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D Aunis

Publications and source records attributed to D Aunis.

At least 127 records · Page 7Linked to original sources
PubMed1991

Depolarization produces an acidification of adrenal gland perfusates.

Stimulation of adrenal glands with a variety of agonists or high potassium produced an acidification of the perfusion medium. The magnitude of the transient pH decrease was similar to that found in other nervous structures, and depended on the buffering capacity of the perfusion medium. However, no alkaline transient could be detected in this tissue. This acidification required Ca2+ and occurred under conditions producing catecholamine release. Since firstly the acidification could not be directly correlated with the amount of catecholamine released and secondly the time-course of both phenomena was different, suggesting that the acidification did not only result from the exocytosis of the acidic content of the chromaffin vesicles. The extracellular acidification may in addition originate: (a) partly from the proton release from acidic proteins, (b) from an output of acid equivalents from the cytoplasmic medium, either by the Na+/H+ antiporter present in all animal cells [24], or another mechanism yet to be determined.

Acetylcholine↗
PubMed1991

Dystrophin in central nervous system: a developmental, regional distribution and subcellular localization study.

Dystrophin, the protein encoded by the Duchenne muscular dystrophy gene has been shown to be expressed in central nervous system. In the present study, polyclonal antibodies raised against 3 fusion proteins constructed from different structural domains of dystrophin were used to identify dystrophin in protein extracts from rat and mdx mouse brain. The developmental expression of the protein, its regional distribution in rat brain and its localization in rat brain subcellular fractions were also examined. We found that dystrophin or a 'dystrophin-related protein' is expressed in mdx mouse brain. Dystrophin is detectable at very early stages of rat brain development and is expressed in all adult brain regions examined, although quantitative regional differences were found. Subcellular distribution analysis indicates that dystrophin is absent in mitochondrial and synaptic vesicle-enriched fractions but is recovered in the synaptic plasma membrane fraction.

Animals↗
PubMed1991

A pertussis-toxin-sensitive protein controls exocytosis in chromaffin cells at a step distal to the generation of second messengers.

The role of GTP-binding proteins (G-proteins) in the secretory process in chromaffin cells was investigated by studying the effects of pertussis toxin (PTX) on catecholamine release and generation of various second messengers. PTX was found to stimulate the catecholamine secretion induced by nicotine, 59 mM-K+ or veratridine. PTX also potentiated Ca2(+)-evoked catecholamine release from permeabilized chromaffin cells, suggesting that PTX substrate(s) regulate the exocytotic machinery at a step distal to the rise in intracellular Ca2+. We have investigated the possible intracellular pathways involved in the stimulation of secretion by PTX. PTX did not modify the translocation of protein kinase C (PKC) to membranes in intact or permeabilized cells; in addition, neither inhibitors nor activators of PKC had any effect on catecholamine release induced by PTX. Thus it seems unlikely that the effect of PTX on secretion is mediated by activation of PKC. The effect of PTX is also cyclic AMP-independent, as PTX did not change cytoplasmic cyclic AMP levels. The relationship between PTX treatment and arachidonic acid release was also examined. We found that an increase in cytoplasmic arachidonic acid concentration enhanced Ca2(+)-evoked catecholamine release in permeabilized cells, but arachidonic acid did not mimic the effect of PTX on the Ca2(+)-dose-response curve for secretion. Furthermore, PTX did not significantly modify the release of arachidonic acid measured in resting or stimulated chromaffin cells, suggesting that the stimulatory effect of PTX on secretion is not mediated by an activation of phospholipase A2. Taken together, these results suggest that PTX may modulate the intracellular machinery of secretion at a step distal to the generation of second messengers. In alpha-toxin-permeabilized cells, full retention of the PTX-induced activation of secretion was observed even 30 min after permeabilization. In contrast, when chromaffin cells were permeabilized with streptolysin-O (SLO), there was a marked progressive loss of the PTX effect. We found that SLO caused the rapid leakage of three G-protein alpha-subunits which are specifically ADP-ribosylated by PTX. We propose that a PTX-sensitive G-protein may play an inhibitory role in the final stages of the Ca2(+)-evoked secretory process in chromaffin cells.

Adenosine Diphosphate Ribose↗
PubMed1991

Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion.

