Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning

A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.     

Differential expression of two cathepsin Es during metamorphosis-associated remodeling of the larval to adult type epithelium in Xenopus stomach

Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.     

Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.     

Cloning and expression of xP1-L,a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H⁺/K⁺-ATPase β subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.     

Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.     

The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme

The Na+/K+-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of Na+ and K+. It is comprised of at least two subunits, a large catalytic alpha subunit that mediates ATP hydrolysis and ion transport, and an ancillary beta subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the alpha subunit have been extensively studied, little is known about the participation of the beta subunit in conformational changes of the enzyme. To elucidate the role of the beta subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep beta1 subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the beta1 subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the Na+/K+-ATPase through a fluorophore-labeled beta subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the beta subunit to certain reaction steps of the Na+/K+-ATPase, which involve changes in the occupancy of the two principle conformational states, E1P and E2P. From these experiments, evidence is provided that the beta1-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in beta1 isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Expression of active alpha-3 subunit of rat brain Na+,K+-ATPase from the messenger RNA injected into Xenopus oocytes

Cloned cDNA encoding the so-far uncharacterized alpha-3 subunit of rat brain Na+,K+-ATPase (Hara et al. (1987) J. Biochem. 102, 43-58, Shull et al. (1986) Biochemistry 25, 8125-8132) was incorporated into a vector carrying the SP6 promoter. The mRNA produced in vitro was injected into Xenopus oocytes with the mRNA encoding the Na+,K+-ATPase beta subunit of Torpedo electroplax. Increased Na+,K+-ATPase activity in the oocyte membrane was observed. This newly expressed activity was inhibited by ouabain (Ki = 1.5 x 10(-7) M), suggesting that the alpha-3 subunit of rat brain Na+,K+-ATPase is a highly ouabain-sensitive catalytic subunit.     

The ontogeny of rat gastric H+/K+-ATPase

The ontogeny of rat H+/K+-ATPase was studied between foetal day 18 and neonatal day 18, using a specific monoclonal antibody (95-111 mAb). The H+/K+-ATPase content of gastric subcellular membranes was assayed and the ATPase subunits were characterized by Western blot. The epithelium density in parietal cells was measured by immunohistochemistry. H+/K+-ATPase was present in the 18-day-old foetuses and parietal cells were detected on foetal day 19. The H+/K+-ATPase concentration remained stable from foetal day 18 to neonatal day 1, while the parietal cell density increased 2.5-fold. The H+/K+-ATPase concentration increased by 2.5-fold on day 6, then remained constant up to day 18. The parietal cell density remained unchanged during this period, suggesting that the concentration increase on day 6 was due to an increase in parietal cell ATPase content. The 95-111 mAb recognized a 95 kDa single band on foetal day 18 and a doublet at all the other stages of development. Previous studies had demonstrated that acid secretion drops critically at day 12 post partum in the rat and that H+/K+-ATPase activity is lost. The present study demonstrates that the H+/K+-ATPase is, however, present on day 12.     

Origin of the gamma polypeptide of the Na+/K+-ATPase

The Na+/K+-ATPase purified from lamb kidney contains a gamma polypeptide fraction which is a collection of fragments derived from the alpha and beta polypeptides of the enzyme. This fraction has the solubility characteristics of a proteolipid and was isolated either by high performance liquid chromatography (size exclusion chromatography) in 1% sodium dodecyl sulfate or by sequential organic extraction of purified lamb kidney Na+/K+-ATPase. Formation of gamma polypeptide(s) from detergent solubilized holoenzyme was accelerated by sulfhydryl containing reagents and was unaffected by addition of inhibitors of proteolytic enzymes. Treatment of the holoenzyme with the photoaffinity reagent N-(2-nitro-4-azidophenyl)[3H]ouabain ([3H]NAP-ouabain) labeled the alpha polypeptide and the gamma polypeptide fraction but not the beta polypeptide. Amino acid sequence analysis of one gamma polypeptide preparation revealed homology of one component of this fraction with the N-terminus of the beta subunit of the Na+/K+-ATPase. Amino acid analysis of two preparations of proteolipid showed similar amino acid compositions with a peptide derived from the alpha subunit. The insolubility and complexity of the gamma polypeptide(s)/proteolipid fraction appears to preclude a conclusive sequence analysis of all components of this fraction.     

Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha1-subunit (alpha1) expressed in Xenopus oocytes, with different beta-subunits. Our results show that betaHK and beta1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha1/beta1 NK and alpha1/betaHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta-isoforms influence the protease sensitivity of AL1 and alpha1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha-subunit.     

Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells

We studied whether K+-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.     

Isoforms of Na+, K+-ATPase in human prostate; specificity of expression and apical membrane polarization

The cellular distribution of Na+, K+-ATPase subunit isoforms was mapped in the secretory epithelium of the human prostate gland by immunostaining with antibodies to the alpha and beta subunit isoforms of the enzyme. Immunolabeling of the alpha1, beta1 and beta2 isoforms was observed in the apical and lateral plasma membrane domains of prostatic epithelial cells in contrast to human kidney where the alpha1 and beta1 isoforms of Na+, K+-ATPase were localized in the basolateral membrane of both proximal and distal convoluted tubules. Using immunohistochemistry and PCR we found no evidence of Na+, K+-ATPase alpha2 and alpha3 isoform expression suggesting that prostatic Na+, K+-ATPase consists of alpha1/beta1 and alpha1/beta2 isozymes. Our immunohistochemical findings are consistent with previously proposed models placing prostatic Na+, K+-ATPase in the apical plasma membrane domain. Abundant expression of Na+, K+-ATPase in epithelial cells lining tubulo-alveoli in the human prostate gland confirms previous conclusions drawn from biochemical, pharmacological and physiological data and provides further evidence for the critical role of this enzyme in prostatic cell physiology and ion homeostasis. Na+, K+-ATPase most likely maintains an inwardly directed Na+ gradient essential for nutrient uptake and active citrate secretion by prostatic epithelial cells. Na+, K+-ATPase may also regulate lumenal Na+ and K+, major counter-ions for citrate.     

Action of palytoxin on apical H+/K+-ATPase in rat colon.

Palytoxin stimulated a cation-dependent short-circuit current (Isc) in rat distal and proximal colon in a concentration-dependent fashion when applied to the mucosal surface of the tissue. The distal colon exhibited a higher sensitivity to the toxin. The palytoxin-induced Isc was blocked by vanadate but was resistant to ouabain or scilliroside, suggesting the conversion of a vanadate-sensitive H+/K+-ATPase into an electrogenic cation transporter. Cation substitution experiments with basolaterally depolarized tissues suggested an apparent permeability of the palytoxin-induced conductance of Na+>K+>Li+. Immunohistochemical control experiments confirmed the absence of the Na+/K+-ATPase in the apical membrane. Consequently, the pore-forming action of palytoxin is not restricted to Na+/K+-ATPase but is also observed with the colonic H+/K+-ATPase.     

Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080.

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.