Biomedical subjects
Publications and source records attributed to D Aunis.
Immunocytochemical staining with anti-tubulin antibodies showed cold-stable microtubules in astrocytes in culture. On rewarming, microtubule-organizing centre (MTOC) could be visualized. MTOCs showed structural complexity, from 2-3 fibres to a mass of interweaving fibres. Only one MTOC per cell was found in cultures of both polygonal and stellate-bearing cells.
Vimentin and glial fibrillary acidic (GFA) protein in rat astrocytes in primary culture were identified with their respective specific antisera on electroblots of one-dimensional and two-dimensional polyacrylamide gels. Each antiserum revealed respectively vimentin (58 kDa, pI 5.3), GFA protein (50 kDa, pI 6-5.8) and several more acidic, lower molecular mass vimentin-immunoreactive and GFA protein-immunoreactive polypeptides. These were more apparent when astrocyte proteins were extracted in the absence of EGTA and in the presence of Ca2+ ions suggesting that the lower immunoreactive polypeptides result from a Ca2+-sensitive proteolytic degradation of vimentin and GFA protein. The extent of proteolysis of GFA protein was higher when the differentiation of astrocytes was induced with 1,N6-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP). In contrast, the proteolysis of vimentin was not affected by the state of differentiation of astrocytes. The results indicate the existence of at least two distinct degradative pathways for intermediate filament proteins in astrocytes in primary culture and suggest that in differentiated astrocytes the activity of proteinase(s) involved in the degradation of GFA protein increases.
The ultrastructural localization of the glycoprotein D2 in rat adrenal gland was investigated using immunohistochemical methods, and D2 localization in cultures of adult bovine chromaffin cells was studied by immunofluorescence. D2 was found to be situated on nerve fibers passing through the adrenal cortex and in the medulla zone, and also on the surface of all chromaffin cells. In addition, it was strongly expressed on the surface of glial (Schwann) cells. Cortical cells were unreactive to the antiserum. In cultures, all adrenalin and noradrenalin [dopamine-beta-hydroxylase (DBH)-positive] cells were surface labelled for D2. A less frequent second cell type was recognized in vitro which was DBH negative but D2 positive. Such cells were presumed to be Schwann cells. These data are discussed in terms of the developmental origin of the cells and with regard to the putative functional rôle of D2 in cell adhesion phenomena.
The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F-actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F-actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F-actin. In addition, vasopressin-containing granules from neurohypophysial tissue were shown to bind F-actin and to increase the viscosity of F-actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin-like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane-associated spectrin-like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD ), the alpha-subunit (240 kD, pHi5 .5) being recognized by the antibody. Nonionic detergents such as Triton X-100 or Nonidet P40 failed to release fully active spectrin-like protein. In contrast, Kyro EOB , a different nonionic detergent, was found to release spectrin-like protein while keeping intact F-actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin-like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F-actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles.
Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.
When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.
After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.
Cytochrome b-561 in chromaffin granules interacts with antimycin and its alpha-peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b-561 in secretory granules other than its accredited energy conserving electron transport property is projected.
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We describe the interaction of pure brain tubulin with purified membranes specialized in different cell functions, i.e., plasma membranes and mitochondrial membranes from liver and secretory granule membranes from adrenal medulla. We studied the tubulin-binding activity of cellular membranes using a radiolabeled ligand-receptor assay and an antibody retention assay. The tubulin-membrane interaction was time- and temperature-dependent, reversible, specific, and saturable. The binding of tubulin to membranes appears to be specific since acidic proteins such as serum albumin or actin did not interfere in the binding process. The apparent overall affinity constant of the tubulin-membrane interaction ranged between 1.5 and 3.0 X 10(7) M-1; similar values were obtained for the three types of membranes. Tubulin bound to membranes was not entrapped into vesicles since it reacted quantitatively with antitubulin antibodies. At saturation of the tubulin-binding sites, the amount of reversibly bound tubulin represents 5-10% by weight of membrane protein (0.4-0.9 nmol tubulin/mg membrane protein). The high tubulin-binding capacity of membranes seems to be inconsistent with a 1:1 stoichiometry between tubulin and a membrane component but could be relevant to a kind of tubulin assembly. Indeed, tubulin-membrane interaction had some properties in common with microtubule formation: (a) the association of tubulin to membranes increased with the temperature, whereas the dissociation of tubulin-membrane complexes increased by decreasing temperature; (b) the binding of tubulin to membranes was prevented by phosphate buffer. However, the tubulin-membrane interaction differed from tubulin polymerization in several aspects: (a) it occurred at concentrations far below the critical concentration for polymerization; (b) it was not inhibited at low ionic strength and (c) it was colchicine-insensitive. Plasma membranes, mitochondrial membranes, and secretory granule membranes contained tubulin as an integral component. This was demonstrated on intact membrane and on Nonidet P-40 solubilized membrane protein using antitubulin antibodies in antibody retention and radioimmune assays. Membrane tubulin content varied from 2.2 to 4.4 micrograms/mg protein. The involvement of membrane tubulin in tubulin-membrane interactions remains questionable since erythrocyte membranes devoid of membrane tubulin exhibited a low (one-tenth of that of rat liver plasma membranes) but significant tubulin-binding activity. These results show that membranes specialized in different cell functions possess high-affinity, large-capacity tubulin-binding sites...
