Role of ALADIN in Human Adrenocortical Cells for Oxidative Stress Response and Steroidogenesis

Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.     

Cyclic Guanosine Monophosphate in Primary Cultures of Glial Cells

Cyclic GMP was found in primary cultures of glial cells obtained by dissociation of newborn mouse brain hemispheres. Its basal level (0.52 pmoles/mg cell protein) was as high as that found in adult mouse brain cortex but 10 times lower than in cerebellum. When glia were grown in the presence of dBcAMP, astrocytes changed their morphology; cGMP level increased and reached about 8 to 10 times the basal value. This increase was dose dependant with cAMP and was enhanced by the presence of 5mM Theophylline. Two hypothesis are discussed, either a direct action oc cAMP on glial cGMP metabolism or an indirect one on the protein activator of cGMP phosphodiesterase.     

Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.     

Attachment of Pneumocystis carinii to Primary Cultures of Rat Alveolar Epithelial Cells

Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type H alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 ± 1.2% on Day 2 to 18.4 ± 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 ± 2.5% to 9.4 ± 1.9% (P < 0.01). Addition of antibodies to the α_v and α₅ integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of α_v and α₅ integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both α_v and α₅ integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells.     

Glia Modulate NMDA-Mediated Signaling in Primary Cultures of Cerebellar Granule Cells

Abstract: Excessive activation of N-methyl-d -aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca²⁺ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.     

Complexity of Depolarization-mediated ERK Phosphorylation in Cerebellar Granule Cells in Primary Cultures

We previously showed that cultured mouse cerebellar granule cells during incubation in glutamine-replete medium respond to 45 mM [K⁺]_e after 20 and 60 min incubation with extracellular-signal regulated kinase 1 and 2 (ERK_1/2) phosphorylation which is mainly, but probably not exclusively, secondary to glutamate release and transactivation of epidermal growth factor (EGF) receptors. In the present study the response after 20 min was shown to be abolished by protein kinase C (PKC) inhibition, whereas that at 60 min was PKC-independent. Addition of 50 μM glutamate to the cells caused ERK_1/2 phosphorylation already after 5 min most of which was sensitive to PKC inhibition although a minor part was PKC inhibition-resistant. Exposure to [K⁺]_e during incubation in glutamine-depleted medium caused no stimulated release of glutamate but a transactivation-independent ERK_1/2 phosphorylation at 20 and 60 min. The response at 20 min was insensitive to PKC inhibition. The potential importance of these complex responses for synaptic plasticity is discussed. Special issue article in honor of Dr. Frode Fonnum.     

Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal–glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10–P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-βIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and βIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and βIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal–glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.     

Growth of Trachoma Agent in Primary Cultures of Rabbit Corneal Endothelial Cells

Primary cultures of rabbit corneal endothelial cells supported growth of trachoma agent, as evidenced by the production of inclusions and an increase of infectivity titers.     

Effect of Acetylcholine Precursors on Proliferation and Differentiation of Astroglial Cells in Primary Cultures

Effects of acetylcholine and of the cholinergic precursors choline, cytidine 5′-diphosphocholine (CDP-choline) and α-glyceril-phosphorylcholine (α-GPC) on transglutaminase (TG) and cyclin D1 expression were studied in primary astrocyte cultures by confocal laser microscopy (CLSM) with monodansyl-cadaverine uptake as a marker of enzyme activity and by immunochemistry (Western blotting). CLSM analysis showed an increased cytofluorescence in 0.1 μM choline-treated astrocytes. Treatment with CDP-choline dose-dependently increased TG. A total of 1 μM CDP-choline exposure in 14 days in vitro (DIV) astrocyte cultures increased cytofluorescence. A total of 1 μM α-GPC 24 h-treated cultures revealed increased cytofluorescence both in cytosol and nuclei. Western blot analysis showed an increased TG expression in cultures exposed for 24 h to 1 μM choline or α-GPC, whereas in 24 h 1 μM CDP-choline and acetylcholine-treated astrocytes TG expression was unaffected. Treatment with 1 μM acetylcholine reduced TG expression at 21 DIV. In cultures at 14 and 35 DIV cholinergic precursor treatment for 24 h induced a marked down-regulation of cyclin D1 expression, with reduced cyclin D1 expression in 1 μM α-GPC treated astrocytes. Our data suggest a role of cholinergic precursors investigated independent from acetylcholine on maturation and differentiation of astroglial cells in vitro, rather than on their growth, proliferation and development in culture. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Pitavastatin Strengthens the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

