Protein metabolism in an astroglial primary culture

Protein metabolism was studied in astroglial primary cultures, grown for different time periods. Removal of fetal calf-serum for two days led to a morphological differentiation consisting of retraction of cell soma and extension of processes. There was a prominent decrease in total soluble protein and a decrease in [³H]valine incorporation into soluble protein. Dibutyrylcyclic-3-5-adenosine monophosphate (dB-cAMP)-treatment for two days also changed morphology in a similar way, but had no effect on [³H]valine incorporation into protein. After addition of soluble brain extract to the cultures an increased [³H]valine incorporation into soluble protein was seen together with a morphological differentiation, more pronounced in the presence than in the absence of fetal calf-serum. Proteins were secreted from the cells into the incubation medium and studied by electrophoresis. The more prominent protein bands had m.w. in the region of 10,000–100,000 daltons. The amount of newly synthesized proteins released into the medium was unchanged (or decreased slightly in 14 and 16 day old cultures) after addition of dB-cAMP or soluble brain extract, and was much reduced after removal of fetal calf-serum.     

Inhibition by n-3 fatty acids of arachidonic acid metabolism in a primary culture of astroglial cells

Docosahexaenoic acid (226 n-3) was present in low concentrations in a primary culture of rat brain astroglial cells, when compared to brain cortex. We have thus supplemented these cells with this fatty acid and investigated the effects of its incorporation in cell phospholipids on the conversion of arachidonic acid, 204 n-6, through the cyclo and lipoxygenase pathways, after cell stimulation. Docosahexaenoic acid-enriched cells produced less thromboxane B₂ and 6-keto-Prostaglandin F₁ and markedly less 12-hydroxyeicosatetraenoic acid than unsupplemented cells, after stimulation with the Ca²⁺-ionophore A23187. The production of 15-hydroxyeicosatetraenoic acid from arachidonic acid was slightly increased in docosahexaenoic acid-supplemented cells. We have also supplemented these cells with eicosapentaenoic acid (205 n-3) and, in addition to accumulation of this fatty acid in cell phospholipids, we found elevation of 225 n-3 and some increment of 226, confirming that glial cells are able to convert eicosapentaenoic acid to the long chain, more unsaturated derivatives. In conclusion, n-3 fatty acids, when supplemented to glial cells, appear to modulate the arachidonic acid cascade and to be converted through the elongation and desaturation pathways.     

Synthesis and secretion of nerve growth factor by mouse astroglial cells in culture

Astroglial cells cultured from the mouse brain have been found to synthesize and secrete a material(s) with nerve growth factor-like immunoreactivity (NGF-LI) into their culture medium. A material(s) with NGF-LI showed identical properties to those of beta NGF purified from the mouse submaxillary gland in immunoreactivity, molecular weight, isoelectric point, and neurite outgrowth stimulatory activity. These results indicate that astroglial cells cultured from mouse brain are able to synthesize and secrete beta NGF in culture.     

Transport of valproate and its effects on GABA uptake in astroglial primary culture

The antiepileptic drug Na⁺-valproate (VPA) is a broadspectrum anticonvulsant. It has been proposed to be involved in the inhibitory mechanisms of GABA-ergic systems. In this study, transport of the drug and possible influence on the GABA uptake were investigated in primary astroglial cell cultures from newborn rat cerebral cortex. The results show a Na⁺ and K⁺ independent high affinity uptake for VPA, withK _m andV _max not significantly different from those observed for the GABA uptake. In the presence of the drug, the K_m-value of the GABA uptake increased. The GABA uptake inhibitors guvacine, (RS)-Cis-4-OH-nipecotic acid and 4,5,6,7-tetrahydroisoxazolo (4,5-c) pyridin-3-ol (THPO) did not influence upon the uptake of VPA, suggesting a transport mechanism for the drug, separated from the GABA uptake carrier.     

