Different expression and activity of the α1 and α4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

K, Wagoner; Sánchez, Gladis; An, Nguyen Hai; Gc, Enders; Blanco, Gustavo

doi:10.1530/rep.1.00806

Cited by 62 publications

(120 citation statements)

References 75 publications

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“…cDNA was generated by reverse transcription using the SuperScript First-Strand Synthesis System (Invitrogen) and oligo (dT) primers as described previously (33). The resulting first-strand cDNA was amplified using Na,K-ATPase isoform specific primers and under PCR conditions that ensure no cross-reactivity among the Na,K-ATPase isoforms.…”

Section: Reverse Transcriptase-pcr Analysismentioning

confidence: 99%

“…Cell proteins (30 g) from homogenates of NHK and ADPKD cells that were grown to confluence on filter supports or plastic were analyzed by 8% SDS-PAGE for the ␣ and ␤ isoforms or 16% tricine gels for the ␥ subunit and blotted onto nitrocellulose membranes (Osmonics, Minnetonka, MN) as described (33,34). Primary antibodies that are specific for each Na,K-ATPase isoform were incubated overnight at 4°C.…”

Section: Immunoblot Analysis Of Nak-atpase Isoformsmentioning

confidence: 99%

“…Dose-response relations for the ouabain inhibition of Na,K-ATPase activity were fitted by Table 1. Characteristics of the primers that were used for RT-PCR analysis of Na,K-ATPase isoforms in normal and cystic human kidney epithelial cells equations assuming the presence of one or two enzyme populations with different affinity for ouabain as described previously (33). The validity of using a two versus a single component regression model for ouabain binding was statistically supported by applying the Snedecor F test (36).…”

Section: Statistical Analysesmentioning

confidence: 99%

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Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Nguyen¹,

Wallace²,

Blanco³

2007

Journal of the American Society of Nephrology

108

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In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the ␣1 and ␤1 subunits. The ␥ variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

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Section: Reverse Transcriptase-pcr Analysismentioning

confidence: 99%

Section: Immunoblot Analysis Of Nak-atpase Isoformsmentioning

confidence: 99%

Section: Statistical Analysesmentioning

confidence: 99%

See 1 more Smart Citation

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Nguyen¹,

Wallace²,

Blanco³

2007

Journal of the American Society of Nephrology

108

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“…The α4 polypeptide is the Na,K-ATPase isoform that is least abundantly expressed across the body. It has been found in male germ cells of the testis, where it predominates in the sperm flagellum [8,9]. The α4 isoform has particular affinities for Na + , K + and ATP [10,11] and is important for maintaining membrane potential, pH and the intracellular Na + and Ca +2 levels of sperm [12].…”

Section: Introductionmentioning

confidence: 99%

“…This shows that other independent experimental approaches will be important to precisely understand the tissue and cell type specific distribution of Na,K-ATPase α4. In addition, while studies of RNA, protein and activity have helped follow the pattern of expression of the α4 isoform [8,9], a complete characterization of the expression of α4 during different stages of development has not yet been accomplished. Comprehending α4 expression pattern during development is an important step in deciphering the biological relevance of this Na,K-ATPase isoform in male reproduction and fertility.…”

Section: Introductionmentioning

confidence: 99%

Green fluorescence protein driven by the Na,K-ATPase α4 isoform promoter is expressed only in male germ cells of mouse testis

McDermott

Sánchez

Chennathukuzhi

et al. 2012

J Assist Reprod Genet

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Purpose Expression of the Na,K-ATPase α4 isoform is required for sperm motility and fertility and is controlled by the Atp1a4 promoter. Here, we have investigated the specific tissue, cell type and developmental regulation of expression mediated by the Atp1a4 promoter. Methods We have inserted the green fluorescent protein (GFP), downstream of the endogenous Atp1a4 promoter, in place of the Na,K-ATPase α4 gene, and used it as a marker for α4 expression in mice (Atp1a4 null(GFP) mice). Results Replacement of α4 by GFP completely disrupted α4 expression and activity, produced sperm morphological and functional abnormalities, and caused infertility of Atp1a4 null (GFP) male mice. Immunoblot analysis of Atp1a4 null (GFP) mouse tissues showed GFP expression in testis. This particular expression pattern was found in adult, but not in mouse embryos or in 7, 18 day old mice. In agreement with expression of GFP, adult Atp1a4 null(GFP) mouse testis displayed the typical fluorescence of GFP. Immunocytochemistry of testis identified GFP in more differentiated male germ cells, but not in spermatogonia, Leydig or Sertoli cells. Further analysis, using immunoblot of fluorescently sorted testis cells with cell specific markers, detected GFP only in spermatocytes, spermatids and spermatozoa. While epididymis showed GFP expression, this was confined to the spermatozoa within the epididymal tubules.Conclusions Our results show that the Atp1a4 promoter drives GFP expression exclusively in male germ cells of the testis, where it restricts it to post-meiotic stages of spermatogenesis. These findings highlight the exquisite spatial and temporal control of expression exerted by the Atp1a4 promoter on Na, K-ATPase α4, which is particularly well suited to fulfill the special functions of spermatozoa.

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The transcription factor CREMτ and cAMP regulate promoter activity of the Na,K‐ATPase α4 isoform

Rodova

Nguyen

Blanco

2006

Molecular Reproduction Devel

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The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.

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Different expression and activity of the α1 and α4 isoforms of the Na,K-ATPase during rat male germ cell ontogeny

Cited by 62 publications

References 75 publications

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

Green fluorescence protein driven by the Na,K-ATPase α4 isoform promoter is expressed only in male germ cells of mouse testis

The transcription factor CREMτ and cAMP regulate promoter activity of the Na,K‐ATPase α4 isoform

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