UNIPROT:P67775 - FACTA Search

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Query: UNIPROT:P67775 (alpha isoform)
797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the renal cortical collecting duct (CCD), mineralocorticoid hormones, like aldosterone, augment the abundance of Na/K-ATPase molecules. It has been postulated that this response involves an isoform switch of the Na/K-ATPase catalytic subunit, alpha, as the molecular basis for the differential regulation of mineralo-corticoid-induced and constitutively expressed Na/K-ATPase pools. In opposition to this attractive hypothesis, three lines of independent evidence are presented which demonstrate that the CCD exclusively expresses the alpha 1 form despite mineralocorticoid-mediated changes in functional Na/K pump density. First, aldosterone increased [3H]ouabain binding in CCD 2.5-fold without changing the ouabain dissociation constant. Second, an electrophysiological assay for pump activity revealed that aldosterone increased maximum Na/K pump current in parallel with the change in ouabain binding without altering the apparent sodium affinity. Third, Western blot analysis with alpha isoform-specific, antipeptide antibodies demonstrated that aldosterone exclusively increased the total chemical pool of the alpha 1 form of the pump without inducing other alpha subunit isoforms. In summary, aldosterone increases the abundance of Na/K-ATPase molecules in the CCD which are pharmacologically, physiologically, and chemically indistinguishable from those that are normally expressed.
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PMID:Aldosterone-mediated Na/K-ATPase expression is alpha 1 isoform specific in the renal cortical collecting duct. 822 73

Na,K-ATPase (Na,K-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na,K-ATPase alpha and beta subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels ([Na+]i) in cardiocytes using a Na(+)-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Na+]i in cardiocytes; from 12.8 +/- 0.3 to 28.8 +/- 1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three- to fourfold increase in alpha 1, alpha 2, and alpha 3 mRNA accumulation, and an approximate two-fold increase in beta 1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced alpha 1 mRNA accumulation was still observed in the Ca(2+)-free culture medium. Exposure of cardiocytes to 10 microM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in alpha 1 mRNA accumulation. The increased alpha 1 mRNA expression by 1 mM ouabain was associated with a fourfold increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1, alpha 2, and alpha 3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each alpha system. These results suggest that Na+ directly regulates Na,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na(+)-responsive elements are located within the 5'-flanking sequences of each alpha isoform gene.
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PMID:Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes. 840 40

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.
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PMID:Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats. 920 63

Using biopsies of rumen epithelium papillae a net influx of [86Rb+] was measured corresponding to a high concentration of Na+, K(+)-pumps found in [3H]ouabain-binding studies (Kristensen et al. 1995). In the present study the Na+, K(+)-ATPase in papillae homogenates is compared with purified (Na+, K+)-ATPase from different sources, immunochemically characterized with respect to the isoform of the hydrolytic alpha subunit and the concentration of pumps substantiated by a novel immunochemical method. Na+, K(+)-ATPase purified from bovine kidney was shown to contain one homogeneous high-affinity population of [3H]ouabain-binding sites (Kd 1.37 nM). The ouabain-binding capacity was 0.82 nmol (mg protein)-1. Site-directed polyclonal antibodies raised to isoform-specific sequences of the three known alpha-subunit isoforms and monoclonal alpha 1-specific antibodies were used for isoform characterization on western blots of peptides separated by SDS-polyacrylamide gel electrophoresis. All three isoforms were present in Na+, K(+)-ATPase prepared from bovine brain. The alpha isoform of bovine kidney Na+, K(+)-ATPase and of rumen epithelium homogenate appeared to be alpha 1 whereas alpha 2 and alpha 3 were undetectable. Using an alpha 1-specific antibody and 125I-labelled antimouse IgG the content of (Na+, K+)-ATPase in rumen epithelium was determined by comparison of the signal from known amount of bovine kidney Na+, K(+)-ATPase on western blots. By this method rumen epithelium was found to contain 2.6 nmol Na+, K(+)-ATPase (g wet wt)-1, i.e. a similarly high or even higher concentration than previously seen in ouabain-binding studies on biopsies.
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PMID:Isoform of Na+, K(+)-ATPase from rumen epithelium identified and quantified by immunochemical methods. 964 39

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.
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PMID:Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography. 969 Jul 35

In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na+ and upregulates the alpha-1 and alpha-2 isoform expression of Na+,K+-ATPase, and that the increase of intracellular Na+ and upregulation of the alpha-2 isoform expression are blocked by Gd3+, which blocks entry of ions (including Na+) through stretch-activated channels. The present study was designed to investigate the role of intracellular Na+ in Na+,K+-ATPase regulation by increasing intracellular Na+ with chronic ouabain treatment. In parallel experiments, we measured Na+,K+-ATPase alpha isoform expression, Na+-pump activity and intracellular Na+ in cultured aortic smooth muscle cells after treatment with two concentrations of ouabain for various time periods. Treatment with 100 nM ouabain resulted in a significant elevation in intracellular Na+ after 1 (21%) and 2 h (12%), but the value returned to baseline after 12 h. Both alpha-1 and alpha-2 subunits of Na+,K+-ATPase were significantly upregulated after 1 through 4 days. Na+-pump activity was also stimulated, and the time course of this effect closely followed protein expression. At 200 microM of ouabain, the effects on intracellular Na+, isoform expression and Na+-pump activity at earlier time points (1 h through 1 day) were similar to those with 100 nM treatment, but prolonged treatment (2 and 4 days) resulted in an accumulation of intracellular Na+ and inhibition of the isoform expression and Na+-pump activity, possibly due to general dysfunction of the cells as a result of chronic exposure to high concentrations of ouabain. We conclude that elevated intracellular Na+ can serve as a signal to mediate the alpha isoform upregulation and the regulatory process requires less than one day.
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PMID:Effect of Na+ on Na+,K+-ATPase alpha-subunit expression and Na+-pump activity in aortic smooth muscle cells. 969 12

