Biomedical subjects

A Winkler

Publications and source records attributed to A Winkler.

At least 55 records · Page 3Linked to original sources

PubMed1997

Survival-promoting activity of inhibitors of cyclin-dependent kinases on primary neurons correlates with inhibition of c-Jun kinase-1.

Cyclin-dependent kinases and mitogen-activated protein kinases have been implicated in the regulation of cellular survival and apoptosis. We tested the effect of two mitogen-activated/cyclin-dependent kinase inhibitors, olomoucine and butyrolactone I, on the in vitro survival of chick embryonic neurons. Sensory, sympathetic, and ciliary neurons, when prepared at their respective time point of programmed cell death, could be rescued from apoptosis by both inhibitors in a dose-dependent fashion. In contrast, dividing sympathetic precursors underwent apoptosis when treated with olomoucine, but not butyrolactone I, at the same range of concentration. With similar potency, olomoucine and butyrolactone I inhibited immunocomplex c-Jun kinase activity. Both substances inhibited neurite outgrowth in a dose-dependent manner; developmentally younger neurons were more sensitive to this effect than older ones. These results suggest that certain mitogen-activated/cyclin-dependent kinases associated with cell division in neuronal precursors (i) may become essential components of the apoptotic machinery by the time neurons reach their phase of naturally occurring cell death and (ii) may be necessary for neurite outgrowth during development.

Animals↗

PubMed1997

A novel SOD1 mutation in an Austrian family with amyotrophic lateral sclerosis.

We report on an Austrian pedigree with autosomal dominant amyotrophic lateral sclerosis (ALS), diagnosed in six patients from two generations. The only surviving clinically affected family member was examined in our ALS clinic. Historical information on other affected individuals was obtained from knowledgeable family members. The mean +/- S.D. age of onset of the disease was 54 +/- 6.9 years, with a range of 43-66 years. The duration of the index patient's disease until death was 8 months. Using single strand conformational polymorphism (SSCP) analysis, we studied the index patient's exons 1, 2 and 4 of the Cu/Zn superoxide dismutase gene (SOD1) on chromosome 21. A variant banding pattern was observed for exon 1. Sequencing studies showed a previously undescribed T to A missense mutation at position 8 in exon 1 of the SOD1 gene. This mutation results in the elimination of an Eco57I restriction site. Whereas the index patient was heterozygous for this restriction site, 50 unrelated healthy controls and an unaffected brother were not. The mutation lies in a region involved in dimer contact in the three-dimensional structure of the SOD1 protein. This region comprises other known sites for ALS-causing mutations.

Adult↗

PubMed1997

Integrative erectile biology: the role of signal transduction and cell-to-cell communication in coordinating corporal smooth muscle tone and penile erection.

The contractility of corporal smooth muscle plays a critical role in the entire erectile process in man. Moreover, in the absence of severe vascular disease, or congenital or other structural abnormalities/malformations, relaxation of the corporal smooth muscle is both necessary and sufficient to elicit a sustained erection. As such, understanding the initiation, maintenance and modulation of corporal smooth muscle tone is an absolute prerequisite to the improved understanding, diagnosis and treatment of erectile dysfunction. Despite this fact, identification of both the precise mechanistic basis by which endogenous and exogenous vasomodulators exert their effects on individual corporal smooth muscle cells, and moreover, the process by which these signals are spread among the diverse array of parenchymal cells in the paired corpora, remains somewhat of a physiological enigma. Therefore, the goal of this report is two-fold: first, to review current knowledge of the regulation of corporal smooth muscle tone at the cellular and molecular level; and second, to outline a cogent explanation for the rapid and syncytial integration of the effects of diverse stimuli among corporal smooth muscle cells in the human penis.

Animals↗

PubMed1996

Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes.

An amphipathic model peptide, KLALKLALKALKAAKLA-NH2, and its complete double D-amino acid replacement set was used to analyze the process of peptide binding at lipid vesicles of different surface charge and to determine the structure of the lipid-bound peptides using CD spectroscopy. The relationship between peptide helicity, model membrane permeability, and biological activity has been studied by dye release from liposomes and investigation of antibacterial and hemolytic activity. The accumulation of cationic KLAL peptides at and the membrane-disturbing effect on bilayers of high negative surface charge were found to be dominated by charge interactions. Independent of any structural propensity, the cationic peptide side chains bind to the anionic phosphatidylglycerol moieties. The charge interactions hold the peptides at the bilayer surface, where they may disturb preferentially lipid headgroup organization by formation of peptide-lipid clusters. In contrast, KLAL peptide interaction with bilayers of low negative surface charge is highly dependent on peptide helicity. With decreasing amounts of anionic phosphatidylglycerol in the bilayer the membrane-disturbing effect of KLAL and other helical analogs substantially increases despite drastically reduced binding affinity. Less helical peptides exhibit reduced bilayer-disturbing activity, showing that the hydrophobic helix domain is decisive for binding at and inducing permeability in membranes of low negative surface charge. It is suggested that hydrophobic interactions drive the penetration of the amphipathic peptide structure into the inner membrane region, thus disturbing the arrangement of the lipid acyl chains and causing local disruption. On the basis of the proposed model for membrane disturbance, interactions modulating antibacterial and hemolytic activity are discussed.

