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F Ismail-Beigi

Publications and source records attributed to F Ismail-Beigi.

At least 19 recordsLinked to original sources
PubMed1992

Enhancement of glucose transport in response to inhibition of oxidative metabolism: pre- and posttranslational mechanisms.

Addition of 5 mM sodium azide to Clone 9 cells, a rat liver cell line characterized by intracellular glucose concentrations of less than 10% that of the external medium and limited glycogen stores, results in a 50-80% reduction in cell ATP content within 20 min which then recovers to near-basal levels within 1 h and is subsequently maintained at normal levels for 24 h despite continuing the presence of the inhibitor. Associated with this adaptive response is a striking stimulation of facilitated glucose transport, mediated by the GLUT-1 transporter, that exhibits "early" and "late" phases that appear to be mechanistically different. During the early phase of the response (0-2 h), glucose transport rate is enhanced 12-fold in the absence of any change in cell GLUT-1 or GLUT-1 mRNA content. In contrast, the late phase of the response (8-24 h) is characterized by a further large stimulation of glucose transport (to 1.6 times the 2-h value) that is associated with 2- to 3- and 6- to 10-fold increments in cell GLUT-1 and GLUT-1 mRNA content, respectively. In time course studies an increase in GLUT-1 mRNA content was observed at 4 h and preceded the increment in GLUT-1 which became detectable after 8 h of exposure to azide. A marked induction of GLUT-1 mRNA by azide was also demonstrable in cells incubated in medium containing higher concentrations of glucose (10.6 mM), although the increment was approximately 20% less than when cells were incubated in standard medium (containing 5.6 mM glucose).(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1991

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.

3-O-Methylglucose↗
PubMed1991

Dexamethasone markedly induces Na,K-ATPase mRNA beta 1 in a rat liver cell line.

Exposure of Clone 9 cells, a "nontransformed" rat liver cell line, to 10(-8) M dexamethasone resulted at 3 h in 1.8 +/- 0.2- and 40 +/- 5-fold increases in mRNA alpha 1 and mRNA beta 1 content, respectively, an effect that was not mimicked by 10(-8) M aldosterone. The increments in mRNA alpha 1 and mRNA beta 1 abundances in total cell RNA were half-maximal at 5 x 10(-9) M dexamethasone and persisted for more than 24 h. Na,K-ATPase activity, however, increased only slightly (by 9%) at 24 h. The induction of mRNA beta 1 by dexamethasone was not prevented by the presence of cycloheximide. mRNA beta 1 abundance increased earlier in the nuclear RNA pool (becoming apparent within 45 min) than in the cytoplasmic RNA pool, consistent with a precursor-product relationship. Moreover, putative pre-mRNA beta 1 bands of approximately 4,600 and approximately 12,000 nucleotides accumulated in the nRNA pool after 1 h of exposure to dexamethasone. Incubation in the presence of dexamethasone for 3 h enhanced the incorporation of [3H]uridine into total cell mRNA alpha 1 and mRNA beta 1 by 1.3- and 12-fold, respectively. In nuclear run-on assays, however, transcription of mRNA alpha 1 and mRNA beta 1 was not altered after 30 min of exposure to 10(-8) M dexamethasone. The abundance of mRNA beta 1 in rat liver also increased markedly (greater than 30-fold) in rats treated with the hormone for 6 h. We conclude that dexamethasone causes an induction of Na,K-ATPase subunit mRNAs, an effect that is markedly greater for mRNA beta 1 than for mRNA alpha 1. The increases in subunit mRNA content, however, are associated with, at most, a small increase in Na,K-ATPase activity, suggesting that the increments in mRNA abundances, especially that of mRNA beta 1, do not play a determining role in the regulation of Na,K-ATPase activity in these cells.

Animals↗
PubMed1991

Enhancement of glucose transport in clone 9 cells by exposure to alkaline pH: studies on potential mechanisms.

Incubation of a nontransformed rat liver cell line, Clone 9, at pH 8.5 resulted in an approximately 16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells "depleted" of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K1 approximately 10(-7) M). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated an approximately 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.

3-O-Methylglucose↗
PubMed1991

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium.

Regulation of Na,K-ATPase mRNA alpha isoform and mRNA beta expression by thyroid hormone (T3) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 abundances were nearly constant while mRNA alpha 2 was undetectable. During the interval between postnatal days 5 and 15, mRNA alpha 3 decreased to negligible levels and mRNA alpha 2 became expressed and increased in abundance to account for approximately 20% of the mRNA alpha pool by the 15th postnatal day. To examine the effect of T3 on this developmental program, neonates were injected with 75 micrograms T3/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T3 treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T3 treatment, the abundances of mRNA alpha 1, mRNA alpha 3, and mRNA beta 1 increased, while mRNA alpha 2 continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T3 augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T3 does not act as a "molecular switch" in the developmental expression of the mRNA alpha isoforms in rat myocardium during the first four postnatal days.

