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1.
Ital J Pediatr ; 45(1): 41, 2019 Mar 29.
Article in English | MEDLINE | ID: mdl-30922377

ABSTRACT

BACKGROUND: Vitamin D plays an important role in inflammatory responses after antigen exposure. Interleukin-23 (Il-23) promotes Il-17-dependent inflammation during Pseudomonas aeruginosa (P. aeruginosa) pulmonary infection. We aimed to compare the ability of calcitriol and cholecalciferol to modulate the inflammatory response of the CF airways infected with P. aeruginosa. METHODS: This was a randomized, placebo-controlled, double-blind, cross-over trial. Twenty-three patients with CF (aged 6-19), chronically infected by P. aeruginosa were randomly assigned to: calcitriol group receiving 1,25(OH)2D 0,5 mcg daily or cholecalciferol group receiving cholecalciferol 1000 IU daily for three months. The levels of Il-23 and Il-17A in the exhaled breath concentrate (EBC) were measured. Calcium-phosphorus balance was also evaluated (serum concentration of calcium, phosphorus, 25OHD, parathormone (PTH) and calcium/creatinine ratio in urine). Data were analyzed using means of Stata/Special Edition, release 14.2. A level of P < 0.05 was considered statistically significant. RESULTS: The level of Il-17A in EBC significantly decreased in calcitriol group from 0,475 pg/mL (± SD 0,515 pg/mL) to 0,384 pg/mL (± SD 0,429 pg/mL) (p = 0,008); there was no change in cholecalciferol group (p = 0,074). The level of Il-23 in EBC did not significantly change in calcitriol group (p = 0,086); there was significant decrease in cholecalciferol group from 8,90 pg/mL (± SD 4,07 pg/mL) to 7,33 pg/mL (± SD 3,88 pg/mL) (p = 0,001). In calcitriol group serum phosphorus and PTH significantly decreased (p = 0,021 and p = 0,019 respectively), the concentration of calcium significantly increased (p = 0,001); there were no changes in cholecalciferol group. CONCLUSIONS: Both analogs of vitamin D revealed their anti-inflammatory effect and reduced the level of Il-17A and Il-23 in the airway of CF patients with chronic P. aeruginosa infection. We observed improvement in calcium-phosphorus metabolism after supplementation with calcitriol, without adverse effects. It is recommended to use vitamin D in CF patients.


Subject(s)
Calcitriol/pharmacology , Cholecalciferol/pharmacology , Interleukin-17/metabolism , Interleukin-23/metabolism , Lung/metabolism , Pneumonia, Bacterial/microbiology , Adolescent , Breath Tests , Calcium/blood , Calcium/urine , Child , Creatinine/urine , Cross-Over Studies , Cystic Fibrosis/metabolism , Double-Blind Method , Humans , Male , Parathyroid Hormone/blood , Phosphorus/blood , Pilot Projects , Pseudomonas Infections/complications , Pseudomonas aeruginosa , Vitamin D/analogs & derivatives , Vitamin D/blood , Vitamins/pharmacology , Young Adult
2.
J Physiol Pharmacol ; 61(6): 743-51, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21224506

ABSTRACT

In the present study fifteen male subjects (age: 22.7 ± 0.5 years; BMI: 23.5 ± 0.6 kg x m⁻²; VO2(max) 46.0 ± 1.0 mL x kg⁻¹ x min⁻¹) performed 5 week moderate intensity endurance training. The training resulted in a significant increase in maximal oxygen uptake (VO2(max)) (P=0.048) and power output reached at VO2(max) (P=0.0001). No effect of training on the uncoupling protein 3 (UCP3) content in the vastus lateralis was found (P>0.05). The improvement of physical capacity was accompanied by no changes in cytochrome-c and cytochrome-c oxidase contents in the vastus lateralis (P>0.05). However, the training resulted in an increase (P=0.02) in mitochondrial manganese superoxide dismutase (SOD2) content in this muscle. Moreover, a significant decrease (P=0.028) in plasma basal isoprostanes concentration [F2isoprostanes](pl) accompanied by a clear tendency to lower (P=0.08) gluthatione disulfide concentration [GSSG](pl) and tendency to higher (P=0.08) total antioxidant capacity (TAC) was observed after the training. We have concluded that as little as 5 weeks of moderate intensity endurance training is potent to improve physical capacity and antioxidant protection in humans. Surprisingly, these effects occur before any measurable changes in UCP3 protein content. We postulate that the training-induced improvement in the antioxidant protection at the muscle level is due to an increase in SOD2 content and that therefore, the role of UCP3 in the enhancement of physical capacity and antioxidant protection, at least in the early stage of training, is rather questionable.


