Biomedical subjects

D Aunis

Publications and source records attributed to D Aunis.

At least 91 records · Page 5Linked to original sources

PubMed1996

Human pheochromocytoma: different patterns of catecholamines and chromogranins in the intact tumour, urine and serum in clinically unsuspected cases.

Clinically unsuspected pheochromocytoma is usually discovered either at autopsy or during surgical intervention for unrelated conditions, despite often enormous neoplastic masses producing and storing catecholamine (CA). In order to assess whether these tumours share some common features we have compiled data for six patients admitted to hospital without previous diagnosis of their pheochromocytoma. The clinical variables and the morphological and immunohistochemical characteristics of the tumours revealed that these cases represented quite different expressions of adrenomedullary neoplasms. They differed not only with respect to nuclear ploidity and overall cytoplasmic morphology but also in catecholamine storage and expression of immunoreactive chromogranin A sequences in the intact tissue. In two of the patients hypertension had been overlooked as a diagnostic indicator of their CA-producing tumours. There was no clear relationship between the mean arterial pressure, the tumour content of CA and the serum levels of CA. Processed chromogranin A dominated in the serum of the two hypertensive cases. The 24-h urine values of CA and its main metabolite (vanillin mandelic acid) were, together with the serum values of chromogranin A and B, proportional to tumour mass and provided the most reliable diagnostic indicators for the non-hypertensive as well as the hypertensive cases.

Adrenal Gland Neoplasms↗

PubMed1996

Immunocytochemical localization of chromogranin A in the normal and stimulated hypothalamo-neurohypophysial system of the rat.

Chromogranin A (CGA) is a calcium-binding glycoprotein thought to be the precursor of several peptides with defined biological activity. Chromogranin A has been localized in most endocrine cells and many neurons in the CNS. Here we studied its expression in neurons of the hypothalamo-neurohypophysial system, which secrete the neurohormones oxytocin and vasopressin. Light and electron microscopic immunocytochemistry and immunoblot analysis with antibodies specific for CGA revealed high levels of chromogranin A immunoreactivity throughout the hypothalamo-neurohypophysial system. In the supraoptic and paraventricular nuclei, it was characterized by intracytoplasmic labelling of magnocellular somata and processes and of certain astrocytes. Extensive labelling of fibres and dilatations characterized the internal layer of the median eminence and the neurohypophysis, transit and terminal site of the neurosecretory axons, respectively. Tanycyte-like cells in the median eminence also displayed reaction. Simultaneous immunofluorescence showed that oxytocinergic and vaso-pressinergic neurons contain chromogranin A. Electron microscopy revealed that chromogranin A immunoreactivity (visualized by pre-embedding immunoperoxidase or silver-enhanced colloidal gold techniques) was associated with neuro-secretory granules in hypothalamo-neurohypophysial system neurons. In astrocytes and pituicytes, it was seen over the cytoplasm and glial filaments. In tissue from colchicine-treated or immobilization-stressed rats, it was clear that chromogranin A immunoreactivity in the hypothalamus was confined to the hypothalamo-neurohypophysial system. In rats in which neurohypophysial secretion was strongly stimulated by dehydration, immunocytochemistry showed that hypothalamo-neurohypophysial system immunoreactivity significantly increased in the magnocellular nuclei but decreased in the neurohypophysis. On the other hand, chromogranin A distribution was not markedly affected by stress or lactation. These observations demonstrate that chromogranin A is present in neurons and, to a lesser degree, glial cells of the hypothalamo-neurohypophysial system and that its expression is closely related to that of the neurohypophysial peptides.

Animals↗

PubMed1995

Long term stimulation changes the vesicular monoamine transporter content of chromaffin granules.

Bovine chromaffin cells cultured for 5 days in the presence of depolarizing concentrations of K+ ions show a decreased number of secretory (chromaffin) granules per cell. These cells were still capable of exocytosis. Their contents in catecholamine and chromogranin A, components of the granule matrix, and cytochrome b561, a major protein of the granule membrane, were decreased to 35, 30, and 50% of control cells, respectively. However, in the same cells, the number of [3H]dihydrotetrabenazine binding sites, a specific ligand of the vesicular monoamine transporter, was increased to 180% of controls. In situ uptake of noradrenaline in permeabilized cells indicated that [3H]dihydrotetrabenazine binding sites were associated with a functional vesicular monoamine transporter. When analyzed by isopycnic centrifugation, these sites cosedimented with catecholamine, chromogranin A, and cytochrome b561, in a peak with a density lighter than that from controls. The composition of this peak suggests that it contains incompletely matured secretory granules, with a 3-5-fold increase in the vesicular monoamine transporter content of this membrane. This increase might indicate that an adaptative process occurs which allows a faster filling of the granules in continuously secreting cells.

