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F Ismail-Beigi

Publications and source records attributed to F Ismail-Beigi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of Na/K-ATPase gene expression by thyroid hormone and hyperkalemia in the heart.

Hypothermic hyperkalemic circulatory arrest has been widely used for myocardial protection during heart surgery. Recent data showed that administration of triiodo-L-thyronine (T3) postoperatively enhanced ventricular function. The effect of hyperkalemic arrest in conjunction with thyroid hormone on the plasma membrane enzyme sodium/potassium-adenosine triphosphatase (Na/K-ATPase), was determined in cultured neonatal rat atrial and ventricular myocytes. Exposure of ventricular myocytes to hyperkalemic medium (50 mM KCl) in the absence of T3 increased expression of the Na/K-ATPase catalytic subunit mRNAs, alpha1 and alpha3 isoforms, by 1.9- and 1.5-fold, respectively (p<0.01), which were accompanied by similar increases (1.4- and 1.8-fold) in protein content. Addition of T3 to the hyperkalemic cultures attenuated these increases in Na/K-ATPase mRNA isoforms to levels of expression observed in cells treated with T3 (10(-8) M) alone. Similarly, expression of the alpha1 mRNA isoform in atrial myocytes was increased (p<0.05) by hyperkalemic conditions, and T3 treatment attenuated this effect. In contrast, although expression of the Na/K-ATPase beta1 mRNA in both atrial and ventricular myocytes was significantly increased by hyperkalemia, addition of T3 did not prevent the hyperkalemic response, and in atrial myocytes T3 significantly increased beta1 mRNA expression 1.8-fold. These results show that expression of cardiac Na/K-ATPase is regulated by T3 and hyperkalemia in an isoform and chamber specific manner, and suggest that use of hyperkalemic cardioplegia during heart surgery may alter plasma membrane ion function.

Animals↗
PubMed1998

Activation of Glut1 glucose transporter in human erythrocytes.

Glut1, the only glucose transporter isoform expressed in the human red blood cell (RBC), binds to and is inhibited by cytochalasin B (CB). In the present study we show that incubation of RBC ghost membranes with 10 microM cytochalasin E (CE) results in a 1.8-fold increase in the number of glucose-sensitive cytochalasin B binding sites. Moreover, treatment with CE was associated with no observable change in the protein composition of RBC ghosts determined by SDS-PAGE. Removal of surface ("extrinsic") proteins from RBC ghosts by treatment with 0.2 mM EDTA (pH 12) for 30 min resulted in a similar 1.8-fold increase in the number of glucose-sensitive CB binding sites. Western blot analysis showed that treatment with CE or EDTA resulted in no change in the amount or mobility of Glut1 present in ghost membranes. Glucose transport, measured as CB-inhibitable 3-O-[3H]methylglucose uptake by resealed ghosts, was stimulated in CE-treated resealed ghosts, with the t1/2 to equilibrium decreasing from 6.8 +/- 0.5 to 3.9 +/- 0.3 s (P < 0.05). Treatment of ghosts with CE or EDTA followed by Western blotting of samples in the presence or absence of beta-mercaptoethanol resulted in no change in immunoreactivity or mobility of the major Glut1 band. The above results suggest that a significant fraction of Glut1 transporters exists in an inactive form ("masked") in RBC plasma membranes and that treatment of ghosts with CE or EDTA leads to an apparent activation of Glut1.

Binding Sites↗
PubMed1998

Induction of GLUT1 mRNA in response to azide and inhibition of protein synthesis.