Analysis of Variance↗
PubMed1991

The Journal of Nutrition, Volume 106, 1976: Decreased absorption of calcium, magnesium, zinc and phosphorus by humans due to increased fiber and phosphorus consumption as wheat bread.

During a 20 day period of high fiber consumption in the form of bread made partly from wheaten wholemeal, two men developed negative balances of calcium, magnesium, zinc and phosphorus due to increased fecal excretion of each element. The fecal losses correlated closely with fecal dry matter and phosphorus. Fecal dry matter, in turn, was directly proportional to fecal fiber excretion. Balances of nitrogen remained positive. Mineral elements were well-utilized by the same subjects during a 20 day period of white bread consumption.

Bread↗
PubMed1991

Induction of Na(+)-K(+)-ATPase subunit mRNAs by cycloheximide in a rat liver cell line.

Exposure of confluent Clone 9 cells to 40 microM cycloheximide (CHX), a concentration sufficient to inhibit leucine incorporation by 95% within 5 min, coordinately increased the abundances of Na(+)-K(+)-ATPase subunit mRNAs, mRNA alpha 1 and mRNA beta 1. The CHX-induced increases in mRNA alpha 1 and mRNA beta 1 abundances were, respectively, 1.8- and 1.9-fold at 40 min and 3.0- and 3.3-fold at 6 h. Augmented subunit mRNA contents were also observed after exposure to other protein synthesis inhibitors including 100 microM anisomycin and 100 microM emetine. Upon removal of CHX, the rate of leucine incorporation returned to control values within 1 h, but mRNA alpha 1 and mRNA beta 1 content decreased only slowly and were still elevated at 24 h at 1.7- and 1.8-fold the respective control values. Despite the persistence of increased levels of the subunit mRNAs and normalization of the rate of leucine incorporation, Na(+)-K(+)-ATPase activity was unchanged at 3, 6, 24, and 48 h after removal of CHX. In cells "depleted" of protein kinase C (PKC) activity after a 24-h preincubation in the presence of 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), mRNA alpha 1 and mRNA beta 1 abundances were still inducible by CHX. It is concluded that exposure of Clone 9 cells to CHX and other inhibitors of protein synthesis results in increased abundances of Na(+)-K(+)-ATPase subunit mRNAs independently of PKC activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Serum and growth factor induction of Na(+)-K(+)-ATPase subunit mRNAs in Clone 9 cells: role of protein kinase C.

In a previous study, we found that addition of serum to confluent Clone 9 cells, a nontransformed rat liver cell line, increased the abundance of mRNA alpha 1 and mRNA beta 1 at 3 h by 2- and 2.7-fold, respectively [Bhutada et al. Am. J. Physiol. 258 (Cell Physiol. 27): C1044-C1050, 1990]. We now report that exposure of these cells to 160 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 6 h increases mRNA alpha 1 and mRNA beta 1 by 1.7 +/- 0.2- and 2.1 +/- 0.3-fold, respectively. Incubation in the presence of 160 nM TPA for 24 h reduced high-affinity phorbol dibutyrate-binding sites [dissociation constant (Kd) = 5 nM; maximum binding (Bmax) = 1.2 pmol/mg protein] to undetectable levels. In such cells, exposure to 10% serum for 6 h still resulted in two- and fourfold increment in mRNA alpha 1 and mRNA beta 1 abundances, respectively, while further addition of TPA to these protein kinase C (PKC)-depleted cells resulted in no change in the subunit mRNA abundances. The increments in mRNA alpha 1 content in response to 10% serum and 160 nM TPA at 6 h were additive, whereas the increments in mRNA beta 1 were not. The following agents increased mRNA alpha 1 and mRNA beta 1 abundance in both control and PKC-depleted cells: epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, dexamethasone, and hypothyroid calf serum. In contrast, N6,2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate and aldosterone had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Thyroidal enhancement of rat myocardial Na,K-ATPase: preferential expression of alpha 2 activity and mRNA abundance.