Animals↗

PubMed1995

Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614-626 with antibacterial activity.

Chromogranins constitute a family of acidic soluble proteins widely distributed in endocrine cells and neurons. Chromogranin A, the major soluble component in bovine adrenal medullary secretory granules in chromaffin cells, has been shown to be actively processed to peptide fragments [Metz-Boutigue, M. H., Garcia-Sablone, P., Hogue-Angeletti, R. & Aunis, D. (1993) Eur. J. Biochem. 217, 247-257]. In the present paper, the structural features of the proteolytic degradation mechanism of chromogranin B/secretogranin I have been characterized with regard to the possible function of this protein as a precursor of biologically active peptides. Chromogranin-B-derived fragments present in bovine chromaffin granules were identified by microsequencing after separation by two-dimensional gel electrophoresis or high-performance liquid chromatography. A similar approach was performed to characterize chromogranin-B-derived fragments released into the extracellular space from depolarized bovine cultured chromaffin cells. In chromogranin B, 18 cleavage sites were identified along the protein chain and chromogranin B/secretogranin I fragments were generated by proteolytic attack at both the N-terminus and C-terminus. A major fragment corresponding to residues 614-626 of the C-terminal sequence, was identified in the extracellular space; this peptide was found to share sequence and structural similarities with the lytic domain of cecropins and, as expected from this similarity, to display potent antibacterial properties. Endogenous and synthetic peptides were active on Micrococus luteus, killing bacteria in the micromolar concentration range. The synthetic peptide slows the growth of Bacillus megaterium and was inactive towards Escherichia coli. In addition, the synthetic peptide was unable to induce hemolytic activity. This antibacterial function might be of biological significance in the neuroendocrine system of living organisms. We propose to name this peptide secretolytin.

Adrenal Medulla↗

PubMed1995

Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides.

Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system. Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity. The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein. The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis. Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence. This E. coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human. Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA. A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa. Four components (molecular mass range 12-18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA1-113) while the two others (molecular mass range 7-8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA1-76). From protein staining the ratio vasostatins II/I was 10:1. The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments. An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.

Adrenal Medulla↗

PubMed1995

Biological effects of encapsulated insulin on transfected Chinese hamster ovary cells.

Oral administration of insulin incorporated into the wall of isobutylcyanoacrylate nanocapsule to diabetic rats induces a long-lasting normalization of their fasting glycaemia. In this study, we examined the biological action of encapsulated insulin on DNA and glycogen syntheses in Chinese hamster ovary cells transfected with the human insulin receptor gene. In the 10(-11) mol/l-10(-9) mol/l concentration range, encapsulated insulin elicited responses comparable to those induced by native insulin: at 10(-9) mol/l, the rates of glycogen and DNA synthesis were enhanced by factors 3 and 2.5, respectively. Encapsulated insulin at 10(-7) mol/l evoked receptor desensitization although it did not induce receptor down-regulation and did not alter receptor recycling for up to 6 h. Chloroquine decreased the action of native insulin on glycogen synthesis, but did not affect the dose-response characteristics of encapsulated insulin. Acid-washing of the cells after 1 h of stimulation decreased maximal insulin responsiveness and provoked a dose response curve for encapsulated insulin similar to that of the native hormone. Direct measurement of effective insulin binding activity showed that encapsulated insulin (at 10(-8) and 10(-7) mol/l) was withdrawn from the incubation medium 5-8 times less efficiently than native insulin. These data are in agreement with previous results showing that the polymeric wall protects encapsulated insulin from degradation. Persistence of intact encapsulated insulin inside and outside the cell may result in modifying signalling events and thus be responsible for the observed cellular desensitization.

Animals↗

PubMed1995

Induction of c-fos, jun B and egr-1 expression by haloperidol in PC12 cells: involvement of calcium.