Microtubules and microfilament patterns in cultured astrocytes were revealed by using indirect immunofluorescent microscopy in conjunction with anti-tubulin immune serum and anti-actin immunoglobulins respectively. In flat epitheloid astroglial cells (either polygonal or elongated) colchicine-sensitive immunofluorescent fibres, which correspond to bundles of microtubules, extend from the perinuclear cytoplasm into the cell periphery by running for long distances through the different focal planes. These patterns of organization differ markedly from the patterns of organization of microfilaments which are arranged in fibres parallel to each other and often oriented along the cell boundary. In response to the combined treatments of serum withdrawal and administration of dBcAMP, flat epitheloid astrocytes adopt a morphology similar to that of the mature astrocytes in situ in the CNS, that is of stellate process-bearing cells. This is prevented or is reverted by the administration of colchicine at the appropriate times. There are strong suggestions indicating that during cell processes formation the microtubular network is reorganized and microtubules assembled into dense bundles which are oriented along the axis of the cell processes. In view of these results, we suggest that, in contrast to microfilaments, microtubules are not determinant for the maintenance of cellular shape in elongated or polygonal flat epitheloid astroglial cells but they are required for both the formation and maintenance of processes in stellate astrocytes.
Actin antibodies were purified by affinity chromatography from the serum of rabbits immunized with actin isolated from bovine skeletal myofibrils. By the indirect immunofluorescence technique the pattern of organization of actin was studied in cultured astroglial cells prepared from cerebral hemispheres of newborn rats. Labelling with monospecifc actin antibody revealed that the flat epitheloid astrocytes contained bundles of actin fibres arranged in characteristic patterns. Actin fibres disaggregated in round cells produced by trypsinization and reorganized when cells returned to their elongate or polygonal form. The retraction of cell bodies produced by treatment with cytochalasin B was associated with the disappearance of actin filament bundles. Reversion to the former flattened morphology and organization of fibres occurred after removal of the drug. In response to either dibutyryl adenosine 3'5'-monophosphate, 3-isobutyl-l-methylxanthine, rat brain extract or to lesser extent to norepinephrine, flat epitheloid cells took on a stellate appearance with extensive processes, resembling more closely mature astrocytes. The conversion of flat epitheloid cells into stellate cells was associated with the disappearance of immunofluorescent fibres and the appearance of diffuse immunofluorescence indicating the structural reorganization of actin from a highly organized fibrillar state into a loosely organized actin network. It is concluded that the respect to actin patterns, the behaviour of flat epithelioid astrocytes in culture does not differ from that of culture non-muscle cells of mesenchymal origin. The pattern of organization of actin in stellate process-bearing astrocytes is compatible with the involvement of actin in typical astroglial motility and in establishment of characteristic stellate shape.
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Immunofluorescence studies of bovine chromaffin cells in culture with specific antibodies against dopamine-beta-hydroxylase gave a distinct punctate pattern of labelling, reflecting the distribution of chromaffin granules. There was strong staining of cell extensions and growth ones. Linear arrays of fluorescent dots were observed, suggesting an association of granules with a filamentous cytoskeleton. Labelling of neuritic processes was periodic, perhaps indicative of a packaging of secretory granules. Chromaffin cells stained strongly with specific anti-actin antisera. Fine filament bundles were observed, and also diffuse staining, some punctate labelling and staining of the plasma membrane or sub-membranous cytoplasm. Growth cones and non-terminal cytoplasmic varicosities contained significant amounts of actin. Colchicine (5 X 10(-5) M) caused retraction of neuritic extensions and formation of lateral growth cones. Cytochalasin (10 microgram/ml) caused ballooning of terminal growth cones and non-terminal cytoplasmic varicosities. Phalloidin (10(-4) M) stimulated microspike formation. The results are discussed in terms of the role of the cytoskeleton in growth cone formation, cell-substratum contacts and the transport of chromaffin granules.
Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.
1 Secretion of catecholamines (CA) evoked by ouabain, chlormadinone acetate (CMA), phenoxybenzamine (Pbz) and vanadate, four agents known to inhibit Na(+), K(+)-dependent Mg(2+)-activated adenosine triphosphatase (ATPase) activity has been studied in suspensions of bovine isolated adrenal medullary cells.2 Acetylcholine (ACh) evoked a 5 fold increase of the basal CA secretion from isolated cells suspended in oxygenated Krebs-bicarbonate solution kept at 27 degrees C. Secretion was antagonized by Ca(2+)-deprivation or hexamethonium, indicating good functional viability of the cells.3 Ouabain (10(-7) to 10(-4) M) evoked a progressive, dose-dependent release of CA from cell suspensions. Study of the time course of the secretory response for 2 h allowed the separation of two components in the secretory response at all doses studied: a slow initial component (0.011 pg/min CA) and a second faster component (0.032 pg/min CA).4 CMA evoked a clear-cut CA secretory response. The ED(50) for CMA was 10(-4) M, as compared to 3 x 10(-6) M for ouabain. Pbz and vanadate did not induce CA release.5 [(3)H]-ouabain was taken up and bound to intact isolated cells by a non-saturable binding process. However, in semi-purified plasma membranes from bovine adrenal medulla a saturable specific [(3)H]-ouabain binding process was observed with a K(D) of 8.1 nM. Binding to the membranes was ATP-dependent and antagonized by K(+).6 [(3)H]-ouabain specific binding to membranes was antagonized by ouabain and CMA, but not by Pbz or vanadate; the ID(50) for ouabain and CMA were 10(-6) and 10(-5) M respectively.7 Ouabain partially inhibited, in a dose-dependent manner, Na(+), K(+)-Mg(2+) ATPase activity of the semi-purified plasma membranes.8 The results demonstrate a good correlation between the ability of different drugs, known to inhibit ATPase activity, to displace [(3)H]-ouabain binding to adreno-medullary plasma membranes and their capacity to evoke a CA secretory response from isolated chromaffin cells. The data also suggest that the CA secretory effects of ouabain may not be due simply to inhibition of the Na(+) pump and the subsequent ionic redistribution across the plasma membrane; a second mechanism may also be involved.
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