Sodium and potassium uptake in primary cultures of proliferating rat astroglial cells induced by short-term exposure to an astroglial growth factor

Primary cultures of rat astroglial cells were maintained in a serum-free medium. After 8–10 days of cultivation the cells were exposed to an astroglial growth factor (AGF2) for short periods (1–120 min). Subsequently, uptake of²²Na⁺ and⁴²K⁺ into control and AGF2-pretreated cells was studied. Assay of the Na⁺ and K⁺ values in the cells was also performed by atomic absorption spectrometry. Treatment of rat astroglial cells with AGF2 resulted in a significant increase of the uptake of both Na⁺ and K⁺ depending on the duration of the exposure period. To reach the maximum increase of cation uptake, 6–10 min and 30 min of AGF2 pretreatment were needed for Na⁺ and K⁺, respectively. Amiloride blocked this increase of Na⁺ and K⁺ uptake elicited by AGF2 pretreatment, but the control cells were amiloride resistant. Treatment with AGF2 increased the ouabain sensitivity of the K⁺ uptake as that: 10^–4 M ouabain inhibited K⁺ uptake of the AGF2-treated cells to the same degree as 5×10^–3 M ouabain with the control cells. The Na⁺ uptake of AGF2-treated cells, however, exhibited no relevant changes in the presence of ouabain. A significant part of the AGF2-induced K⁺ uptake could be inhibited by both ouabain and amiloride, but a ouabain-resistant and amiloride-sensitive component also was revealed. The furosemide sensitivity of both Na⁺ and K⁺ uptake into cultured astroglial cells was also significantly increased by AGF2. Our findings suggest that short-term exposure of cultured glial cells to AGF2 induces these very early ionic events: 1) The appearance of a relevant amiloride-sensitive Na⁺/H⁺ exchange, and as a consequence of increased Na⁺ entry into the cells, secondary activation of the ouabain-sensitive K⁺ uptake via the Na⁺,K⁺-pump. 2) A direct effect of AGF2 on the Na⁺,K⁺-pump assembly in the membrane, resulting in increased Na⁺ sensitivity of the inner pump sites and enhanced ouabain sensitivity of the external K⁺-binding sites. 3) An increase of ouabain-resistant but amiloride- or furosemide-sensitive Na⁺ and K⁺ uptake.Some of the results reported here were presented as a lecture at a Symposium on Na⁺/H⁺ exchange of the Second European Congress on Cell Biology, Budapest, Hungary, 1986.     

Active accumulation and exchange transport of chloride in astroglial cells in culture

Chloride transport in primary cultures of astroglial cells from rat brain shows saturation kinetics, is inhibited by SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), and exchanges with HCO-3. These properties are similar to those of the anion exchange system in erythrocytes. Estimated of intracellular C1- concentration([C1-]i) in the primary cultures give values in the range of 31 to 43 mM, which are 3- to 5-times greater than predicted from equilibration with an average measured membrane potential of -70 mV, suggesting that these cells also actively accumulate Cl-.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

Adrenergic and cholinergic stimulation of arachidonate and phosphatidate metabolism in cultured astroglial cells

Primary cultures of neonatal rat brain polygonal astroglia, or cells of the C62B glioma line, were incubated with [1-¹⁴C]arachidonic acid (AA) in culture for 18 hr. In both culture systems, more than 80% of the added [1-¹⁴C]AA was taken up into cellular glycerolipids; less than 1% of the radioactivity in the cells was present in an unesterified form. When prelabeled C62B cells were stimulated with acetylcholine (ACh), there was a rapid accumulation of arachidonyl-phosphatidic acid (PA) accompanied by a liberation of [1-¹⁴C]AA. A variety of other neurotransmitters failed to activate this response in C62B glioma cells, primary astroglia generated PA and liberated [1-¹⁴C]AA in response to several neurotransmitters (i.e., ACh, norepinephrine, glutamate, and histamine) Treatment of astroglia with a combination of norephrine, ACh, and histamine resulted in a greater production of PA and free [1-¹⁴C]AA than did treatment with any one of these neurotransmitters alone. The results suggest that cultures of astroglia can respond to several different neurotransmitters with specific changes in AA and PA metabolism. Thus, a variety of neurotransmitters initiate cascades of lipid metabolism which may be of physiological significance in glial function.Special Issue dedicated to Prof. Eduardo De Robertis.     