Incubation of Clone 9 cells, a nontransformed rat liver cell line, in the presence of 5 mM azide results in an induction of GLUT1 mRNA which becomes detectable after approximately 3 h of continuous exposure to the agent. In examining the role of on-going protein synthesis in this response, we found that: (i) the induction of GLUT1 mRNA by azide was not inhibited by anisomycin, (ii) exposure to anisomycin alone also resulted in increased GLUT1 mRNA content, and (iii) the increments in GLUT1 mRNA content in the presence of both azide and anisomycin were additive. Following exposure to 30 microM anisomycin, the increase in GLUT1 mRNA content became evident at 1 h, reached a maximum level of approximately 7-fold at 3 h, then slowly decreased but remained elevated at approximately 2-fold control levels at 12 h. Transcription of the GLUT1 gene, estimated by nuclear run-on assay, was stimulated 1.4 +/- 0.1 and 1.6 +/- 0.2-fold in cells exposed to anisomycin for 1 and 2 h, respectively (p < 0.05 for both). Upon inhibition of RNA synthesis by actinomycin D, GLUT1 mRNA content decreased with a half-life of 1.9 +/- 0.4 h in control cells, while in contrast, GLUT1 mRNA half-life was 4.6 +/- 0.8 h in cells exposed to anisomycin. The induction of GLUT1 mRNA by anisomycin was half-maximal at approximately 3 microM, whereas inhibition of leucine incorporation and stimulation of Stress Activated Protein Kinase (SAPK), measured as c-Jun N-terminal kinase activity, were half-maximal at approximately 0.3 and approximately 0.05 microM anisomycin, respectively. GLUT1 mRNA content was also increased by the protein synthesis inhibitor emetine, and the effect was associated with no stimulation of SAPK activity. Finally, SAPK activity was minimally stimulated in cells exposed to azide. It is concluded that: (1) on-going protein synthesis is not necessary for the induction of GLUT1 mRNA content in response to azide, (2) the induction of GLUT1 mRNA by anisomycin is related to its activity to inhibit protein synthesis, and (3) under basal conditions, a rapidly turning-over putative protein exerts a negative regulatory effect on GLUT1 mRNA expression.

Animals↗
PubMed1998

Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis.

Results of previous studies indicated that treatment of diabetic rats (induced by streptozotocin) with cobalt chloride (CoCl2) resulted in a significant decrement in serum glucose concentration. The present study was designed to determine the potential role of enhanced glucose uptake vs. decreased glucose production in the above response. The rate of systemic appearance of glucose, measured under fasting conditions using [3-3H]glucose tracer, was reduced from 35.5 +/- 2.5 to 17.5 +/- 1.8 micromol . kg-1 . min-1 in diabetic rats treated with 2 mM CoCl2 added to the drinking water for 10-14 days (P < 0.01). Tissue accumulation of intravenously administered 2-deoxy-[14C]glucose was significantly reduced in kidney and eye of diabetic rats treated with CoCl2, whereas the uptake remained unchanged in several other tissues including cerebrum, red and white skeletal muscle, heart, and liver. The relative content of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased 3.1-fold in livers of diabetic compared with normal rats (P < 0.001), and treatment of diabetic rats with CoCl2 decreased hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the liver was decreased by 33% in CoCl2-treated normal rats (P < 0.05). Treatment with CoCl2 resulted in no change in cAMP levels in the livers of either diabetic or normal rats. These results suggest that the glycemia-lowering effect of CoCl2 is mediated by reductions in the rate of systemic appearance of glucose and hepatic gluconeogenesis.

Animals↗
PubMed1997

Glycemia-lowering effect of cobalt chloride in the diabetic rat: increased GLUT1 mRNA expression.

