Ionic determinants of spontaneous activity in clusters of cultured cardiac cells from newborn rats.

The spontaneous activity of cell clusters derived from ventricle cells of newborn rats was studied using a recording television microscope. The influence of varying concentrations of sodium, potassium, calcium, tetrodotoxin (TTX), and that of 2 mM MnCl2 was tested. The spontaneous activity of the cell clusters persisted in TTX but it was abolished by Mn. The beating rate increased when [Ca]0 and [Na]0 were changed from 0.3 mM to 3.0 mM and from 30 mM to 75 mM; it decreased with a change of [Na]0 from 75 mM to 142 mM. It is concluded that electrogenesis in their behavior to very young embryonic rat heart cells or cells of the rabbit sinoauricular node.     

Iodothyronine-5'-deiodinase activity in progenies of hypothyroid rats

Iodothyronine-5'-deiodinase activity (I-5'DA) was measured in the progenies of control rats, hypothyroid (Tx) rats, and hypothyroid treated with ovine GH (Tx + GH) during gestation. The enzyme was measured in cerebral cortex and cerebellum at 22 days gestation and at 5, 10, 30 and 60 days postpartum. In addition, the pituitary I-5'-DA was assessed in the postnatal animals. The experiments were undertaken because the tissues of the progenies of rats that were hypothyroid during pregnancy appeared in many ways to resemble those of hypothyroid animals, even at ages when serum thyroxine (T4) and triiodothyronine (T3) levels were normal. It was found that the progenies of Tx mothers had low liver 5'-deiodinase activities. This is a likely cause of the low serum T3 levels with normal T4 levels seen in these progenies in the neonatal period. Cerebral and cerebellar 5'-deiodinase activities were low in these progenies during the thyroid hormone-dependent perinated period of brain development. The progenies of GH-treated Tx dams had higher enzyme activities than the progenies of untreated Tx dams. These pups from GH-treated Tx mothers have been shown previously to have significantly less neurological impairment than the progenies of untreated Tx mothers. As most of the brain intracellular T3 is produced in situ, a functional thyroid deficiency could result from such a 5'-deiodinase deficiency. As the deiodinase deficiency was still seen in the progenies of Tx mothers at 60 days of age, such a deficiency could explain why, even though serum T4 and T3 levels were normal, brain metabolism was in many ways characteristic of hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of 5'-deiodinase activity in rats differing in hepatic lipogenic activity

Studies of the relationship of hepatic 5'-deiodinase activity to hepatic lipogenic capacity were conducted. Rats of the Zucker, BHE, and Sprague-Dawley strains were used. BHE and Sprague-Dawley rats were starved and refed a 65% glucose diet, whereas lean and obese Zucker rats were fed a stock diet; the rats were thus different in hepatic lipogenic capacity. After hepatic 5'-deiodinase activity was determined, we found that rats genetically predisposed to increased hepatic lipogenesis had less deiodinase activity than rats without this genetic feature. The role of the interaction between the thyroid hormones and glucocorticoid in the activity of hepatic deiodinase was also studied. Adrenalectomized (ADX) or intact BHE and Sprague-Dawley rats were injected with saline, thyroxine, or triiodothyronine and either saline or glucocorticoid. The normal Sprague-Dawley rats made predictable adjustments to their deiodinase activity when their hormonal status was manipulated, whereas the BHE rats responded as though these manipulations were corrections rather than additions or deletions.     

Comparison of isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate stimulation of thyroxine 5'-deiodinase activity in cultured pineal glands from euthyroid and hypothyroid rats

Thyroxine 5'-deiodinase was increased by isoproterenol and dibutyryl adenosine cyclic 3',5'-monophosphate in a dose- and time-related manner in cultured rat pineal gland. Basal and stimulated activity was higher in glands from hypothyroid than from euthyroid animals. Our data suggest direct beta-adrenergic stimulation of intracellular cyclic AMP may be involved in the regulation of pineal thyroxine 5'-deiodinase activity.     

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Impaired basal and noradrenaline-induced iodothyronine 5'-deiodinase activity in brown adipose tissue from pregnant and lactating rats

Basal iodothyronine 5'-deiodinase activity is lowered in brown fat from 20-day pregnant, 5 and 15-day lactating rats when compared with virgin controls. Acute noradrenaline treatment caused a seven fold increase in 5'-deiodinase activity in brown fat from virgin control rats. Late pregnant and lactating rats showed a reduction in noradrenaline-induced 5'-deiodinase activity in brown adipose tissue and the maximum impairment was observed in 15-day lactating rats. Lowered 5'-deiodinase activity in brown fat during late pregnancy and lactation correlates with the known reduction in the thermogenic activity of the tissue during these situations and agrees with the proposal that the rate of 3,5,3'-triiodothyronine generated in situ because of thyroxine 5'-deiodination could be an essential event related to thermogenesis in brown fat. Even though the relationship between 3,5,3'-triiodothyronine generation in the tissue and the specific thermogenic mechanisms of brown fat is unknown, present results indicate a close link between the thermogenic and 5'-deiodinase activities in physiological situations when brown adipose tissue needs to adapt to a low activity, such as that of the breeding cycle.     

Effect of manganese on the development of glial cells cultured from prenatally alcohol exposed rats

Maternal alcohol abuse is known to produce retardation in brain maturation and brain functions. Using cultured glial cells as a model system to study these effects of alcohol we found an alcohol antagonizing property for manganese (Mn). Mn was added to the alcohol diet (MnCl₂ 25 mg/l of 20% v/v ethanol) of pregnant rats. Glial cells were cultured during 4 weeks from cortical brain cells of pups born to these mothers. Several biochemical parameters were examined: protein levels, enzymatic markers of glial cell maturation (enolase and glutamine synthetase), superoxide dismutase a scavenger of free radicals produced during alcohol degradation. The results were compared to appropriate controls. A beneficent effect of Mn was observed for the pups weight which was no more significantly different from the control values. Protein levels, enolase and glutamine synthetase activities were increased mainly during the proliferative period when Mn was added to the alcohol diet compared to the only alcohol treated animals. This Mn effect was not found for superoxide dismutase in cultured glial cells but exists in the total brain of the 2 week-old offspring. In the total 2 and 4 week-old brain the alcohol induced decrease of enolase and glutamine synthetase was also antagonized by the Mn suplementation. Our data suggest that Mn may act as a factor overcoming at least partially some aspects of alcohol induced retardation of nerve cell development.     

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000- 70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.     

Microgravity-induced apoptosis in cultured glial cells

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.     

GABA metabolism in cultured glial cells

GABA-transaminase has been characterized in cultured astrocytes. It is identical to the synaptosomal and perikaryal enzyme in terms of charge, molecular weight, and stability, but it differs in its affinity for GABA, which is much higher in the glial compartment. GABA-transaminase has been shown to be inducible by high GABA concentrations, which suggests that astrocytes have the possibility not only to transport GABA but also to metabolize the amino acid which is taken up.     

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.     

Suspension culture of myocardial cells from newborn rats.

Myocardial cells from newborn rats were held in a spinner type culture for 2 days and then explanted into culture flasks. Three main cell types were observed: single multipolar cells of embryonic type, cell aggregates containing 10 to 50 connected cells, and bipolar cells retaining some adult characteristics. Except for the latter, up to 95% of intact cells settled and were beating 6 hours after explantation. The percentage of fibroblast-like cells was drastically reduced when compared with conventional cultures. Cell debris could be removed 2 hours after explantation by changing the culture medium, or more effectively by a density step centrifugation using Lymphoprep or Lymphoprep-Ficoll mixtures.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Characteristics and adaptive regulation of glycine transport in cultured glial cells.

The transport of glycine in C6 glioma cells takes place mainly in a heterogeneous Na+-dependent manner which can be resolved into different components. A Na+- and Cl(-)-dependent component with high affinity for glycine is pH-sensitive and inhibited by sarcosine, all these characteristics corresponding to System Gly. The low-affinity component of the transport of glycine can be discriminated as two components, namely System A and System ASC. The main proportion of glycine transport through the low-affinity system is carried out by the ASC System, which appears to be constitutively expressed by the cells. The adaptive response of the low-affinity Na+-dependent transport of glycine to amino acid deprivation was identified with System A on the basis of its ion-dependency, pH-sensitivity and by inhibition analysis. The possible physiological role of the high- and low-affinity components of the transport system for glycine in glial cells is discussed.     

Increased pituitary thyroxine 5'-deiodinase activity in adult rats rendered hyper- or hypo-thyroid during perinatal life

Perinatal thyroid dysfunction in the rat leads to permanent alterations in pituitary TSH secretion in the adult animal. Thus, neonatal hyperthyroidism (NH) and perinatal hypothyroidism (PH) both result in apparent increased pituitary sensitivity to the feedback effects of thyroid hormones in the adult rat. To determine if increased intrapituitary generation of triiodothyronine (T3) might account for these observations, we measured thyroxine (T4) 5'-deiodinase activity in pituitary homogenates of adult NH and PH rats. NH was induced by injecting neonatal rats with 12 daily sc injections of T4 (0.4 microgram/g body weight (BW]. Control rats received vehicle alone. PH was induced by administering 0.05% 6-n-propylthiouracil in the drinking water to pregnant dams from the 16th day of gestation through the 12th day postpartum. Thereafter, a normal water supply was substituted. NH and PH rats were allowed to mature and were sacrificed at 105 days of age. Serum T4, T3, and TSH concentrations were measured by radioimmunoassay. Pituitary T4 5'-deiodinase activity was assessed by the measurement of T3 formation by pituitary homogenates incubated in the presence of 0.65 microM T4 and 100 mM dithiothreitol at 37 degrees C for 90 min. Body weights of adult NH and PH rats were slightly but not significantly decreased compared with control rats. Relative pituitary gland weight (milligrams per 100 g BW) was significantly decreased in adult PH rats (P less than 0.005) but not in adult NH rats. In adult NH rats, serum T4 and T3 concentrations were significantly decreased (P less than 0.01) compared with control rats. Serum TSH concentrations were similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.