Chromogranin A (CGA) is a ubiquitous 48-kDa secretory protein present in adrenal medulla, anterior pituitary, central and peripheral nervous system, endocrine gut, thyroid, parathyroid, and endocrine pancreas. Recently, we have demonstrated that the protein could be a precursor of bioactive peptides capable of modulating catecholamine secretion from cultured adrenal medullary chromaffin cells. Here we cleaved CGA purified from bovine chromaffin granules with endoproteinase Lys-C, and we isolated and partially sequenced the peptide inhibiting catecholamine secretion from cultured chromaffin cells. A corresponding synthetic peptide composed of the first 20 N-terminal amino acids produced a dose-dependent inhibition in the 10(-9) to 10(-6) M range (with an ID50 of 5 nM) of the catecholamine secretion evoked by carbamoylcholine or by potassium at a depolarizing concentration. This peptide affected secretagogue-induced calcium fluxes but did not alter sodium fluxes. It was found to increase desensitization of cell responses and to modify the kinetics of catecholamine release. Our results indicate that the peptide is extracellularly generated from CGA by a calcium-dependent proteolytic mechanism. We suggest that this peptide, named chromostatin, may be an endocrine modulator of catecholamine-associated responses.

Adrenal Medulla↗
PubMed1991

Protein kinase C-induced redistribution of the cytoskeleton and phosphorylation of vimentin in cultured brain macrophages.

The phorbol ester 12-O-tetradecanoyl-acetate (TPA) induced prominent and transient changes in the organization of the cytoskeleton in cultured amoeboid microglial cells including redistribution of actin toward the center of the cells and in the subplasmalemmal region, appearance of fine actin filaments, retraction of microtubules (MT), and rearrangement of intermediate filaments (IF) containing vimentin. The possible implication of protein kinase C (PKC) in mediating the effects of TPA was suggested by a parallel shift of PKC activity from the soluble to membrane fractions and phosphorylation of several microglial proteins. The rearrangement of IF closely correlated with increased vimentin phosphorylation, detected by pulse labeling of intact cells. Two monoclonal antivimentin antibodies, B3 and V9, showed different staining patterns. Immunoreactivity with the antibody B3 was more restricted and could be abolished by treatment of fixed, permeabilized cells with alkaline phosphatase, thus suggesting that the antibody reacts with a phosphorylated epitope. Using this antibody, rearrangement of IF involving vimentin phosphorylation was detected within 15 to 60 min of treatment with 50 nM TPA and consisted in the appearance of intense perinuclear fluorescent label. This perinuclear fluorescence persisted up to 24 hr after TPA removal and gradually diminished during the following 2 to 3 days. Immunochemical analysis of nonionic detergent-soluble and -insoluble extracts from untreated and TPA-treated cells revealed no differences in vimentin solubility suggesting that TPA induced vimentin phosphorylation does not result in notable vimentin filament disassembly. However the extent of vimentin degradation was more prominent in TPA-treated cultures indicating a higher sensitivity of vimentin to proteolytic degradation. The data show that PKC-mediated phosphorylation of vimentin results in precise spatial and temporal rearrangement of IF which are not associated with altered vimentin solubility, but possibly changes the mechanical properties and interactions of vimentin filaments.

Actins↗
PubMed1991

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C.

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.

Adenosine Triphosphate↗
PubMed1991

Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.

Actin Cytoskeleton↗
PubMed1991

[Chromogranin A. Prohormonal function and synthesis regulation in neuroendocrine cells].

Chromogranins, which were originally found in adrenal medullary chromaffin cells, are a family of proteins exclusively localized in secretory granules of endocrine cells and neurons. Studies on primary structure have shown the presence of basic amino acid pairs which are putative cleavage sites. Recently, two chromogranin A-derived peptides, pancreastatin and chromostatin, have been characterized which supports the assumption of chromogranin A to be a prohormone. These two peptides have autocrine and paracrine biological functions. Mechanisms which regulate chromogranin synthesis appear to be highly complex depending on the stimulated receptor and involving protein kinase C and cyclic AMP. The promoter region of the chromogranin A gene possesses numerous consensus transcriptional control elements (TATA box, cyclic AMP responsive element, SP1 site, phorbol ester regulatory element, oestrogen regulatory element,...), showing the complexity of the mechanisms regulating the expression of this gene, which is tissue- and neuroendocrine cell-specific.

Animals↗
PubMed1990

Effect of tuamine, heptaminol and two analogues on uptake and release of catecholamines in cultured chromaffin cells.

The effects of tuamine (1-methylhexylamine), a sympathomimetic compound with hypertensive properties, heptaminol (6-amino-2-methyl-2-heptanol), an aliphatic amine with pressor properties, and two structural analogues of tuamine on high-affinity Na(+)-dependent noradrenaline uptake and on nicotine-evoked release were examined in bovine chromaffin cells maintained in primary culture for 3 to 6 days. Tuamine was found to be a potent competitive inhibitor of noradrenaline uptake with an effect similar to that of cocaine. Its inhibition constant (Ki) was 1.1 +/- 0.1 microM while Ki values of heptaminol, of 1-methylamino-5-pentanol oxalate and of 5-amino-2-methylhexanol oxalate, which were also found to be competitive inhibitors of noradrenaline uptake, were 60 +/- 2 microM, 260 +/- 28 microM and 48 +/- 76 microM, respectively. Tuamine, hepataminol and 5-amino-2-methyl-2-hexanol were also shown to be inhibitors of nicotine-induced release of catecholamines, with IC50 values of 26 +/- 2 microM, 650 +/- 11 microM and 500 +/- 10 microM, respectively. Tuamine and hepataminol did not inhibit noradrenaline release evoked by 59 mM K+, suggesting that it acts at a step prior to calcium entry. The pharmacological properties of heptaminol as an anti-hypotension agent may partially account for its inhibitory effect on catecholamine uptake and release.

