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J B Lingrel

Publications and source records attributed to J B Lingrel.

At least 19 recordsLinked to original sources
PubMed1992

Chimeric rat Na,K-ATPase alpha 1/alpha 3* isoforms. Analysis of the structural basis for differences in Na+ requirements in the alpha 1 and alpha 3* isoforms.

Na,K-ATPase molecules containing the alpha 1, alpha 2*, and alpha 3* isoforms expressed in HeLa cells exhibit a two- to threefold difference in their K0.5 for Na+ (alpha 1 = alpha 2* < alpha 3*). To investigate the structural basis for this difference, chimeric alpha 1/alpha 3* isoform cDNAs were constructed and expressed in HeLa cells. Na,K-ATPase containing each alpha isoform chimera was analyzed for its Na+ dependence properties. Results of these experiments do not reveal a region in the alpha 1 or alpha 3* isoform that is clearly responsible for the apparent affinity for Na+. It is possible that molecular interactions involving amino acids that span virtually the entire Na,K-ATPase molecule contribute to the determination of this parameter.

Amino Acid Sequence↗
PubMed1992

Na,K-ATPase: isoform structure, function, and expression.

An interesting feature of the Na,K-ATPase is the multiplicity of alpha and beta isoforms. Three isoforms exist for the alpha subunit, alpha 1, alpha 2, and alpha 3, as well for the beta subunit, beta 1, beta 2, and beta 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the alpha subunit are present in the brain, while only alpha 1 is present in kidney and lung, and alpha 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na+ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell. Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the alpha subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Human gamma- to beta-globin gene switching using a mini construct in transgenic mice.

The developmental regulation of the human globin genes involves a key switch from fetal (gamma-) to adult (beta-) globin gene expression. It is possible to study the mechanism of this switch by expressing the human globin genes in transgenic mice. Previous work has shown that high-level expression of the human globin genes in transgenic mice requires the presence of the locus control region (LCR) upstream of the genes in the beta-globin locus. High-level, correct developmental regulation of beta-globin gene expression in transgenic mice has previously been accomplished only in 30- to 40-kb genomic constructs containing the LCR and multiple genes from the locus. This suggests that either competition for LCR sequences by other globin genes or the presence of intergenic sequences from the beta-globin locus is required to silence the beta-globin gene in embryonic life. The results presented here clearly show that the presence of the gamma-globin gene (3.3 kb) alone is sufficient to down-regulate the beta-globin gene in embryonic transgenic mice made with an LCR-gamma-beta-globin mini construct. The results also show that the gamma-globin gene is down-regulated in adult mice from most transgenic lines made with LCR-gamma-globin constructs not including the beta-globin gene, i.e., that the gamma-globin gene can be autonomously regulated. Evidence presented here suggests that a region 3' of the gamma-globin gene may be important for down-regulation in the adult. The 5'HS2 gamma en beta construct described is a suitable model for further study of the mechanism of human gamma- to beta-globin gene switching in transgenic mice.

Aging↗
PubMed1992

Isoforms of the alpha subunit of Na,K-ATPase and their significance.

Recent studies of the Na,K-ATPase have demonstrated that multiple isoforms of both the alpha and beta subunits exist and that these are expressed in a tissue and developmental specific manner. In the case of the alpha subunit, there are three known isoforms, alpha 1, alpha 2 and alpha 3. We have examined adult human heart for the presence of these isoforms and found that all three exist in approximately equal amounts. This is in contrast to the adult rat heart which contains only alpha 1 and alpha 2 isoforms. The difference in abundance of various isoforms in various tissues could result from the necessity to express Na,K-ATPase with different properties at various developmental stages or in specific cell types. For example, enzymes with differences in Na+ or K+ affinity or the ability to respond to various effector molecules may be required. Alternatively, the presence of three isoforms may simply result from the triplication of the alpha subunit gene and the divergence of expression of these genes during evolution. In this case the isozymes would not confer a specific function to the Na,K-ATPase. In order to provide information with respect to these two alternatives, cell lines producing rat alpha 1, alpha 2 and alpha 3 were developed and the enzymatic properties of the resulting enzyme determined. The results indicate that Na,K-ATPase carrying the alpha 1 are alpha 2 isoforms are fairly similar while enzyme with the alpha 3 isoform differs in its apparent affinity for sodium. The K0.5 for Na+ is approximately three fold lower for this isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Discordant segregation of Na+,K(+)-adenosine triphosphatase alleles and essential hypertension.