We have recently shown that expression of the GLUT1 glucose transporter isoform is augmented in cells exposed to cobalt chloride [Co(II)], an agent that stimulates the expression of hypoxia-responsive genes (Behrooz, A., Ismail-Beigi, F., 1997. J. Biol. Chem. 272, 5555-5562.). Here, we examine the effect of Co(II) on glycemia and tissue GLUT1 mRNA content of normal and diabetic rats. The addition of 2 mM Co(II) in the drinking water reduced the glycemia of streptozotocin-induced diabetic rats by day 3 from 32.3 +/- 2.1 to 21.0 +/- 1.9 mM (non-fasting). Co(II) resulted in no change in serum insulin levels of normal or diabetic rats. Treatment with 4 mM Co(II) was more effective than 2 mM Co(II) in reducing the glycemia of diabetic rats, while 6 mM Co(II) was associated with severe toxicity. GLUT1 mRNA content increased significantly in ventricular myocardium, renal cortex, skeletal muscle, cerebrum and liver of normal and diabetic rats treated with 2 mM cobalt chloride (ranging from 1.3- to 2.9-fold in the different tissues). It is concluded that: (1) treatment with Co(II) decreases the glycemia of diabetic rats, and (2) the glycemia-lowering effect of Co(II) is associated with, and may be mediated by, enhanced expression of GLUT1 mRNA.

Animals↗
PubMed1997

Dual control of glut1 glucose transporter gene expression by hypoxia and by inhibition of oxidative phosphorylation.

glut1 gene expression and glucose transport are stimulated in a variety of cells and tissues in response to hypoxia. glut1 is also up-regulated by inhibitors of oxidative phosphorylation (such as azide) in the presence of oxygen. Here, we test the hypothesis that hypoxia stimulates glut1 gene expression independent of its inhibitory effect on oxidative phosphorylation. We examined the effect of cobalt chloride, a known stimulator of genes responsive to reduced oxygen concentration per se, on GLUT1 expression under normoxic conditions and compared the results with the response to azide. Exposure of a rat liver cell line (Clone 9) to 250 microM cobalt chloride increases GLUT1 mRNA content, which becomes evident at 2 h, reaches a maximal value of approximately 12-fold at 8 h, and remains elevated at approximately 8-fold at 24 h. GLUT1 mRNA was the only GLUT isoform expressed in control cells and in cells exposed to cobalt chloride or azide. The induction of GLUT1 mRNA by cobalt chloride is associated with a approximately 10-fold stimulation of cytochalasin B-inhibitable 3-O-methyl-D-glucose transport at 24 h. In contrast to the rapid decrease in cell ATP levels and the stimulation of glucose transport in response to azide, cell ATP content and glucose transport remained unaltered during the initial 1-h period of exposure to cobalt chloride. The effect of cobalt chloride on GLUT1 mRNA content is mimicked by Ni(II) or Mn(II) but not by Fe(II). Employing actinomycin D, we found no increase in the approximately 1.5-h half-life of GLUT1 mRNA in cobalt chloride-treated cells, suggesting that the effect of cobalt chloride on GLUT1 mRNA content is largely mediated at the transcriptional level; in contrast, GLUT1 mRNA half-life increased to >8 h in azide-treated cells. In transient transfections we found that approximately 6 kilobase pairs (kbp) of 5'-flanking region of the rat glut1 promoter confers both cobalt chloride- and azide-inducibility to a reporter gene. Deletion of approximately 2, 500 base pairs (bp) from the 5' end of the approximately 6-kbp DNA fragment results in a reduction of the response to cobalt chloride and a complete loss of the response to azide. A 666-bp DNA segment located approximately 6.0 kbp upstream of the transcription start site was found to be necessary for the increase in reporter gene expression in response to azide, whereas a 480-bp segment located at approximately -3.5 kbp mediated the response to cobalt chloride. The 480-bp segment is highly homologous to the previously reported mouse glut1 enhancer and contains several potential regulatory elements, including a hypoxia-inducible element; an additional hypoxia-inducible element is present in the 666-bp segment. Our results suggest that glut1 gene expression is regulated in a dual fashion by hypoxia per se and in response to inhibition of oxidative phosphorylation.

3T3 Cells↗
PubMed1997

Ouabain-sensitive Na+,K(+)-ATPase activity in toad brain.

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.

Animals↗
PubMed1996

Stimulation of glucose transport in Clone 9 cells by insulin and thyroid hormone: role of GLUT-1 activation.

