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R Levenson

Publications and source records attributed to R Levenson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Regional, cellular, and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain.

The pathways governing signal transduction in the mesocortical and nigrostriatal dopamine systems of the brain are of central importance in a variety of drug actions and neurological diseases. We have analyzed the regional, cellular, and subcellular distribution of the closely related D1 and D5 subtypes of dopamine receptors in the cerebral cortex and selected subcortical structures of rhesus monkey using subtype specific antibodies. The distribution of D1 and D5 receptors was highly differentiated in subcortical structures. In the neostriatum, both D1 and to a lesser extent D5 antibodies labeled medium spiny neurons, while only D5 antibodies labeled the large aspiny neurons typical of cholinergic interneurons. In the caudate nucleus, D1 labeling was concentrated in the spines and shafts of projection neurons, whereas D5 antibodies predominantly labeled the shafts, and less commonly, the spines of these cells. The D1 receptor was abundantly expressed in the neuropil of the substantia nigra pars reticulata while the D5 antibodies labeled only a few scattered cell bodies in this structure. Conversely, D5 antibodies labeled cholinergic neurons in the basal forebrain more intensely than D1 antibodies. Within the cerebral cortex and hippocampus, D1 and D5 antibody labeling was prominent in pyramidal cells. Double-label experiments revealed that the two receptors were frequently coexpressed in neurons of both structures. Ultrastructurally, D1 receptors were especially prominent in dendritic spines whereas dendritic shafts were more prominently labeled by the D5 receptor. The anatomical segregation of the D1 and D5 receptors at the subcellular level in cerebral cortex and at the cellular level in subcortical areas suggest that these closely related receptors may be preferentially associated with different circuit elements and may play distinct regulatory roles in synaptic transmission.

Animals↗
PubMed1994

On the origin of mRNA encoding the truncated dopamine D3-type receptor D3nf and detection of D3nf-like immunoreactivity in human brain.

A truncated dopamine D3-receptor-like mRNA, named D3nf, predicts a protein that differs from the D3-receptor only in the carboxyl terminus. However, such a protein has lost the predicted membrane topology typically found for G protein-coupled receptors. Results presented here show that D3nf mRNA arises from the D3-encoded primary transcript via alternative splicing. This splicing, however, appears to involve cleavage of an unusual 3' splice site. Therefore, we tested the possibility that D3nf mRNA results from a splicing error. If this were the case, D3nf mRNA would be expected to be present in the cytoplasm only at very low amounts, and it would not be expected to be translated into protein. However, the relative abundance of cytoplasmic D3/D3nf mRNA in human cortical tissues was found to be similar. Furthermore, we raised polyclonal antisera against the predicted carboxyl-terminal peptide sequence of D3nf that reacts specifically with a protein expressed in stably D3nf mRNA-expressing COS 7 cells. The use of this antiserum also revealed the presence of a approximately 68 kDa D3nf-like immunoreactive protein in human brain, suggesting that the atypically processed D3nf mRNA is translated.

Aged↗
PubMed1994

Neurons and astroglia express distinct subsets of Na,K-ATPase alpha and beta subunits.

We have analyzed the expression pattern of Na,K-ATPase alpha and beta subunit isoforms within the rodent and primate central nervous system. Membrane fractions prepared from rat cerebral cortical type-1 astrocytes and rat cerebellar granule and hippocampal neurons were characterized by immunoblot analyses using a panel of alpha and beta subunit isoform-specific antisera. Each cell type was found to express the alpha 1 isoform but showed differences in the expression of other subunits. Cortical astrocytes displayed alpha 2 and beta 2 subunits, whereas cerebellar granule neurons showed expression of alpha 3 and beta 1 subunits. All three alpha subunit isotypes were detected in hippocampal neurons. A survey of the immunofluorescent staining pattern of the alpha 3 subunit in rat and monkey brain confirmed that expression of this Na,K-ATPase alpha subunit isoform was restricted exclusively to neurons. These results suggest that both neurons and astrocytes express multiple, yet distinct, Na,K-ATPase isoenzymes. The identification of cell types expressing limited combinations of alpha and beta subunits should provide a framework for understanding the physiological significance of Na,K-ATPase isoenzyme diversity and may provide useful tools for the analysis of cell lineage in the mammalian central nervous system.