OBJECTIVES: To determine whether the alpha 2 and or beta 1 isoforms of the Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) are involved in the pathogenesis of essential hypertension. DESIGN: Segregation analysis of polymorphic DNA markers was used to test the involvement of Na+,K(+)-ATPase in essential hypertension. PARTICIPANTS: Children with persistent hypertension having one parent with essential hypertension were included in the study. Criteria for persistent hypertension were blood pressure readings with systolic and/or diastolic levels exceeding the 95th percentile based upon age and sex. The diagnosis of hypertension for adults, including parents and older siblings, was confirmed using criteria recommended in the 1988 report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. RESULTS: In three essential hypertensive families consisting of 18 members including 11 hypertensives, several obligate recombinants between the Na+,K(+)-ATPase alpha 2 isoform marker and the hypertension phenotype were observed. Similarly, in one hypertension family consisting of four members, obligate recombinants between the beta 1 isoform marker and the disease were observed. CONCLUSIONS: The discordant segregation of the alpha 2 and beta 1 isoform markers and essential hypertension suggests that neither the Na+,K(+)-ATPase alpha 2 nor beta 1 isoform genes play a primary role in the pathogenesis of hypertension in the families studied.

Adult↗
PubMed1991

Expression of Na,K-ATPase isoforms in human heart.

The expression pattern of the multiple isoforms of Na,K-ATPase was examined in the human heart. Isoform specific oligonucleotide probes for the alpha 1, alpha 2, alpha 3 and beta 1 subunits were used to probe Northern blots. The adult human ventricle expresses mRNAs for all three alpha subunit isoforms in addition to beta 1 subunit mRNA.

Adult↗
PubMed1991

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.

Animals↗
PubMed1991

Identification of sequences mediating interleukin-6 induction of a rat T kininogen gene.

Interleukin-6 is a pleiotropic cytokine that has a major role in the coordination of the hepatic acute phase response. In order to more fully understand this role, we have examined the interleukin-6 induction of T kininogen, a cysteine protease inhibitor and a major acute phase reactant in the rat. Using deletional analysis and site-directed mutagenesis of T kininogen-chloramphenicol acetyltransferase fusion constructs transfected into HepG2 hepatoma cells, we have identified two similar interleukin-6 response elements within 250 base pairs of the transcription start site. These two response elements are functionally interdependent. The sequences of these two elements match the consensus sequence for the previously described Type B interleukin-6 response element. Interleukin-6 signal transduction via two Type B elements has not been observed previously in vivo. A DNA fragment encompassing these response elements forms the same protein complex with nuclear extracts from both untreated and interleukin-6-treated cells.

Animals↗
PubMed1991

Transcription factor OTF-1 interacts with two distinct DNA elements in the A gamma-globin gene promoter.

A DNA region (site II) in the promoter of the human A gamma-globin gene (-182 to -168) is involved in transcriptional regulation. At least two nuclear proteins bind to this region: the erythroid-specific factor NF-E1/GF-1 and another factor present in many cell lines. In the present study, we demonstrate that the ubiquitous factor binding to site II has immunological identity with the octamer transcription factor OTF-1, which has been implicated in the regulation of expression of genes such as histone H2b and small nuclear RNA. In addition, we show that OTF-1 binds to site I (-291 to -267), a purine-rich region upstream of site II. Interestingly, OTF-1 binds to sites I and II with equal affinity. This was unexpected since the 14 bp site I binding site AAGAATAAATTAGA (-291 to -278), determined by methylation interference, does not show obvious similarities to the canonical octamer binding site for OTF-1 in site II (ATGCAAAT). Interaction of OTF-1 with functionally active binding sites in the gamma-globin promoter suggests that this factor has a role in gamma-globin transcription.

Base Sequence↗
PubMed1991

Developmental and tissue-specific expression of rat T-kininogen.

The ontogeny of T-Kininogen expression in the rat liver was examined. Levels approximately 6 fold higher than seen in the adult liver are present during the perinatal period. Elevated levels are also seen in the maternal liver, beginning 5 days prior to parturition. The timing of induction in the fetal and maternal liver is distinct, suggesting independent regulation. While T-Kininogen mRNA is mainly synthesized in the liver, low but significant expression is seen in other tissues, including lung and kidney.

Aging↗
PubMed1990

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit.

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.

Animals↗
PubMed1990

Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes.

The Na,K-ATPase alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes exhibit a complex pattern of expression during heart development. To identify possible molecular signals that regulate the differential expression of these genes, isolated neonatal rat myocardial and non-myocardial cells were cultured in chemically defined medium and the responses of the multiple Na,K-ATPase subunit mRNAs to various hormones were tested. Myocardiocytes in control cultures express primarily alpha 1 and beta mRNAs. Triiodothyronine (T3) induced the expression of alpha 2, alpha 3, and beta mRNAs without influencing alpha 1 mRNA levels. Dexamethasone (DEX) treatment similarly induced alpha 2 mRNA levels, but the abundance of the other subunit transcripts remained unaltered. T3 and DEX together caused increases in alpha 2 and beta mRNA, increments similar to that observed with T3 alone. However, DEX specifically repressed the induction of alpha 3 mRNA by T3. Both hormones stimulated corresponding changes in the sarcolemma concentration of these Na,K-ATPase isozymes. Addition of norepinephrine to the cultures had little appreciable effect on expression of the alpha isoform and beta mRNAs. Although characterized less extensively, control cultures of non-myocardiocytes expressed alpha 1, alpha 3, and beta mRNAs, of which only the beta mRNA was stimulated by T3. These data indicate that thyroid and glucocorticoid hormones differentially regulate the expression of multiple alpha isoform and beta subunit mRNAs of Na,K-ATPase in cardiocytes in vitro and, therefore, may also be important physiological modulators in vivo.