In hypothyroid rat myocardium, the low-ouabain-sensitivity Na,K-ATPase activity had a KI = 10(-4) M and accounted for approximately 95% of the enzyme activity, while the high-ouabain-sensitivity activity contributed approximately 5% to the total activity, with a KI = 3 x 10(-7) M. mRNA alpha 1 was 7.2- and 5.5-fold more abundant than mRNA alpha 2 and mRNA beta, respectively, in hypothyroid ventricles while mRNA alpha 3 was undetectable. Administration of T3 increased total Na,K-ATPase activity 1.6-fold; the low-ouabain-sensitivity activity increased 1.5-fold while high-ouabain-sensitivity activity was stimulated 3.2-fold. T3 increased the number of high-affinity ouabain-binding sites 2.9-fold with no change in Kd (approximately 2 x 10(-7) M). The abundances of mRNA alpha 1, mRNA alpha 2, and mRNA beta (per unit RNA) following T3 treatment increased 3.6-, 10.6-, and 12.7-fold, respectively. The larger increments in subunit mRNA abundances than in Na,K-ATPase activity suggests the involvement of translational and/or post-translational regulatory steps in Na,K-ATPase biogenesis in response to T3. It is concluded that T3 enhances myocardial Na,K-ATPase subunit mRNA abundances and Na,K-ATPase activity, and that the expression of the high- and low-ouabain-sensitivity activities are probably a reflection of the abundances of the alpha 2 and alpha 1 isoforms, respectively. The physiological role played by the beta subunit remains uncertain.

Animals↗
PubMed1990

Stimulation of glucose transport in Clone 9 cells by exposure to alkaline pH.

Incubation of a rat liver cell line (Clone 9) for 2 h at pH 8.5 was found to result in a profound (5- to 8-fold) stimulation of cytochalasin B-inhibitable glucose transport. The enhancement of glucose transport after exposure to elevated external pH (achieved by lowering the CO2 tension in a bicarbonate-containing medium) was demonstrable within 15 min, was half-maximal at pH 8.0, and was near-maximal at pH 8.6. Intracellular pH rose linearly with incremental changes in external pH, from pH 7.45 to 8.6 with a slope of 0.6. The increase in transport activity in response to incubation at alkaline pH was accompanied by a parallel increase in lactate production and persisted for more than an hour after external pH was restored to normal. During the latter period, intracellular glucose concentration (less than 10% of that of the external medium under control conditions) increased greater than 10-fold to approximate that in the extracellular medium. Incubation of these cells at pH 8.5 for 2 h resulted in a complete resistance of cell ATP levels to challenge with 5 mM cyanide, suggesting that the adaptive facilitation of glucose transport was of sufficient magnitude to permit a marked stimulation of glycolytic ATP synthesis on inhibition of oxidative phosphorylation. The enhancement of glucose transport was attributable to an increase in the maximum velocity (Vmax) rather than to any change in the Michaelis constant (Km) for transport and was not prevented by cycloheximide. It is concluded that the marked stimulation of glucose transport resulting from exposure of these "low-glucose" cells to alkaline pH reflects either an increase in the abundance of functional glucose transporters in the plasma membrane or an increase in their catalytic turnover rate.

3-O-Methylglucose↗
PubMed1990

Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line.

The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Induction of Na(+)-K(+)-ATPase and its subunit mRNAs by serum in a rat liver cell line.

Addition of serum to confluent Clone 9 cells increased the protein, RNA, and DNA content per plate of cells; the increments became manifest within 3-6 h and were sustained for the 48-h duration of study. After the addition of serum, Na(+)-K(+)-ATPase activity was stimulated 1.25- and 1.45-fold at 6 and 12 h, respectively. In cells preincubated in the absence of serum for 24 h, addition of serum increased the abundances of Na(+)-K(+)-ATPase subunit mRNAs, mRNA alpha 1 and mRNA beta, coordinately by approximately 2- and 2.7-fold at 3 h, an effect that preceded the stimulation of Na(+)-K(+)-ATPase activity. The serum-induced increments in subunit mRNA abundances were further enhanced by the combined presence of serum and cycloheximide; mRNA alpha 1 and mRNA beta abundances were also augmented by cycloheximide alone (approximately 2.5- and 9.2-fold at 6 h, respectively). In cells incubated in the absence of serum, the half-lives of mRNA alpha 1 and mRNA beta, estimated from decrements in their abundances after the addition of actinomycin D, were 12 and 10 h, respectively. These data demonstrate that serum enhances Na(+)-K(+)-ATPase subunit mRNA abundance and enzyme activity in Clone 9 cells. Comparison of the estimated half-lives of Na(+)-K(+)-ATPase subunit mRNAs with the observed increments in their abundances at 3 h suggests that the serum-induced increases in mRNA alpha 1 and mRNA beta abundances are in large part due to enhanced synthesis of these mRNAs.

Animals↗
PubMed1990

Na(+)-K(+)-ATPase in adipocyte differentiation in culture.