Thyroid hormone (T3) and insulin are both shown to stimulate glucose transport in Clone 9 cells, a rat liver cell line in which the utilization of glucose is limited by transport rate and in which only the GLUT-1 transporter isoform is expressed. Pre-treatment of these cells with T3 moreover substantially enhances the stimulatory effect of insulin such that at maximally effective hormone concentrations the effects of T3 and insulin on glucose transport are more than additive and indeed nearly multiplicative, suggesting that the mechanisms mediating the enhancement of glucose transport differ between the two hormones. Cell surface biotinylation followed by Western-blot analysis of plasma membrane fractions showed that the stimulatory effects of T3 and insulin on glucose transport, whether acting singly or in combination, exceed the attendant increases in the abundance of GLUT-1 in the plasma membrane. It is suggested that activation of GLUT-1 molecules pre-existing in the plasma membrane plays a major role in mediating the stimulatory effects of T3 and insulin on glucose transport in this cell line.

Animals↗
PubMed1996

Stimulation of GLUT1 glucose transporter expression in response to inhibition of oxidative phosphorylation: role of reduced sulfhydryl groups.

Treatment of Clone 9 cells incubated in the absence of serum with 5 mM azide for 24 h results in an 8- and 3-fold induction in GLUT1 mRNA and GLUT1 protein, respectively. To explore the pathways mediating the induction of GLUT1 mRNA, we first examined whether inhibition of oxidative phosphorylation by other agents results to a similar response. Exposure of cells to 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 0.15 microM oligomycin B, or 5 mM azide resulted in near-equivalent increases in GLUT1 mRNA content. The inhibition of oxidative phosphorylation is associated with increased cell lactate content and in extracellular lactate to pyruvate ratio, reflecting a rise in cytosolic NADH/NAD+ ratio. We next tested the possibility that an increase in cell SH/SS ratio mediates the enhancement of GLUT1 mRNA in response to azide. We show that treatment of cells with 10 mM mercaptoethanol, an agent that increases cell SH/SS ratio, results in a approximately 6-fold increase in GLUT1 mRNA content. Moreover, incubation of cells in the presence of 0.3 mM diamide, a known intracellular sulfhydryl oxidizing agent, completely abolishes the induction of GLUT1 mRNA by azide. The results suggest that an increase in cell SH/SS ratio plays a critical role in the induction of GLUT1 mRNA in response to inhibition of oxidative phosphorylation.

Animals↗
PubMed1996

Effect of GLUT1 glucose transporter overexpression on the stimulation of glucose transport in response to inhibition of oxidative phosphorylation.

Glucose transport is markedly stimulated in response to inhibition of oxidative phosphorylation by cyanide or azide in Clone 9 cells, a rat liver cell line in which only the GLUT1 isoform of glucose transporters is expressed. Here, we examine the possibility that the stimulation of glucose transport by azide is similarly observed in cells exhibiting high basal rates of glucose transport. We stably transfected Clone 9 cells with an expression plasmid containing full-length rat GLUT1 cDNA; nontransfected cells and cells transfected with plasmid alone served as controls. Two clones of cells transfected with the GLUT1-cDNA-containing insert, labeled A and B, respectively, expressed 8- and 20-fold higher levels of GLUT1 mRNA, contained 11- and 23-fold higher levels of GLUT1, and manifested 11- and 17-fold higher rates of glucose transport in the basal state. Upon incubation with 5 mM azide for 2 h, the rate of glucose transport was markedly stimulated in both clones. Moreover, the transient fall in cell ATP content following exposure to azide did not correlate with the magnitude of the glucose transport response. We conclude that in GLUT1-overexpressing Clone 9 cells (i) GLUT1 content and glucose transport parallel cellular GLUT1 mRNA content, suggesting no major translational or posttranslational control of GLUT1 expression and function in the basal state, and (ii) the rate of glucose transport in cells overexpressing GLUT1 is markedly stimulated by exposure to azide. These results indicate that the stimulation of glucose transport in response to inhibition of oxidative phosphorylation is maintained in cells with very high basal rates of glucose transport.