Acute injection of haloperidol, a dopamine D2 receptor antagonist, is known to increase immediate early gene expression of the fos and jun families in rodent striatal neurons. A set of gene induction, including c-fos, jun B and TIS8/egr-1, was found when haloperidol was added to PC12 cells in culture. Electrophoretic mobility-shift assays show that haloperidol-evoked gene induction was accompanied by a transient and dose-dependent increase in AP1 and EGR-1 binding activities in these cells. Gene expression is tentatively explained by the rapid and transient increase in cytosolic free Ca2+ concentration observed upon haloperidol addition. The cytosolic calcium rise and AP1 binding activation elicited by haloperidol were dependent on extracellular Ca2+, suggesting that haloperidol exerted its effects by promoting Ca2+ entry into PC12 cells. The haloperidol-induced increase in AP1 binding activity and intracellular Ca2+ was not reproduced by two other dopamine D2 receptor antagonists, sulpiride and (+)-butaclamol.

Animals↗

PubMed1995

5'-(N-ethylcarboxamido)adenosine inhibits Ca2+ influx and activates a protein phosphatase in bovine adrenal chromaffin cells.

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.

Adenosine↗

PubMed1995

Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells.

Human immunodeficiency virus type 1 (HIV-1) infection of the neuronal and astroglial cells of the central nervous system has been proposed to contribute to HIV-1-associated dementia. Recently it was shown that differences in the nucleotide sequence of the long terminal repeat (LTR) of different HIV-1 strains govern the tissue-specific pattern of viral expression. The LTR from central nervous system-derived HIV-1 strains JR-FL and JR-CSF directs expression in the neurons of transgenic mice, in contrast with the lymphotropic LAI strain. By in vitro footprinting, gel retardation, and methylation interference experiments, we have studied the interactions of host cell proteins from human neuronal, glial, HeLa, and Jurkat T cells with the LTRs from the neurotropic JR-FL and JR-CSF strains, compared with the LAI strain. Proteins belonging to the nuclear receptor family bind with different affinities to variant -352 to -324 sites. Gel supershift assays with Jun and Fos antibodies showed that the AP-1 transcription factor present in the various cell types was unable to recognize the -352 to -324 and -306 to -285 AP-1 putative binding sites. Interestingly, Jun and Fos components of AP-1 interact with the variant TGGCTCA sequence located in the -247 to -222 region of both neurotropic strains. These interactions were cell type specific, since they were detected only with extracts from glial and HeLa cells and not from neuronal or Jurkat cells. Cotransfection experiments further revealed that the -247 to -222 sequence is able to mediate AP-1-induced transcriptional activation in glial and not neuronal cells.

Animals↗

PubMed1995

Circulating chromogranin A and catecholamines in human fetuses at uneventful birth.

Chromogranin A (CGA), a large acidic 48-kD protein, costored and coreleased by exocytosis with catecholamines, has been shown to be a precursor of peptides that exert feedback regulatory control on catecholamine secretion. In plasma, CGA levels increase in response to a large-amplitude physical stimulation in adult subjects and may be related to catecholamine levels. Any skin information is not yet available when the sympathoadrenal system is highly active during birth. This activation is strongly related to parturition circumstances such as the mode of delivery. The aim of our study was to determine CGA plasma levels in infants delivered vaginally or by elective cesarean section and to investigate the possible correlation between CGA and catecholamine concentrations. Plasma levels of catecholamines (norepinephrine and epinephrine) and CGA were assessed by HPLC with electrochemical detection and immunoenzymology, respectively. CGA and norepinephrine concentrations were significantly higher (p < 0.0002 and p < 0.02) in infants vaginally born than in the group delivered by elective cesarean section. A significant relationship (p < 0.04) was found between CGA and norepinephrine levels. However, for epinephrine, no significant difference was found between both groups. These results demonstrate the fetus' ability to corelease CGA and norepinephrine massively in response to stress of birth.

Adult↗

PubMed1995

Immunocytochemical localization of protein kinases Yes and Src in amoeboid microglia in culture: association of Yes kinase with vimentin intermediate filaments.

