Julio Ávila

Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na+, K+-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na+, K+-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na+, K+-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the α subunit isoformspossess considerably different kinetic properties and modes of regulationand the β subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na+, K+-ATPase isozymes. This review focuseson recent developments in Na+, K+-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa+, K+-ATPase function.

Expression of the β-subunit isoforms of the Na, K-ATpase in rat embryo tissues, inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K‐ATPase β‐subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform‐specific antisera against the human Na, K‐ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β‐isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N‐endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso‐form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β‐subunit mRNA expression (Watts AG, Sanchéz‐Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β‐isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15‐day‐old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ears semicircular ducts and in the cuboidal cells of the choroid plexus

Expression of the β1 and β2(AMOG) subunits of the Na,K-ATPase in neural tissues: Cellular and developmental distribution patterns

Abstract We have used isoform-specific antisera against the Na,K-ATPase β1 (SpETb1) and β2(AMOG) (SpETb2) subunit isoforms in order to establish their specific cellular and subcellular localization in several developmental stages of the rat central nervous system. Immunocytochemical preparations revealed β1 isoform protein in most neural cells, being predominantly located in the soma of neurons and astrocytes, with no appreciable developmental variations. In the newborn rat, β2(AMOG) immunoreactivity was present in cellular processes of astroglia and in the somas of neurons and decreasing in intensity with maturation until adulthood, where no β2 isoform was detected in neurons. The diffenential location of these isoforms, both developmentally and at the cellular level suggest a complex regulation of their genes expression and mechanisms of subcellular distribution, as well as functional differences.

Cloning and disruption of the YNR1 gene encoding the nitrate reductase apoenzyme of the yeast Hansenula polymorpha

The nitrate reductase gene (YNR1) from the yeast H. polymorpha was isolated from a lambda EMBL3 genomic DNA library. As probe a 350 bp DNA fragment synthesized by PCR from H. polymorpha cDNA was used. By DNA sequencing an ORF of 2,577 bp was found. The predicted protein has 859 amino acids and presents high identity with nitrate reductases from other organisms. Chromosomal disruption of YNR1 causes inability to grow in nitrate. Northern blot analysis showed that YNR1 expression is induced by nitrate and repressed by ammonium.

Regeneration influences expression of the Na+, K+-atpase subunit isoforms in the rat peripheral nervous system.

Neural injury triggers changes in the expression of a large number of gene families. Particularly interesting are those encoding proteins involved in the generation, propagation or restoration of electric potentials. The expression of the Na+, K+-ATPase subunit isoforms (alpha, beta and gamma) was studied in dorsal root ganglion (DRG) and sciatic nerve of the rat in normal conditions, after axotomy and during regeneration. In normal DRG, alpha1 and alpha2 are expressed in the plasma membrane of all cell types, while there is no detectable signal for alpha3 in most DRG cells. After axotomy, alpha1 and alpha2 expression decreases evenly in all cells, while there is a remarkable onset in alpha3 expression, with a peak about day 3, which gradually disappears throughout regeneration (day 7). beta1 Is restricted to the nuclear envelope and plasma membrane of neurons and satellite cells. Immediately after injury, beta1 shows a homogeneous distribution in the soma of neurons. No beta2 expression was found. Beta3 Specific immunofluorescence appears in all neurons, although it is brightest in the smallest, diminishing progressively after injury until day 3 and, thereafter, increasing in intensity, until it reaches normal levels. FXYD7 is expressed weakly in a few DRG neurons (less than 2%) and Schwann cells. It increases intensely in satellite cells immediately after axotomy, and in all cell types at day 3. Transient switching of members of the Na+, K+-ATPase isoform family elicited by axotomy suggests variations in the sodium pump isozymes with different affinities for Na+, K+ and ATP from those in intact nerve. This adaptation may be important for regeneration.

A second Zn(II)2Cys6 transcriptional factor encoded by the YNA2 gene is indispensable for the transcriptional activation of the genes involved in nitrate assimilation in the yeast Hansenula polymorpha

Nitrate assimilation genes encoding a nitrate transporter (YNT1), nitrite reductase (YNI1), a Zn(II)2Cys6 transcriptional factor involved in nitrate induction (YNA1) and the nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. A second gene, termed YNA2 (yeast nitrate assimilation), was located seven nucleotides away from the 3′ region of YNR1 gene. The cluster is flanked by an ORF encoding a protein with similarity to glutathione‐S‐transferase on the YNT1 side and an ORF with similarity to Saccharomyces cerevisiae Rad3p on the YNA2 side. The disruption of YNA2 confers the resulting null mutant strain with inability to grow in nitrate. The YNA2 gene encodes a putative protein of 618 residues bearing in the N‐terminus the consensus sequence Cys–X2–Cys–X6–Cys–X5–16–Cys–X2–Cys–X6–8–Cys characteristic of the Zn(II)2Cys6 transcriptional factors. YNA2 is therefore a member of the H. polymorpha nitrate assimilation gene cluster which is transcribed in the opposite direction to the rest of the members. Yna2p shares about 27% similarity with the H. polymorpha Yna1p Zn(II)2Cys6 transcriptional factor involved in nitrate induction. Unlike the wild‐type, the yna2::URA3 strain showed no expression of the nitrate assimilation genes when incubated in nitrate for 2 h. With regard to YNA2 expression, similar YNA2 transcript levels were observed in ammonium and in ammonium plus nitrate, but about a four‐fold higher expression was observed in nitrate. However, this induction by nitrate of the YNA2 gene was not observed in the Δyna1::URA3 strain. On the contrary, the pattern of YNA1 expression was the same in the wild‐type as in the yna2::URA3 strain, indicating that YNA2 does not affect YNA1 expression. The nucleotide sequence Accession No. for YNA2 is AJ223294. Copyright