Adenosine Triphosphate↗
PubMed1996

Induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation: role of increased [Ca2+]i.

Exposure of Clone 9 cells (a rat liver cell line expressing only the GLUT-1 isoform) to 5 mM azide or to 3 microM ionomycin for 12 h results in 3.7 +/- 0.3- and 4.9 +/- 0.4-fold increases in GLUT-1 mRNA content, respectively, suggesting the hypothesis that a rise in cytosolic free calcium concentration ([Ca2+]i) mediates the induction of GLUT-1 mRNA by azide. Five lines of evidence were employed to test this hypothesis. 1) Exposure of cells to 0-3 microM of ionomycin increased [Ca2+]i from 83 +/- 9 to 504 +/- 20 nM (half-maximal effect at 0.1 microM ionomycin), whereas half-maximal increase in GLUT-1 mRNA occurred at 1 microM ionomycin, with the increase in the mRNA being negligible at [Ca2+]i below 400 nM. Exposure of cells to 5 mM azide, however, increased [Ca2+]i to maximal value of 174 +/- 22 nM at 15 s, suggesting that the magnitude of the increase in [Ca2+]i by azide may not be adequate for the response. 2) The increase in GLUT-1 mRNA content by azide was fully preserved in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 3) GLUT-1 mRNA content increased within 30 min of exposure to ionomycin, whereas the mRNA increased after a "delay" period of 2 h in cells exposed to 5 mM azide. 4) A brief (2-min) rise in [Ca2+]i by ionomycin was sufficient to increase GLUT-1 mRNA content, whereas continuous exposure to azide for > 1 h was necessary for a subsequent induction of the mRNA. 5) Treatment with ionomycin, A-23187, and thapsigargin caused larger increases in glucose-regulated protein 78 and 94 and in 70-kDa heat shock protein mRNAs than in GLUT-1 mRNA, whereas treatment with azide resulted in greater induction of GLUT-1 mRNA. These results strongly suggest that, whereas increased [Ca2+]i enhances GLUT-1 mRNA expression and azide increases [Ca2+]i, the rise in [Ca2+]i does not mediate the induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1995

Modulation of GLUT1 intrinsic activity in clone 9 cells by inhibition of oxidative phosphorylation.

Brief (1-2 h) exposure of Clone 9 cells to inhibitors of oxidative phosphorylation such as azide is known to markedly increase glucose uptake. Clone 9 cells express GLUT1 but not GLUT2, -3, and -4, and the azide effect was not accompanied by any increase in cellular or plasma membrane GLUT1 level. To identify the molecular event underlying this apparent increase in GLUT1 intrinsic activity, we studied the acute effects of azide on the substrate binding activity of GLUT1 in Clone 9 cells by measuring glucose-sensitive cytochalasin B binding. The glucose-displaceable, cytochalasin B binding activity was barely detectable in membranes isolated from Clone 9 cells under control conditions but was readily detectable after a 60-min incubation of cells in the presence of 5 mM azide showing a 3-fold increase in binding capacity with no change in binding affinity. Furthermore, the cytochalasin B binding activity of purified human erythrocyte GLUT1 reconstituted in liposomes was significantly reduced in the presence of cytosol derived from azide-treated Clone 9 cells but not in the presence of cytosol from control cells; this effect was heat-labile and abolished by the presence of the peptide corresponding to the GLUT1 COOH-terminal sequence. These results suggest that a cytosolic protein in Clone 9 cells binds to GLUT1 at its COOH-terminal domain and inhibits its substrate binding and that azide-induced metabolic alteration releases GLUT1 from this inhibitory interaction. Studying the binding of cytosolic proteins derived from 35S-labeled Clone 9 cells to glutathione S-transferase fusion protein containing glucose transporter COOH-terminal sequences, we identified 28- and 70-kDa proteins that bind specifically to the cytoplasmic domain of GLUT1 and GLUT4 in vitro. We also found a 32P-labeled, 85-kDa protein that binds to GLUT4 but not to GLUT1 and only in cytosol derived from azide-treated cells. The roles, if any, of these glucose transporter-binding proteins in the azide-sensitive modulation of GLUT1 substrate binding activity in Clone 9 cells are yet to be determined.

Animals↗
PubMed1995

Stimulation of GLUT-1 glucose transporter expression in response to exposure to calcium ionophore A-23187.

We tested the hypothesis that an increase in cytosolic calcium concentration stimulates glucose transporter isoform (GLUT-1) gene expression. Exposure of a rat liver cell line (Clone 9) to 3 microM A-23187 for 12 h resulted in 3-, 5-, and 10-fold increases in cytochalasin B-inhibitable 3-O-methyl-D-glucose transport, GLUT-1 protein, and GLUT-1 mRNA content, respectively. The induction of GLUT-1 mRNA in response to A-23187 is not preceded by a significant decrease in cell ATP content. This induction is prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in conjunction with ethylene glycol-bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid. To investigate the mechanism of GLUT-1 mRNA induction, we found that exposure to A-23187 stabilized GLUT-1 mRNA: with the employment of actinomycin D, GLUT-1 mRNA had a half-life of 1.5 and 5.5 h in control and A-23187-treated cells, respectively. In nuclear run-on assays, the rate of GLUT-1 gene transcription was stimulated 1.5- to 1.7-fold in nuclei isolated from cells exposed to A-23187 for either 30 min or 2 h. These results demonstrate that exposure to A-23187 stimulates GLUT-1 gene expression and that the increase in GLUT-1 mRNA content is mediated in part by enhanced GLUT-1 gene transcription as well as decreased GLUT-1 mRNA degradation. The increase in GLUT-1 mRNA content, in turn, is associated with increased cell GLUT-1 content and enhanced glucose transport.

Animals↗
PubMed1994

Post-transcriptional regulation of rat alpha cardiac myosin heavy chain gene expression.

The cardiac myosin heavy chain genes, alpha and beta, have been shown to change their patterns of expression rapidly and dramatically in response to a variety of stimuli. A major means of achieving these changes in gene expression is transcriptional control; however, the role of post-transcriptional regulation in cardiac myosin gene expression has not been investigated. We have identified two post-transcriptional events in rat alpha cardiac myosin heavy chain (alpha-MHC) gene expression and investigated their regulatory significance in different developmental and thyroid hormone states. The polyadenylation of alpha-MHC mRNA occurs at three different sites: 12, 18, and 23 bases downstream from a single polyadenylation signal. Hyperthyroid hearts did not demonstrate any change in the proportion of the three alpha-MHC mRNA subspecies. Hypothyroid hearts (which have a decreased amount of total alpha-MHC mRNA) showed a significant increase in the proportion of the longest subspecies and a decrease in the shortest subspecies. The second post-transcriptional event in alpha-MHC gene expression which was demonstrated was the inclusion or exclusion of a codon, CAG, encoding glutamine at position 1931, resulting from alternate splicing of the alpha-MHC transcript. The ratio of CAG+ and CAG- forms of mRNA in the adult euthyroid hearts is 40:60% which was unchanged in hypo- and hyperthyroid states. This is the first example of alternate splicing in a vertebrate sarcomeric myosin heavy chain gene. We conclude that the rat alpha-MHC gene transcript is post-transcriptionally modified.

Amino Acid Sequence↗
PubMed1994

Role of enhanced Na+ entry in the control of Na,K-ATPase gene expression by serum.