Subject(s)
Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Muscle Proteins/metabolism , Oxidative Stress/physiology , Physical Endurance/physiology , Quadriceps Muscle/metabolism , Superoxide Dismutase/metabolism , Cytochromes c/metabolism , Electron Transport Complex IV/metabolism , Glutathione Disulfide/blood , Humans , Isoprostanes/blood , Male , Mitochondria, Muscle/metabolism , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , Oxygen Consumption/physiology , Quadriceps Muscle/enzymology , Uncoupling Protein 3 , Young Adult
3.
Clin Exp Allergy ; 39(12): 1830-41, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19817753

ABSTRACT

BACKGROUND: The possibility of additional strategies to enhance the effectiveness of specific immunotherapy (SIT) is highly attractive. AIM: The aim of our study was to assess the influence of oral corticosteroids and oral corticosteroids combined with vitamin D(3) on the early clinical and immunological effects of SIT. METHODS: It was a randomized, double-blind, placebo-controlled trial conducted in 54 asthmatic children allergic to house dust mites. Intervention was based on receiving a single dose of oral steroid, with or without vitamin D(3), or placebo only on the day of the build-up phase of SIT. RESULTS: After 12 months of SIT, the median daily inhaled corticosteroid (ICS) dose, which controls the symptoms of asthma, was reduced by 25% in the steroid group. However, a 50% reduction of the median daily ICS dose was observed in the control group. The clinical effects of SIT were not affected in the steroid+D(3) group. Concomitantly, we found that intervention with prednisone significantly impaired the induction of T regulatory lymphocytes. Importantly, the clinical and immunological effects of SIT were not affected by intervention with steroids administered with vitamin D(3). CONCLUSIONS: Our study failed to show a beneficial effect of oral corticosteroids on allergen-specific immunotherapy. We observed that the combined administration of a corticosteroid drug and allergen extract suppressed the early clinical and immunological effects of SIT and that vitamin D(3) prevented this 'adverse' influence of steroids.


Subject(s)
Antigens, Dermatophagoides/therapeutic use , Asthma/therapy , Cholecalciferol/therapeutic use , Desensitization, Immunologic/methods , Steroids/therapeutic use , Adrenal Cortex Hormones/administration & dosage , Adrenal Cortex Hormones/therapeutic use , Animals , Antigens, Dermatophagoides/administration & dosage , Antigens, Dermatophagoides/immunology , Arthropod Proteins , Asthma/diagnosis , Asthma/immunology , Asthma/physiopathology , Calcifediol/blood , Child , Cholecalciferol/administration & dosage , Combined Modality Therapy/methods , Dermatophagoides farinae/immunology , Dermatophagoides pteronyssinus/immunology , Double-Blind Method , Female , Forced Expiratory Volume/physiology , Forkhead Transcription Factors/genetics , Gene Expression/genetics , Gene Expression/immunology , Humans , Interleukin-10/metabolism , Interleukin-13/metabolism , Interleukin-5/metabolism , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Male , Prednisone/administration & dosage , Prednisone/therapeutic use , Steroids/administration & dosage , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta/metabolism , Treatment Outcome
5.
J Membr Biol ; 193(2): 79-90, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12879156

ABSTRACT

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2',7'-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/blood , Erythrocyte Membrane/metabolism , Anions , Fluoresceins , Fluorescent Dyes , Humans , In Vitro Techniques , Ion Transport
6.
Free Radic Biol Med ; 30(12): 1418-25, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11390187

ABSTRACT

Cell culture media (RPMI 1640, Dulbecco's Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5'-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.


