K-ATPase mRNA Research Articles

Increased Association of Deamidated \u03b1A-N101D with Lens membrane of transgenic \u03b1AN101D vs. wild type \u03b1A mice: potential effects on intracellular ionic imbalance and membrane disorganization

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.MethodsLenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.ResultsCompared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice.ConclusionsThe results show that an increased lens membrane association of αAN101D-−relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Dexamethasone‐stimulation of Na‐transport differs across lung epithelia

Dexamethasone (DEX) stimulates alveolar reabsorption by stimulating the expression of ENaC and Na/K‐ATPase. Nasal potentials (NP) have been used repeatedly to estimate changes in alveolar reabsorption. However, in a clinical trial on DEX‐treatment (Maggiorini AIM 2006) we found no effect of DEX on NP. Therefore we tested whether a similar DEX‐treatment of rats affects both NP and the expression of Na‐transporters in nasal epithelium, trachea, primary alveolar epithelial cells (AII), and in leukocytes. Rats were treated with DEX (2mg/kg/day; i.p.) for up to three days. NP was measured after anesthesia, and tissues were removed for mRNA preparation. In a separate series of experiments lung reabsorption was measured after fluid instillation. Total NP was increased (+2mV) after 48hDEX but the amiloride‐sensitive component was not affected. DEX did not affect. α,β,γ‐ENaC in nasal epithelium and leukocytes but increased it in trachea and AII cells (50–100%). DEX did not affect α1,β1‐Na/K‐ATPase mRNA in nasal epithelium but increased α1 mRNA in trachea (100%). Leukocyte α1 mRNA was decreased by 50%. CFTR, nedd‐4 and sgk‐1 mRNAs were not affected. DEX stimulated alveolar fluid reabsorption by increasing its amiloride‐sensitive component. These results indicate that a) NP do not reflect alveolar ion transport activity upon treatment with DEX, and that b) DEX stimulation of Na‐transport varies across lung epithelia.

Chronic pulmonary artery occlusion increases alveolar fluid clearance in rats

We had observed that pulmonary artery ligation for 14 days did not induce lung infiltration in a patient who had undergone a lobectomy for lung cancer. Our hypothesis was that long-term pulmonary artery ligation decreased lung water volume and/or increased alveolar fluid clearance. We determined the mechanism responsible for lung water balance in rats with chronic pulmonary artery occlusion for 14 days. Sprague-Dawley rats (n = 45) were used. Through a left thoracotomy, the left pulmonary artery was ligated for 14 days. Then, we measured lung water volume, alveolar fluid clearance, the effects of beta-adrenergic agonist and antagonist, mRNA expression, and protein expression in the lungs. Chronic left pulmonary artery occlusion increased both lung water volume and alveolar fluid clearance in the left lungs, but not in the right lungs with pulmonary perfusion. Neither a beta-agonist nor a beta-antagonist changed the increase in alveolar fluid clearance. Real-time polymerase chain reaction revealed an increase in alpha1-Na,K-ATPase mRNA and a decrease of beta2-adrenoreceptor mRNA, but no change in beta1-Na,K-ATPase mRNA and alpha-, beta-, gamma-epithelial sodium channel mRNA, in the left lung without pulmonary perfusion. Western blot analysis revealed an increase in alpha1-Na,K-ATPase subunit, but no change in beta1-Na,K-ATPase subunit. Chronic pulmonary artery occlusion increases alveolar fluid clearance via alpha1-Na,K-ATPase overexpression in rats.

Mechanisms responsible for acute pancreatitis‐stimulated alveolar fluid clearance capacity in rat lungs