Sodium and potassium uptake in primary cultures of rat astroglial cells induced by long-term exposure to the basic astroglial growth factor (AGF2)

Astroglial cell cultures were derived from newborn rat forebrain and cultured for 5 days in serum containing-, and for an additional 4 days in a serum-free, defined medium. At the end of this 9-day-long period, basic astroglial growth factor (AGF2) was administered to the culture medium (10 ng per ml). Cells were subsequently cultured in AGF2 containing serum-free, defined medium for further two weeks. At definite intervals of culturing, unidirectional influx of both Na⁺ and K⁺ (I_Na and I_K, respectively) was determined by applying²²Na and⁴²K. The AGF2-treated cultures showed highly increased, amiloride-sensitive I_Na at the early exposure period (2–8 hours), similar to that we have reported about cultured astroglia exposed to AGF2 for minutes. They also exhibited significant furosemide-sensitive-, while relatively poor ouabain-sensitive component of I_Na. However, at later periods of exposure to AGF2, I_Na was significantly reduced, particularly due to the decrease of its amiloride-sensitive component, while its furosemide-sensitive component further increased with the time of AGF2 treatment. In contrast to I_Na, the I_K in the cultures exposed to AGF2 increased significantly in the course of the long-term exposure period, particularly the ouabain-, and furosemide-sensitive-components, while its amiloride-sensitive component, similarly to that of I_Na, decreased. Our findings show that the initial activation of the Na⁺/H⁺ (or K⁺/H⁺) exchange, what characterized the cation transport changes by short-term exposure of astroglial cells to AGF2 in our previous study, comes relatively soon to a cessation but activation of the Na⁺, K⁺-pump and the furosemide-sensitive Na⁺ and K⁺ influxes further increases. Thus, they suggest the possibility that furosemide-sensitive cation movements play a role, besides the Na⁺, K⁺-pump, in the control of glial cell differentiation.Cente de Neurochimie du CNRS.Special issue dedicated to Dr. Paola S. Timiras.     

Characterization of spermine uptake by Ehrlich tumour cells in culture

Summary. Spermine is taken up by Ehrlich ascites tumour cells through a specific, saturable, temperature and energy-dependent transport system with a remarkably low affinity constant for spermine (around 1 μM). In the absence of a potassium ion gradient through the plasma membrane, spermine uptake remains saturable but the value of the K_m for spermine is much higher (153 μM). Difluormethylornithine treatment (3 mM for 48 h) induces significant increases in V_max values (up to 9-fold) and changes in the K_m values with scarce statistical significance. Among the biogenic amines tested, only spermidine and, partly, agmatine seem to share the same transport system with spermine. No difference is observed in the rate of spermine transport when assays are carried out in the presence of 50-fold excess of ornithine or calcium, or 100-fold excess of glutamine. Received April 25, 2000 Accepted November 1, 2000     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