Animals↗
PubMed1990

Characterization of the 5'-flanking region of the human and rat Na,K-ATPase alpha 3 gene.

Genomic clones containing the 5'-flanking region and exon 1 of the human and rat Na,K-ATPase alpha 3 isoform gene have been isolated and characterized. The nucleotide sequences of 1.6 kb of the rat gene and 2.8 kb of the human gene in the 5'-flanking region were determined. Mapping of transcription initiation sites by primer extension and S1 nuclease protection analyses indicates that transcription is initiated in the same region in both genes although the rat gene has a greater number of initiation sites. Neither gene has a canonical TATA box, having instead a ATAT sequence preceding the transcription initiation sites. There is a perfect CCAAT sequence, in the reverse orientation, approximately 30 bp upstream of the potential TATA box in both genes. We have identified potential binding sites for transcription factors Sp-1, AP-1, AP-2, and AP-4, as well as for glucocorticoid and thyroid hormone receptors in the 5'-flanking regions. These are conserved in both human and rat alpha 3 isoform genes.

Animals↗
PubMed1990

The human Na, K-ATPase alpha 1 gene: characterization of the 5'-flanking region and identification of a restriction fragment length polymorphism.

We have determined the sequence of the 5'-flanking region and first three exons of the human Na,K-ATPase alpha 1 gene, ATP1A1. Primer extension and S1 nuclease protection analyses of RNA from human kidney, brain, and skeletal muscle indicate that transcription initiates 273 nucleotides upstream of the translation start site. The promoter region contains a potential TATA box at position -27 relative to the transcription initiation site; however, no CCAAT sequence is observed. The 5'-untranslated and 5'-flanking regions are G + C rich. Five sequence elements exhibiting similarity to binding sites for the transcription factor Sp1 are located within the 5'-flanking region. This region also contains potential binding sites for the transcription factors AP-1, AP-2, AP-3, and NF-1, as well as a site which exhibits perfect identity to an 8-bp sequence element important for calcium induction. A comparison of the 5'-flanking region of the alpha 1 and alpha 2 genes reveals differences in potential transcription factor and hormone receptor binding sites which may be important in mediating the tissue- and developmental stage-specific expression of these genes. We have also identified an intragenic DNA probe which detects a restriction fragment length polymorphism at the alpha 1 locus. This marker should facilitate genetic linkage studies designed to evaluate the role of the sodium pump in human disease.

Base Sequence↗
PubMed1990

Molecular genetics of Na,K-ATPase.

Researchers in the past few years have successfully used molecular-genetic approaches to determine the primary structures of several P-type ATPases. The amino-acid sequences of distinct members of this class of ion-transport ATPases (Na,K-, H,K-, and Ca-ATPases) have been deduced by cDNA cloning and sequencing. The Na,K-ATPase belongs to a multiple gene family, the principal diversity apparently resulting from distinct catalytic alpha isoforms. Computer analyses of the hydrophobicity and potential secondary structure of the alpha subunits and primary sequence comparisons with homologs from various species as well as other P-type ATPases have identified common structural features. This has provided the molecular foundation for the design of models and hypotheses aimed at understanding the relationship between structure and function. Development of a hypothetical transmembrane organization for the alpha subunit and application of site-specific mutagenesis techniques have allowed significant progress to be made toward identifying amino acids involved in cardiac glycoside resistance and possibly binding. However, the complex structural and functional features of this protein indicate that extensive research is necessary before a clear understanding of the molecular basis of active cation transport is achieved. This is complicated further by the paucity of information regarding the structural and functional contributions of the beta subunit. Until such information is obtained, the proposed model and functional hypotheses should be considered judiciously. Considerable progress also has been made in characterizing the regulatory complexity involved in expression of multiple alpha-isoform and beta-subunit genes in various tissues and cells during development and in response to hormones and cations. The regulatory mechanisms appear to function at several molecular levels, involving transcriptional, posttranscriptional, translational, and posttranslational processes in a tissue- or cell-specific manner. However, much research is needed to precisely define the contributions of each of these mechanisms. Recent isolation of the genes for these subunits provides the framework for future advances in this area. Continued application of biochemical, biophysical, and molecular genetic techniques is required to provide a detailed understanding of the mechanisms involved in cation transport of this biologically and pharmacologically important enzyme.

Amino Acid Sequence↗