Amines↗
PubMed1990

Subcellular distribution of acetylcholinesterase forms in chromaffin cells. Do chromaffin granules contain a specific secretory acetylcholinesterase?

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (Ga1, Ga2 and Ga4), and one non-amphiphilic form (Gna4). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N-linked and O-linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol-specific phospholipases C converted the Ga2 form, but not the other amphiphilic forms, into non-amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. Gna4 was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine-binding sites and Gna4 AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of Gna4 during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.

Acetylcholinesterase↗
PubMed1990

Proteolytic processing of chromogranin A in cultured chromaffin cells.

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.

Animals↗
PubMed1990

Importance of catecholamine release for the functional action of intrastriatal implants of adrenal medullary cells: pharmacological analysis and in vivo electrochemistry.

The aim of the present experiments was to test whether adrenal chromaffin cells implanted into the striatum of rats could exert a functional effect through a release of catecholamines. A cell suspension obtained from bovine adrenal medulla was implanted unilaterally into the striatum. The striatal dopaminergic input was extensively destroyed beforehand to preclude the possibility of reinnervation of the striatum by endogenous dopaminergic neurons. The functional influence of the implant was tested through the measurement of drug-induced rotation, while catecholamine release was measured subsequently in the same animals by in vivo electrochemistry. Transplant survival, as shown by the immunohistochemical analysis performed at the end of the in vivo experiments, was highly variable. Surviving chromaffin cells maintained their endocrine morphology and no reinnervation of the host striatum could be detected. Rotation of the animals evoked by apomorphine (0.1 mg/kg, sc) or amphetamine (5.0 mg/kg, ip) following the lesion was left uninfluenced following transplantation, even when a large transplant was recovered. On the other hand, nicotine (0.5 mg/kg, sc) evoked a strong contraversive rotational response in the transplant-bearing animals. This response could not be ascribed to the central effect of substances released peripherally and entering the nervous system through the blood-brain barrier opened by the implantation procedure, as it could not be found in animals bearing implants of other peripheral endocrine tissue, viz, pituitary. The effect of nicotine was not blocked by the pretreatment of the animals with either the opiate antagonist naloxone (2.5 mg/kg, 10 min) or the dopamine receptor blocker pimozide (0.5 mg/kg, 1 h), although the latter pretreatment blocked the amphetamine-evoked rotation. No spontaneous catecholamine release could be detected from the implanted chromaffin cells by in vivo electrochemistry, while treatment with amphetamine or nicotine did evoke a release. The results suggest that the functional effects of such intrastriatal grafts of chromaffin cells, reported in previous studies, cannot be explained by the secretion from the grafted cells of catecholamines into the denervated striatum. On the other hand the results obtained following the pharmacological stimulation of these cells indicate that adrenal grafts can, under suitable conditions, influence the functioning of the host nervous system.

Adrenal Medulla↗
PubMed1990

Chromogranin A: osmotically active fragments and their susceptibility to proteolysis during lysis of the bovine chromaffin granules.

Osmotically active fragments of chromogranin A (Chr A) were studied in lysates from bovine chromaffin granules (CG) disrupted in the presence or absence of inhibitors of endogenous proteolytic activities. The effects of various methods of lysis were examined by micro-osmometry, PAGE-SDS electrophoretic techniques and immunoblots with polyclonal anti-Chr A sera. Osmotically active 'small' Chr A fragments (below 30 kDa) were conspicuous in lysates containing cocktails of leupeptin, pepstatin A, pHMB, PMSF and aprotinin. The osmotically inactive native Chr A in the 68-100 kDa range and the osmotically active fragments below 47 kDa were degraded in lysates at neutral or acid pH in the absence of inhibitors. However, degradation of the native Chr A and intermediates below 47 kDa could be prevented by extraction directly from intact CG, notably in cold or boiling distilled water. On the other hand, the main product after large-scale extraction of CG in 1 M acetic acid (pH 1.9, 100 degrees C) was a novel, osmotically active fragment (22 kDa), immunostaining only for the N-terminal sequence (Chr A1-40). The heat-stable fraction (Mr,n 23 kDa) exhibited concentration-independent colloid osmotic pressures even in the absence of phosphate, a property which may distinguish this N-terminal-containing fragment from the larger intermediates, probably containing the pancreastatin sequence, and other regions at the C-terminal side of the prohormone molecule. The functional roles of these osmotically active intermediates in the processing of Chr A are not yet known.