We determined whether acute pancreatitis affected alveolar fluid clearance in rat lungs. Acute pancreatitis was induced by a retrograde injection of 5% taurocholate sodium (0.2 ml) into the common bile duct. Alveolar fluid clearance was estimated by the increase of the alveolar albumin concentration in the isolated rat lungs 4, 24, and 48 hrs after pancreatitis. Effects of endogenous catecholamine on alveolar fluid clearance and expression of sodium channel and Na,K-ATPase mRNA were determined. We found that acute pancreatitis increased epinephrine and norepinephrine levels in blood and stimulated alveolar fluid clearance by 180 and 120% at 4 and 24 hrs after pancreatitis, respectively. Prazosin (α1-antagonist), yohimbine (α 2-antagonist), and adrenalectomy, but not propranolol (β-antagonist), inhibited pancreatitis-stimulated alveolar fluid clearance in part. A combination of prazosin and yohimbine abolished the effect of pancreatitis. Pancreatitis also increased the expression of α-, β-, γ-ENaC mRNA and α1-, β 1-Na,K-ATPase mRNA. In conclusion, acute pancreatitis increases alveolar fluid clearance via α-adrenoceptors-mediated mechanism. (Grant from Kanazawa Medical University H2005-7, Grand-in-Aid for Scientific Research from MEXT Japan 17591492)

Inhibition of rat lung fluid absorption by αENaC silencing in vivo

The relative importance for ENaC during baseline and βAR-stimulated conditions has not been fully tested. We used siRNA specifically generated to αENaC and measured AFC during baseline and terbutaline-stimulated conditions in normal rat lungs. siRNA-generating pDNA against αENaC (pSi-4) or an irrelevant pDNA (pSi-0) was instilled intratracheally (100 μg/kg body wt) into the lungs of adult rats. After 24 h, AFC was measured by mass balance with and without terbutaline. In pSi-0-instilled rats, terbutaline stimulated AFC by 165%, while terbutaline-stimulation of AFC was attenuated in pSi-4-instilled rats. Baseline AFC was reduced by ~30% in pSi-4-instilled rats compared to pSi-0. We measured lung αENaC, βENaC, and α1-Na,K-ATPase mRNA and protein expression from pSi-4- and pSi-0-instilled rats. Both αENaC mRNA and protein were undetectable in pSi-4-instilled lungs, while normal in pSi-0-instilled lungs. We then isolated alveolar epithelial type II (ATII) cells 24 h after in vivo pSi-4 and pSi-0 pretreatment. In ATII cells, αENaC mRNA was undetectable, thus demonstrating that alveolar epithelial ENaC expression was attenuated. A dose-response curve for siRNA-silencing of αENaC was carried out. The dose-response curve demonstrated that, in order to reach 50% inhibition (IC50) for αENaC mRNA expression, 32 μg/kg body wt pSi-4 was needed and to reach IC50 for αENaC protein expression, 59 μg/kg body wt pSi-4 was needed. We tested for organ-, ENaC subunit-, and protein specificity and found no changes in kidney αENaC and βENaC mRNA and protein, lung βENaC, or lung α1-Na,K-ATPase expression. Thus, we provide conclusive evidence that βAR stimulation of AFC depends exclusively on ENaC, while baseline AFC seems less ENaC-dependent. (Supported by a research grant from the March of Dimes Foundation for Birth Defects (Grant No. 6-FY03-64)

Overexpression of regucalcin enhances its nuclear localization and suppressesL-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport-related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild-type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24-72 h in a medium containing either vehicle, aldosterone (10(-8) or 10(-7) M), or parathyroid hormone (PTH) (1-34) (10(-8) or 10(-7) M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K(+) channel (ROMK) mRNA expression, while it caused a remarkable decrease in L-type Ca(2+) channel and calcium-sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K-ATPase (alpha-subunit), Type II Na-Pi cotransporter (NaPi-IIa), angiotensinogen, Na(+)-Ca(2+) exchanger, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10(-8) or 10(-7) M) caused a significant increase in ENaC, Na, K-ATPase, and ROMK mRNA expressions in the wild-type cells. Those increases were weakened in the transfectants. Culture with PTH (10(-8) or 10(-7) M) significantly decreased NaPi-IIa mRNA expression in the wild-type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild-type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10(-8) or 10(-7) M) caused a significant decrease in L-type Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L-type Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.

Changes in brain Na, K-ATPase isoform expression and enzymatic activity after aortic constriction

The objective of the present study was to test the hypothesis that brain Na, K-ATPase expression and/or activity is altered following an increase in blood pressure produced by constriction of the abdominal aorta just proximal to the renal arteries. A suprarenal constriction (SRC) was made to conform to the diameter of a 19-gauge (19-G) or 20-gauge (20-G) needle, while in a sham-operated group (Sham) the aorta was exposed surgically but not constricted. Within 1 week of SRC, mean arterial pressure was increased and remained elevated at 4 weeks post surgery. At 1 week, whole-brain Na, K-ATPase mRNA levels were depressed for all isoforms (α1∼β1>α2>α3). No changes were observed in the hypothalamus. At 4 weeks, the mRNA levels of all α isoforms were significantly increased in the whole brain and these changes were paralleled by an increase of α2 and α3 transcript in the hypothalamus. β1 mRNA expression was increased in the hypothalamus only. The α-isoform protein expression generally changed in the same direction as mRNA changes at both 1 and 4 weeks, as did α1 enzyme activity at 1 week and the combined α2/α3 enzyme activities at 4 weeks. Since inhibition of brain Na, K-ATPase increases sympathetic nervous system (SNS) activity and blood pressure, the decreases in brain Na, K-ATPase expression and activity at 1 week post SRC may contribute to the hypertension during its developmental phase, while the increase in the α2/α3 brain expression and activity at 4 weeks may be a compensatory response to established hypertension.

Gastrin interferes with the differentiation of gastric pit cells and parietal cells.

Gastrin is known to have stimulatory effects on gastric mucosa; however, long-term effect of gastrin stimulation is not well known. To investigate the long-term effect of hypergastrinaemia, we established hypergastrinaemic transgenic mice by introducing a mutated human gastrin gene. Homozygously transgene-expressing mice showed serum gastrin levels of more than 600 pg/mL. Neither progastrin nor glycine-extended gastrin titre elevation were observed in hypergastrinaemic transgenic mice. Stomachs from the 30-35-week-old transgenic mice were 30-50% heavier and their mucosa were markedly thicker than those of the controls. The hypertrophic gastric mucosa of hypergastrinaemic transgenic mice consisted of elongated pits with widespread proliferative zones, and comprised depleted glandular regions. In situ hybridization study indicated that expression of H, K-ATPase mRNA in parietal cells of hypergastrinaemic transgenic mice was markedly decreased. By gastrin binding assay in vivo, specific gastrin binding sites were observed in the mid-glandular region of hypergastrinaemic transgenic mice that consisted mainly of prepit cells. These results suggest that long-term stimulation of gastrin increases the expression of CCK-B/gastrin receptors in the less-differentiated pit cells that are the main component of elongated gastric units, and lessens the well-differentiated characteristics of parietal cells.

Aldosterone directly induces Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of rat.

The change of blood pressure and the induction of Na, K-ATPase alpha 1-subunit mRNA have been investigated in the renal cortex of aldosterone-treated hypertensive rat. The increase of blood pressure by aldosterone-treatment for 25 days was decreased by the treatment of amiloride or spironolactone. The level of Na, K-ATPase alpha 1-subunit mRNA of the renal cortex in aldosterone-treated rat was increased than that in the control, and its increase was repressed by treatment of spironolactone, but not altered by the treatment of amiloride. This result suggests that the increase of Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of aldosterone-treated hypertensive rat may be related with the direct induction of Na, K-ATPase mRNA without the increase of Na-traffic through Na-channel.

Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells.

Decrease in alveolar oxygen tension may induce acute lung injury with pulmonary edema. We investigated whether, in alveolar epithelial cells, expression and activity of epithelial sodium (Na) channels and Na,K-adenosine triphosphatase, the major components of transepithelial Na transport, were regulated by hypoxia. Exposure of cultured rat alveolar cells to 3% and 0% O2 for 18 h reduced Na channel activity estimated by amiloride-sensitive 22Na influx by 32% and 67%, respectively, whereas 5% O2 was without effect. The decrease in Na channel activity induced by 0% O2 was time-dependent, significant at 3 h of exposure and maximal at 12 and 18 h. It was associated with a time-dependent decline in the amount of mRNAs encoding the alpha-, beta-, and gamma-subunits of the rat epithelial Na channel (rENaC) and with a 42% decrease in alpha-rENaC protein synthesis as evaluated by immunoprecipitation after 18 h of exposure. The 0% O2 hypoxia also caused a time-dependent decrease in (1) ouabain-sensitive 86Rubidium influx in intact cells, (2) the maximal velocity of Na,K-ATPase on crude homogenates, and (3) alpha1- and beta1-Na,K-ATPase mRNA levels. Levels of rENaC and alpha1-Na,K-ATPase mRNA returned to control values within 48 h of reoxygenation, and this was associated with complete functional recovery. We conclude that hypoxia induced a downregulation of expression and activity of epithelial Na channels and Na,K-ATPase in alveolar cells. Subsequent decrease in Na reabsorption by alveolar epithelium could participate in the maintenance of hypoxia-induced alveolar edema.