Statins have a neuroprotective effect in neurological diseases, a pleiotropic effect possibly related to blood–brain barrier (BBB) function. We investigated the effect of pitavastatin on barrier functions of an in vitro BBB model with primary cultures of rat brain capillary endothelial cells (RBEC). Pitavastatin increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), at a concentration of 10^?8 M, and decreased the endothelial permeability for sodium fluorescein through the RBEC monolayer. The increase in TEER was significantly reduced in the presence of isoprenoid geranylgeranyl pyrophosphate, whereas farnesyl pyrophosphate had no effect on TEER. Our immunocytochemical and Western blot analyses revealed that treatment with pitavastatin enhanced the expression of claudin-5, a main functional protein of TJs. Our data indicate that pitavastatin strengthens the barrier integrity in primary cultures of RBEC. The BBB-stabilizing effect of pitavastatin may be mediated partly through inhibition of the mevalonate pathway and subsequent up-regulation of claudin-5 expression.     

Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells

The potency of ethanol to inhibit N-methyl-D-aspartate (NMDA) receptor functions may depend on the subunit composition of the NMDA receptors. We used a NR2A-B subunit-selective NMDA receptor agonist, homoquinolinic acid (HQ), and a subunit-unselective agonist, NMDA, to induce neurotoxicity in cerebellar granule cells and examined the neuroprotective actions of ethanol, as well as NR2A- and NR2B-subunit selective antagonists, respectively. HQ was a more potent neurotoxic agent than NMDA, as measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. NR2A- and NR2B-selective NMDA receptor antagonists displayed quite similar neuroprotective potencies against the NMDA- and HQ-produced cell death, indicating that the higher potency of HQ to induce neurotoxicity cannot be simply explained by NR2A- or NR2B-subunit selectivity. As expected, ethanol (25 and 50 mM) attenuated the NMDA-induced neurotoxicity in a non-competitive manner by significantly reducing the maximum neurotoxicity produced by NMDA. By contrast, ethanol inhibited the HQ-induced neurotoxicity in a manner resembling a competitive-like interaction significantly increasing the EC₅₀ value for HQ, without reducing the maximum neurotoxicity produced by HQ. These results suggest that HQ reveals either a novel site or a not previously observed mechanism of interaction between ethanol and NMDA receptors in rat cerebellar granule cell cultures.     

Opiate Receptor-Mediated Inhibition of Catecholamine Release in Primary Cultures of Bovine Adrenal Chromaffin Cells

Abstract: Primary cultures of chromaffin cells from bovine adrenal medulla were used to evaluate the ability of several opiates to reduce the release of catecholamines induced by stimulation of nicotinic receptors. Etorphine, β-endorphin, Met-enkephalin[Arg⁶,Phe⁷], and the synthetic peptide [d -Ala²,Me-Phe⁴,Met(O)^s-ol]enkephalin inhibited the acetylcholine-induced release of catecholamines with an IC₃₀ varying from 10^?7 to 1 × 10^?6M. The effect was stereospecific because levorphanol (IC₃₀= 7.5 × 10^?7M) was approximately two orders of magnitude more potent than dextrorphan. Morphine (μ-receptor agonist), [d -Ala², d -Leu⁵]enkephalin (δ-receptor agonist), ethylketazocine (k -receptor agonist), and N-allylnormetazocine (σ-receptor agonist) were at least 100–1000 times less potent than etorphine. Diprenorphine (IC₅₀= 5 × 10^?7M) and naloxone (IC₅₀= 10^?6M) antagonized the effect of etorphine. High-affinity, saturable, and stereospecific binding sites for [³H]etorphine, [³H]dihydromorphine, [³H-d -Ala²,d -Leu⁵]enkephalin, [³H]ethylketazocine, and for [³H]N-allylnormetazocine, [³H]diprenorphine, and [³H]naloxone were detected in chromaffin cell membranes and in membranes obtained from adrenal medulla homogenates. However, the number of binding sites for [³H]etorphine and [³H]diprenorphine was 10–70 times higher than the number of sites measured with the other ³H ligands. The rank order of potency of these compounds for the displacement of [³H]etorphine binding correlates (r = 0.90) with the rank order of potency of the same compounds for the inhibition of acetylcholine-induced catecholamine release. These data suggest that a stereoselective opiate receptor (different from the classic μ-, δ-, k -, or σ-receptor) with high affinity for etorphine, diprenorphine, β-endorphin, and Met-enkephalin[Arg⁶,Phe⁷] modulates the function of the nicotinic receptor in adrenal chromaffin cells.     

Eimeria tenella: Vitamin Requirements for Development in Primary Cultures of Chicken Kidney Cells

SYNOPSIS. Development of Eimeria tenella was studied in primary cultures of chicken kidney cells maintained in Medium 199 lacking each of the following: vitamin A. biotin, p -aminobenzoic acid, folic acid, nicotinamide, Ca pantothenate, pyridoxine, pyridoxal, riboflavin, thiamin, ascorbic acid, calciferol, α-tocopherol, and menadione. Data obtained concerning numbers of mature schizonts or total numbers of parasites or both indicated that all of the vitamins are needed for 1st- and 2nd-generation schizogony, and all except calciferol and folic acid are needed for gametogony.     

