Up-regulation of Na,K-ATPase beta 1 transcription by hyperoxia is mediated by SP1/SP3 binding.

The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,K-ATPase beta(1) gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase beta(1) transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5'-deletions of the rat Na,K-ATPase beta(1) promoter-luciferase fusion gene demonstrated promoter activation by hyperoxia between -102 and +151. The hyperoxia response was localized to a 7-base pair region between -62 and -55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at -55 had higher affinity for SP proteins.

Regulation of Na,K-ATPase beta 1 subunit gene transcription by low external potassium in cardiac myocytes. Role of Sp1 AND Sp3.

Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K(+). Our previous data showed that exposure of cardiac myocytes to low K(+) increased the steady-state abundance of Na,K-ATPase beta1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K(+) augmented Na,K-ATPase beta1 gene expression at a transcriptional level and that this effect required extracellular Ca(2+). The stimulatory effect of low K(+) on Na,K-ATPase beta1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase beta1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta1 promoter contained a low K(+) response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta1 promoter decreased basal and low K(+)-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K(+) effect on beta1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the beta1 promoter showed enhanced binding of two complexes in response to low K(+). The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K(+) response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase beta1 subunit gene promoter mediates beta1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K(+).

Regulation of Na/K-ATPase beta1-subunit gene expression by ouabain and other hypertrophic stimuli in neonatal rat cardiac myocytes.

Partial inhibition of Na/K-ATPase by ouabain causes hypertrophic growth and regulates several early and late response genes, including that of Na/K-ATPase alpha3 subunit, in cultured neonatal rat cardiac myocytes. The aim of this work was to determine whether ouabain and other hypertrophic stimuli affect Na/K-ATPase beta1 subunit gene expression. When myocytes were exposed to non-toxic concentrations of ouabain, ouabain increased beta1 subunit mRNA in a dose- and time-dependent manner. Like the alpha3 gene, beta1 mRNA was also regulated by several other well-known hypertrophic stimuli including phenylephrine, a phorbol ester, endothelin-1, and insulin-like growth factor, suggesting involvement of growth signals in regulation of beta1 expression. Ouabain failed to increase beta1 subunit mRNA in the presence of actinomycin D. Using a luciferase reporter gene that is directed by the 5'-flanking region of the beta1 subunit gene, transient transfection assay showed that ouabain augmented the expression of luciferase. These data support the proposition that ouabain regulates the beta1 subunit through a transcriptional mechanism. The effect of ouabain on beta1 subunit induction, like that on alpha3 repression, was dependent on extracellular Ca2+ and on calmodulin. Inhibitions of PKC, Ras, and MEK, however, had different quantitive effects on ouabain-induced regulations of beta1 and alpha3 subunits. The findings show that partial inhibition of Na/K-ATPase activates multiple signaling pathways that regulate growth-related genes, including those of two subunit isoforms of Na/K-ATPase, in a gene-specific manner.

Characterization of a negative thyroid hormone response element in the rat sodium, potassium-adenosine triphosphatase alpha3 gene promoter.

The thyroid hormone L-T3 elicits either a stimulatory or an inhibitory effect on expression of the Na,K-adenosine triphosphatase alpha3-subunit gene in primary cultures of neonatal rat cardiac myocytes. The present study was undertaken to characterize a negative thyroid hormone response element present within the rat Na,K-adenosine triphosphatase alpha3-subunit gene proximal promoter. Transient transfection assays indicated that the DNA-binding domain of thyroid hormone receptor was essential for mediating repression of alpha3 gene transcription by thyroid hormone. This negative effect of thyroid hormone was enhanced in the presence of cotransfected retinoid X receptor and its ligand 9-cis-retinoic acid. Inhibition of alpha3 chimeric gene expression by thyroid hormone was dependent on the initial cell plating density. The negative thyroid hormone response element was localized to a region between nucleotides -68 to -6 of the alpha3 gene. Electrophoretic mobility shift assays showed that thyroid hormone receptor binds in a synergistic manner as a heterodimer with retinoid X receptor to two sites at positions -62 to -41 and -39 to -17 of the alpha3 gene promoter. The upstream and downstream heterodimer binding sites coexist with CAAT and TATA elements, respectively.

Alterations in cardiac gene expression during ventricular remodeling following experimental myocardial infarction.

