ATPase mRNA Research Articles

Renal Expression of the Essential Genes Associated with Sodium Transport Following a Total Proctocolectomy in Rats

The water and electrolyte balance is regulated by the renin-angiotensin-aldosterone system in the kidney. We previously reported that the levels of circulating aldosterone dramatically increased following a total proctocolectomy in rats. However, there is no direct evidence regarding whether renal adaptation is accelerated by the induction of aldosterone-associated molecules. To explore this question, Sprague-Dawley rats underwent total proctocolectomies and then were killed 8 weeks later. We investigated the renal expression of 11beta-hydroxysteroid dehydrogenase type 2, the alpha-, beta-, and gamma-subunits of the epithelial sodium channel, and the alpha1- and beta1-subunits of Na+,K+-ATPase mRNAs because those molecules are responsible for the aldosterone specificity for mineralocorticoid receptor, amiloride-sensitive sodium absorption, and sodium extrusion from distal tubules, respectively. A Northern blot analysis demonstrated the kidney to exhibit mRNA induction for all of these molecules, thus supporting the idea that renal adaptation following a total proctocolectomy depends, at least in part, upon the molecular induction which is principally regulated by circulating aldosterone.

754. Different Regulation of Myocardial Atrial and Ventricular Calcium Pump Activity (Ca-ATP-ase) during Cardiac Arrest

Ca++-ATP-ase activity is a sensitive marker of myocardial metabolic integrity and ischemia. We hypothetized that changes occur in atria (A) and ventricle (V) Ca++ATP-ase mRNA expression of this gene during cardiac arrest protected by continuous warm blood cardioplegia (CWBCP).

Effects of amphotericin B on ion transport proteins in airway epithelial cells

Topical intranasal application of the antifungal Amphotericin B (AmphoB) has been shown as an effective medical treatment of chronic rhinosinusitis. Because this antibiotic forms channels in lipid membranes, we considered the possibility that it affects the properties and/or cell surface expression of ion channels/pumps, and consequently transepithelial ion transport. Human nasal epithelial cells were exposed apically to AmphoB (50 microM) for 4 h, 5 days (4 h daily), and 4 weeks (4 h daily, 5 days weekly) and allowed to recover for 18-48 h. AmphoB significantly reduced transepithelial potential difference, short-circuit current, and the amiloride-sensitive current. This was not due to generalized cellular toxicity as judged from normal transepithelial resistance and mitochondrial activity, but was related to inhibitory effects of AmphoB on ion transport proteins. Thus, cells exposed to AmphoB for 4 h showed decreased apical epithelial sodium channels (ENaC) activity with no change in basolateral Na(+)K(+)-ATPase activity and K(+) conductance, and reduced amount of alphaENaC, alpha1-Na(+)K(+)-ATPase, and NKCC1 proteins at the cell membrane, but no change in mRNA levels. After a 5-day treatment, there was a significant decrease in Na(+)K(+)-ATPase activity. After a 4-week treatment, a decrease in basolateral K(+) conductance and in alphaENaC and alpha1-Na(+)K(+)-ATPase mRNA levels was also observed. These findings may reflect a feedback mechanism aimed to limit cellular Na(+) overload and K(+) depletion subsequently to formation of AmphoB pores in the cell membrane. Thus, the decreased Na(+) absorption induced by AmphoB resulted from reduced cell surface expression of the ENaC, Na(+)K(+)-ATPase pump and NKCC1 and not from direct inhibition of their activities.

Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

We investigated whether depressed muscle Na(+)-K(+)-ATPase activity with exercise reflected a loss of Na(+)-K(+)-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na(+)-K(+)-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at approximately 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na(+)-K(+)-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na(+)-K(+)-ATPase content via [(3)H]ouabain binding sites, and Na(+)-K(+)-ATPase alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, beta(2)- and beta(3)-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [(3)H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated alpha(1)-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Delta3-O-MFPase(rest-fatigue)) (r = -0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) alpha(1)-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Delta3-O-MFPase(rest-fatigue) (r = -0.56, P = 0.08). Exercise elevated alpha(2)-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Delta3-O-MFPase(rest-fatigue) (r = -0.60, P = 0.05). The average postexercise alpha(2)-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Delta3-O-MFPase(rest-fatigue) (r = -0.68, P < 0.05). Nonsignificant correlations were found between %Delta3-O-MFPase(rest-fatigue) and other isoforms. Thus acute exercise transiently decreased Na(+)-K(+)-ATPase activity, which was correlated with increased Na(+)-K(+)-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na(+)-K(+)-ATPase activity with exercise.