22Na+ Uptake and Catecholamine Secretion by Primary Cultures of Adrenal Medulla Cells

The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+-dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor-linked ion conductance channel, blocked nicotine-stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine-stimulated catecholamine secretion and had no effect on veratridine-stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine-stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine-stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin-sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+-dependent catecholamine secretion. However, These channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor-associated ion conductance channel.     

Intracellular Metabolism of 4-Hydroxynonenal in Primary Cultures of Rabbit Synovial Fibroblasts

The intracellular metabolism of 4-hydroxynonenal (HNE), a secondary product of lipid peroxidation and mediator of inflammation, which was found in the joints of patients with rheumatoid arthritis, was investigated in primary cultures of rabbit synovial fibroblasts. A consumption rate of 27.3 nmol/min × 10⁶ cells was measured for the cultivated fibroblasts. It could be shown, that 4-hydroxynonenal enters the synovial fibroblasts and is metabolized mainly oxidatively to 4-hydroxynonenoic acid, intermediates of the tricarboxylic acid cycle and water and by formation of the glutathione-HNE adduct. The share of protein-bound HNE was about up to 8% of the total added HNE after 10 min of incubation. All metabolites accumulates intracellularly within the incubation time except of 4-hydroxynonenal itself. An increase of 4-hydroxynonenoic acid could be detected also extracellularly during the intracellular metabolism of 4-hydroxynonenal. Therefore, an involvement of synovial fibroblasts in the secondary antioxidant defense system of the joints during conditions of higher HNE concentrations like rheumatoid arthritis is suggested. © 1997 Elsevier Science Inc.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

TGF-β Modulates the Expression of Retinoic Acid-Induced RAR-β in Primary Cultures of Embryonic Palate Cells

We have previously shown that both transforming growth factor-β (TGF-β) and retinoic acid (HA) regulate the expression of cellular retinoic acid binding proteins (CRABP) I and II and TGF-β3 mRNAs in primary cultures of murine embryonic palate mesenchyreal (MEPM) cells. We now describe additional crosstalk between the RA and TGF-β signal transduction pathways—the ability of TGF-β, including the endogenous form(s), to modulate the expression of the nuclear retinoic acid receptor-β (RAR-β). Northern blot hybridization revealed that RA induced the expression of RAR-β mRNA, there being little or no detectable expression in untreated MEPM cells. Induction by 3.3 μM RA was abrogated by simultaneous treatment with TGF-β1 (5 ng/ml). TGF-β1 alone had no effect on RAR. mRNA expression. Determination of RAR-β mRNA half-life by treatment with actinomycin D indicated that TGF-β1 did not alter the stability of RAR-β mRNA. Conditioned medium (CM) from MEPM cells contained little active TGF-β protein; heat treatment of the CM dramatically increased the amount of active TGF-β as assessed by the mink lung epithelial cell bioassay. Furthermore, heat- or acid-activated CM also inhibited CRABP-I and RA-induced RAR-β expression. The effect of heat-activated conditioned medium could be abrogated with panspecific neutralizing antibodies to TGF-β, confirming that endogenous TGF-β is the biologically active factor in heat-activated CM. These results provide evidence for complex interactions between TGF-β and RA in the regulation of gene expression in embryonic palatal cells and suggest a role for endogenous TGF-β in the regulation of expression of genes encoding elements of the RA signal transduction pathway.     

Cellular Localization and Regulation of Glutamine Synthetase in Primary Cultures of Brain Cells from Newborn Mice

The cellular distribution of glutamine synthetase was determined by indirect immunofluorescence in cultures of dissociated brain cells from newborn mice. The enzyme could be detected in about 40% of all cells, among which cells with astrocytic morphology were clearly identified. Treatment with the glucocorticoid dexamethasone led to a strong increase in the number of positivity stained cells. Enzyme induction by dexamethasone was maximal after 36 h and at a concentration of 0.1 micrometer. Under these conditions glutamine synthetase specific activity was elevated about six fold. Steroid hormones other than corticosteroids had no effects. The basal activity in these cultures was near that found in brains of newborn mice, but far below the activity in adult brains, showing that in culture the normal development of these cells is disturbed. A comparison of glial and neuronal cell lines showed that glutamine synthetase is present in both types of cell lines at a very low specific activity. Inducibility of this enzyme by dexamethasone was found in glial but not in neuronal cell lines.