Subject(s)
Culture Media/radiation effects , Light , Reactive Oxygen Species , Azides/pharmacology , Culture Media/chemistry , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , Fluoresceins/radiation effects , Fluorescent Dyes/radiation effects , Folic Acid/radiation effects , Hydrolysis , Hydroxyl Radical , Oxidation-Reduction , Oxygen , Phenolsulfonphthalein/radiation effects , Photochemistry , Piperazines/pharmacology , Pyridoxine/radiation effects , Rhodamines/radiation effects , Riboflavin/radiation effects , Singlet Oxygen , Spin Labels , Superoxides/analysis , Tryptophan/radiation effects , Tyrosine/radiation effects
7.
Toxicol Lett ; 120(1-3): 43-9, 2001 Mar 31.
Article in English | MEDLINE | ID: mdl-11323160

ABSTRACT

The co-ordinated glucuronidation and export of compounds from cells is an important determinant in the detoxification of many compounds in vivo. Many UDP-glucuronosyltransferases (UGTs) and several multidrug resistance proteins (MRPs) have been cloned and individually expressed to assess specificity of glucuronidation and transport. However, to further understand the interplay between glucuronidation and transport we are developing stable cell lines that express different combinations of UGT and MRP isoforms. V79 cells expressing both UGT1A1 and MRP1 have been established. The ability of these cell lines to both glucuronidate and transport compounds was assessed ex vivo using estradiol and bilirubin as substrates.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Glucuronosyltransferase/physiology , ATP-Binding Cassette Transporters/analysis , Animals , Bilirubin/metabolism , Biological Transport , Cell Line , Estradiol/metabolism , Glucuronides/metabolism , Glucuronosyltransferase/analysis , Multidrug Resistance-Associated Proteins
8.
Blood Cells Mol Dis ; 27(5): 894-900, 2001.
Article in English | MEDLINE | ID: mdl-11783953

ABSTRACT

A method to fluorometrically monitor efflux of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was developed. Genistein, daidzein, sophoraisoflavone A, and licoisoflavone A induced 50% inhibition (IC(50)) of BCPCF efflux at 15-70 microM. The IC(50) value of the most efficient isoflavone, licoisoflavone A (15-25 microM), was comparable to that of indomethacin (approximately 10 microM) and markedly lower than for probenecid (100-200 microM), both known MRP1 inhibitors. Our results indicate that the human erythrocyte is a useful cell model in screening potential MRP inhibitors, that BCPCF is a good substrate for MRP, and that some isoflavones at low concentrations inhibit MRP-mediated efflux.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Erythrocytes/metabolism , Isoflavones/pharmacology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Dose-Response Relationship, Drug , Fluoresceins/pharmacokinetics , Fluorescent Dyes , Hemolysis/drug effects , Humans , Inhibitory Concentration 50 , Isoflavones/toxicity , Kinetics
10.
Acta Biochim Pol ; 47(3): 763-72, 2000.
Article in English | MEDLINE | ID: mdl-11310975

ABSTRACT

The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years. Further studies identifled the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP). Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.


Subject(s)
ATP-Binding Cassette Transporters/blood , Erythrocytes/metabolism , Anions/blood , Carrier Proteins/blood , Drug Resistance, Multiple , Glutathione/analogs & derivatives , Glutathione/blood , Glutathione Disulfide/blood , Humans , In Vitro Techniques , Ion Transport , Kinetics , Membrane Transport Proteins , Models, Biological , Multidrug Resistance-Associated Proteins
11.
Arch Biochem Biophys ; 354(2): 311-6, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9637741