Amino Acid Sequence↗

PubMed1996

p21ras supports the survival of chick embryonic motor neurones.

Various trophic factors present in muscle extract can promote the survival of cultured motor neurones. However, little is known about the signal transduction pathways used in these cells. The proto-oncogene product p21ras has been shown to play an important role in proliferative and differentiative signalling pathways. We report here that cytoplasmic introduction of its oncogenic form, p21ras(G12V), fully supports the in vitro survival of chick embryonic motor neurones. The proto-oncogenic form of p21ras also showed a dose-dependent survival effect, while a C-terminally truncated counterpart of p21ras(G12V) was ineffective. These results suggest an involvement of p21ras in signal transduction pathways leading to motor neurone survival and may be of relevance for the development of therapeutic strategies for motor neurone disease.

Animals↗

PubMed1996

Recombinant human ligand for MPL, megakaryocyte growth and development factor (MGDF), stimulates thrombopoiesis in vivo in normal and myelosuppressed baboons.

Megakaryocyte growth and development factor (MGDF) is a ligand for c-mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5-fluorouracil (5-FU). rHuMGDF was administered to normal baboons as a single s.c. injection at doses of 1, 10, 25 and 50 micrograms/kg/day for 10 days and, as a control, heat-inactivated MGDF was administered at a dose of 10 micrograms/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat-inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7-, 3.4-, 5.1- and 4.0-fold above baseline in animals administered 1, 10, 25 and 50 micrograms/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 micrograms/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in the circulation. Following a single dose of 5-FU (120 mg/kg) animals were given either saline or pegylated (PEG) rHuMGDF (25 micrograms/kg/day) for 14 days. Platelet counts recovered to baseline by 13.8 +/- 1.8 days for PEG-rHuMGDF-treated baboons compared with 16.8 +/- 0.6 days for saline treated controls. Marrow biopsies revealed more rapid recovery of overall marrow cellularity and megakaryocytes in PEG-rHuMGDF-treated animals compared with controls. Thus, rHuMGDF specifically stimulates thrombopoiesis in normal and myelosuppressed baboons. rHuMGDF may be useful for stimulating thrombopoiesis in humans in clinical settings after myelosuppression.

Animals↗

PubMed1996

Swelling, intracellular acidosis, and damage of glial cells.

Cerebral ischemia and severe head injury among others are associated with a limited availability of oxygen, leading to cell catabolism as well as anaerobic glycolysis. Resulting metabolites, such as arachidonic- and lactic acid, can be expected to leak into perifocal brain areas, contributing there to cytotoxic swelling and damage of neurons and glia. Since elucidation of mechanisms underlying cell swelling and damage in the brain is difficult in vivo, respective investigations were carried out in vitro using suspended glial cells. Thereby, effects of arachidonic acid (AA) and of lactacidosis on glial cell volume, intracellular pH (pHi), and cell damage were analyzed utilizing flow cytometry. AA led to an immediate, dose dependent swelling and intracellular acidosis of glial cells. A concentration of 0.1 mM increased cell volume to 110% of control and decreased pHi to 7.05. Whereas glial swelling was permanent, pHi recovered to baseline after 90 min. Cell viability of 90% remained unchanged after addition of AA up to 0.1 mM, while at 0.5 mM it was significantly decreasing. Glial swelling from AA was nearly completely inhibited by the aminosteroid U-74389F or by using a Na(+)-free suspension medium for the experiment. Acidification of the medium to pH 6.8 or 6.2 led to a cell volume of 110% or 120% of control without affecting cell viability. The cells were not capable to defend their normal pHi during lactacidosis of the suspension medium but became acidotic as well. Addition of amiloride or utilization of Na(+)-free medium inhibited cell swelling from lactacidosis, while intracellular acidosis was even more pronounced. The results indicate that AA as well as acidosis are potent mediators of glial swelling and damage at levels found under pathophysiological conditions in the brain in vivo. Whereas intracellular acidification caused by AA was reversible, glial cells were unable to regulate their pHi during maintenance of extracellular acidosis. Concerning the mechanisms of glial swelling by AA, the production of oxygen- and lipid radicals might play a major role in the swelling process. The results indicate a role of the Na+/H(+)-antiporter in acidosis-induced glial swelling, whereas the exchanger has a limited significance for maintenance of pHi. As seen, the final pathway of glial swelling from both, AA and lactacidosis, requires a net influx of Na(+)-ions, probably together with Cl-ions, and osmotically obliged water.