Structure and expression of the human Na,K-ATPase β2-subunit gene

We cloned and characterized the human Na,K-ATPase b2-subunit gene. The gene encompasses over 8 kb at chromosome 17 in the human genome and is composed of seven exons. Primer extension analysis identified a major transcription initiation site 529 bases upstream of the translation start site. The 5ae-flanking region of the gene harbors a potential TATA sequence, located 94 bases upstream of the transcription initiation site and a number of potential promoter and regulatory elements, among them a Sp1 site, at position ’120. A functional Sp1 site has also been found in the rat Na,K-ATPase b2-subunit gene ( Kawakami, K., Watanabe, Y., Araki, M., Nagano, K., 1993). Sp1 binds to the adhesion molecule on glia regulatory element that functions as a positive transcription regulatory element in astrocytes. (J. Neurosci. Res. 35, 138‐146). Putative AATAAA and TG sequences were found at positions 7018 and 7068, respectively. These signals delimit the origin of the the poly(A) tail and mark the end of the sequence that completes the 3ae-UT downstream sequence of the human cDNA. An Alu repetitive sequence is located between positions 5961 and 6274. The gene is expressed as a single mRNA species, of 3.36 kb, which is present in cerebrum, cerebellum, kidney and heart, being more abundant in neural tissues. Structural analyses of this and other of the P-type ATPase b subunit genes reveal that they evolved from a common ancestor.

Angiotensin II induces apoptosis in human mural granulosa-lutein cells, but not in cumulus cells.

OBJECTIVE To test whether angiotensin II (AngII) could modulate apoptosis of human granulosa-lutein (GL) cells from gonadotropin-stimulated follicles. DESIGN In vitro assays on mural and cumulus granulosa cells. SETTING University laboratory and private IVF practice. PATIENT(S) One hundred six consecutive women undergoing 113 IVF cycles. INTERVENTION(S) Purified human GL mural or cumulus cells were cultured in serum-free media in the presence or absence of AngII with or without the AngII receptor blockers saralasin and CGP42112A. MAIN OUTCOME MEASURE(S) Detection of apoptosis using a fluorescent in situ marker for activated caspases. RESULT(S) Mural cells had approximately eightfold the amount of apoptosis compared with cumulus cells (average 0.23 vs. <0.03, respectively). With mural cells, AngII increased GL cell apoptosis versus untreated control samples (AngII 10(-)11 mol/L +6.5%; AngII 10(-9) mol/L +13.3%, and AngII 10(-7) mol/L +11.3%), an effect which was blocked by concurrent incubation with AngII receptor blockers. The AngII receptor blockers produced a significant decrease of apoptosis compared with control cultures (saralasin: 19.4%; CGP42112A: 28.9%). Neither AngII nor blockers had effect on cumulus cells. CONCLUSION(S) Preovulatory concentrations of AngII, most likely via AT2 receptors, increase apoptosis of cultured mural GL cells but have no effect on cumulus cells. Granulosa cells appear to be differentially regulated by AngII.

Expression and cellular localization of Na,K‐ATPase isoforms in the rat ventral prostate

To determine the expression and plasma membrane domain location of isoforms of Na,K‐ATPase in the rat ventral prostate.

Na,K-ATPase Isozymes in Colorectal Cancer and Liver Metastases

The goal of this study was to define Na,K-ATPase α and β subunit isoform expression and isozyme composition in colorectal cancer cells and liver metastases. The α1, α3, and β1 isoforms were the most highly expressed in tumor cells and metastases; in the plasma membrane of non-neoplastic cells and mainly in a cytoplasmic location in tumor cells. α1β1 and α3β1 isozymes found in tumor and metastatic cells exhibit the highest and lowest Na+ affinity respectively and the highest K+ affinity. Mesenchymal cell isozymes possess an intermediate Na+ affinity and a low K+ affinity. In cancer, these ions are likely to favor optimal conditions for the function of nuclear enzymes involved in mitosis, especially a high intra-nuclear K+ concentration. A major and striking finding of this study was that in liver, metastasized CRC cells express the α3β1 isozyme. Thus, the α3β1 isozyme could potentially serve as a novel exploratory biomarker of CRC metastatic cells in liver.