Animals↗
PubMed1993

Transmembrane organization of the Na,K-ATPase determined by epitope addition.

The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (alpha) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the alpha subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat alpha 1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the alpha subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.

Amino Acid Sequence↗
PubMed1993

A murine genomic DNA fragment amplifies ouabain-induced Na,K-ATPase alpha/beta-subunit mRNA up-regulation and confers ouabain resistance.

Transfection of primate cells with a 6.4-kilobase murine genomic DNA fragment (called ouabain resistance gene or MOR6.5) has been shown previously to confer ouabain resistance (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493). The mechanism by which this sequence can transfer ouabain resistance remains unclear. In order to further investigate this mechanism, we determined the full-length nucleotide sequence of MOR6.5. Other than mouse repetitive domains, this DNA does not have significant homology with any coding sequence in GenBank. Although potential open reading frames and polyadenylation signals were found, we were unable to detect an MOR6.5 transcript in CV-1 or COS-1 cells transfected with this DNA, either at early or late times following transfection. We show that in early passages of MOR6.5 transfectants which were under ouabain-selective pressure and still contained MOR6.5 DNA sequence, mRNAs for both alpha 1- and beta 1-subunits of the Na,K-ATPase were amplified approximately 10-fold, compared to parental CV-1 cells. These results suggest that MOR6.5 may rescue the cells from ouabain toxicity by inducing transient up-regulation of the messages for the Na,K-ATPase. This might prolong cell survival on ouabain until mutations in the alpha 1-subunit occur, which permanently reduce ouabain inhibition of the pump (Cantley, L. G., Zhou, X.-M., Cunha, M., Epstein, J., and Cantley, L. C. (1992) J. Biol. Chem. 267, 17271-17278). Possible mechanisms for the up-regulation of transcription based on sequence similarities found between MOR6.5 and the 5'-flanking regions of alpha 1- and beta 1-subunit genes are discussed.

Animals↗
PubMed1993

Genes encoding the H,K-ATPase alpha and Na,K-ATPase alpha 3 subunits are linked on mouse chromosome 7 and human chromosome 19.

We have used linkage analysis and fluorescence in situ hybridization to determine the chromosomal organization and location of the mouse (Atp4a) and human (ATP4A) genes encoding the H,K-ATPase alpha subunit. Linkage analysis in recombinant inbred (BXD) strains of mice localized Atp4a to mouse Chromosome (Chr) 7. Segregation of restriction fragment length polymorphisms in backcross progeny of Mus musculus x Mus spretus mating confirmed this assignment and indicates that Atp4a and Atp1a3 (gene encoding the murine Na,K-ATPase alpha 3 subunit) are linked and separated by a distance of approximately 2 cM. Analysis of the segregation of simple sequence repeats suggested the gene order centromere-D7Mit21-D7Mit57/Atp1a3-D7Mit72/Atp 4a. A human Chr 19-enriched cosmid library was screened with both H,K-ATPase alpha and Na,K-ATPase alpha 3 subunit cDNA probes to isolate the corresponding human genes (ATP4A and ATP1A3, respectively). Fluorescence in situ hybridization with gene-specific cosmid clones localized ATP4A to the q13.1 region, and proximal to ATP1A3, which maps to the q13.2 region, of Chr 19. These results indicate that ATP4A and ATP1A3 are linked in both the mouse and human genomes.

Animals↗
PubMed1992

Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity.

We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.

Amino Acid Sequence↗
PubMed1991

Structural organization and transcription of the mouse gastric H+, K(+)-ATPase beta subunit gene.