Amoeboid microglia isolated from primary cultures of neonatal rat brain correspond to a transient form of activated microglia, a resident population of macrophage-like cells. In order to understand the molecular aspects of microglial activation, we have studied amoeboid microglia in primary culture for the presence of Yes and Src protein tyrosine kinases, two kinases which have been implicated in signal transduction process during monocyte/macrophage activation. Immunofluorescence with an antibody raised against the peptide from unique N-terminal domains of Yes and Src demonstrated that Yes and Src kinases are enriched in perinuclear areas of amoeboid microglia. In addition, the antibody to c-yes peptide had a cytoplasmic distribution which coincided with the distribution of vimentin-containing intermediate filaments. Preadsorption of anti-c-yes antibody with an excess of antigenic peptide inhibited anti-c-yes immunofluorescence, while vimentin immunofluorescence remained unchanged. Double immunofluorescence images analyzed with the two-dimensional intensity distribution program (2-D scattered histograms) on Zeiss confocal scanning laser microscope demonstrate the colocalization of c-yes with vimentin. The extent of colocalization was more prominent after exposure of intact cultured microglia to dibutyryl cyclic AMP (dBcAMP), or to phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) or to okadaic acid, an inhibitor of protein phosphatases. The findings suggest that vimentin might serve as molecular support for Yes kinase and, since previous studies have shown that vimentin in amoeboid microglia is one of the major protein substrates of serine/threonine protein kinases, this function could be regulated by phosphorylation.

Animals↗

PubMed1994

GAP-43 controls the availability of secretory chromaffin granules for regulated exocytosis by stimulating a granule-associated G0.

Besides having a role in signal transduction, heterotrimeric G proteins may also be involved in membrane trafficking events as suggested by their presence in specific intracellular compartments. In chromaffin cells, G alpha 0 is associated with secretory organelles, and its activation inhibits exocytosis. Although plasma membrane-bound G proteins are activated by cell-surface receptors, the intracellular proteins controlling organelle-associated G proteins are currently unknown. GAP-43, a neuronal protein enriched in axonal growth cones and presynaptic terminals, is one possible candidate since it can directly stimulate purified G0. We have investigated the interaction of adrenal medullary GAP-43 with chromaffin granule-associated G0 and its effect on catecholamine secretion. Cytosolic and depalmitoylated membrane-extracted GAP-43 were found to stimulate guanine nucleotide binding and exchange activity in chromaffin granule membranes. In permeabilized chromaffin cells, both forms of GAP-43 blocked calcium-dependent exocytosis, and this effect was inhibited by specific antibodies against G alpha 0. A synthetic peptide corresponding to the GAP-43 domain that interacts with G0 inhibited catecholamine secretion. This effect could be selectively reversed by the COOH-terminal peptide of G alpha 0. These results indicate that GAP-43 may be an endogenous pseudoreceptor for the secretory granule-bound form of G0 and can thereby control calcium-regulated exocytosis in chromaffin cells.

Adrenal Medulla↗

PubMed1994

Expression of GAP-43 (neuromodulin) during the development of the rat adrenal gland.

The 'growth-associated protein', GAP-43 was originally considered to be a neuron-specific protein associated with plasticity. However, we have recently shown that GAP-43 is expressed by noradrenergic, but not by adrenergic chromaffin cells in the adult rat adrenal gland. In this study, we examine the expression of GAP-43 during embryonic and post-natal development of the adrenal gland using immunohistochemical techniques. In parallel, antibodies directed against two neuroendocrine markers, the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were employed to permit identification of the developing chromaffin cell phenotypes. At embryonic day 15.5, GAP-43 was predominately localized in sympathoadrenergic precursor cells in the extra-adrenal blastema, and also in nerve fibers within the adrenal gland. At later embryonic stages, GAP-43 was expressed by nearly all intra-adrenal chromoblasts. Two subsets of chromoblasts can be distinguished even at early stages. A strong GAP-43-positive immunoreaction was observed in those chromoblasts organized in a few large compact clusters which weakly expressed TH and did not express PNMT. A generally weaker GAP-43 immunoreaction was observed in a second type of intra-adrenal chromoblasts which were organized in small isolated groups and characterized by a PNMT-positive, and strong TH-positive immunoreactivity. GAP-43 immunoreactivity was still associated with many PNMT-positive adrenergic chromoblasts at birth, but decreased to undetectable levels during the first post-natal week. By the second post-natal week, GAP-43 was restricted, as in the adult, to noradrenergic chromaffin cells which expressed TH, but not PNMT, in addition to nerve fibers and their associated glial cells in the gland. An immunoblot analysis confirmed a decrease in GAP-43 protein during the post-natal period. In agreement with these observations, a three-fold decrease in GAP-43 mRNA in the adrenal gland was measured between late embryogenesis and the second post-natal week. During development, the spatiotemporal expression of GAP-43 suggests a possible role in the migration and aggregation of chromaffin cell precursors into the medullary region of the adrenal gland.

Adrenal Glands↗

PubMed1994

Okadaic acid induces activator protein 1 activity and immediate early gene transcription in rat pheochromocytoma cells. Mechanism of action.