ABSTRACT

Molecular masses of functional units of two components of 2, 4-dinitrophenyl-S-glutathione (DNP-SG) transport across the erythrocyte membrane determined by radiation inactivation were 437 +/- 69 kDa for the high-affinity component and 466 +/- 67 kDa for the low-affinity component. These results confirm that the multidrug resistance-associated protein (MRP) 1 is responsible for the high-affinity DNP-SG transport across the erythrocyte membrane and suggest that MRP1 exists in the membrane as a dimer. The molecular size of the low-affinity transporter is similar if not identical to that of MRP1. Moreover, while the molecular mass of the DNP-SG-ATPase activity of the erythrocyte membrane corresponds also to that of MRP (375 +/- 36 kDa), the molecular mass of the functional unit of dinitrophenol-stimulated ATPase is significantly lower (232 +/- 26 kDa), which suggests that thisactivity is linked to a different protein, perhapsaminophospholipid translocase.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Erythrocyte Membrane/chemistry , 2,4-Dinitrophenol/metabolism , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphatases/metabolism , Adult , Biological Transport/radiation effects , Dimerization , Erythrocyte Membrane/metabolism , Erythrocyte Membrane/radiation effects , Glutathione/analogs & derivatives , Glutathione/metabolism , Humans , In Vitro Techniques , Multidrug Resistance-Associated Proteins
12.
Biochem Mol Biol Int ; 44(1): 97-105, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9503152

ABSTRACT

A hypothesis of the flippase nature of the glutathione S-conjugate transport is presented. Experimental premises for this hypothesis include interaction of glutathione S-conjugates with the membrane, as demonstrated by their effects on membrane fluidity, quenching of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene fluorescence and induction of echinocytosis by 2,4-dinitrophenyl-S-glutathione (DNP-SG). This hypothesis can rationalize, i. a., observations of the enhancement of DNP-SG transport by butanol and stimulation of erythrocyte membrane Mg(2+)-ATPase activity by albumin-coupled DNP-SG.


Subject(s)
Carrier Proteins/blood , Membrane Proteins/blood , Phospholipid Transfer Proteins , 1-Butanol/pharmacology , Biological Transport/drug effects , Ca(2+) Mg(2+)-ATPase/blood , Cell Size/drug effects , Dinitrophenols/blood , Dinitrophenols/pharmacology , Erythrocyte Membrane/enzymology , Glutathione/analogs & derivatives , Glutathione/blood , Glutathione/pharmacology , Humans , Membrane Transport Proteins , Serum Albumin, Bovine/pharmacology
13.
Biochem Mol Biol Int ; 37(1): 73-9, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8653090

ABSTRACT

Pig erythrocyte membrane Mg2+-ATPase activity was stimulated by various glutathione S-conjugates. For alkyl S-conjugates, the Km for the stimulation was lower, the more hydrophobic was the conjugate. 2,4-Dinitrophenyl-S-(N-acetyl)cysteine also stimulated the Mg2+-ATPase activity, suggesting a low specificity of the ¿glutathione S-conjugate pump¿. The Km values for the stimulation by 2,4-dinitrophenyl conjugates were lower than predictable on the basis of hydrophobicity which indicates a high affinity of the transporter for these conjugates.


Subject(s)
Acetylcysteine/analogs & derivatives , Adenosine Triphosphatases/metabolism , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/enzymology , Glutathione/analogs & derivatives , Glutathione/pharmacology , Magnesium/metabolism , Acetylcysteine/chemical synthesis , Acetylcysteine/pharmacology , Adenosine Triphosphatases/drug effects , Animals , Chromatography, High Pressure Liquid , Erythrocyte Membrane/metabolism , Kinetics , Solubility , Structure-Activity Relationship , Substrate Specificity , Swine
14.
Neuropharmacology ; 31(3): 279-91, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1321358