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa

BackgroundThe intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.MethodsPseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.ResultsThe infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.ConclusionsThese results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.

Proposed Function of the Accumulation of Plasma Membrane-Type Ca 2+ -ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca2+-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca2+-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca2+-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined.

An interdisciplinary approach to assess the functional diversity of free-living microscopic eukaryotes

Free-living microbes are arguably ubiquitously dispersed. Molecular phylogeographies have been used to assess microbial distributions but can be difficult to interpret. Combinations of molecular and ecophysiological data have, however, proven useful in assessing the influence of dis- persal, local adaptation and historical contingency on distributions. A logical extension of this approach is to develop molecular markers for ecophysiological traits, which would allow the assess- ment of adaptations in large numbers of environmental isolates without requiring extensive cultur- ing. To assess this approach, we compared enzyme activity and gene expression of Na + /K + ATPase in response to salinity, and compared Na + /K + ATPase mRNA sequences, in 3 Brachionus plicatilis (Rotifera) sibling species. B. plicatilis siblings display different salinity tolerances, which in turn influence their distributions. Na + /K + ATPase is an important component of salinity tolerance and a potentially useful marker for ecophysiological variation. In all cases, Na + /K + ATPase enzyme activity and gene expression increased in response to salinity (5 to 50‰) and paralleled growth rate differ- ences: highest enzyme activity/gene expression occurred in the sibling species that displayed high- est growth. However, sequence variation in Na + /K + ATPase mRNAs was minor (~4%) and did not match gene expression patterns; thus, differences between siblings in Na + /K + ATPase occurred as a result of differential expression of highly similar gene transcripts. While we were not able to develop functional molecular markers for salinity tolerance in B. plicatilis, we highlight that the application of phylogenetic and functional markers will be a powerful tool for assessing the distributions of free- living microorganisms.

Cysteamine increases expression and activity of H+-K+-ATPase of gastric mucosal cells in weaning piglets

To determine the in vivo and in vivo effects of cysteamine (CS) on expression and activity of H(+)-K(+)-ATPase of gastric mucosal cells in weaning piglets. Eighteen litters of newborn Xinhuai piglets were employed in the in vivo experiment and allocated to control and treatment groups. From 12 d of age (D12), piglets in control group were fed basal diet, while the treatment group received basal diet supplemented with 120 mg/kg CS. Piglets were weaned on D35 in both groups. Six piglets from each group (n = 6) were slaughtered on D28 (one week before weaning), D35 (weaning), D36.5, D38, D42, and D45 (36 h, 72 h, one week and 10 d after weaning), respectively. Semi-quantitative RT-PCR was performed to determine the levels of H(+)-K(+)-ATPase mRNA in gastric mucosa. H(+)-K(+)-ATPase activity in gastric mucosa homogenate was also determined. Gastric mucosal epithelial cells from piglets through primary cultures were used to further elucidate the effect of CS on expression and activity of H(+)-K(+)-ATPase in vivo. Cells were treated for 20 h with 0.001, 0.01, and 0.1 mg/mL of CS (n = 4), respectively. The mRNA expression of H(+)-K(+)-ATPase and somatostatin (SS) as well as the H(+)-K(+)-ATPase activity were determined. in vivo, both mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of control group exhibited a trend to increase from D28 to D45, reaching a peak on D45, but did not show significant age differences. Furthermore, neither the mRNA expression nor the activity of H(+)-K(+)-ATPase was affected significantly by weaning. CS increased the mRNA expression of H(+)-K(+)-ATPase by 73%, 53%, 30% and 39% on D28 (P = 0.014), D35 (P = 0.017), D42 (P = 0.013) and D45 (P = 0.046), respectively. In accordance with the mRNA expression, H(+)-K(+)-ATPase activities were significantly higher in treatment group than in control group on D35 (P = 0.043) and D45 (P = 0.040). In vivo, CS exhibited a dose-dependent effect on mRNA expression and activity of H+-K+-ATPase. Both H(+)-K(+)-ATPase mRNA expression and activity in gastric mucosal epithelial cells were significantly elevated after 20 h of exposure to the moderate (H(+)-K(+)-ATPase expression: P=0.03; H(+)-K(+)-ATPase activity: P = 0.014) and high concentrations (H(+)-K(+)-ATPase expression: P=0.017; H(+)-K(+)-ATPase activity: P = 0.022) of CS. Significant increases in SS mRNA expression were observed to accompany the elevation of H(+)-K(+)-ATPase expression and activity induced by the moderate (P = 0.024) and high concentrations (P = 0.022) of CS. Low concentration of CS exerted no effects either on expression and activity of H(+)-K(+)-ATPase or on SS mRNA expression in cultured gastric mucosal epithelial cells. No significant changes are observed in mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of piglets around weaning from D28 to D45. CS increases expression and activity of gastric H(+)-K(+)-ATPase in vivo and in vivo. SS is involved in mediating the effect of CS on gastric H(+)-K(+)-ATPase expression and activity in weaning piglets.