Utilization of mannose by astroglial cells

Uptake and metabolism of mannose were studied in astroglia-rich primary cultures derived from neonatal rat brains. A saturable component of mannose uptake was found with half-maximal uptake at 6.7±1.0 mM mannose. In addition, a non-saturable component dominated the uptake at high concentrations of mannose. Glucose, cytochalasin B, or phloretin in the incubation buffer inhibited the carrier-mediated uptake of mannose. Within the astroglial cells mannose is phosphorylated to mannose-6-phosphate. In cell homogenates, the K_M value of mannose-phosphorylating activity was determined to be 24±7 M. The V_max value of this activity is only 40% that of glucose-phosphorylating activity. Mannose-6-phosphate was converted to fructose-6-phosphate by mannose-6-phosphate isomerase. The specific activity of this enzyme in homogenates of astroglial cultures was higher than that of hexokinase. Two products of mannose utilization in astroglial cells are glycogen and lactate. The amounts of each of these products increased with increasing concentrations of mannose. In contrast to the generation of lactate, that of glycogen from mannose was enhanced in the presence of insulin. In conclusion, we suggest that mannose is taken up into the cells of astroglia-rich primary cultures by the glial glucose transporter and is metabolized to fructose-6-phosphate within the astroglial cells.     

The uptake of norepinephrine by rat uterine subepithelial cells under normal and experimental conditions

On specified days of the estrus cycle, pregnancy, pseudopregnancy, or following administration of estradiol-17 beta, progesterone, or both, nulliparous, ovariectomized rats were assigned to 1 of 3 treatment groups. Group 1 received nialamide and norepinephrine 5 hours and 15 minutes, respectively, before killing. Group 2 received nialamide alone 5 hours before killing. Group 3 received L-dihydroxyphenylalanine 2 hours before killing. Pregnant, pseudopregnant, and progesterone-treated rats from Group 1 showed a greenish-yellow fluorescence in uterine subepithelial cells. Groups 2 and 3 did not show this fluorescence. It was suggested that this fluorescence was due to the extraneuronal uptake of norepinephrine, and that progesterone is necessary for the uptake of norepinephrine by subepithelial cells since no florescence was observed in the rat estrus cycle but ovariectomized rats treated with progesterone showed florescence.     

Inhibition of the adrenergic phenotype in cultured neural crest cells by norepinephrine uptake inhibitors

Tricyclic antidepressants in combination with in vitro clonal analysis of quail neural crest cells were used to examine the role the norepinephrine uptake mechanism might play in the development of adrenergic neural crest derivatives. Norepinephrine (NE) uptake inhibitors blocked expression of the adrenergic phenotype by neural crest cells. The degree of inhibition of phenotypic expression correlated with the potency and specificity of the uptake inhibitors. The drugs acted during the early phase of in vitro development, i.e., several days before overt expression of the adrenergic phenotype in clonal culture. They were nontoxic, and a chronic exposure of the cells to NE uptake inhibitors was necessary to cause an effect. These observations suggest that norepinephrine and possibly related neurotransmitters play a direct or indirect role in the expression of the adrenergic phenotype by neural crest cells and that tricyclic antidepressants may affect neurogenesis during sensitive stages of embryonic development. The data may reflect in vivo mechanisms, since there are neurotransmitters present in the migratory pathway of presumptive sympathetic neurons and the norepinephrine uptake system is expressed in the embryo by these cells before they synthesize and accumulate catecholamines.     

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na⁺-dependent Ca²⁺ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na⁺ could be prepared by incubating them in Ca²⁺-free medium containing various concentrations of Na⁺. Cells pre-loaded with various concentrations of Na⁺ were incubated in medium containing Ca²⁺ and ⁴⁵Ca. The resulting ⁴⁵Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na⁺. Lineweaver-Burk plots of the initial rate of Ca²⁺ uptake against the external concentration of Ca²⁺ fitted well to straight lines obtained by linear regression (r > 0.95). This result shows that Ca²⁺ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na⁺-dependent Ca²⁺ uptake was accompanied by Na⁺ release and the ratio of Na⁺ release to Ca²⁺ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na⁺; (2) the coincidence of the apparent Michaelis constants for Ca²⁺ (0.1 mM); (3) the similarities of the K_i values for Co²⁺, Sr²⁺ and Mg²⁺ (Co²⁺ < Sr²⁺ < Mg²⁺) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about $\text{1}\text{100}$ that of myocardial cells. The rate of Na⁺-dependent Ca²⁺ uptake by non-excitable cells was much lower than that by myocardial cells.