Biomedical subjects

S R Max

Publications and source records attributed to S R Max.

At least 19 recordsLinked to original sources

PubMed1996

Co-expression of tyrosine hydroxylase and glutamic acid decarboxylase in dopamine differentiation factor-treated striatal neurons in culture.

We have previously shown that dopamine differentiation factors (DDF) can stimulate the novel expression of tyrosine hydroxylase (TH) in the phenotypically plastic neurons of the embryonic mouse striatum (Du et al., J. Neurosci., 14 (1994) 7688-7694; Du and Iacovitti, J. Neurosci., 15 (1995) 5420-5427). The present study sought to determine whether TH induction required down-regulation of an existing GABAergic trait in striatal neurons or whether enzymes of both neurotransmitter systems were simultaneously expressed. Immunocytochemical analysis revealed that, following treatment with DDFs, TH and the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) were co-expressed in the same neurons. Moreover, GAD enzyme activity was not affected by the dramatic increase in TH. Thus, the induction of a novel neurotransmitter phenotype in brain neurons does not appear to occur at the expense of the existing phenotype.

Animals↗

PubMed1995

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture.

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM-1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10(-7)-10(-5) M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.

Analysis of Variance↗

PubMed1993

A glutamatergic mechanism for aluminum toxicity in astrocytes.

The effect of aluminum on the metabolism of glutamate and glutamine in astrocytes was studied to provide information about a possible biochemical mechanism for aluminum neurotoxicity and its potential contribution to neurodegenerative disease. Exposure of cultured rat brain astrocytes for 3-4 d to 5-7.5 mM aluminum lactate increased glutamine synthetase activity by 100-300% and diminished glutaminase activity by 50-85%. Increased glutamine synthetase enzyme activity was accompanied by an elevated level of glutamine synthetase mRNA. Alterations in glutaminase and glutamine synthetase following aluminum exposure caused increased intracellular glutamine levels, decreased intracellular glutamate levels, and increased conversion of glutamate to glutamine and the release of the latter into the extracellular space. The results of these changes may alter the availability of neurotransmitter glutamate in vivo and may be a mechanism for the aluminum neurotoxicity observed in individuals exposed to the metal during dialysis procedures and other situations.

Amino Acids↗

PubMed1993

Unsaturated fatty acid modulation of glucocorticoid receptor binding in L2 cells.

Glucocorticoids stimulate fatty acid synthesis during late fetal lung development by inducing fatty acid synthetase. To determine whether fatty acids modulate glucocorticoid receptor binding, we investigated the in vitro effect of fatty acids on [3H]triamcinolone acetonide (TA) binding to the cytosolic glucocorticoid receptor in L2 cells, a cell line cloned from the adult rat type II cell. The L2 cell glucocorticoid receptor exhibited specific binding of [3H]TA which was saturable and appeared to be a single species of binding sites with an apparent KD = 4.9 +/- 3.7 nM and Bmax = 395.4 +/- 84.4 fmol/mg protein. The receptor had the ligand specificity typical of a physiologically relevant glucocorticoid receptor. Long-chain unsaturated fatty acids (oleic acid [18:1], linoleic acid [18:2], and arachidonic acid [20:4]) markedly inhibited [3H]TA specific binding in a dose-dependent manner, but long-chain saturated fatty acids (myristic, 14:0; palmitic, 16:0; and stearic acid, 18:0) and phospholipids had no effect. Scatchard analysis revealed a noncompetitive type of inhibition by unsaturated fatty acids. This suggests that unsaturated fatty acids modulate L2 cell glucocorticoid receptor by binding to sites different from the glucocorticoid binding sites in the receptor. We propose that unsaturated fatty acids may act as negative feedback modulators of glucocorticoid-receptor binding in the lung.

Animals↗

PubMed1991

Alterations in protein synthesis in rat liver cells by in vitro and in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Alterations in protein synthesis in rat liver cells were examined following in vitro and in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary cultured rat liver parenchymal cells were exposed to 1 nM TCDD for 23 and 47 hr. Synthesis of two proteins with molecular weights (Mr) of 26,000 and 39,000 (designated 26k-P and 39k-P, respectively), other than cytochrome P450, was increased markedly in the cells. These proteins did not have the same antigens as cytochromes P450IA1 and P450IA2. Synthesis of three proteins with Mrs of 24,000, 25,000 and 29,000, respectively, was decreased by TCDD. TCDD was administered to rats at a dose of 100 micrograms/kg body weight. The amount of five proteins (two proteins with Mr of 26,000, one of 36,000 and two of 39,000) was increased in TCDD-treated rat liver. However, the proteins increased in vivo by TCDD were distinguishable from 26k-P and 39k-P by two dimensional gel electrophoresis.

Animals↗

PubMed1990

Transcriptional regulation of glutamine synthetase gene expression by dexamethasone in L6 muscle cells.

Dexamethasone increases glutamine synthetase activity and mRNA abundance in L6 muscle cells in culture, apparently by a glucocorticoid receptor-mediated process. The data in this report reveal that increased glutamine synthetase mRNA abundance is attributable at least in part to an enhanced rate of transcription of the glutamine synthetase gene. "Nuclear runoff" assays of glutamine synthetase gene expression were performed with purified myonuclei from dexamethasone-treated or untreated L6 skeletal muscle cells. These assays showed glutamine synthetase transcription to be increased approximately 2-fold as early as 1 h after incubation of cells with dexamethasone (10(-7) M); there was no increase in the rate of transcription of the beta-tubulin gene, which served as a control. The increase in glutamine synthetase gene transcription correlates with increased glutamine synthetase enzymatic activity after dexamethasone treatment. Studies with actinomycin D indicated that the half-life of glutamine synthetase mRNA (7-8 h) is not altered by dexamethasone. Therefore, the degradation of glutamine synthetase mRNA is not affected by dexamethasone, and the increased glutamine synthetase mRNA level is attributable to increased transcription. The dexamethasone-mediated increase in glutamine synthetase mRNA abundance is glucocorticoid receptor-mediated; RU38486 (a glucocorticoid receptor blocker) completely blocked the effect of dexamethasone. The dexamethasone-mediated increase in glutamine synthetase gene transcription and steady-state mRNA level was not blocked by cycloheximide, indicating a direct effect.

Blotting, Northern↗

PubMed1990

Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture.

Metabolite control of glutamine synthetase expression (by glutamine) was studied in L6 skeletal muscle cells. Depletion of glutamine from the culture medium for 24 hours resulted in a 3-4-fold increase in glutamine synthetase activity. This effect was blocked by cycloheximide but not by actinomycin D. Addition of glutamine to L6 cells maintained in glutamine-free medium caused a rapid return of glutamine synthetase activity to the control level. As reported, dexamethasone caused a striking increase in the glutamine synthetase mRNA level. In contrast, neither depletion nor addition of glutamine caused a change in the glutamine synthetase mRNA level. Therefore, regulation of glutamine synthetase by glutamine is exerted at a post-transcriptional level.

Animals↗

PubMed1990

Induction of glutamine synthetase by 8-bromo cyclic AMP in primary cultures of rat brain astrocytes.

Glutamine synthetase (GS) is the key enzyme in cerebral glutamine production. Understanding the regulation of the expression of GS is important for definition of the control of glutamine metabolism in brain. Therefore, we studied the control of GS expression by 8-bromo cyclic AMP in primary cultures of astrocytes prepared from brains of neonatal rats. GS activity was increased by 8-bromo cyclic AMP in a dose- and time-dependent manner. This increase was associated with a corresponding increase in the steady-state level of GS mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes.

Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

ACTH1-24 stimulates muscle cell glucose uptake.

3H-2-deoxyglucose (2-DG) uptake was measured in L6A-1 rat skeletal muscle cells (a rapidly fusing subclone of L6), following addition of several concentrations (10(-16) to 10(-9)M) of the N-terminal fragment of ACTH1-24 to cells deprived of serum and insulin for 21 hours, but maintained in the presence of (5 micrograms/ml) insulin (stimulated state). There was a marked dose-dependent increase of 2-DG uptake at the various ACTH1-24 (P less than 0.001). There was no correlation between the time of exposure of the cells to serum-free conditions and the rate of uptake of 2-DG at the various ACTH1-24 concentrations both in the basal and insulin-stimulated states. Addition of catochalasin B (50 microM) to the cells, which inhibited both basal and insulin-stimulated uptake of 2-DG (by 70% and 91%, respectively) completely eliminated the enhancement of both of these uptake rates to 10(-12)M ACTH1-24. The results suggest that: 1) ACTH1-24 stimulates carrier-mediated uptake of glucose in skeletal muscle cells. 2) The site of action of ACTH1-24 is on the non-insulin mediated glucose uptake (NIMGU) system. 3) ACTH1-24 may be a useful probe to delineate some of the events associated with the NIMGU pathway.

Animals↗

PubMed1990

Neural control of glutamine synthetase activity in rat skeletal muscles.

The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighted, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased approximately twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoid-mediated glutamine synthetase induction previously described by us.

Animals↗

PubMed1990

Effect of diabetes on glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression in muscles from normal and diabetic (streptozotocin-treated) rats was studied. Muscle and body weights were markedly reduced in diabetic animals. Glutamine synthetase activity was significantly (2-fold) elevated 7 days after induction of diabetes. Increased enzyme activity persisted for at least 14 days after induction of diabetes, and it was apparent in both slow (soleus) and fast (plantaris) muscles. The diabetes-induced increase in enzyme activity was reflected in an increased steady-state level of glutamine synthetase mRNA. The increases in glutamine synthetase activity and mRNA level in muscle from diabetic rats were reversed by insulin administration. Increased expression of glutamine synthetase may be important for accelerated glutamine production by muscle from diabetic rats.

Adrenalectomy↗

PubMed1990

Glucocorticoid-mediated induction of glutamine synthetase in skeletal muscle.

We studied the effect of glucocorticoids on glutamine synthetase in rat skeletal muscle in culture and in vivo. Dexamethasone, a synthetic glucocorticoid, caused striking, receptor-mediated increases in both glutamine synthetase activity and the steady-state glutamine synthetase mRNA level. This effect was observed in rat skeletal muscle cells in culture, as well as in rat muscles in vivo. Glucocorticoid-mediated induction of glutamine synthetase was blocked by androgenic/anabolic steroids at high doses, suggesting that anabolic steroids might have an anticatabolic mode of action in enhancing skeletal muscle mass in athletes. Further studies of the control of glutamine synthetase expression may shed light on mechanisms of muscle atrophy and hypertrophy.

Anabolic Agents↗

PubMed1990

[Studies on enzyme activities relating to amino acid mobilization in biopsied muscles].

We evaluated glutamine synthetase (GS) and alanine aminotransferase (GPT) activities in biopsied muscle from 40 cases of various neuromuscular diseases. GS and GPT catalyze the synthesis of glutamine and alanine, respectively, from amino acids derived in part from the breakdown of muscle proteins. The subjects were 7 cases of muscular dystrophy; 1 Duchenne type (DMD), 3 limb-girdle type, 2 facioscapulohumeral type (FSH), 1 Fukuyama type (FCMD); and 1 myotonic dystrophy (MyD); 5 mitochondrial myopathies; 11 inflammatory myopathies including 6 polymyositis and 3 myopathy associated with collagen disease; 5 endocrinological myopathies including 2 periodic paralysis; and, 11 cases of neurogenic amyotrophies [4 amyotrophic lateral sclerosis (ALS), 4 spinal progressive muscular atrophy (SPMA) and 3 other types]. Control subjects were 8 patients with thigh operations. Biopsied muscle was homogenized and assayed for GS activity by the method of Smith et al.; GPT was assayed by commercial kit. Protein was assayed by the method of Lowry et al. Enzyme activities between mean -2SD and mean +2SD of controls were considered to be the normal range. GS activity in control subjects was 28.22 +/- 7.13 (mean +/- SD) nmol glutamine formed/mg protein/hr. Fifteen of 40 cases showed increased enzyme activity, including DMD and FCMD, the acute phase of polymyositis, and periodic paralysis. GPT activity in controls was 16.56 +/- 4.05 IU/mg protein. Sixteen of 40 patients showed increased enzyme activity: FCMD, FSH, MyD, inflammatory and endocrinological myopathy, and ALS. On the other hand, mitochondrial myopathy showed significantly decreased activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1989

Enzymes of glutamine metabolism in inflammation associated with skeletal muscle hypertrophy.

Glutamine synthesis and utilization were studied in the plantaris muscle after removal of its functional synergists, the soleus and gastrocnemius muscles. Rat plantaris muscle was compared with unoperated controls at 7, 14, and 30 days after synergist ablation and induction of hypertrophy. Glutamine synthetase activity increased from 6.17 +/- 1.77 to 33.92 +/- 2.23 nmol.h-1.mg protein-1, and glutaminase activities increased from 98.63 +/- 23.05 to 478.70 +/- 64.17 nmol.h-1.mg protein-1 7 days after surgery and remained elevated at 14 and 30 days. Sham-operated controls examined 7 days after surgery did not exhibit significantly increased glutamine synthetase activity. Histological examination revealed a large proliferation of connective tissue cells, as well as cells involved in tissue repair and inflammation; this influx was maximal 1 wk after surgery. The activity of the oxidative enzymes of the pentose phosphate pathway increased from 3.08 +/- 4.31 to 20.86 +/- 1.13 nmol.min-1.mg protein-1 1 wk after surgery. The time course of changes in pentose phosphate pathway enzymes was similar to that of the increases in glutamine synthetase, glutaminase, and cellular infiltration. Increases in muscle wet weight followed a different time course than changes in glutamine synthetase, glutaminase, and pentose phosphate pathway activities. It is concluded that the initial increases in plantaris muscle weight are probably due to edema, connective tissue proliferation, and cells involved in tissue repair and inflammation. The increase in glutamine synthetase activity appears to occur in skeletal muscle, whereas the changes in glutaminase and pentose phosphate pathway activities appear to represent infiltrating inflammatory cells. Furthermore, the increase in glutamine synthetase activity may serve to support the infiltrating cells, which appear to lack substantial capacity for glutamine production. These results represent a functional relationship between skeletal muscle glutamine synthesis and utilization by cells mediating inflammation and connective tissue repair and synthesis.

Animals↗

PubMed1988

Nuclear glucocorticoid receptor binding in L6 skeletal muscle cells in culture.

Mechanisms underlying glucocorticoid hormone actions on skeletal muscle remain incompletely understood. This problem may be amenable to solution with a simple cell culture system in which the hormonal environment can be controlled. In this report, we demonstrate that the L6 muscle cell line may provide such a system. These cells, which possess many morphological and functional characteristics of skeletal muscle, originate as mononuclear myoblasts, which fuse to form multinucleated myotubes. L6 myoblasts and myotubes contain an intracellular glucocorticoid receptor that has binding parameters and ligand specificity similar to those of glucocorticoid receptors of classical glucocorticoid target tissues. A major advantage of the use of cultured cells is ease of isolation of myonuclei that display specific glucocorticoid receptor binding. L6 muscle cells should provide a valuable model system for further studies of the mechanisms of glucocorticoid hormone actions on muscle.

Animals↗

PubMed1988

Regulation of viral and cellular genes in a human neuroblastoma cell line latently infected with herpes simplex virus type 2.

A latent state of the herpes simplex virus type 2 genome was established in a human neuroblastoma cell line (SMS-KCNR) to initiate studies on the mechanism by which host cells interact and regulate latent viral genes. To establish viral latency, it was necessary to prevent virus replication by briefly exposing the infected cells to antiherpetic acycloguanosine (20 microM) and human interferon (120 U/ml). Subsequently however, these cells could be propagated without any antiherpetic agents and almost 60% of the cell population contained viral genome. While these cells did not produce any infectious virus, immunoblot analysis revealed two intracellular polypeptides with molecular weights of 87.5 kDa and 67 kDa, respectively, that interacted with hyperimmune anti-HSV2 rabbit serum. Two cellular enzymes, acetylcholinesterase and choline acetyltransferase, involved in metabolism of neurotransmitters were expressed at a higher level in the latently infected cells than in the mock-infected control cells. Infectious HSV-2 could be reactivated from these cells only after the cells had undergone massive morphological differentiation and maturation to flat cell types by extensive treatment with 20 micron bromodeoxyuridine.

Acetylcholinesterase↗

PubMed1988

Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression by dexamethasone was studied in rat skeletal muscles. Daily administration of dexamethasone caused striking enhancement of glutamine synthetase activity in plantaris, soleus, and diaphragm muscles. Northern blot analysis revealed that the dexamethasone-mediated increase of glutamine synthetase activity was associated with dramatically increased levels of glutamine synthetase mRNA. Both glutamine synthetase activity and mRNA levels were significantly elevated in plantaris muscle at 0.5 mg.kg-1.day-1 of dexamethasone, a dose that approximates endogenous corticosteroid levels in animals under severe stress. Quantification of changes in glutamine synthetase mRNA on the basis of total mRNA (by oligo dT hybridization) also revealed a major increase in glutamine synthetase mRNA. Dexamethasone was without effect on beta-tubulin mRNA levels, indicating that glutamine synthetase induction is not part of a global response to glucocorticoids. Dexamethasone treatment resulted in only an approximately 15% increase in glutamine synthetase activity in heart; there was no change in glutamine synthetase mRNA level in this tissue. Thus glucocorticoids regulate glutamine synthetase gene expression in rat skeletal muscles.

Animals↗