Acid-Base Equilibrium↗

PubMed1996

Interaction of the receptor tyrosine kinase p145c-kit with the p210bcr/abl kinase in myeloid cells.

The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210 kDalton, p210bcr/abl, which stimulates a variety of cytosolic signalling intermediates. The effects of bcr/abl on the activity of growth factor receptors are less well known. In order to investigate interaction of p210bcr/abl with the receptor tyrosine kinase p145c-kit, we used two myeloid, factor-dependent cell lines, MO7 and 32D, to generate bcr/abl positive sublines, MO7p210 and 32Dp210, by transfection with the bcr/abl gene. Since 32D and 32Dp210 cells did not express p145c-kit, a c-kit retrovirus was used to generate c-kit positive cell lines (32Dkit, 32Dp210kit). In contrast to MO7 and 32Dkit cells, MO7p210 and 32Dp210kit cells were factor independent and did not respond to the growth-promoting effects of recombinant human Steel factor (rhSF). Preincubation with a monoclonal antibody (MAb) neutralizing the binding of SF to p145c-kit did not affect the growth of MO7p210 cells, thus eliminating the possibility of an autocrine SF secretion. 32Dkit cells transfected with bcr/abl containing an inactivating point mutation (Lys-->Arg271) in the Abl kinase domain (32Dp210(Arg271)kit) retained their responsiveness to the effects of rhSF. Immune complex kinase assays showed that the kinase activity of p145c-kit was several-fold higher in MO7p210 and 32Dp210kit cells than in MO7, 32Dkit and 32Dp210(Arg271)kit cells, suggesting that Abl kinase activity was necessary to activate p145c-kit. Co-immunoprecipitation experiments with anti-Kit and anti-Abl MAbs demonstrated that p145c-kit and p210bcr/abl were associated in an intracellular complex in human bcr/abl positive, c-kit positive cell lines (MO7p210; GM/SO). Finally, colony assays with bone marrow from bcr/abl positive CML patients showed that the haemopoietic progenitors of three of four patients did not respond to rhSF. Taken together, the results suggest that p145c-kit can be activated by p210bcr/abl via an Abl-kinase dependent mechanism involving the complex formation of both proteins. These findings could explain some clinical features (basophilia, increase of immature myeloid cells) of chronic-phase CML.

Cell Division↗

PubMed1996

Plasma and urine salsolinol in humans: effect of acute ethanol intake on the enantiomeric composition of salsolinol.

The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R-enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S-enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.

Adult↗

PubMed1996

[Not Available].

Explore the source record for details and available documents.

Austria↗

PubMed1995

Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells.

Although substance P is known to take part in the regulation of the anterior pituitary, no conclusive evidence for the expression of the tachykinin NK1 receptor has been found yet in the pituitary or pituitary derived cells. With the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the low abundant transcripts of the NK1 receptor in the rat pituitary and in the AtT20 cell line (clone D16v). Furthermore, the functional expression of the NK1 receptor in AtT20 cells was confirmed by activation of the phosphatidylinositol-calcium second messenger system when the cells were treated with substance P. In addition, binding studies also indicated the functional expression of this receptor in AtT20 cells. Thus we provide the first evidence that the NK1 receptor is expressed in AtT20 cells and the rat pituitary.

Animals↗

PubMed1995

Epiluminescence microscopy. A useful tool for the diagnosis of pigmented skin lesions for formally trained dermatologists.