We have cloned and characterized the mouse gene encoding the beta subunit of H+, K(+)-ATPase (EC 3.6.1.36). The entire 10.5-kilobase transcription unit of the H+,K(+)-ATPase beta subunit gene was cloned in three overlapping cosmids encompassing approximately 46 kilobases of genomic DNA. A tight cluster of transcription initiation sites has been localized 24-25 nucleotides upstream of the translation start site and 28-29 nucleotides downstream of a TATA-like sequence. The H+, K(+)-ATPase beta subunit gene is split into seven exons encoding predicted structural domains of the beta subunit protein. The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons, and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the mouse H+,K(+)-ATPase beta subunit gene is identical to that of the mouse Na+,K(+)-ATPase beta 2 subunit gene. The conservation of genomic organization, together with the high sequence homology, indicates that the mouse H+,K(+)-ATPase beta and Na+,K(+)-ATPase beta 2 subunit genes originated from a common ancestral gene.

Amino Acid Sequence↗
PubMed1991

Cell-specific expression of mRNAs encoding Na+,K(+)-ATPase alpha- and beta-subunit isoforms within the rat central nervous system.

We have used in situ hybridization histochemistry to analyze the subcellular distribution of mRNAs encoding Na,K-ATPase alpha- and beta-subunit isoforms in the rat central nervous system. Substantial differences in the cell-specific pattern of expression were found for the genes encoding three isoforms of the alpha subunit. Transcripts of alpha 1-subunit gene were detected in virtually all cell types and structures examined. Expression of alpha 2-subunit mRNA was characteristic of glia, whereas alpha 3-subunit transcripts were predominant in neurons. Transcripts encoding the beta 1 subunit were detected in neurons, whereas beta 2-subunit mRNA expression was characteristic of glia. mRNA encoding both beta-subunit isoforms was present in choroidal epithelial cells. The distribution pattern of alpha- and beta-subunit mRNAs in structures throughout the central nervous system is consistent with the possibility of six structurally distinct Na+,K(+)-ATPase isoenzymes.

Animals↗
PubMed1991

Evolution of the Na,K- and H,K-ATPase beta subunit gene family: structure of the murine Na,K-ATPase beta 2 subunit gene.

We have cloned and characterized the mouse Na,K-ATPase beta 2 subunit gene (Atp1b2). The gene spans approximately 7 kb and is split into seven exons. The transcription initiation site has been mapped and consensus TATA and putative CAAT sequences have been found at positions -23 and -137, respectively. Discrete structural domains of the beta 2 subunit protein are encoded by separate exons: The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the beta 2 subunit gene closely resembles that of the H,K-ATPase beta subunit gene, suggesting that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the mouse and rat beta 2 subunit genes reveals a remarkably high degree of homology within a 788-nucleotide segment immediately upstream of the transcription start site. This observation suggests that elements that serve to regulate the cell-specific expression of the beta 2 subunit gene are likely to be located within this conserved region.

Adenosine Triphosphatases↗
PubMed1991

Three brain sodium channel alpha-subunit genes are clustered on the proximal segment of mouse chromosome 2.

We have used long-range physical mapping and restriction fragment length polymorphisms between two mouse species to determine the chromosomal organization and location of the genes encoding three distinct isoforms of the alpha-subunit of the brain sodium channel. Physical mapping by pulsed-field gel electrophoresis has established that Scn2a and Scn3a (genes encoding type II and type III sodium channel alpha-subunit isoforms) are physically linked and are separated by a maximum distance of 600 kb. The segregation of restriction fragment length variations in backcross progeny of a Mus musculus and Mus spretus mating indicates that Scn 1 a (gene encoding the type I sodium channel alpha subunit) and Scn2a are tightly linked and are separated by a distance of 0.7 cM. Linkage analysis in backcross and recombinant inbred (BXD and AKXD) strains of mice localized the three sodium channel genes to the proximal segment of mouse chromosome 2 and suggested the probable gene order centromere-Hc-Neb-Pmv7-Scn2a/Scn3a-Scn1a-Mpmv 14. These results indicate that the three isoforms of the brain sodium channel alpha-subunit are encoded by three distinct genes that share a common ancestral origin.