The role of enhanced Na+ entry in the induction of Na,K-ATPase subunit mRNAs by serum was investigated in a "nontransformed" rat liver cell line, Clone 9. Exposure of cells to 10% calf serum resulted in a 1.5-fold increase in the rate of Na+ entry associated with a transient rise in cell Na+ content (twofold at 15 min) and a sustained 1.15-fold rise in cell K+ content. After 6 hr of exposure to serum mRNA alpha 1 and mRNA beta 1 content increased by 1.8- and 2.6-fold, respectively. In nuclear run-on assays, serum stimulated the transcription of the alpha 1 gene approximately 1.9-fold while the transcription rate of the beta 1 gene remained unchanged. In cells incubated in Na(+)-free medium where NaCl was replaced by choline chloride, the induction of mRNA alpha 1 by serum was fully preserved, whereas the increase in mRNA beta 1 was prevented. An unexpected finding was that incubation of cells in Na(+)-free medium alone for 6 hr increased mRNA alpha 1 but not mRNA beta 1 content. These results indicate that Na,K-ATPase subunit mRNAs are differentially induced by serum, and that the induction of mRNA alpha 1, in contrast to that of mRNA beta 1, is transcriptionally mediated and does not require the presence of Na+ in the extracellular medium.

Animals↗
PubMed1993

Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in clone 9 cells.

Exposure of Clone 9 cells to inhibitors of oxidative phosphorylation results in a rapid and striking stimulation of facilitated glucose transport (7.5-fold at 2 h) that is mediated by the GLUT-1 transporter. We have previously shown that this rapid stimulation of glucose transport occurs in the absence of any detectable increase in cell GLUT-1 or GLUT-1 mRNA content. To determine whether this early enhancement of transport is attributable to a translocation of glucose transporters to the plasma membrane, or instead to an activation of transporters already present in the plasma membrane, we have employed four different approaches to determine whether the stimulation of transport is accompanied by a corresponding increase in plasma membrane GLUT-1 sites: 1) immunofluorescence microscopy; 2) quantitation of GLUT-1 sites in plasma membrane fractions isolated by differential centrifugation and subsequent Western blotting; 3) cell surface biotinylated followed by isolation of plasma membranes and quantitation of GLUT-1 sites by Western blotting; and 4) quantitation of GLUT-1 sites in plasma membrane fractions by [3H]cytochalasin B binding. Each of these experimental approaches led to the same conclusion, namely that the large stimulation of glucose transport observed during the early phase of the response to azide is associated with only a slight increase in the abundance of GLUT-1 sites in the plasma membrane. These results strongly suggest that activation of GLUT-1 sites pre-existing in the plasma membrane is the dominant mechanism mediating the early glucose transport response to inhibition of oxidative phosphorylation.

Animals↗
PubMed1993

Na,K-ATPase in several tissues of the rat: tissue-specific expression of subunit mRNAs and enzyme activity.

The relative contents of Na,K-ATPase subunit mRNAs in rat renal cortex, ventricular myocardium, skeletal muscle (hind limb), liver and brain (cerebrum) were measured. Expressed per unit DNA, mRNA alpha 1 content was approximately 2-fold greater in the kidney and brain as compared to either heart, skeletal muscle or liver. The hierarchy of mRNA alpha 2 expression was brain > skeletal muscle > heart, whereas mRNA alpha 3 was restricted to brain. Beta 1 subunit mRNA content in both kidney and brain exceeded the abundance of liver mRNA beta 1 by approximately 7-fold. In all tissues examined, the combined abundances of the alpha subunit mRNAs exceeded the content of mRNA beta 1. The hierarchy of Na,K-ATPase activity expressed per unit DNA was brain > kidney > skeletal muscle = heart > liver. The sum of mRNA alpha as well as mRNA beta 1 content, expressed per g of tissue, was highest in brain and kidney. A statistically significant correlation between mRNA beta 1 content and Na,K-ATPase activity was evident.

Animals↗