BACKGROUND AND DESIGN: Epiluminescence microscopy (ELM) is a noninvasive technique that, by employing the optical phenomenon of oil immersion, makes subsurface structures of the skin accessible for in vivo examination and thus provides additional criteria for the clinical diagnosis of pigmented skin lesions. At present, almost all studies about the value and clinical importance of ELM are based on data derived from ELM experts (ie, dermatologists specifically trained in this technique). In the present study, we attempt to determine whether the clinical diagnosis of pigmented skin lesions is significantly improved using ELM and whether ELM-trained individuals and dermatologists not trained in this technique profit equally from this technique. Randomly selected histologically proven pigmented skin lesion specimens, photographed with (ELM) and without oil immersion (surface microscopy) were presented by slide projection to six ELM experts and 13 ELM nonexperts (ie, dermatologists not formally trained in ELM) for diagnosis. To evaluate the diagnostic performance of ELM experts and nonexperts with and without the oil immersion technique (ie, ELM vs surface microscopy), the following parameters were obtained: intraobserver and interobserver agreement by kappa statistics and sensitivity and specificity of diagnostic performance. RESULTS: Our results show that by using the ELM technique the ELM experts reach a substantially better intraobserver agreement than nonexperts (median kappa, 0.56 vs 0.36). The interobserver agreement was markedly increased in the ELM experts group (average gain, 7%) but decreased in the ELM nonexperts group (average loss, 6%). The sensitivity of diagnosis was significantly increased in the ELM experts group (average gain, 10%), but decreased in the nonexperts group (average loss, 10%). Finally, the specificity of diagnosis was excellent in the ELM experts group, both with and without oil immersion (0.91) and was somewhat improved by ELM in the nonexperts group (0.77 vs 0.85). CONCLUSIONS: We conclude that the ELM technique increases sensitivity in formally trained dermatologists, but may decrease the diagnostic ability in dermatologists not formally trained in the ELM technique. Consequently, formal broad-based training in ELM should be offered to the dermatologic community.

Clinical Competence↗

PubMed1995

DNA polymerase delta of Physarum polycephalum.

DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.

Amino Acid Sequence↗

PubMed1995

Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes.

Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolites were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF2 alpha and LTB4 by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36 +/- 0.055 mM were found at 20 min, while 0.275 +/- 0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF2 alpha and LTB4. Following addition of 0.01 or 0.5 mM, concentrations of PGF2 alpha increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB4 rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1995

Effects of voluntary ethanol ingestion on the POMC gene expression in the rat pituitary and on the plasma beta-endorphin content.

Studies investigating the influence of chronic ethanol treatment on the beta-endorphin content and the proopiomelanocortin (POMC) gene expression in the rat pituitary revealed contradictory results. Because of this we decided to start a more complex study to investigate the effects of isolation stress, chronic ethanol treatment and voluntary ethanol consumption on the POMC mRNA level in the rat pituitary. The immediately prepared total RNA from rat pituitaries was used in hybridization experiments (Northern- and Dot-blots). The results suggest a correlation between the POMC gene expression and the different fashions of 'living conditions' tested. So the POMC gene expression in long-term alcohol-treated animals was decreased supporting the theory of beta-endorphin deficiency in alcoholism. More interestingly, data obtained from the group of voluntary ethanol consumption suggest an inverse correlation between total ethanol ingestion and POMC gene expression. This indicates the importance of the method of ethanol administration. Consistent with a decreased POMC gene expression in the pituitary during chronic ethanol treatment are previous studies showing a decrease in the plasma beta-endorphin content in such situations. Surprisingly, in the present study the plasma beta-endorphin levels measured by radioimmunoassay were only slightly decreased in chronically ethanol-treated rats. This may be due to dysregulatory effects of ethanol on post-translational processing, degradation and/or release of beta-endorphin.

Alcohol Drinking↗

PubMed1995

[Clinical diagnosis in sleep laboratory patients based on ICD-10, DSM-III-R and ICSD classification criteria].

For the diagnosis of sleep disorders, 3 different standardized classification systems are available: the International Statistical Classification of Diseases and Related Health Problems (ICD-10), the Diagnostic and Statistical Manual of Mental Disorders (DSM-III-R/DSM-IV) and the International Classification of Sleep Disorders (ICSD). These 3 classification schemata were comparatively evaluated in 50 sleep-disturbed patients who were admitted within 1 year to a non-specialized sleep laboratory for diagnostic evaluation and treatment. 17 female and 33 male sleep-disturbed patients, aged 54 +/- 12 years, were recorded polysomnographically in 3 subsequent nights (adaptation night, baseline/diagnosis night, treatment night) for measuring objective sleep quality. The subjective sleep quality as well as the subjective and objective awakening quality was assessed by means of rating scales, as well as psychometric and psychophysiological test battery. During the day, EEG, EEG-mapping, psychodiagnostic tests as well as, in many cases, pulmonary function, otolaryngological, CT, MRT and pharyngometric investigations were carried out. Psychic disorders were the leading cause for sleep problems in all 3 classification systems. Based on the ICD-10, the most frequent diagnosis was non-organic insomnia (46%), followed by sleep apnea (18%) and other organic sleep disorders (14%). Based on the DSM-III-R, 46% of the patients were diagnosed as insomnias based on another mental disorder, 38% as organic hypersomnias and 14% as parasomnias. Based on the ICSD Classification, sleep disorders associated with anxiety disorders were leading (30%), followed by sleep disorders based on affective disorders (16%), obstructive snoring (14%), primary snoring (8%) and sleep disorders based on neurological disorders (6%). While the broader ICD-10 and DSM-III-R diagnoses are syndrome-etiologically oriented and may be easily utilized by the practicing physician, the more narrowly defined, extensive, pathogenetically oriented polysomnographic features including ICSD diagnoses are suited better for the specialist.

Adult↗

PubMed1994

Mechanisms of glial swelling by arachidonic acid.

The effect of arachidonic acid (AA, 20:4) was analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in an incubation chamber under continuous control of pH, pO2, and temperature. Cell swelling was quantified by flow cytometry. After a control period, the suspension was added with AA at concentrations of 0.01 to 1.0 mM. Administration of AA induced an immediate, dose dependent swelling in C6 glioma cells or astrocytes. AA-concentrations of 0.01 mM led to an increase of the glial cell volume to 103.0 +/- 1.0% of control, 0.1 mM to 110.0 +/- 1.5%, and 1.0 mM to 118.8 +/- 1.5% within 10 min. The swelling response to linoleic acid (18:2) was only about half of what was found when AA was administered at a concentration of 0.1 mM, whereas stearic acid (18:0) did not induce any cell volume changes. Inhibition of the cyclo- and lipoxygenase pathway by BW 755C did not prevent glial swelling from AA, whereas it was reduced by SOD, or almost completely abolished by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Replacement of Na(+)- and Cl- -ions in the suspension medium by choline chloride was also associated with complete abolishment of cell swelling from AA. The results demonstrate an impressive efficacy of arachidonic acid to induce glial swelling which might be attributable to activation of lipid peroxidation by the fatty acid, leading to an increased Na(+)-permeability and subsequent influx of water into the cells.

Animals↗

PubMed1994

Swelling, acidosis, and irreversible damage of glial cells from exposure to arachidonic acid in vitro.

Swelling and damage of C6 glioma cells and of primary cultured astrocytes were analyzed in vitro during incubation with arachidonic acid (AA; 20:4). The cells were suspended in a physiological medium supplemented with AA at concentrations of 0.001-1.0 mM. Cell swelling was quantified by flow cytometry with hydrodynamic focusing. Flow cytometry was also utilized for assessment of cell viability by exclusion of the fluorescent dye propidium iodide and for measurement of the intracellular pH (pHi) by 2',7'-bis-(2-carboxyethyl)-5(and -6)carboxy-fluorescein. Administration of AA caused an immediate dose-dependent swelling of C6 glioma cells, even at a concentration of 0.01 mM. At this level cell volume increased within 20 min to 105.0% of control, at 0.1 mM to 111.0%, while at 1.0 mM to 123.7%. Following a phase of rapid cell volume increase, swelling leveled off during the subsequent observation period of 70 min. Viability of the C6 glioma cells was 90% under control conditions. It remained unchanged after raising AA concentrations to 0.1 mM. At 0.5 mM, however, cell viability fell to 72.8%, and at 1.0 mM to 32.7%. pHi of the glioma cells was 7.3 under control conditions. In parallel with the early swelling phase, AA led to a dose-dependent decrease of the intracellular pH and an elevated lactate production of the cells. During incubation with 0.1 mM AA, pHi decreased to 7.06 after 5 min, but recovered to normal subsequently. In addition, swelling-inducing properties of linoleic (18:2) or stearic (18:0) acid were analyzed for evaluation of the specificity of glial swelling induced by AA. Whereas stearic acid (0.1 mM) failed to induce a swelling response, linoleic acid (0.1 mM) was found to be effective. The volume increase of the glial cells, however, was only half of that found during exposure to AA at the same concentration. Further, glial swelling from AA or linoleic acid was completely inhibited by the aminosteroid U-74389F, an antagonist of lipid peroxidation. Finally, omission of Na+ ions in the suspension medium with replacement by choline led also to inhibition of the cell volume increase by AA. Experiments using astrocytes from primary culture confirmed the swelling-inducing properties of AA at a quantitative level, whereas vulnerability of the cells to AA was increased. The present results demonstrate an important role of AA in cytotoxic swelling and irreversible damage of glial cells at concentrations that occur in vivo in cerebral ischemia or trauma.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