Abnormalities, Multiple↗
PubMed1991

Co-localization and polarized distribution of Na,K-ATPase alpha 3 and beta 2 subunits in photoreceptor cells.

Na,K-ATPase plays a central role in the visual sensitivity of photoreceptors by driving the dark current of vision. The alpha 3 and beta 2 isoforms of Na,K-ATPase were previously shown to be the major alpha and beta subunit mRNAs expressed in photoreceptors. Here we compared the distribution of beta-subunits of the enzyme in the retina and kidney, using electron microscopic immunocytochemistry with specific antibodies against alpha 3, beta 1, and beta 2 isoforms as well as with an antibody (Ax2) that binds to alpha 2 and/or alpha 3 isoforms. Both the alpha 3 and beta 2 isoforms were localized to photoreceptor inner segments at highest labeling density between the base of the connecting cilium and the outer limiting membrane (OLM). Quantitative analysis of Ax2 antibody binding to alpha 3 revealed a significant decrease in labeling density below the OLM and above the base of the connecting cilium. Although the beta 2-subunit has been reported to have adhesive functions in glial cells in cerebellum, we detected beta 2 in the photoreceptor, a cell of neural origin, but not in the Mueller cell, the glial cell of the retina. Moreover, anti-beta 2 antibodies bound maximally to portions of photoreceptor cells not involved in cell-cell contact.

Animals↗
PubMed1990

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits.

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.

Adenosine Triphosphatases↗
PubMed1990

The Na,K-ATPase beta 2 subunit is expressed in rat brain and copurifies with Na,K-ATPase activity.

We have used a cloned fusion protein as antigen to generate an antiserum specific for the rat Na,K-ATPase beta 2 subunit. Utilizing this antiserum, we analyzed some of the structural features and tissue distribution of the beta 2 subunit. Treatment of a rat brain microsomal membrane fraction with N-glycanase F revealed that the beta 2 subunit is composed of an approximately 32 kDa core protein and at least two N-linked carbohydrate chains. The beta 2 subunit also was found to copurify with ouabain-inhibitable Na,K-ATPase activity from rat brain. Western blot analysis of rat tissue microsomes showed that beta 2 subunits were expressed in brain, pineal gland, and thymus. However, no beta 2 subunits were detected in kidney, heart, spleen, liver, mammary gland, or lung. These results suggest that the beta 2 subunit is a functional component of the rat brain Na,K-ATPase. The restricted tissue distribution of beta 2 subunits may reflect important differences in the functions of individual beta subunit isoforms.

Animals↗
PubMed1990

Expression of Na,K-ATPase alpha and beta subunit genes during preimplantation development of the mouse.

Na,K-ATPase is a plasma membrane enzyme that plays a critical role in eutherian blastocoel formation (cavitation) by pumping Na+ into the extracellular space enclosed by the trophectoderm. Previous experiments with the mouse had shown that the alpha (catalytic) subunit of the enzyme becomes detectable by immunocytochemistry in the late morula, just prior to the onset of cavitation. In the present study we have used cDNAs corresponding to three mRNA isoforms of the alpha subunit and a beta subunit to determine which genes are expressed during preimplantation development and to explore the timing of their expression. Of the three alpha subunit cDNAs tested by Northern blot hybridization with blastocyst RNA, only alpha 1 produced a hybridization signal, recognizing a single mRNA about 4 kb in length. This mRNA is relatively abundant in zygotes but barely detectable by the 2-cell stage and then accumulates steadily thereafter to reach its preimplantation maximum in blastocysts. The beta 1 cDNA detected mRNA of about 2.6-2.8 kb. This mRNA is present in zygotes but could not be detected in 2-, 4-, or 8-cell stages; it is present at a low level in late morulae and is abundant in blastocysts. The temporal profile of accumulation of beta 1 mRNA thus matches more closely than does alpha 1 the timing of appearance of the catalytic subunit. This suggests that the beta subunit may regulate production of the holoenzyme and hence the timing of cavitation.

Animals↗