermore, the potentiation of calcium-induced secretion in alpha-toxin-permeabilized cells following activation of protein kinase C either directly with TPA or indirectly with GTP analogues provides additional support for the concept that protein kinase C may exert a positive control directly on the intracellular exocytotic machinery.

Adenosine Triphosphate↗
PubMed1989

gamma-Aminobutyric acid uptake and localization in bovine chromaffin cells in primary culture.

gamma-Aminobutyric acid (GABA) uptake was studied in bovine chromaffin cells maintained in primary culture. Uptake was found to be dependent on Na+, but not on K+ and Ca2+ ions; it was found that 2 Na+ ions were necessary for each molecule of GABA transported. 2,4-Dinitrophenol, ouabain and vanadate inhibited GABA uptake showing the energy dependency of the system. Two affinity sites were demonstrated, a high affinity site and a low affinity site with Km values of 10 microM and 170 microM, respectively. While the low affinity site did not show large variations with culture age, the Km of the high affinity site increased from 1 microM in freshly isolated cells to 10 microM in 3-9 day-old cells. GABA uptake was unaffected by glutamic acid, aspartic acid, glycine and catecholamines, while taurine, beta-alanine, nipecotic acid and L-2,4 diaminobutyric acid inhibited GABA uptake. Nipecotic acid and L-2,4 diaminobutyric acid acted as competitive inhibitors modifying Km values of the high affinity site. Subcellular studies performed on [3H]GABA-loaded chromaffin cells showed that GABA was not in secretory granules but was recovered in the 100,000 g soluble fraction. The GABA uptake process associated with chromaffin cells may be an important mechanism for regulating the modulation of catecholamine secretion. In addition, the presence of GABA in the cytosol indicates that this molecule may be an effector of chromaffin cell activity in addition to modulating catecholamine secretion.

Adrenal Medulla↗
PubMed1989

Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C.

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.

Adenylyl Cyclases↗
PubMed1989

Chromogranin A (CgA) in the gastro-entero-pancreatic (GEP) endocrine system. II. CgA in mammalian entero-endocrine cells.

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.

Animals↗
PubMed1989

Mechanism of action of Clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells.

Toxin B, an exotoxin produced by Clostridium difficile, induces the rounding-up and arborization of cultured mammalian cells, a typical effect which resembles that provoked by cytochalasins. In this study, the effect of toxin B was examined on astroglial cells grown in primary culture. A specific antiserum to toxin B was used to investigate its mechanisms of action. We found that the toxin exerts its effects on cell morphology after its incorporation into cells. The internalization of toxin B requires the presence of calcium ions in the extracellular medium. Replacement of NaCl with sucrose or with potassium glutamate prevents the internalization of the toxin. The direct introduction of calcium ions into cells by the calcium ionophore A23187 stimulates toxin-induced morphological changes. In contrast, toxin-induced morphological transformations were prevented in cells treated with tumor-promoting phorbol. esters or with dibutyryl-cAMP, although such treatment did not abolish the internalization of the toxin. As in the other cell types, the earliest effect of toxin B on astrocyte cytoskeleton is the disruption of actin filaments, without no visible alteration of intermediate filament nor microtubule networks. As astrocytes with toxin-induced stellate morphology survive toxin treatment, the progression of cell morphology and cytoskeleton organization were followed for several weeks. Twenty-six days after exposure to toxin B, stellate astrocytes have processes which were markedly longer and much more branched than those of cells freshly exposed to toxin. At that time, cells are still devoid of F-actin as assessed with rhodamine-conjugated phalloidin and only 70% contain vimentin while all astrocytes present in control cultures express vimentin. Some flat epithelioid astrocytes with prominent bundles of microfilaments reappear during the second week after toxin treatment. Our results show that Clostridium difficile toxin B is internalized into brain astrocytes in culture where it acts by modifying cytoskeletal elements. Its cytopathic effects are reversible. Although actin-related components of the cytoskeleton are the major target of toxin B, other cytoskeletal elements also seem to be affected.

Animals↗
PubMed1988

Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.

Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms.

Animals↗
PubMed1988

Alpha-fodrin (brain spectrin) immunocytochemical localization in rat vestibular hair cells.

The presence of a spectrin-related protein in rat vestibular sensory receptors was demonstrated by immunocytochemistry and immunoblotting using affinity purified anti alpha-fodrin antibodies. Intense immunoreactivity was found in the apical pole of sensory hair cells where it seems to be concentrated in the cuticular plate. In contrast, alpha-fodrin immunoreactivity was absent from the stereocilia. We suggest that a spectrin-related protein participates in the organization of the cuticular plate of vestibular hair cells by cross-linking actin filaments as well as by anchoring the cuticular plate to the apical cell membrane.

Animals↗
PubMed1988

Acquisition of vimentin in astrocytes cultured from postnatal rat brain.

Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin-GFAP transition which occurs during glial development in situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.

Animals↗
PubMed1988

Induction of neurofilament phosphorylation in cultured chromaffin cells.

The distribution, structural organization and state of phosphorylation of neurofilaments have been examined in chromaffin cells from adult bovine adrenal medulla cultured under various conditions using a series of monoclonal antibodies directed against phosphorylated and nonphosphorylated epitopes of the 200,000 mol. wt subunit. Nonphosphorylated neurofilament epitopes were detected immunocytochemically to varying extents in chromaffin cells maintained under standard culture conditions for up to 3 weeks. Staining was usually limited to a perinuclear region from which fine filaments sometimes appeared to radiate around the nucleus. In marked contrast, none of the antibodies directed against phosphorylated neurofilament epitopes stained these structures. When cells were cultured under conditions favouring neurite outgrowth, in conditioned medium derived from intermediate lobe cultures, there was a more extensive expression of the nonphosphorylated neurofilament epitopes. In addition, phosphorylation of neurofilaments was induced. The phosphorylated neurofilament epitopes were restricted to the neurite, whereas the nonphosphorylated neurofilament epitopes were localized in both neurite extensions and perikarya. These results demonstrate that conditioned medium from intermediate lobe cells of the hypophysis not only provokes neurite outgrowth from chromaffin cells, but also supports neuronal maturation as demonstrated by the phosphorylation of neurofilaments in neurites.

Adrenal Medulla↗
PubMed1988

Secretion from chromaffin cells is controlled by chromogranin A-derived peptides.

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.

Adrenal Medulla↗
PubMed1988

The cytoskeleton as a barrier to exocytosis in secretory cells.

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.

Animals↗
PubMed1988

Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells.

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.

Actins↗
PubMed1987

Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis.

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.

Adrenal Medulla↗
PubMed1987

Presence of three pertussis toxin substrates and Go alpha immunoreactivity in both plasma and granule membranes of chromaffin cells.

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Gi alpha. Significant immunoreactivity with anti-Go alpha antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

Adenosine Diphosphate Ribose↗
PubMed1987

Influence of hypertonic solutions on catecholamine release from intact and permeabilized cultured chromaffin cells.

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.

Adrenal Medulla↗
PubMed1987

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes.

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.

Adrenal Gland Neoplasms↗
PubMed1987

Membrane recycling after exocytosis: an ultrastructural study of cultured chromaffin cells.

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.

Adrenal Medulla↗