Na, K-ATPase isoform gene expression in normal and hypertrophied dog heart.

The catalytic alpha subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms; the expression of these isoforms is tissue-specific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression. hypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immunofluorescence, and 3H-ouabain binding. RNA blotting indicated that the alpha 3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed alpha 1 and alpha 3 protein, but no detectable alpha 2; immunoreactive alpha 1 and alpha 3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all alpha isoform mRNA's in hypertrophied dog LV, resulting in a greater proportion of alpha 1. The expression level of the alpha 3 isoform mRNA and protein was lower in hypertrophied hearts. These results indicate a greater proportion of alpha 1 isoform pumps in experimental canine hypertrophy. Thus, shifts in NA, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.

Decreased Na,K-ATPase gene expression in cardiomyopathic hamster hearts

We studied Na, K-ATPase mRNA expression in cardiomyopathic (Bio 14.6) and normal (F1b) Syrian hamster ventricles. In Northern blot analysis, Na,K-ATPase α1, α2, α3 and β1 isoform mRNAs were detected in 3-week-old Bio 14.6 and F1b hamster venticles. We then investigated the expression of α1 subunit mRNA in Bio 14.6 hamster ventricles at the ages of 3 weeks prehypertrophic and 30 weeks hypertrophic, and in age-matched F1b hamster ventricles. The α1 subunit mRNA levels in Bio 14.6 hamster ventricles were approximately 50% lower than those in F1b hamster ventricles at both 3 and 30 weeks of age. Na,K-ATPase activity measured in membrane fractions from the venticles of 3-week-old Bio 14.6 hamsters was also approximately 20% lower than that of F1b hamsters, suggesting that the differences in the mRNA level were associated with the differences in the protein level. We conclude that Na,K-ATPase mRNA expression and enzyme activity are significantly decreased in the hearts of Bio 14.6 hamsters even before the onset of hypertrophy and cardiomyopathy, suggesting that the altered expression of Na,K-ATPase gene is an early event in the pathogenesis of cardiomyopathy in this animal model.

Altered Na, K-ATPase mRNA Expression in Spontaneously Hypertensive Rats.

We investigated the expression of Na, K-ATPase α and β isoform mRNAs in spontaneously hypertensive rat (SHR) hearts, aortae and kidneys, compared with findings in normotensive Wistar-Kyoto rat (WKY), to obtain insight into the role of these isoforms in the development of hypertension in SHR. We used 4-, 8-, and 16-week-old SHR and WKY. Systolic blood pressure was recorded by tail sphygmomanometry. The expression of Na, K-ATPase α and β isoform mRNAs was analyzed by Northern blot hybridization, using isoform specific cDNA probes. In the ventricles, pre-hypertensive 4-week-old SHR showed a significant 3 to 4-fold increase in the α1 and α2 isoform mRNA accumulation compared with age-matched WKY. The α3 isoform mRNA expression was detected only in 4-week-old SHR hearts. The pattern of β1 mRNA expression resembled that of α1 or α2 mRNAs, and a significant 7-fold increase was observed in 4-week-old SHR compared with age-matched WKY. In the aortae, the α1 and α2 isoform mRNAs were expressed, and 4-week-old SHR exhibited a significant 5-fold increase in the α1 and α2 isoform mRNA levels compared with age-matched WKY. In the kidneys, only the α1 isoform mRNA was expressed, and that of SHR showed a significant 2-fold increase compared with age-matched WKY. We conclude that the expression of Na, K-ATPase α and β isoform mRNAs is significantly increased in the hearts, aortae and kidneys of SHR before the onset of hypertension. These findings suggest that, in this model, the altered expression of Na, K-ATPase gene is an early, if not a primary, event in the development of hypertension. (Hypertension Res 1993; 16: 269-274)