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.

Increased cardiac workload by closure of the ductus arteriosus leads to hypertrophy and apoptosis rather than to hyperplasia in the late fetal period.

It is generally thought that adult mammalian cardiomyocytes compensate for an increased workload by hypertrophy, whereas fetal myocardium grows by cellular proliferation. We analyzed the response of late-fetal rat hearts upon an increased workload imposed by premature constriction of the ductus arteriosus with indomethacin. Initially the fetal heart responds by proliferative growth, as both wet weight and labeling index (bromodeoxyuridine incorporation) of the ventricles increased, whereas neither a change in the fibroblast fraction, ploidy and nucleation in the ventricles is observed. However, this hyperplastic growth is abrogated by a subsequent burst in apoptosis and followed by a hypertrophic response as based on a decrease in DNA and increase in both RNA and protein concentration. This hypertrophic growth was accompanied by a 1.4-fold increase in the volume of the cardiomyocytes. Changes in the molecular phenotype characteristic of pressure-overload hypertrophic growth accompany the process. Thus, the levels of expression of beta-myosin heavy chain and atrial natriuretic factor mRNA increased, of sarcoplasmic/endoplasmic reticulum ATPase (SERCA2) mRNA decreased, and of alpha-myosin heavy chain, phospholamban, and calsequestrin mRNA did not change. In situ hybridization showed that the pattern of mRNA expression changed first in the right ventricular wall and subsequently in the left ventricular free wall as well. It is concluded that pressure-overload imposed on the late-fetal heart induces limited proliferative growth complemented by extensive hypertrophic growth.

17-$beta; Estradiol and 4-nonylphenol delay smolt development and downstream migration in Atlantic salmon, Salmo salar

The effect of 17-β estradiol (E2) and 4-nonylphenol (4-NP) on smoltification and downstream migration of Atlantic salmon was studied in an integrated laboratory and field study. In a stock of hatchery-raised 1-year-old salmon, smoltification progressed from February until late May as judged by increased gill Na + ,K + -ATPase activity and 24 h sea water (SW)-tolerance. Starting late March, three groups of 150 fish were each given 6 serial injections over 20 days of 2 μg/g body weight E2, 120 μg/g 4-NP dissolved in peanut oil or peanut oil (4 μl/g) as control. After the last injection, all fish were individually tagged (Passive Integrated Transponder tags) and a non-lethal gill biopsy was taken. Two days later (8 April), 100 fish per group were transported to the field site and released into a small stream. Smolt migration was registered by measuring arrival time at a trap downstream of the release site. Serum vitellogenin levels increased several-fold in both male and female E2- and 4-NP-treated fish. Overall, E2- and 4-NP-treatment impaired smolting as judged by elevated condition factor, reduced gill Na + ,K + -ATPase activity and α-subunit Na + ,K + -ATPase mRNA level, reduced muscle water content and increased mortality following 24 h SW-challenge. After release, control fish initiated downstream migration immediately, with 50% of the total number of migrants appearing in the trap within 10 days. E2- and 4-NP-treated fish appeared in the trap with a delay in comparison to controls of 6 and 8 days, respectively. After the smolt run, no fish were registered by electro-fishing upstream of the trap. The total number of fish reaching the trap and thus post-release survival was in the order control (81%), E2 (53%), 4-NP (12%). Representatives from all treatment groups held under simulated natural conditions in the laboratory survived 100% through the migration period, suggesting that a combination of behavioural and in-stream factors (predation by herons) may contribute to the differential mortality. The study indicates that short-term exposure to natural and environmental estrogens may impair smolt development and survival and delay subsequent downstream migration in Atlantic salmon.