ABSTRACT

Babies born to mothers abusing the psychotomimetic drug phencyclidine (PCP), often show profound deficits in CNS function. It is now reported that PCP can cause progressive degeneration and death in human fetal cerebral cortical neurons in culture and sublethal levels of PCP can inhibit axon outgrowth. In cerebral cortical cell cultures established from 14 week fetuses, exposure to 500 microM PCP resulted in a progressive degeneration of neurons over a 2-8 day period, characterized by early reversible vacuolation of the soma and later irreversible neurite fragmentation and cell death. A sublethal concentration of PCP (100 microM) suppressed axon outgrowth. The adverse effects of PCP on neurons were apparently not due to actions on N-methyl-D-aspartate (NMDA) receptors because: the neurons were not responsive to NMDA during the first 3 weeks in culture; concentrations of PCP that should block NMDA receptors maximally (1-50 microM) did not affect axon outgrowth or cell survival; and another NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not cause neurodegeneration or affect axon outgrowth. Exposure of cultures to the sigma receptor ligand, pentazocine (10 microM), did not significantly affect the survival of neurons and haloperidol did not reduce PCP-induced neurodegeneration, indicating that the effects of PCP were not mediated by sigma receptors. Degenerative effects, similar to those elicited by PCP, were observed in cortical neurons exposed to the K+ channel blockers, 4-aminopyridine and tetraethylammonium, a finding consistent with the possibility that the degenerative actions of PCP were mediated by its known inhibitory effects on K+ channels. In support of the latter possibility, it was found that PCP caused a progressive elevation in intracellular levels of calcium during several days of exposure. In addition to affecting neurons, PCP and K+ channel blockers caused vacuolation and degeneration of astrocytes. Taken together with previous data, indicating that PCP can be concentrated and retained in the fetal CNS, the present data suggest that high levels of PCP can disrupt the normal development of neural circuitry in the human fetus, which would be expected to result in profound functional impairments.


Subject(s)
Axons/drug effects , Cerebral Cortex/drug effects , Phencyclidine/toxicity , Astrocytes/drug effects , Astrocytes/metabolism , Axons/physiology , Calcium/metabolism , Cerebral Cortex/cytology , Female , Fetus/physiology , Fura-2 , Humans , Potassium Channels/drug effects , Pregnancy , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, Opioid/drug effects , Receptors, sigma
15.
Mol Chem Neuropathol ; 15(2): 117-42, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1663746

ABSTRACT

Considerable evidence suggests that altered neuronal calcium homeostasis plays a role in the neuronal degeneration that occurs in an array of neurological disorders. A reduction in microtubules, the accumulation of 8-15 nm straight filaments, and altered antigenicity toward antibodies to the microtubule-associated protein tau and ubiquitin, as well as granulovacuolar degeneration, are observed in many human neurodegenerative disorders. Progress toward understanding how and why human neurons degenerate has been hindered by the inability to examine living human neurons under controlled conditions. We used cultured human fetal cerebral cortical neurons to examine ultrastructural and antigenic changes resulting from elevations in intracellular calcium levels. Elevation of intracellular calcium by exposure to a calcium ionophore or a reduced level of extracellular Na+ for periods of hours to days caused a loss of microtubules, an increase in 8-15 nm straight filaments, and increased immunostaining with Alz-50 and 5E2 (tau antibodies) and ubiquitin antibodies. Granulovacuolar degeneration was also observed. Antigenic changes in tau were sensitive to phosphatases, and the electrophoretic mobility of tau was altered in cells exposed to calcium ionophore, indicating that tau was excessively phosphorylated as the result of elevated intracellular calcium levels. Colchicine also caused an accumulation of straight filaments and altered tau immunoreactivity, suggesting that a disruption of microtubules secondary to altered calcium homeostasis may be a key event leading to altered tau disposition and neuronal degeneration. These data demonstrate that aberrant rises in intraneuronal calcium levels can result in changes in the neuronal cytoskeleton similar to those seen in neurodegenerative disorders, and suggest that this experimental system will be useful in furthering our understanding of the cellular and molecular mechanisms of human neurological disorders.


Subject(s)
Calcimycin/pharmacology , Calcium/physiology , Cerebral Cortex/physiology , Cytoskeleton/physiology , Neurons/physiology , Ubiquitins/metabolism , tau Proteins/metabolism , Antibodies , Antibodies, Monoclonal , Blotting, Western , Cells, Cultured , Cytoskeleton/ultrastructure , Embryo, Mammalian , Humans , Microtubules/drug effects , Microtubules/ultrastructure , Neurons/cytology , Neurons/drug effects , Phosphorylation
16.
Mech Ageing Dev ; 60(2): 171-87, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1745073

ABSTRACT

A problem that has not been addressed directly at the cellular and molecular levels concerns the basic mechanisms governing rates of development and aging in the nervous system. We have begun to examine this problem by employing parallel cell cultures of cerebral cortical neurons taken from rat and man, two species with markedly different developmental periods and life spans. Cortical cultures were established from rat and human fetuses and were maintained under identical conditions. Long-term neuronal survival in human cortical cultures was strikingly greater than in rat cortical cultures, and axonal outgrowth was significantly slower in the human neurons. These differences were consistently observed in a variety of culture media. Exchanges of culture medium between cultures of the two species did not alter the differences in neuronal survival and axon outgrowth suggesting that the differences between human and rat neurons were probably not due to factors released into the culture medium. Furthermore, the differences were not related to developmental stage at the time of culture initiation since similar results were obtained in cultures established from fetuses of different gestational ages. These initial data indicate that differences in the developmental time courses and life spans of human and rat cerebral cortex are reflected at the level of the individual neuron, and provide a system in which to explore the mechanisms responsible for these differences.


Subject(s)
Aging/pathology , Cerebral Cortex/cytology , Animals , Axons/ultrastructure , Cell Differentiation , Cell Survival , Cells, Cultured , Cellular Senescence , Cerebral Cortex/growth & development , Humans , Neurons/cytology , Rats , Species Specificity
17.
Brain Res ; 542(1): 97-106, 1991 Feb 22.
Article in English | MEDLINE | ID: mdl-2054664

ABSTRACT

Although there has been a large body of literature from animal studies concerning neuronal excitatory amino acid (EAA) receptors and their possible roles in brain development, function, and pathology, essentially no direct information on actions of EAAs in humans has previously been available. We now report on experiments in cell cultured human embryonic cerebral cortical neurons which directly addressed the actions of EAAs in the developing human brain. In cultures established from 14-week fetuses, neurons were insensitive to glutamate neurotoxicity during the first 30 days in culture. After 30 days in culture increasingly more neurons became vulnerable to glutamate acting at the N-methyl-D-aspartate and kainate type receptors. The development of calcium responses to glutamate (as measured with the calcium indicator dye fura-2) preceded sensitivity to excitotoxicity by several weeks in the human neurons. Glutamate-induced rises in intracellular calcium and neurotoxicity developed much more rapidly in rat cortical neurons. Studies of dynamic aspects of calcium responses to calcium ionophore A23187 in human and rat cortical neurons demonstrated a direct relation between calcium buffering ability and resistance to EAA neurotoxicity. Interestingly, the human neurons were better able to buffer a calcium load than were rat neurons, suggesting that species-specific and/or developmental stage-specific differences in calcium-buffering systems are likely to play roles in determining neuronal vulnerability to EAAs. These initial observations indicate that human cortical neurons become sensitive to EAAs during the prenatal period, and suggest that EAAs may play important roles in both normal human brain development and neurodegenerative processes.


Subject(s)
Amino Acids/pharmacology , Calcium/metabolism , Cerebral Cortex/embryology , Neurons/metabolism , Buffers , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Humans , Intracellular Membranes/metabolism , Neurons/drug effects , Time Factors
18.
Neuron ; 6(1): 41-51, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1670921

ABSTRACT

Neuronal systems for calcium homeostasis are crucial for neuronal development and function and may also contribute to selective neuronal vulnerability in adverse conditions such as exposure to excitatory amino acids or anoxia, and in neurodegenerative diseases. Previous work demonstrated the presence and differential distribution of calcium-binding proteins in the CNS. We now report that a subpopulation of neurons in dissociated cell cultures of embryonic rat hippocampus expresses calbindin-D28k (Mr 28,000 calcium-binding protein) immunoreactivity and that these neurons are relatively resistant to neurotoxicity induced by either glutamate or calcium ionophore. Direct comparisons of dynamic aspects of intracellular calcium levels and calbindin-D28k immunoreactivity in the same neurons revealed that calbindin-D28k-positive neurons were better able to reduce free intracellular calcium levels than calbindin-D28k-negative neurons. These findings indicate that the differential expression of calbindin-D28k in hippocampal neurons occurs early in development and may be one determinant of selective neuronal vulnerability to excitotoxic insults.