Differentiation of 3T3-L1 cells from a fibroblast to an adipocyte phenotype results in an approximately 50% decline in Na(+)-K(+)-ATPase activity and ouabain-sensitive 86Rb uptake. Kinetic analysis revealed a K 1/2 for Na+ of approximately 14 mM, a Km for ATP of approximately 0.4 mM, and maximal activation by sodium dodecyl sulfate at a 0.05 (wt/wt) detergent/protein ratio in both mature fibroblasts and adipocytes. Both fibroblasts and adipocytes exhibited Na(+)-K(+)-ATPase activity with an inhibition constant (Ki) for ouabain of approximately 10(-4) M. In addition, adipocytes exhibited a second component representing 30% of total activity with a Ki of approximately 5 x 10(-7) M. The emergence of biphasic ouabain inhibition kinetics in adipocytes raised the possibility of a change in alpha-subunit isoform composition with cytodifferentiation. This inference was evaluated by isoform-specific mRNA analysis (Northern blots) and by alpha-isoform-specific immunoassays (Western blots). Northern blots revealed a modest decrease in mRNA alpha 1, a striking increase in mRNA alpha 2, and a significant loss of mRNA beta content with differentiation of fibroblasts to adipocytes. By immunoassay, fibroblasts exhibited the alpha 1-isoform. Adipocytes exhibited an admixture of alpha 1- and alpha 2-isoforms, with alpha 2 being the more abundant isoform. There was no one-to-one correspondence either between the mRNA isoform and alpha-subunit abundances or between alpha-subunit abundances and enzymatic activity, suggesting that regulation occurs at multiple levels in this system. Findings indicate, however, that a shift in alpha-isoform composition accompanied by a change in ouabain inhibition kinetics occurs with cytodifferentiation.

Adipose Tissue↗
PubMed1989

Enhanced glucose transport in response to inhibition of respiration in Clone 9 cells.

An acceleration of ATP synthesis by anaerobic glycolysis provides important compensation for interference with respiration in a variety of cells. Effective compensation for an inhibition of respiration, however, can occur in cells in which glucose entry is rate limiting only if sufficient glucose becomes available through an enhancement of transport. We present here a detailed study of the effects of inhibition of respiration in Clone 9 cells, a continuous cell line characterized by low internal glucose concentrations (less than 10% that of the external medium) and minimal stores of glycogen. Exposure of these cells to 5 mM cyanide results in a 90% fall in cell ATP and a twofold rise in cell Na+ within 20 min. By the end of 1 h, however, there is a 4.5- to 7-fold increase in cytochalasin B-inhibitable glucose transport that is accompanied by a parallel increase in the rate of lactate production, a partial recovery of cell ATP, and no further rise in cell Na+. The acute fall in ATP resulting from a submaximally effective concentration of cyanide (0.5 mM) is moreover followed by a time-dependent recovery of cell ATP to near-normal levels and subsequent resistance to challenge with even 5 mM cyanide. The stimulation of facilitative glucose transport resulting from exposure to cyanide is attributable to an increase in maximal velocity rather than to a change in Km and persists for more than 2 h after removal of the inhibitor. These results demonstrate that, in these cells characterized by low internal glucose concentrations, regulation of glucose entry is of central importance in ATP homeostasis and that a major component of the adaptive response to an inhibition of respiration is a time-dependent increase in glucose transport.

3-O-Methylglucose↗
PubMed1988

Thyroidal regulation of rat renal and hepatic Na,K-ATPase gene expression.

Na,K-ATPase activity, Na,K-ATPase alpha- and beta-subunit mRNA abundance (mRNA alpha and mRNA beta), and gene transcription rates were determined in kidney cortex and liver of hypothyroid and triiodothyronine (T3)-treated rats. In hypothyroid rats, Na,K-ATPase activity (expressed per unit of DNA) was 3.6-fold greater in kidney cortex than liver, and the abundance of mRNA alpha and mRNA beta in kidney cortex exceeded that of liver by 2.8- and 5.2-fold, respectively. In vitro nuclear run-on analysis revealed similar rates of Na,K-ATPase alpha and beta gene transcription in nuclei isolated from either kidney cortex or liver. Administration of T3 for 72 h elicited a 2.3-fold stimulation of renal Na,K-ATPase activity that was associated with a 3.1- and 2.6-fold increase of mRNA alpha and mRNA beta content, respectively. In contrast, T3 induced a 1.3-fold stimulation of liver Na,K-ATPase activity accompanied by a 7.3-fold increase in mRNA alpha and no change in mRNA beta abundance. Transcription rates of alpha and beta genes (assayed by nuclear run-on) in renal cortex were both stimulated 1.8-fold in response to T3 injection. Similarly in liver nuclei, T3 treatment produced a 1.4- and 1.3-fold stimulation in the rate of alpha and beta gene transcription, respectively. These results indicate that significant discrepancies exist in the quantitative relationships between control and T3-induced changes in renal and hepatic enzyme activity, mRNA abundance and rate of gene transcription, and imply that the T3-induced increase in Na,K-ATPase abundance is mediated at both transcriptional and post-transcriptional steps.

Actins↗
PubMed1988

Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line.

Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (KI = 1 x 10(-4) M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ("back-door") phosphorylation, such that the calculated catalytic turnover number of approximately 8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. approximately 1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the alpha-I isoform is expressed under control and low-K+ conditions.

Animals↗