Acids↗
PubMed1990

Modulation of secretion by dopamine involves decreases in calcium and nicotinic currents in bovine chromaffin cells.

1. Catecholamine secretion from cultured bovine adrenal chromaffin cells was decreased in a dose-dependent manner by the D2 dopamine agonists apomorphine and LY 17 1555. 2. 45Ca2+ uptake was similarly inhibited and whole-cell Ca2+ currents were reduced by apomorphine. 3. These inhibitory effects of D2 agonists depended on the secretagogue used, being much more pronounced for nicotine-evoked responses compared to high K+ stimulation, indicating another possible site of action of apomorphine up-stream of Ca2+ entry. 4. Inhibition by apomorphine of nicotine-evoked responses could not be explained by competitive antagonism against nicotine or DMPP (1,1-dimethyl-4-phenyl-piperazinium iodide). 5. Apomorphine caused reductions of inward whole-cell nicotinic current evoked by ACh and nicotine. 6. Inhibition of nicotine-evoked secretion and 22Na+ influx by apomorphine were not affected by tetrodotoxin, and voltage-dependent, whole-cell Na+ currents were unaltered by apomorphine. 7. No evidence was obtained for increases in K+ conductance by apomorphine. 8. Action potentials recorded in whole-cell current clamp were blocked by apomorphine when they were triggered by nicotinic depolarization but not when they were elicited by direct electrical stimulation. 9. Inclusion of GDP-beta-S in the pipette internal solution did not affect apomorphine-dependent inhibition of nicotinic-evoked responses, while the decrease in whole-cell Ca2+ current induced by apomorphine was completely inhibited in the presence of GDP-beta-S. 10. Increases in cyclic AMP caused by cholera toxin and forskolin did not change the apomorphine-dependent inhibitory effects on nicotine-evoked secretion, indicating that changes in cyclic AMP levels caused by dopamine receptor stimulation are probably not involved.

Action Potentials↗
PubMed1990

Neurofilament expression in bovine chromaffin cells.

Neurofilament (NF) expression was examined in adult bovine adrenal chromaffin cells by immunocytochemistry using a series of monoclonal antibodies directed against either nonphosphorylated or phosphorylated epitopes of the heavy NF subunit. In situ, this NF subunit was not detected in chromaffin cells. However, chromaffin cells grown in primary culture under standard conditions contained NF proteins, but only in a nonphosphorylated state. Phosphorylation of NFs could be induced under culture conditions favouring the development of a neuronal phenotype--40% of the cells developed neurites within a week while NF phosphorylation occurred later. Phosphorylated NFs were restricted to neurites, unlike nonphosphorylated NFs which were observed in both perikarya and neurites.

Adrenal Medulla↗
PubMed1990

Effects of phorbol esters on cytoskeletal proteins in cultured bovine chromaffin cells: induction of neurofilament phosphorylation and reorganization of actin.

Bovine chromaffin cells normally express mostly nonphosphorylated neurofilaments (NFs) in primary culture, and thus provide a unique model for examining the kinase capable of phosphorylating these proteins in situ. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C induced NF phosphorylation both in the perikaryon and in neuritic extensions of neurite-bearing cells as judged by immunofluorescence using monoclonal anti-NF antibodies which distinguish between phosphorylated and nonphosphorylated epitopes. NF phosphorylation was suppressed by pretreating the cells with sphingosine, an inhibitor of protein kinase C, and was not observed in the presence of the phorbol ester. 4 alpha-phorbol-12,13-didecanoate (PDD) which does not activate protein kinase C, arguing that protein kinase C was responsible for the observed phosphorylation. Immunochemical analysis of cytoskeletal extracts indicated that TPA induced a 3 to 6-fold increase in NF phosphorylation and showed that the 150,000 dalton NF subunit was the principal protein kinase C substrate. In addition to the TPA effect on NF phosphorylation, TPA provoked a reversible membrane ruffling, which eventually resulted in a flattening of chromaffin cells. These morphological alterations were linked with actin patching and the development of stress fibers, respectively. Sphingosine blocked the TPA-induced membrane ruffling and actin patching, and these phenomena were correlated with increased protein kinase C activity. In contrast, there was no change in the localization of microtubules and NFs. The actin reorganization and NF phosphorylation induced by TPA suggest that at least two distinct proteins of the neuronal cytoskeleton are susceptible to the influence of protein kinase C activation. It remains to be established whether protein kinase C plays a role in the regulatory mechanism controlling actin organization and neurofilament phosphorylation during neuronal differentiation.

Actins↗