17-β Estradiol and 4-nonylphenol delay smolt development and downstream migration in Atlantic salmon, Salmo salar

The effect of 17-β estradiol (E2) and 4-nonylphenol (4-NP) on smoltification and downstream migration of Atlantic salmon was studied in an integrated laboratory and field study. In a stock of hatchery-raised 1-year-old salmon, smoltification progressed from February until late May as judged by increased gill Na +,K +-ATPase activity and 24 h sea water (SW)-tolerance. Starting late March, three groups of 150 fish were each given 6 serial injections over 20 days of 2 μg/g body weight E2, 120 μg/g 4-NP dissolved in peanut oil or peanut oil (4 μl/g) as control. After the last injection, all fish were individually tagged (Passive Integrated Transponder tags) and a non-lethal gill biopsy was taken. Two days later (8 April), 100 fish per group were transported to the field site and released into a small stream. Smolt migration was registered by measuring arrival time at a trap downstream of the release site. Serum vitellogenin levels increased several-fold in both male and female E2- and 4-NP-treated fish. Overall, E2- and 4-NP-treatment impaired smolting as judged by elevated condition factor, reduced gill Na +,K +-ATPase activity and α-subunit Na +,K +-ATPase mRNA level, reduced muscle water content and increased mortality following 24 h SW-challenge. After release, control fish initiated downstream migration immediately, with 50% of the total number of migrants appearing in the trap within 10 days. E2- and 4-NP-treated fish appeared in the trap with a delay in comparison to controls of 6 and 8 days, respectively. After the smolt run, no fish were registered by electro-fishing upstream of the trap. The total number of fish reaching the trap and thus post-release survival was in the order control (81%), E2 (53%), 4-NP (12%). Representatives from all treatment groups held under simulated natural conditions in the laboratory survived 100% through the migration period, suggesting that a combination of behavioural and in-stream factors (predation by herons) may contribute to the differential mortality. The study indicates that short-term exposure to natural and environmental estrogens may impair smolt development and survival and delay subsequent downstream migration in Atlantic salmon.

Differential gene expression in infarct scar and viable myocardium from rat heart following coronary ligation.

Post-myocardial infarction (MI) remodeling of cardiac myocytes and the myocardial interstitium results in alteration of gross ventricular geometry and ventricular dysfunction. To investigate the mechanisms of the remodeling process of the heart after large MI, the expression of various genes in viable left ventricle and infarct scar tissue were examined at 16 weeks post-MI. Steady-state expression of Na(+)-K+ ATPase alpha-1 and -2, phospholamban (PLB), alpha-myosin heavy chain (alpha-MHC), ryanodine receptor (Rya) and Ca2+ ATPase (Serca2) mRNAs were decreased in the infarct scar vs noninfarcted sham-operated controls (P < 0.05). On the other hand, Gialpha2 and beta-MHC mRNAs were upregulated (P < 0.05, respectively) in the infarct scar whereas Na(+)-K+ ATPase-beta, Na(+)-Ca2+ exchanger and Gs mRNAs were not altered vs control values. In viable left ventricle, the alpha-1 subunit of Na(+)-K+ ATPase, alpha-3, beta-isoforms, Rya, beta-MHC, Gialpha2, Gs and Na(+)-Ca2+ exchanger were significantly elevated while expression of the alpha-2 subunit of Na(+)-K+ ATPase, PLB and Serca2 were significantly decreased compared to controls. Expression of CK2alpha mRNA was elevated in noninfarcted heart (145 +/- 15%) and diminished in the infarct scar (66 +/- 13%) vs controls. Expression of beta-MHC mRNA was elevated in both viable and infarct scar tissues of experimental hearts (140 +/- 31% and 183 +/- 30% vs. controls, respectively). These results suggest that cardiac genes in the infarcted tissue and viable left ventricle following MI are differentially regulated.

The gill and homeostasis: transport under stress.

in his recent cannon lecture, Cowley ([3][1]) pointed out the parallels between Cannon's time early in the last century and our own, i.e., the challenge facing us now, as then, is that of “systems integration” and understanding how organisms actually achieve homeostasis. We have begun to

Do untranslated introns control Ca 2+-ATPase isoform dependence on CaM, found in TN and PM?