Subject(s)
Calcium/metabolism , Hippocampus/metabolism , Neurons/metabolism , S100 Calcium Binding Protein G/physiology , Animals , Calbindin 1 , Calbindins , Calcimycin/pharmacology , Cells, Cultured , Glutamates/pharmacology , Glutamic Acid , Hippocampus/drug effects , Hippocampus/embryology , Immunoenzyme Techniques , Neuroglia/physiology , Neurons/drug effects , Rats
19.
Brain Res ; 522(2): 204-14, 1990 Jul 09.
Article in English | MEDLINE | ID: mdl-2224523

ABSTRACT

Past knowledge of the human brain at the cellular and molecular levels has come largely from studies of postmortem fixed tissue or by way of extrapolation from studies of lower mammalian species. The ability to study living human brain neurons would provide a new avenue for further insight into mechanisms operative in human brain development, function, and disease. The present study established procedures for the cryopreservation and culture of human fetal cerebral cortical and hippocampal neurons, and characterized the development of the cells in culture. The predominant cell type in both cortical and hippocampal cultures was pyramidal-like neurons that extended one long axon-like process and a few minor dendrite-like processes. Bipolar and stellate cells, as well as astrocyte-like glia were also present in cultures from both brain regions. Fibroblast growth factor (FGF), but not nerve growth factor (NGF), enhanced long-term neuronal survival in both cerebral cortical and hippocampal cultures. Immunocytochemistry demonstrated the presence of both FGF- and NGF-like immunoreactivities in neurons and glia, from both cerebral cortex and hippocampus, suggesting that these endogenous growth factors may play roles in human fetal brain development. The ability to cryopreserve large numbers of viable dissociated human fetal brain neurons, and subsequently study them in cell cultures, provides new opportunities to understand dynamic aspects of the human brain at the cellular and molecular levels.


Subject(s)
Cerebral Cortex/embryology , Fibroblast Growth Factors/physiology , Hippocampus/embryology , Nerve Growth Factors/physiology , Neurons/cytology , Cells, Cultured , Cerebral Cortex/cytology , Cryopreservation , Embryonic and Fetal Development/physiology , Hippocampus/cytology , Humans , Immunoenzyme Techniques
20.
Int J Dev Neurosci ; 8(4): 399-415, 1990.
Article in English | MEDLINE | ID: mdl-2251934

ABSTRACT

Low density cell cultures of embryonic rat hippocampus containing astrocyte-like glia and neurons were used to test the hypothesis that glia can alter 'natural' and excitatory amino acid (EAA)-induced neuronal death. Neurons contacting glia survived for longer time periods than did neurons not contacting glia. Neurons associated with glia were also protected against glutamate and kainate neurotoxicity. Fibroblast growth factor (FGF)-like immunoreactivity was associated with the glia. Addition to the cultures of an antiserum raised against an internal peptide fragment of FGF greatly reduced the protective effect of glia against both spontaneous and EAA-induced neurotoxicity. Contact with glia, or exposure to exogenous FGF, also protected the hippocampal neurons against Ca2+ ionophore-induced degeneration indicating that FGF enhanced the ability of neurons to handle a Ca2+ load. Taken together, these results suggest that glia surface-associated FGF may play important roles in hippocampal development, and in neurodegenerative conditions that involve EAAs.


Subject(s)
Amino Acids/pharmacology , Fibroblast Growth Factors/pharmacology , Hippocampus/cytology , Nerve Degeneration/drug effects , Neuroglia/physiology , Neurons/drug effects , Animals , Calcium/metabolism , Cell Communication , Cell Survival , Neurons/metabolism , Neurotoxins/pharmacology , Time Factors
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