Following myocardial Infarction (MI) the heart undergoes a process of remodeling characterized by considerable hypertrophy of the non-infarcted myocardium. We have recently characterized the molecular basis of key electrophysiologic alterations that may provide insight into the arrhythmogenecity of post-MI remodeled hypertrophied myocardium. To further characterize other key alterations in the pattern of cardiac gene expression in a time-dependent manner, we have measured mRNA and immunoreactive protein levels of selective cardiac genes in the remodeled hypertrophied left-ventricular (LV) myocardium of rats, 3 and 21 days after left-coronary ligation and compared the results with sham-operated rats. RNase protection assay was performed to assess the expression of c-fos, atrial natriuretic factor (ANF), brain natriuretic factor (BNF), alpha2/3 isoform of Na-K ATPase, cardiac alpha/beta isoform of myosin heavy chain (MHC). Compared to the sham group, the expression of c-fos was increased 10-fold (P<0.02) in the MI group on day 3, but unlike other overload hypertrophy models, the expression remained elevated by three-fold on day 21. Similar to other overload models, the ANF and BNF expression increased significantly. No alterations were observed in the expression of cardiac alpha-actin. There was reexpression of the fetal isogene form of MHC and Na-K ATPase after MI. The beta-MHC mRNA levels, the fetal isoform of MHC, returned to basal levels after 21 days. After an initial five-fold decrease the adult isoform of alphaNa-K ATPase, alpha2 Na-K ATPase mRNA, returned to control levels and similar changes were seen in the corresponding protein levels. These findings indicate that during LV remodeling and hypertrophy following MI, there is an upregulation of early response genes and fetal isogene expression. The pattern of activation, however, is distinct from that observed in other overload models, indicating the possible involvement of alternate signal transduction pathways.

Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells.

Na-K-ATPase plays a central role in a variety of physiological processes, including ion transport and regulation of cell volume. Our previous data showed that hyperoxia increased the expression of Na-K-ATPase alpha 1 and beta 1 mRNA in lung type II cells. We similarly show that hyperoxia (> or = 95% O2 for 24-48 h) increased steady-state mRNA levels in both Na-K-ATPase subunits in Madin-Darby canine kidney (MDCK) cells. The mechanism of gene regulation by hyperoxia was assessed. Stability of the Na-K-ATPase mRNA levels of both subunits was unchanged in hyperoxia-exposed MDCK cells. To determine whether gene transcription was augmented by hyperoxia, MDCK cells were transfected with a beta 1-subunit promoter-reporter construct. Transfection with the wild-type promoter (beta 1-817) revealed a 1.9 +/- 0.2-fold increase in promoter activity. Transfection with 5' deletion constructs identified a 61-base pair (bp) region between -102 and -41 that was necessary for this increase in promoter activity by hyperoxia. Incorporation of this 61-bp region into a minimal promoter (mouse mammary tumor virus) similarly increased promoter activity 2.3-fold in the presence of hyperoxia. This increase in promoter activity was not seen when MDCK cells were incubated with various concentrations of hydrogen peroxide. In summary, hyperoxia increased Na-K-ATPase beta 1-subunit mRNA steady-state level due to increased transcription in MDCK cells. A region necessary for this hyperoxic effect on beta 1 transcription is located between base pairs -102 and -41 on the promoter.

Selective induction of Na,K-ATPase alpha3 subunit mRNA abundance in cardiac myocytes by retinoic acid.

Retinoic acid (RA) is a high affinity ligand for a nuclear receptor which regulates transcription in target cells. Specific effects of RA on cardiac development and myocardial cell hypertrophy have been demonstrated; however, little information exists concerning RA-mediated regulation of cardiac genes. This study was initiated to investigate whether RA regulates Na,K-ATPase subunit gene expression in primary cultures of neonatal rat cardiac myocytes. Northern blot analyses demonstrated that NA, K-ATPase alpha3 subunit mRNA content was stimulated three-fold by RA. The effect of RA on alpha3 subunit gene expression was selective as RA treatment had no effect on either Na,K-ATPase alpha1, alpha2 or beta1 subunit mRNAs. A stimulatory effect of RA on Na,K-ATPase alpha3 gene transcription was not evident in either transient transfection or nuclear run-on studies, suggesting that augmentation of alpha3 mRNA content by RA was due to a post-transcriptional mechanism. Finally, RA diminished the magnitude of the thyroid hormone (T3)-mediated increase in Na,K-ATPase beta1 subunit mRNA, while RA had no effect on the stimulation of alpha3 mRNA content by T3.

Negative regulation of the rat Na-K-ATPase alpha 3-subunit gene promoter by thyroid hormone.

Na-K-ATPase alpha 3-subunit mRNA levels are both positively and negatively controlled by thyroid hormone [3,5,3'triiodothyronine (T3)] in primary cultures of neonatal rat cardiac myocytes. In this study, transient transfection analysis indicated that two regions of the rat alpha 3 gene between nucleotides -116 and -6 and -6 and +80 conferred T3-mediated inhibition of reporter gene expression. Electrophoretic mobility shift assays showed specific binding of T3 receptor monomers and T3 receptor-retinoid X receptor heterodimers at each alpha 3 gene negative T3-response region. The alpha 3 gene region from -116 to -6 base pairs also mediates repression in response to retinoic acid (RA) and binds RA receptor. In the absence of ligand, reporter gene expression driven by the -116 to -6-base pair region is repressed with cotransfection of T3 receptor, whereas it is unaffected by overexpression of RA receptor. These data demonstrate that the proximal promoter of the rat Na-K-ATPase alpha 3 gene contains sequence motifs that mediate repression of alpha 3 gene transcription in response to either T3 or RA in neonatal rat cardiac myocytes.