The serine/threonine protein phosphatase inhibitor okadaic acid (OA) was found to enhance mRNA transcripts of c-fos and of the jun family of proto-oncogenes including c-jun, jun B and jun D in cultured pheochromocytoma PC12 cells. This expression remained elevated for more than 8 hr. An increase in the binding of the transcription factor activator protein 1 (AP1) to its DNA consensus sequence that occurred prior to early gene transcription was observed. Enhanced AP1 activity was still observed when OA was added to the cells together with the transcription inhibitor actinomycin D, or with the protein synthesis inhibitor cycloheximide, indicating that it is actually AP1 activation due to posttranslational modifications that triggers transcription of the fos and jun genes. AP1 was activated through serine/threonine phosphorylation since its activation was abolished when nuclear extracts of OA-treated cells were incubated with protein phosphatase-1 or, to a lesser extent, with protein phosphatase-2A. C-Jun and Jun D proteins are likely candidates for being phosphorylated, since they were shown to constitute the AP1 complex at the time when it was activated (2 hr after OA addition).

Animals↗

PubMed1994

Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein.

We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.

Adenosine Triphosphate↗

PubMed1994

Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells.

The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.

Calcium↗

PubMed1994

Bacterial endotoxin induces [Ca2+]i transients and changes the organization of actin in microglia.

We have employed amoeboid microglia purified from primary cultures of neonatal rat brain to examine the effect of bacterial lipopolysaccharide (LPS), a potent activator of immune cells, on intracellular calcium concentration ([Ca2+]i) in brain macrophages. In single brain macrophages loaded with indo 1, pulse administration of LPS elicited a rapid and transient increase in [Ca2+]i. From a total of 70 cells examined, all responded to LPS with a similar [Ca2+]i transient, indicating a good homogeneity of the cell population with regard to the LPS response. It was concluded that the rise of cytosolic [Ca2+]i originated from intracellular stores because the response to LPS occurred similarly in the presence or in the absence of extracellular Ca2+. A second administration of LPS to the same cells resulted in a second but reduced [Ca2+]i transient. In contrast to the first response to LPS, this second response was totally dependent on the presence of Ca2+ in the extracellular medium. The first response to LPS was strongly inhibited by ruthenium red and could be suppressed in a reversible manner by preincubating the cells with caffeine in the absence of Ca2+ in the extracellular medium. These results indicate that caffeine-sensitive intracellular Ca2+ stores may be the major source of Ca2+ in the response of brain macrophages to LPS. The possible release of Ca2+ from phosphatidylinositol(3,4,5)-trisphosphate (IP3)-sensitive stores in brain macrophages was also evaluated by stimulating cells with the IP3-mobilizing agonist histamine. Brain macrophages were heterogeneous with regard to the histamine response since histamine induced a [Ca2+]i rise in only 30% of cells examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1994

cAMP and bFGF negatively regulate tropomyosin expression in rat cultured astroblasts.

We have examined the expression of tropomyosin (TM) messenger RNAs (mRNAs) and protein isoforms in primary cultures of rat astroblasts during morphological changes. Three messenger RNA bands of 2.5, 1.8 and 1.2 kilobase pairs (kb) were detected by Northern blot. Using an antibody cross-reacting with all tropomyosin isoforms, we found that rat cerebellar neonatal astroblasts expressed three tropomyosin protein isoforms termed TM-As1, TM-As2 and TM-As3 (As for Astroblast) with respective molecular masses of 38,000, 33,000 and 31,000. Treatment of cells with agents which promote or mimick the action of cyclic AMP, or with growth factors, is known to induce astroblast morphological alteration from flat, polygonal epitheloid cells into star-shaped, process-bearing cells. In the presence of dibutyryl cAMP (dBcAMP), forskolin or basic fibroblast growth factor (bFGF), these morphological changes were found to be associated with dramatic decreases of the three mRNA transcripts and also of the three protein isoforms. This decrease was reversed upon removal of the drugs. The pattern of the tropomyosin protein isoforms in cultured astroblasts showed that TM-Asl, the most immunoreactive isoform recovered in the cytoskeletal insoluble cell fraction, had a developmental profile similar to that of F-actin. Therefore this isoform, which belongs to the high-molecular-mass family of proteins known to interact strongly with F-actin, could specifically be involved in the regulation/control of F-actin stability and thus be associated with the plasticity of astroblasts.

Animals↗