The role of protein kinase C (PKC) in nitric oxide (NO)-mediated peripheral nerve disturbance in lipopolysaccharide (endotoxin, LPS)-treated rat was studied. The impaired Na+,K+ -ATPase activities in sciatic nerves from LPS-treated rats were prevented by aminoguanidine (NO synthase inhibitor) and corrected by PKC agonist in vitro. Using Western blot to determine PKC isoforms alpha and beta polypeptide levels in LPS-treated rat sciatic nerves, we found that alpha isoform was markedly reduced in the particulate fraction, but the beta isoform was unaffected. The alpha and beta isoforms in the cytosolic fractions were not significantly different as compared with control. This diminished particulate PKC alpha isoform was prevented by the treatment of aminoguanidine. Moreover, the motor nerve conduction velocity was significantly reduced in endotoxemic rats and corrected by aminoguanidine. These results indicate that the alteration of PKC alpha isoform in Na+,K+ -ATPase-enriched fraction of sciatic nerve may be related to the NO-mediated peripheral nerve disturbance in endotoxemic rats.
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PMID:Involvement of protein kinase C in the nitric oxide-mediated peripheral nerve disturbance in endotoxemic rats. 1002 67

Purified Na+/K+-ATPase (EC 3.6.1.37) isolated from the rectal gland of Squalus acanthias was characterized in ouabain-binding studies and with respect to isoform(s) of the alpha peptide. To avoid enzyme inactivation [3H]ouabain equilibrium binding was carried out at 20 degrees C. The heterogeneity of Na+/K+-ATPase isolated from shark rectal gland was similar in [3H]ouabain binding as previously seen in hydrolytic studies. The binding isotherms were compatible with the existence of a high-affinity (Kdis 0.69 nM) and a low-affinity (Kdis 42 nM) component of 1.46 and 0.79 nmol.(mg protein)-1, respectively. In Western blots the alpha peptide of the enzyme hybridized with an isoform-specific polyclonal antibody raised to an alpha3-specific region of the large intracellular domain of rat Na+/K+-ATPase, but not with the supposed alpha3-specific monoclonal antibody MA3-915 with its epitope near the N-terminus. Semi-quantitative analysis of the reaction of the alpha3-specific polyclonal antibody with the alpha peptide from the shark enzyme compared to the reaction with alpha peptide from rat brain enzyme indicated that this region is not exactly the same in the two species. The alpha peptide of shark enzyme was not recognized by alpha1- or alpha2-specific polyclonal antibodies, or by the alpha1-specific monoclonal antibodies 3B and F6. The large intracellular domain of Na+/K+-ATPase from shark rectal gland thus seems to be alpha3-like and no alpha isoform heterogeneity seems able to account for the heterogeneity seen in ouabain binding.
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PMID:Heterogeneity of Na+/K+-ATPase from rectal gland of Squalus acanthias is not due to alpha isoform diversity. 1008 63

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue-specific isoforms exist for each of these, alpha1, alpha2 and alpha3 and beta1, beta2 and beta3. We have proposed that an additional alpha isoform, alpha4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative alpha4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in alpha4 isoform cDNA transfected 3T3 cells. Using an alpha4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical KD values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.
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PMID:Characterization of the fourth alpha isoform of the Na,K-ATPase. 1022 50

The H+,K+-ATPases belong to the X+,K+-ATPase subfamily of P-type cation-transporting ATPases. While these H+,K+-ATPase isoforms share approximately 60%-70% amino acid identity, they exhibit discrete kinetic and pharmacological properties. The colonic alpha isoform (HKalpha2) is insensitive to Sch-28080, an inhibitor of the gastric H+,K+-ATPase, and is sensitive to high concentrations of ouabain. This profile contrasts with the sensitivities attributed to HKalpha2 in transport studies. HKalpha2 mRNA and protein abundance appear to be both site-specifically upregulated in response to chronic hypokalemia, and have been localized to the outer and inner medulla. To reconcile expressed sensitivities with those reported in vitro in isolated tubules and cells in culture, it requires transformation of the expressed insensitivity of the colonic H+,K+-ATPase to Sch-28080. Although a "unique" beta subunit has been reported recently, this beta subunit ("betac"), is identical at the amino acid level to the recently cloned beta3-Na+,K+-ATPase. Moreover, while HKalpha2 can assemble indiscriminately with any X+,K+-ATPase beta subunit, HKalpha2 has been reported to assemble stably with beta1-Na+,K+-ATPase in the renal medulla and in the distal colon. It is conceivable that subunit assembly could be tissue-specific and might respond to different physiological and pathophysiological stimuli. Recent studies have suggested that the H+,K+-ATPase is both Na+-dependent and localized to the apical membrane in the distal colon. Future studies will be needed to resolve these discrepancies by determining if a unique, yet undiscovered H+,K+-ATPase isoform exists in the kidney, or if posttranslational modifications of the alpha and/or beta-subunits could account for these functional diversities.
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PMID:Contrasting functional and regulatory profiles of the renal H+,K+-ATPases. 1051 79

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