Low external K+ regulates Na,K-ATPase alpha 1 and beta 1 gene expression in rat cardiac myocytes.

Incubation of mammalian cells in medium containing low K+ is often associated with an increase in Na,K-ATPase activity and content. In the present studies, we investigated the effect of low K+ on the regulation of Na,K-ATPase alpha 1 and beta 1 gene expression in primary cultures of neonatal rat cardiac myocytes. Northern blot hybridization analysis indicated that exposure of cardiocytes to a medium containing 0.3 mmol/L K+ for 72 h resulted in 1.8- and 3.3-fold increases in the abundance of alpha 1 and beta 1 mRNAs, respectively, compared with control cells exposed to 5.4 mmol/L K+. To investigate the molecular mechanism of the low K effect on beta 1 mRNA content, we constructed chimeric genes with a full-length and deleted portion of the 5' end of the rat beta 1 gene linked to the firefly luciferase gene. Transient transfection experiments using these constructs indicated that beta 1 gene sequences between -102 and +151 base pairs (bp) are required for low K(+)-induced trans-activation of reporter gene expression. Taken together, these results indicate that low K+ induction of beta 1 mRNA abundance in neonatal rat cardiac myocytes is mediated by regulatory DNA sequences in close proximity to the site of transcription initiation of the beta 1 gene.

Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes.

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.

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.

Regulation of Na,K-ATPase beta 1 mRNA content by thyroid hormone in neonatal rat cardiac myocytes.

Incubation of primary cultures of neonatal rat cardiac myocytes in the presence of 100 nM triiodothyronine (T3) resulted in a three- to fivefold increase in the content of Na,K-ATPase beta 1 subunit mRNA which was maximal at 1 d of exposure to hormone. To investigate the mechanism by which T3 stimulates the abundance of beta 1 mRNA, transient transfection experiments were conducted with a chimeric gene containing a portion of the 5' end of the rat beta 1 gene linked to a luciferase reporter gene. We found no effect of T3 on chimeric gene activity either in the absence or presence of cotransfected T3 receptor. The effect of T3 on the transcription rate of the endogenous beta 1 gene was quantitated by the nuclear run-on assay. T3 had no effect on beta 1 gene transcription following either 1 or 3 d of exposure and yielded a 1.3-fold increase at 6 d. These data indicate that T3 induction of Na,K-ATPase beta 1 mRNA content in neonatal rat cardiac myocytes in vitro is primarily mediated at a post-transcriptional site.

Characterization of the 5' flanking region of the rat Na+/K(+)-ATPase beta 1 subunit gene.

We have isolated the 5' end of the rat Na+/K(+)-ATPase beta 1 subunit gene. A genomic fragment containing 817 bp of the 5' flanking sequence, exon 1 and 479 bp of intron 1 was sequenced. The 5' flanking region contains a potential TATA box and several putative CAAT and GC boxes. Potential binding sites for thyroid and glucocorticoid receptors were identified together with multiple sequence motifs which exhibit homology to calcium and serum responsive elements.

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.

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)

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.

Increased abundance of Na+-K+-ATPase mRNAs in response to low external K+.

Exposure of ARL 15 cells, an established line from adult rat liver, to external K+ concentrations less than 1 mM for 24 h increases Na+-K+ pump abundance (Na+-K+-ATPase) (J. Gen. Physiol. 87:591-606, 1986). We found that treatment of confluent monolayers of ARL 15 cells with low-K+ medium (0.65 mM) caused a 100% increase in total RNA content per plate after 24 h, as well as a 25% increase in DNA and protein content per plate. Concomitant with this growth effect, low-K+ exposure for 6 h elicited 60% increases in mRNA alpha and mRNA beta, the mRNAs that encode the constituent subunits of the Na+-K+-ATPase, in a polyadenylated RNA fraction. At 24 h, however, the abundance of mRNA alpha increased by 290%, whereas mRNA beta increased by only 70%. Moreover, in both control and low-K+-treated cells, mRNA alpha was 30-fold or more greater in abundance than mRNA beta. This discrepancy in abundance was also present in rat liver, but not in cultured MDCK cells. The differences in abundance of mRNA alpha and mRNA beta suggest that the liver may have an unusual subunit composition or biosynthetic mechanism. Nevertheless, the increases in the abundance of mRNA alpha and mRNA beta are sufficient to account for the observed 70-100% increase in Na+-K+-ATPase activity in response to low external K+.

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.