Transcript splicing characterization of tomato Ca 2+-ATPase ( LCA1 gene) mRNA indicates that two main transcripts are differentiated in the 3 ′ terminal region. One of them contains a sequence of about 90 bp that could correspond to an untranslated intron that displays sequence homology to calmodulin-binding regions. Calmodulin-binding experiments demonstrate that only one of the two isoforms encoded by LCA1 binds to calmodulin. Since the M w calculated for this peptide is 3.7 kDa, it is suggested that the presence of this intron is accounted for by the difference in the sizes of the two 116- and 120-kDa isoforms, and it determines calmodulin regulation. This represents a new strategy for a single gene to produce two isoforms that are localized differently (TN and PM), and which are either dependent on or independent of the calmodulin, which in turn is either regulated by the presence or by the absence of a 90 bp untranslated intron.

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells.

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.

The age-associated decline in the intestinal uptake of glucose is not accompanied by changes in the mRNA or protein abundance of SGLT1

Studies performed using human and animal models offer conflicting results regarding the effect of age on nutrient absorption. The objectives of this study were to determine (1) the effects of aging on the in vitro uptake of glucose in rats; and (2) the molecular mechanisms of these age-associated changes. Male Fischer 344 rats aged 1, 9 and 24 months were fed a standard laboratory diet (PMI # 5001). The uptake of 14 C -labelled d-glucose was determined in vitro using the intestinal sheet method. Northern blotting, Western blotting and immunohistochemistry were used to determine the effects of age on the BBM sodium-dependent glucose transporter, SGLT1, and the BLM Na +K +-ATPase. When expressed on the basis of intestinal weight, mucosal weight or surface area, there was a reduction in glucose uptake in the 24-month-old animals. SGLT1, GLUT2 and Na +K +-ATPase mRNA and protein abundance did not parallel the changes seen in glucose uptake. These results indicate that (1) age reduces in vitro intestinal glucose uptake in the rat; and (2) this age-associated decline in glucose uptake was not explained by alterations in SGLT1, GLUT2 or Na +K +-ATPase.

Activity and Protein Expression of Na+/K+ATPase Are Reduced in Microvillous Syncytiotrophoblast Plasma Membranes Isolated from Pregnancies Complicated by Intrauterine Growth Restriction

In contrast to classical transporting epithelia, the Na(+)/K(+) ATPase is distributed to both the microvillous membrane (MVM) and the basal membrane (BM) of the placental syncytiotrophoblast. Na(+)/K(+) ATPase is important in maintaining the electrochemical gradient for Na(+), which represents the driving force for Na(+)-coupled transport of nutrients. We hypothesized that syncytiotrophoblast Na(+)/K(+)-ATPase activity is reduced in intrauterine growth restriction (IUGR). We isolated MVM and BM from control (n = 10) and IUGR placentas (n = 11). The protein expression of Na(+)/K(+)-ATPase alpha(1)-subunit was determined by Western blotting and found to be slightly reduced in MVM isolated from IUGR (-10%; P < 0.05) placentas. Na(+)/K(+) ATPase activity was measured as the ouabain-sensitive, K(+)-dependent cleavage of the fluorescent pseudosubstrate 3-O-methylfluorescein phosphate and was reduced by 35% in MVM obtained from IUGR placentas (P < 0.02). To assess the transcriptional levels of Na(+)/K(+)-ATPase mRNA, real time PCR was used. No significant changes in steady state mRNA levels for Na(+)/K(+)-ATPase were detected. The expression of the Na(+)/K(+)-ATPase alpha(1)-subunit and Na(+)/K(+)-ATPase activity in the BM were unaffected in cases of IUGR. These data suggest that Na(+)/K(+)-ATPase activity is reduced in the MVM of placentas from IUGR pregnancies. These changes might impair the function of Na(+)-coupled transporters and contribute to the reduced growth of these fetuses.

Dexamethasone stimulates transcription of the Na+-K+-ATPase beta1 gene in adult rat lung epithelial cells.

Na+-K+-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na+-K+-ATPase activity and mRNA levels. We sought to define the mechanism of Na+-K+-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased beta1-subunit Na+-K+-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5' deletion mutants of a beta1 promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5' deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of Na+-K+-ATPase beta1 gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites.